UMA COLETA DE SANGUE NO MEU DENTISTA?

Logo você irá fazer uma cirurgia na nossa clínica.

Pode ser que seu dentista fará uma coleta de sangue no dia da cirurgia.

Porque? Seu sangue será tirado logo antes da cirurgia e será posteriormente
centrifugado. Seu dentista irá retirar um pequeno concentrado (um tecido de
fibrina, muito rico em fatores de crescimento).

Para que? Esses tecidos irão protejer suas feridas, promover cicatrização, redu-
zir a dor pós-operatória, diminuir o risco de infeções e acelerar a regeneração
óssea.

Vocês irão coletar muito sangue? No, coletaremos a mesma quantidade ou

menos dos exames de sangue em laboratórios. Não haverá sensação de cansaço
após a cirurgia.

Existem contra indicações? NÃO! Todos os pacientes podem se beneficiar des-

ta técnica. Não existem contra-indicações. Pelo contrário, problemas de saúde
podem fazer desse procedimento essencial para alcançar a cicatrização.

Nada pode substituir seu sangue!

Existem bio materiais humanos, animais e sintéticos, que podem ser adicio-
nados ao seu sangue. Mas somente o seu sangue pode trazer tão eficazmente e
naturalmente todos os fatores de crescimento necessários à cicatrização. Mais
ainda, não existem riscos de rejeição do material a serem temidos, visto que ele
é seu.

Essa técnica natural requer a coleta de sangue que pode ser um pouco des-
confortável, mas irá facilitar muito o processo de recuperação e ajudará o seu
corpo a produzir osso e gengiva nos locais que estes foram reabsorvidos.

Todo o pessoal está disponível para responder suas perguntas.

1. 2. 3. 4.
CUSO DE FIBRINA RICA EM PLAQUETA

Dr. Joseph Choukroun, MD Dra. Elisa CHOUKROUN, DDS

joseph@a-prf.com elisa@a-prf.com

1. Conceito
Princípio: centrifugação de sangue sem anti-coagulantes.
PRF foi lançado no ano de 2000 pelo J. Choukroun (Choukroun J. Et al. 2001)
PRF contém plaquetas, mas também contém alta concentração de fibrina e leucócitos.
PRF é comumente chamada de L-PRF (Leucócito-PRF) pela sua formulação incisa por J.
Choukroun em 2001.
Hoje em dia, houveram modificações na velocidade de centrifugação, na força G e no tempo, que
levaram ao desenvolvimento de um PRF mais bioativo, chamado de A-PRF (Advanced - PRF),
desenvolvido pelo conceito de LSCC (Low Speed Centrifugation Concept, 2014).

Fibrina é componente principal do PRF. Descobertas de cientistas da ciência básica mostraram

que:

- A Fibrina é uma matriz extra celular provisória, totalmente derivada de fontes autogênicas;
- Fibrina contém uma concentração supra-fisiológica de fatores de crescimento humanos;
- Fibrina libera citocinas de maneira lenta e continua por 10 dias, e até mais.
2. Indicações
O uso de PRF é indicado para todos os procedimentos cirúrgicos orais:
- Preservação de alvéolo: PRF é usado como uma matriz tecidual. Preencha os alvéolos com o
máximo que você puder (Comprima os plugs com gaze ou compactador). O alvéolo deve ser
deixado aberto, sem necessidade de fechamento inicial. Suturas são feitas apenas para manter
o PRF no alvéolo. Não existe a necessidade de esperar mais de 3 meses para colocar o
implante. Na maioria dos casos não é necessário a adição de biomateriais.
- Manejo de tecido mole: PRF é usado como uma matriz tecidual. A melhor técnica é a de
tunelização de todo o arco (Tunnel Technique). Mesma regra é aplicada: Use o máximo de PRF
que conseguir.
- Enxerto ósseo: Protocolo de Sticky bone com S-PRF
Senão, corte membranas (1 ou 2) em pequenos pedaços e misture com o biomaterial. Hidrate a
mistura com o exsudato de PRF que restou no fundo do PRF BoX.
- Levantamento de seio:
‣ Proteção da Membrana de Schneider: 2 membranas colocadas inicialmente
‣ PRF misturado com biomaterial
‣ Tratamento de perfurações na Membrana de Schneider;
‣ Fechamento da janela óssea retirada;
‣ PRF pode também ser usado sozinho, mas somente com a colocação do implante
simultaneamente;
‣ Regeneração Ósseo-Guiada: PRF pode ser usado com uma membrana com efeito de
barreira para cobrir a área aumentada e melhorar a vascularização tanto do tecido duro
quanto dos tecidos moles. É ideal posicioná-la especialmente quando nenhuma reabertura é
planejada.

3. Preparação / Protocolos
๏ PRF ou L-PRF (1º Protocolo de 2001): 2700 RPM & 12min
๏ A-PRF+ (Coágulos, membranas, plugs): tubos vermelhos:
(1) Colete as amostras com os tubos (enchimento muito rápido);
(2) Coloque os tubos na centrífuga, diametralmente opostos (use as cores para ajudar)
(3) Inicie o programa A-PRF+: 1300 RPM & 14 min
(4) Ao final da centrifugação: Retire os tubos da máquina, remova a tampa & e coloque os
tubos no suporte.

Aguarde 5 minutos se os coágulos não estiverem completamente sólidos.

Não deixe os tubos na centrífuga, nem os coágulos dentro dos tubos.
[Imagem 1] [Imagem 2] [Imagem 3] [Imagem 4]

Retire o coágulo do tubo e raspe o coágulo vermelho, para manter somente a fibrina.
- Para usar como coágulo: já está pronto;
- Para usar como membrana: Coloque o coágulo dentro do PRF BoX e a bandeja em cima;
- Para usar como plug: Colocar o coágulo no cilindro branco dedicado e pressione com o pistão;
๏ S-PRF: (Sticky bone & Membranas grandes) Tubos Verdes
(1) Colete as amostras com os tubos verdes (coleta rápida) + 2 tubos vermelhos
(2) Coloque os tubos na centrífuga, diametralmente opostos (use as cores para ajudar)
(3) Inicie o programa A-PRF+: 1300 RPM & 14 min
(4) Ao final da centrifugação: Retire os tubos da máquina, remova a tampa & e coloque os
tubos no suporte.
(5) Com o auxílio de uma seringa de 2ml, pegue o líquido dos tubos verdes
(6) Aplique este líquido sobre o biomaterial para formar o sticky bone, ou sozinho na tigela,
para formar grandes membranas;
(7) Para acelerar o processo de coagulação: Adicione 1 coágulo junto ao sticky bone ou à
membrana (dos tubos vermelhos)

Para obter mais líquido, colete mais tubos verdes.

Adicione mais líquido para o seu Sticky bone se você fizer um enxerto grande ou se quiser um
sticky bone mais denso.

[Imagem 1] [Imagem 2] [Imagem 3] [Imagem 4] [Imagem 5] [Imagem 6&7]

Se você quiser aplicar o enxerto e depois coagulá-lo no local:

- Corte 1 ou 2 membranas em pedaços pequenos na tigela;
- Adicione o biomaterial
- Adicione algumas gotas do exsudato que está no fundo do PRF BoX;
- Aplique esas mistura no enxerto, no local;
- Coagule a mistura com gotas do S-PRF
Limitações do protocolo:
- Se você fizer a veno-punção no início da cirurgia (para as membranas de A-PRF), você vai
precisar de uma segunda coleta no momento da enxertia (para o líquido de S-PRF)
- Ou faça a coleta para ambos A-PRF (vermelho) & S-PRF (verde) no momento da enxertia.
๏ i-PRF: Tubos Verdes: Líquido em até 15-20 min.
- Mulheres —> configurações para o i-PRF: 700 RPM (60g) & 3min
- Homens —> configurações para o i-PRF: 700RPM (60g) & 4min
๏ i-PRF+ (Estética e Ortopedia): Tubos lilás: Líquido por 20 min.
- Mulheres —> configurações para o i-PRF+: 700 RPM (60g) & 5min
- Homens —> configurações para o i-PRF+: 700RPM (60g) & 6min

4. Condições Biológicas

Colesterol LDL
O metabolismo do colesterol ocorre principalmente dentro dos osteoblastos.
O colesterol LDL é um oxidante e induz a apoptose celular.
Um alto nível de colesterol LDL deteriorará a saúde do osso.
O exame de colesterol LDL é altamente recomendado: o valor deve ser < 1,4g/L
Prescrição para Laboratório: Colesterol LDL + Colesterol total

Vitamina D
A vitamina D é um dos hormônios mais importantes, necessário para a homeostasia óssea e
também por uma resposta imune eficiente. Muitas falhas e peri-implantites devem ser associadas
ás doenças e falhas metabólicas.
Um exame de laboratório também é recomendado: o valor deve ser > 50ng/mL para entrar em
cirurgia.
Prescrição para Laboratório: 25OH - vit D2 + D3
Suplementação:
- Inicia-se na consulta pré-operatória: 2000 UI/dia
- Faça adaptação de acordo com o nível sérico após os exames de laboratório:
✓ Se o nível > 30ng/mL —> 2000 UI/dia
✓ Se o nível estiver entre 20 & 30ng/mL —> 4000 UI/dia
✓ Se o nível estiver entre 10 & 20ng/mL —> 6000 UI/dia
✓ Se o nível estiver < 10ng/mL —> 10 000 UI/dia
Metronidazol (pó puro)
O uso de metronidazol ajuda a reduzir a contaminação do enxerto ósseo durante a cirurgia.
Metronidazol é a molécula mais eficiente contra bactérias anaeróbias. O pó (puro) é misturado
com o biomaterial, antes de adicionar os i-PRF, A-PRF ou o exsudato de PRF. Apenas algumas
miligramas são necessárias, perto de 50mg (1 copo).

5. Pressão e Tensão
Pressão em um tecido leva a ausência de angiogênese. Essa regra essencial foi publicada por
Mammoto, em 2009. Todas as falhas em cicatrização estão associadas com o déficit de
vascularização.

Tensão
Um fechamento de flap livre de tensão é recomendado. No entanto, a tensão também vem da
vestibular (ao falar, sorrir, tossir, mastigar…). Com a alta tensão, o periósteo se torna isquêmico e
a reabsorção óssea se torna obrigatória.

Soluções:
- Diminuir a tensão do flap ao máximo;
- Suturar o flap com a técnica “Apical Mattress”: profunda sutura na vestibular em forma de U
- Sempre avalie a tensão ao final da cirurgia: puxado os lábios e bochechas.
- Se a tensão persistir (flap móvel mesmo com sutura “Apical Mattress”): Faça incisão na
vestibular.

[Image: apical mattress]

Pressão
A pressão é aplicada pelo flap e irá induzir isquemia do periósteo.

Soluções:
- Parafuso tenda
- Sutura “Apical Mattress”
- Uso de Malhas de Titânio
- Placa de osso cortical
Instalação de implante sem pressão
Quando colocado com alto torque, os implantes podem causar enormes pressões no osso,
especialmente na cortical, levando à isquemia desse osso e sua reabsorção.
O mais apropriado é furar para mais o osso cortical nos primeiros 2 mm de profundidade, ou
realizar uma instalação do implante abaixo da crista, para aliviar o osso de qualquer pressão.

O osso enxertado é menos elástico e, por isso, mais frágil. Qualquer instalação de implante irá
aumentar o estresse aplicado ao osso. Diminuir consideravelmente o torque de instalação é
altamente recomendado. Nesse caso, a carga imediata deve ser evitada.

Suturas
Quando um flap é aberto, a readesão do perióstio é suficiente depois de algumas semanas. As
suturas devem permanecer por pelo menos 3-4 semanas. O uso de mono-filamento é altamente
recomendado. A solução mais fácil é usar um mono filamento reabsorvível.
6. Tratamento de dor

Analgesia
A dor vem da inflamação.
Para reduzir a inflamação, a molécula de cortisona é a mais eficiente.
Sua meia vida biológica é de cerca de 3 dias.
Uma dose já é o bastante para reduzir a inflamação:
Prednisolona: 1mg/kg, ou Dexametasona: 0,20mg/kg

Somente uma dose é suficiente para a maioria dos casos. Se necessário, uma segunda dose
pode ser dada depois de 3 dias. Analgésicos como Ibuprofeno ou Paracetamol podem ser
associados.

Anestesia
Para melhorar uma fraca anestesia (acidez local) é possível aumentar o pH da gengiva com
Bicarbonato de Sódio 1,4%: 5-10mL injetados na área da anestesia, depois da injeção do
anestésico.

Alergia
Os sintomas alérgicos vêm da liberação da Histamina dos macrófagos depois de exposição aos
alérgenos.
É fácil previnir uma reação alérgica, bloqueando a reação de liberação da Histamina, antes da
cirurgia: Uso de drogas Anti-histamínicas como Hidroxizina (Atarax®, 1 comprimido de 25mg/dia,
por 3 dias antes da cirurgia, tomados na hora de dormir).

Sedação
Uso de anti-Histamínicos (ex: Hidroxizina) como Atarax®
Cerca de 1mg/kg para um adulto, com início 2 horas antes da cirurgia.
No caso de alta ansiedade do paciente, dê a mesma quantidade da noite anterior.

Saliva
Sulfato de Atropina 0,25mg: IM, IV, sub-cutânea ou no assoalho da boca.
Se injetado no assoalho: injete algumas gotas do anestésico antes, porque a injeção de Atropina
pode ser dolorosa nesse local.
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017
 Downloaded by [University Town Library of Shenzhen] at 20:50 14 December 2017

View publication stats

Eur J Trauma Emerg Surg
DOI 10.1007/s00068-017-0767-9
ORIGINAL ARTICLE
Reduction of relative centrifugation force within injectable
platelet-rich-fibrin (PRF) concentrates advances patients’
own inflammatory cells, platelets and growth factors: the first
introduction to the low speed centrifugation concept
J. Choukroun1,2 · S. Ghanaati2
Received: 26 October 2016 / Accepted: 23 January 2017
© The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract
Purpose The aim of this study was to analyze system-
atically the influence of the relative centrifugation force
(RCF) on leukocytes, platelets and growth factor release
within fluid platelet-rich fibrin matrices (PRF).
Materials and methods Systematically using peripheral
blood from six healthy volunteers, the RCF was reduced
four times for each of the three experimental protocols (I–
III) within the spectrum (710–44 g), while maintaining a
constant centrifugation time. Flow cytometry was applied
to determine the platelets and leukocyte number. The
growth factor concentration was quantified 1 and 24 h after
clotting using ELISA.
Results Reducing RCF in accordance with protocol-II
(177 g) led to a significantly higher platelets and leuko-
cytes numbers compared to protocol-I (710 g). Protocol-III
(44 g) showed a highly significant increase of leukocytes
and platelets number in comparison to -I and -II. The
growth factors’ concentration of VEGF and TGF-β1 was
significantly higher in protocol-II compared to -I, whereas
protocol-III exhibited significantly higher growth factor
concentration compared to protocols-I and -II. These find-
ings were observed among 1 and 24 h after clotting, as well
as the accumulated growth factor concentration over 24 h.
* J. Choukroun
joseph@a-prf.com
* S. Ghanaati
shahram.ghanaati@kgu.de
1 teins (BMPs) [7, 8]. Although less invasive than autologous
Private Practice, Pain Therapy Center, Nice, France
2
Department for Oral, Cranio-Maxillofacial and Facial Plastic
Surgery, FORM (Frankfurt Orofacial Regenerative Medicine)
Laboratory, University Hospital Frankfurt Goethe University,
Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
Discussion Based on the results, it has been demonstrated
that it is possible to enrich PRF-based fluid matrices with
leukocytes, platelets and growth factors by means of a sin-
gle alteration of the centrifugation settings within the clini-
cal routine.
Conclusions We postulate that the so-called low speed
centrifugation concept (LSCC) selectively enriches leuko-
cytes, platelets and growth factors within fluid PRF-based
matrices. Further studies are needed to evaluate the effect
of cell and growth factor enrichment on wound healing and
tissue regeneration while comparing blood concentrates
gained by high and low RCF.
Keywords Inflammation · Leukocytes · Platelets ·
Platelet-rich fibrin · Tissue engineering · Pain ·
Centrifugation · A-PRF+ · I-PRF
Abbreviations
PRF Platelet-rich-fibrin
LSCC Low speed centrifugation concept
Introduction
In recent years, several concepts have been introduced for
clinically relevant tissue engineering by means of mini-
mally invasive approaches. Currently, well-accepted mod-
els include pure biomaterial application [1–4] and the
combination of biomaterials with autologous bone marrow
aspirates [5, 6] or recombinant bone morphogenetic pro-
bone transplantation, the previously mentioned minimally
invasive bone marrow aspirate methodologies are still
reserved for only experienced surgeons, as these methods
can also be associated with complications such as pain,
13
Vol.:(0123456789)

infection, and damage to the adjacent organs during bone
marrow retrieval. Furthermore, the optimal growth factor
concentration needs to be determined for the use of BMPs.
To resolve these issues, concentrates from the peripheral
blood have been recently proposed as potential supporters
in complex tissue engineering. In this context, several blood
concentrate systems were introduced such as plasma-rich
growth factors (PRGF) [9] and platelet-rich plasma (PRP)
[10]. Both systems require the addition of non-autologous
anticoagulants to generate fluid blood concentrates after
centrifugation [9]. In addition to anticoagulants, multi-step
centrifugation is needed to obtain PRP [11]. Moreover,
PRGF and PRP focus on the advantages of platelets and
their released growth factors to support tissue regeneration
in different medical fields and aim to eliminate leukocytes
from the final blood concentrates [12].
The introduction of platelet-rich-fibrin (PRF) in 2001
exemplified the first step toward the generation of blood-
derived PRF matrices that do not require additional antico-
agulation agents [13]. The centrifugation within a specific
glass-based tube leads to the activation of the physiological
coagulation cascade and, thus, the formation of a fibrin clot
enriched with cells within the peripheral blood i.e., plate-
lets and leukocytes.
Recent developments in cellular and molecular biology
and continuous research have increased the understanding
of the wound healing and regeneration processes. Thus,
the crucial role of leukocytes and their subgroups as main
protagonists to modulate the various phases during wound
healing became obvious [14]. In addition, platelets as well
as the fibrin network are known to play a major role in
the wound healing process [15]. Leukocytes participate in
angiogenesis and lymphogenesis, whereas the fibrin net-
work is a key player in the early stages of wound healing
rendering its synergistic effects with platelets and its func-
tion as a reservoir of cytokines [16–18].
Nevertheless, to generate PRF the application of high
relative centrifugal forces (RCF) (708 g) during the centrif-
ugation process was described as essential. Furthermore,
previous histological analyses of the PRF clot demonstrated
that platelets and inflammatory cells within the fibrin scaf-
fold accumulate mainly in the proximal portion of the PRF
clot [19]. Therefore, we questioned to what extent adjust-
ing the applied RCF could influence the cellular distribu-
tion within the solid PRF matrix, aiming to generate modi-
fied centrifugation protocols to enhance the regenerative
capacity of PRF-based matrices. Thereby, fine tuning of the
centrifugation process in terms of RCF reduction and slight
increase of the centrifugation time resulted in an advanced
PRF with enhanced leukocyte numbers, especially neutro-
philic granulocytes. Moreover, the platelets and leukocytes
within the advanced PRF were evenly distributed over the
entire clot compared to PRF [19].
13
J. Choukroun, S. Ghanaati
Based on these results, the question was raised to what
extent a systematic reduction in RCF within fluid PRF
matrices might have any influence on the increase of plate-
lets and leukocytes as well as growth factors within the
obtained blood concentrates.
In this context, the present study demonstrates a sys-
tematic analysis of the RCF influence on fluid PRF-based
matrices. Based on the first introduced PRF, we halved
the revolutions per minutes (rpm), which according to the
radius of the respective centrifuge (110 mm) led to a reduc-
tion of the RCF by four times in a stepwise approach to
examine a wide RCF range (i.e., 710–44 g) including the
high, medium and low RCF, systematically. Simultane-
ously, the centrifugation time was kept constant to exclude
its impact. To the best of our knowledge, the present study
is the first to analyze the RCF impact on human blood con-
centrates to determine the leukocyte and platelet numbers
as well as the growth factor release potential within PRF-
based matrices. The aim of the study was to determine to
what extent a controlled reduction of the RCF, by modi-
fying the centrifugation setting rpm, could influence the
platelet and leukocyte total number as well as the growth
factor release within fluid PRF-based matrices.
Materials and methods
Systematic protocols for PRF-based matrices
To evaluate the high-, medium- and low-RCF spectrum,
three different experimental centrifugation protocols for
the systematic analysis series were established by decreas-
ing the RCF within a wide range (710–44 g) with a con-
stant centrifugation time. Based on PRF (708 g), a step-
wise reduction of the rpm in half and, thus, a reduction of
the RCF four times were performed for each protocol as
follows:
I: 710 g; 2400 rpm; 8 min
II: 177 g; 1200 rpm; 8 min
III: 44 g; 600 rpm; 8 min
PRF preparation
For this experiment, the blood of six healthy volunteers
(three males and three females) was collected for each
of the evaluated protocols. The tubes were immediately
placed in the centrifuge and prepared according to the pre-
viously mentioned established protocols. A Duo centrifuge
(Process for PRF, Nice, France) was used to perform the
centrifugation procedure. This centrifuge has a fixed angle
rotor with a radius of 110 mm. Written informed consent
was obtained from the volunteers for their samples to be
Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF)…
used in the research. All donors were free of any infectious
disease and did not have any abnormal consumption of nic-
otine or alcohol. None of the subjects used any drugs for
anticoagulation.
Tubes for blood collection
For the purpose of these experiments, sterile plastic tubes
(Process for PRF, Nice, France) with a volume of 10 ml
were used to generate fluid blood concentrates according to
the previously described protocols. This method was used
because a fluid matrix was required for flow cytometry. The
blood was drawn by means of a clinically approved but-
terfly blood collection method. The centrifugation for each
protocol started after the last tube of this group was col-
lected over a total time of 2–3 min maximum.
Automated cell counting
The collected fluid matrices of each protocol were anti-
coagulated using a BD vacutainer with 4 ml of ethylene-
diaminetetraacetic acid (EDTA). This anticoagulation was
only performed for research purposes, as no cell counting
measurements would be possible otherwise. The samples
were further analyzed with ADVIA® LabCell® Automation
Solution (Siemens, France) at a medical laboratory (Laba-
zur laboratory, Nice, France) to detect the number of leuko-
cytes and platelets per microliter. An automated cell count
was performed by means of flow cytometry. This method
enables a multiparameter analysis of the cell number sus-
pended within a liquid. The cell suspension passes through
a laser beam, where one cell per unit of time leads to laser
scattering in different directions according to the cell sizes
and properties. The scattered light is detected by a side and
a forward sensor. The forward scatter is roughly propor-
tional to the cell size, while side scatter is caused by the
cell characteristics such as granularity and structural com-
plexity [20]. These data are automatically further analyzed
to detect the total number of leucocytes and platelets within
the cell suspension, i.e., fluid PRF matrices.
Growth factor quantification with ELISA
After centrifugation, the collected liquid PRF from each
protocol was transferred into a cell culture plate. The
plate was placed in 37 °C degree incubator until all the
samples formed a clot. Afterwards, Dulbecco’s Modified
Eagle Medium (Biochrom GmbH, Berlin, Germany) was
added to all clots and further incubated in 37 °C degree
to allow growth factor release. The supernatant (5 ml)
was collected after 1 h and frozen. The collected superna-
tant was replaced by a fresh cell media and further incu-
bated for 24 h. At the latter time point, the supernatants
were collected. The collected samples at both time points
were analyzed for human vascular epithelial growth factor
(VEGF) and human transforming growth factor (TGF-β1).
Protein quantification was performed by means of ELISA
(DuoSet® ELISA RND system) according to the manufac-
turer’s instructions. Optical density was assessed using a
microplate reader at 450 and 570 nm. The data measured at
570 nm were subtracted from the data measured at 450 nm
for an optical density correction. The measurements were
performed in triplicates for each protocol and donor.
Finally, the quantified data were statistically analyzed.
Statistical analysis
Statistical analysis was performed using Prism Version
6 (GraphPad Software Inc., San Diego, La Jolla, USA).
Data are expressed as the mean and standard deviation.
The significance of the differences between the means was
analyzed using one-way and two way analyses of variance
(ANOVA) with Tukey multiple comparisons test (α = 0.05).
Thereby, significant differences were marked as significant
if P values were less than 0.05 (*P < 0.05) and highly sig-
nificant if P values were less than 0.01 (**P < 0.01), 0.001
(***P < 0.001) or 0.0001 (****P < 0.0001).
Results
Total leukocyte number
The total leukocyte number was analyzed within the exper-
imental PRF protocols. Generally, reducing the RCF led to
a clearly detectable increase of the total leukocyte number
within the PRF-based matrices. The first protocol-I (710 g),
which was centrifuged with the highest RCF, showed the
lowest number of leukocytes among the three evaluated
experimental protocols. The second protocols I–II (177 g),
using a four time slower RCF than protocol-I, showed a sig-
nificantly higher number of leukocytes compared to proto-
col-I (P < 0.001). Finally, the third protocol-III (44 g) with
four times less RCF than protocol-II and 16 times less RCF
than protocol-I revealed the highest number of leukocytes,
which was statistically highly significant compared to pro-
tocol-I (P < 0.0001) and protocol-II (P < 0.0001) (Fig. 1a).
The donor-related leukocyte total number showed simi-
lar results in each individual. All evaluated samples showed
the same curve progression as a frequent observation of an
increased leukocyte number with reduced RCF (Fig. 1b).
Total platelet number
The total platelet number as a result of automated cell
counting showed a tendency towards increasing total
13

Fig. 1 a Number of leukocytes within the different experimental
PRF-based matrices. b Donor-related leukocyte number within the
different experimental PRF-based matrices
platelet number with RCF reduction within the PRF-based
matrices. The first experimental protocol-I (710 g) exhib-
ited the lowest platelet number compared to all other exam-
ined groups. Looking at the second protocol-II (177 g),
a significant increase in the platelet total number was
detected in comparison to protocol-I (P < 0.0001). Moreo-
ver, a further RCF reduction resulted in the highest plate-
let total number in protocol-III (44 g). Statistical analysis
showed significantly higher platelet numbers in protocol-
III compared to protocol-II (P < 0.0001) and protocol-I
(P < 0.0001) (Fig. 2a).
The donor-related values showed very similar reactions
to the exposed RCF influence in the various PRF-based
matrices. The curve shape was reproduced within the single
donor samples, showing an increased number of platelets
with reduced RCF (Fig. 2b).
VEGF concentration
The VEGF concentration was quantified 1 and 24 h
after clotting. At both time points, a clear tendency was
observed. The growth factor concentration increased by
reducing the applied RCF. One hour after clotting, the
VEGF concentration in protocol-I with the highest RCF
13
J. Choukroun, S. Ghanaati
Fig. 2 a Number of platelets within the different experimental PRF-
based matrices. b Donor-related platelet number within the different
experimental PRF-based matrices
showed the lowest concentration compared to the medium
range RCF and low range RCF protocols. At the same time
point, protocol-II, within the medium RCF range, showed
increased VEGF concentration. These results were highly
significant compared to protocol-I (P < 0.0001). Moreo-
ver, protocol-III with the lowest RCF application revealed
the highest VEGF concentration. These data were highly
significant compared to protocol-I (P < 0.0001) and proto-
col-II (P < 0.0001) (Fig. 3a). Similar results were detected
24 h after clotting. At this time point, protocol-I showed
the lowest VEGF concentration. Along with RCF reduc-
tion, the VEGF concentration significantly increased in
protocol-II. Statistical analysis showed a highly significant
increase in protocol-II compared to protocol-I (P < 0.0001).
Finally, protocol-III, which was prepared using the lowest
RCF, showed the highest VEGF concentration which was
highly significant compared to protocol-I (P < 0.0001) and
protocol-II (P < 0.0001) (Fig. 3b).
The accumulated VEGF concentration over 24 h was
calculated in the examined protocols. The released VEGF
concentrations in all protocols increased from 1 to 24 h.
At 24 h, the accumulated concentration in protocol-I was
the lowest within the tested groups. Protocol-II showed
a significantly higher accumulated VEGF concentra-
tion compared to protocol-I (P < 0.01). Additionally, the
Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF)…
Fig. 3 a VEGF concentration within the different experimental
PRF-based matrices 1 h after clotting. b VEGF concentration within
the different experimental PRF-based matrices 24 h after clotting. c
Accumulated VEGF concentration within the different experimental
PRF-based matrices over 24 h
accumulated VEGF concentration in protocol-III was the
highest, which was highly significant compared to proto-
col-I (P < 0.0001) and protocol-II (P < 0.0001) (Fig. 3c).
TGF-β1 concentration
The growth factor concentration for human TGF-β1 was
measured 1 and 24 h after clotting. A general trend was
observed at both time points. Reducing the applied RCF
enhanced the growth factor concentration. Looking at the
results 1 h after clotting, protocol-I showed the lowest
TGF-β1 concentration among all tested protocols. Thereby,
protocol-II, which was prepared within the medium RCF
range, revealed a significantly higher TGF-β1 concentra-
tion when compared to protocol-I (P < 0.0001), which was
Fig. 4 a TGF β-1 concentration within the different experimen-
tal PRF-based matrices 1 h after clotting. b TGF β-1 concentration
within the different experimental PRF-based matrices 24 h after
clotting. c Accumulated TGF β-1 concentration within the different
experimental PRF-based matrices over 24 h
prepared within the high RCF range. Additionally, proto-
col-III, representing the low RCF range, showed the highest
TGF-β1 concentration among the analyzed protocols. This
concentration was highly significant compared to protocol-I
(P < 0.0001) and protocol-II (P < 0.0001) (Fig. 4a).
Twenty-four hours after clotting, the TGF-β1 was detect-
able in all protocols. However, protocol-I showed the lowest
concentration compared to protocols-II and -III. The first
step RCF reduction toward protocol-II within the medium
RCF range showed a highly significant increase of TGF-β1
concentration compared to the high RCF range in protocol-
I (P < 0.0001). Further RCF reduction toward the low RCF
range in protocol-III reached the highest TGF-β1 concen-
tration among all tested protocols. Thereby, this increase
was highly significant compared to protocol-I (P < 0.0001)
and protocol-II (P < 0.0001) (Fig. 4b).
13

The accumulated TGF-β1 concentration increased from
1 to 24 h in all protocols. However, protocol-I showed
the lowest accumulated TGF-β1 concentration among all
tested protocols. Whereas in protocol-II, which was pre-
pared within the medium RCF range, a significantly higher
accumulated TGF-β1 level compared to protocol-I was
observed. Finally, protocol-II, which represents the low
RCF range, showed the highest accumulated TGF-β1 con-
centration. This value was statistically highly significant
compared to protocols-I and -II (Fig. 4c).
Discussion
The present study analyzed the platelets and leukocyte total
number, as well as the growth factor release within fluid
PRF matrices with different preparations of settings. To
the best of our knowledge, this study is the first to inves-
tigate the influence of RCF on the cell number and growth
factor release in fluid PRF-based human blood matrices.
Using specific experimental centrifugation protocols,
which were systematic modifications of the first described
PRF [13], we generated PRF-based fluid matrices with dif-
ferent proportions of platelets and leukocytes. Moreover, a
relation between RCF reduction and growth factor release
was evidenced. The present data provide new insight into
the potential of the centrifugation process in generating
different total cell numbers and growth factor levels of
release within the same blood volume only in relation to
the amount of exposure to specific RCF.
Three experimental protocols were established with a
constant centrifugation time to focus on the impact of RCF
across various ranges, including high, medium and low.
Adapting the RCF spectrum (710–44 g) based on the first
described PRF protocol [13], a systematic reduction of the
RCF was performed by a four time RCF reduction for each
protocol (I–III) as a consequence of stepwise halving the
rpm (revolutions per minute), because the centrifuge radius
used in this study was 110 mm.
Automated cell count by means of flow cytometry was
performed to determine the total leukocyte and platelet
number. The results showed that decreasing RCF up to 16
times less than the first described PRF led to a significant
increase in leukocyte and platelets numbers within the gen-
erated PRF-matrices. The first protocol-I (710 g) showed
the lowest detectable number of leukocytes and platelets
among all samples tested. Interestingly, reduction of RCF
in protocol-II (177 g) resulted in a significantly higher
number of leukocytes and platelets compared to protocol-I
(710 g). Moreover, the second step RCF reduction toward
protocol-III (44 g) revealed a further significant increase
of the leukocyte and platelet total number—with the high-
est value in comparison to all other groups. These data
13
J. Choukroun, S. Ghanaati
emphasize that modifications of RCF contribute to a clearly
significant alteration of the leukocyte and platelet number
toward the lower RCF ranges (177–44 g).
Centrifugation is a widely used technique to separate a
biological mixture within a liquid phase. The principles of
this technique are based on the use of the centrifugal force,
which is much higher than the gravity. During centrifuga-
tion, different forces interact and influence the particle
movement within the liquid including centrifugal force,
gravitational force and the drag force of the particles, i.e.,
cells. This process results in particle migration depending
on their size, density and mass [21]. The present results
suggest that the mass, size and density range of leukocytes
and platelets require a low RCF, which is enough to sepa-
rate them from the rest blood components while not caus-
ing aggregation to the bottom of the tube. However, further
studies are needed to demonstrate to what extent higher
or lower RCF ranges than the presently investigated spec-
trum may have on any further benefits regarding cell and
growth factor accumulation within the fluid PRF matrices.
Thus, the RCF reduction can be used as a “tool” to gener-
ate fluid PRF-based matrices enriched with leukocytes and
platelets. This phenomenon is of high scientific and clinical
relevance, as leukocytes are one of the main drivers of bone
and soft tissue regeneration by contributing to the release
of the angiogenic and lymphogenic factors responsible
for cellular cross-talk in the tissue regeneration process
[18, 22]. Leukocytes are also known to be involved in the
communication between precursor cells and mesenchymal
cells with regard to bone formation [23–26]. Accordingly,
without leukocytes, sophisticated cell–cell communication
for tissue regeneration is not possible. In addition, platelets
are known to harbor potent growth factors (PDGFs) and
platelet-derived growth factors for tissue regeneration such
as vascular endothelial growth factor (VEGF) and trans-
forming growth factor-beta (TGF-ß) [17, 27–29], which
can be released only after platelet aggregation [30, 31]. In
addition, platelets are not the only players in tissue regen-
eration—they require leukocytes for better performance in
their capacity towards tissue regeneration [32–34].
Furthermore, the present study demonstrated that sys-
tematic reduction of the applied RCF results in a clearly
increased tendency in growth factor concentration. Thus,
the determined growth factors VEGF and TGF-β1 were the
lowest in protocol-I, which was prepared within the high
RCF range. However, RCF reduction toward the medium
RCF range led to a significant increase of VEGF and TGF-
β1 concentrations in protocol-II. Furthermore, the RCF
reduction showed the highest VEGF and TGF-β1 in proto-
col-III within the low RCF spectrum. This observation was
evidenced at 1, 24 h as well as for the accumulated growth
factor over 24 h. These findings are in correlation with the
results shown by increasing the number of platelets and
Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF)…
leukocytes. In this context, the increased growth factor
concentration within the medium and low RCF ranges is
probably related to the increased number of platelets and
leukocytes as these cells are important sources of growth
factors. Moreover, it might be that applying a high RCF not
only results in a lower number of platelets and leukocytes
but also affects these cells’ ability to release growth factors.
Consequently, enhancing the growth factor concentrations
within the fluid PRF-based matrices reflects improving the
regenerative capacity of the fluid PRF-based matrices as
an autologous growth factor reservoir. VEGF is one of the
most important signaling molecules for neoangiogenesis,
which is highly required during wound healing [35]. More-
over, TGF-β1 contributes to tissue regeneration rendering
the recruitment of keratinocytes, especially in the early
wound healing stage [36–38].
To date, there are no data showing how many leukocytes
or platelets within the PRF-based matrices are sufficient
to have the best possible physiological condition accord-
ing to which an optimal condition for wound healing or a
basis for a successful soft tissue and bone regeneration can
be generated. The results of the present study highlight,
however, that RCF reduction might be a clinical applicable
tool, in order to tailor the amount of leukocytes within the
PRF-derived blood concentrates according to the required
indication.
Based on the present data, according to which reduction
of RCF results in PRF-matrices enriched with leukocytes
and platelets and enhanced growth factor release, we pos-
tulated the so-called LSCC, which can be used to generate
fluid PRF-based matrices enriched with cells and plasma
proteins from peripheral blood as an autologous source for
a smart tissue regeneration in complex tissue engineering.
In a clinical setting, there is a need to increase the regen-
erative potential of bone substitute materials or bio-mem-
branes. This could be achieved by adding autologous tissue
engineering fluid systems. Accordingly, the presented plas-
tic tubes and the RCF reduction allowed for the generation
of a liquid injectable PRF-based matrices (i-PRF) without
the use of anticoagulants. This i-PRF, prepared according
to the LSCC, is highly enriched with platelets, leukocytes
and growth factors, which could provide a significant ben-
efit for the regeneration process.
Recently, our group has shown that the addition of only
monocytes isolated from peripheral blood contributes to
increasing the in vivo vascularization of synthetic bone
substitute materials [39]. Consequently, looking at PRF
as a complex “physiological” system, which can be gen-
erated by a one step centrifugation, it is assumed that in
this system monocytes, and all other substances and cells
can be enriched. This system might, therefore, be able to
contribute to an improved wound healing condition along
with enhanced vascularization, while remaining within
their cell-specific niche. These data underline that fluid
PRF-based matrices, gained by reduced RCF, can be used
to functionalize biomaterials by means of an autologous
source, i.e., concentrates of peripheral blood to promote
tissue regeneration.
Recent ex vivo work by our group demonstrated
that alerting the RCF within solid PRF-based matrices
advances the cellular distribution and enhances the neu-
trophilic granulocytes number as a leukocytes subgroup
within the advanced PRF clot [19]. However, further
studies are necessary to show the role of RCF reduction
on cell number and growth factor release in solid PRF-
based matrices.
The blood composition is specific and individual
according to the particular donor. Still, the donor-related
values within the different PRF-based matrices were
approximately similar in all groups regarding the plate-
let distribution as well as the leukocytes. These findings
indicate that first of all, PRF-based matrices are repro-
ducible systems and individually applicable regardless of
the donor characteristics. Second, the results also estab-
lish the reproducibility of the LSCC in various blood
samples when comparing the six different blood donors.
In this study, the data demonstrate that it is possible to
apply the LSCC for the enrichment of blood concentrates
with platelets, leukocytes and growth factors. The combi-
nation of leukocytes and platelets plays an important role
in tissue regeneration [40, 41]. Thus, the ability to control
the cell content within blood-derived fluid PRF-based
matrices by only changing the centrifugation settings,
such as rpm, i.e., changing the exposure to a specific
RCF, might serve as a valuable step to have personal-
ized medicine for widespread clinically applicable cell-
based tissue engineering. Thereby, using RCF as a tool
to control the number of the included cells could be used
to adjust the preparation protocol to the specific needs of
individual patients according to the clinical indications.
Based on the present outcome, the LSCC helps to
show that the number of several blood components
responsible for tissue regeneration, i.e., platelets, leuko-
cytes and growth factors, can be selectively enriched by
application of a clinically applicable system through a
single parameter within the centrifugation process. Con-
sidering the complexity of cell isolation and cultivation
in laboratories under sterile conditions, the PRF system
embodies a relatively simple tool to influence the number
of these cells. In this context, further systematic in vivo
and clinical studies that evaluate the benefit of this sys-
tem, i.e., enhancing the autologous regeneration capacity,
are needed to assess the correlation between cell enrich-
ment and the improved potential of tissue regeneration
and wound healing.
13

Conclusions
In the present study, the growth factor release and the leu-
kocyte and platelet total numbers were analyzed in rela-
tion to the systematic variation of the relative centrifuga-
tion force (RCF) exposure for the first time. The present
data demonstrated that reducing the RCF from a high
range toward a low spectrum within autologous PRF-based
matrices leads to a significant increase of the leukocyte
and platelet number, as well as growth factor concentration
(VEGF and TGF-β1). Based on these results, we postulate
the low speed centrifugation concept (LSCC) enhances the
regeneration potential of fluid PRF-based matrices. Con-
sequently, the reduction of RCF by application of LSCC
opens up new avenues for advanced PRF-matrices, in
which the cell–cell communication between platelets and
leukocytes and that of these cells within the recipient tis-
sue might result in improved wound healing and enhanced
tissue regeneration. Thus, further preclinical and clinical
studies are necessary to evaluate this concept to optimize
clinical benefits.
Acknowledgements The authors would like to thank the members
of the FORM-lab for the graphical support of this manuscript.
Compliance with ethical standards
Conflict of interest Joseph Choukroun and Shahram Ghanaati de-
clare that they have no conflict of interest. Choukroun is the owner of
PROCESS. None of the present protocols have been yet approved for
clinical application.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
References
1. Ghanaati S, Kovács A, Barbeck M, Lorenz J, Teiler A, Sadeghi
N, et al. Bilayered, non-cross-linked collagen matrix for regener-
ation of facial defects after skin cancer removal: a new perspec-
tive for biomaterial-based tissue reconstruction. J Cell Commun
Signal. 2016;10:3–15.
2. Tsaryk R, Gloria A, Russo T, Anspach L, De Santis R, Ghanaati
S, et al. Collagen-low molecular weight hyaluronic acid semi-
interpenetrating network loaded with gelatin microspheres for
cell and growth factor delivery for nucleus pulposus regenera-
tion. Acta Mater Inc. 2015;20:10–21.
3. Schlee M, Ghanaati S, Willershausen I, Stimmlmayr M, Scu-
lean A, Sader RA. Bovine pericardium based non-cross linked
collagen matrix for successful root coverage, a clinical study in
human. Head Face Med BioMed Central Ltd. 2012;8:6.
4. Lorenz J, Blume M, Barbeck M, Teiler A, Kirkpatrick CJ,
Sader RA, et al. Expansion of the peri-implant attached
13
J. Choukroun, S. Ghanaati
gingiva with a three-dimensional collagen matrix in head and
neck cancer patients-results from a prospective clinical and
histological study. Clin Oral Investig. 2016.
5. Sauerbier S, Rickert D, Gutwald R, Nagursky H, Oshima T,
Xavier SP, et al. Bone marrow concentrate and bovine bone
mineral for sinus floor augmentation: a controlled, rand-
omized, single-blinded clinical and histological trial-per-proto-
col analysis. Tissue Eng Part A. 2011;17:2187–97.
6. Duttenhoefer F, Hieber SF, Stricker A, Schmelzeisen R, Gut-
wald R, Sauerbier S. Follow-up of implant survival comparing
ficoll and bone marrow aspirate concentrate methods for hard
tissue regeneration with mesenchymal stem cells in humans.
Biores Open Access. 2014;3:75–6.
7. Springer ING, Açil Y, Kuchenbecker S, Bolte H, Warnke
PH, Abboud M, et al. Bone graft versus BMP-7 in a criti-
cal size defect-cranioplasty in a growing infant model. Bone.
2005;37:563–9.
8. Liu Y, Möller B, Wiltfang J, Warnke PH, Terheyden H. Tissue
engineering of a vascularized bone graft of critical size with
an osteogenic and angiogenic factor-based in vivo bioreactor.
Tissue Eng Part A. 2014;20:3189–97.
9. Anitua E, Andia I, Sanchez M. PRGF plasma rich growth fac-
tors. Dent Dialogue. 2004;1–9.
10. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss
JE, Georgeff KR. Platelet-rich plasma: growth factor enhance-
ment for bone grafts. Oral Surg Oral Med Oral Pathol Oral
Radiol Endodontol. 1998;85:638–46.
11. Everts P a M, Knape JT a, Weibrich G, Schönberger JP a M,
Hoffmann J, Overdevest EP, et al. Platelet-rich plasma and
platelet gel: a review. J Extra Corpor Technol. 2006;38:174–87.
12. Anitua E, Sánchez M, Orive G, Andía I. The potential impact
of the preparation rich in growth factors (PRGF) in different
medical fields. Biomaterials. 2007;28:4551–60.
13. Choukroun J, Adda F, Schoeffler C, Vervelle A. An opportu-
nity in paro-implantology: PRF [in French]. Implantodontie.
2001;42:55–62.
14. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound
repair and regeneration. Nature. 2008;453:314–21.
15. Litvinov RI, Weisel JW. What is the biological and clinical rel-
evance of fibrin? Semin Thromb Hemost. 2016;42:333–43.
16. Sahni A, Francis CW. Vascular endothelial growth factor binds
to fibrinogen and fibrin and stimulates endothelial cell prolif-
eration. Blood. 2000;96:3772–8.
17. van Hinsbergh VW, Collen a, Koolwijk P. Role of fibrin matrix
in angiogenesis. Ann N Y Acad Sci. 2001;936:426–37.
18. Soloviev DA, Hazen SL, Szpak D, Bledzka KM, Ballantyne
CM, Plow EF, et al. Dual role of the leukocyte integrin M 2 in
angiogenesis. J Immunol. 2014;193:4712–21.
19. Ghanaati S, Booms P, Orlowska A, Kubesch A, Lorenz J, Rut-
kowski J, et al. Advanced platelet-rich fibrin: a new concept
for cell-based tissue engineering by means of inflammatory
cells. J Oral Implantol. 2014;40:679–89.
20. Quirke P, Dyson JED. Flow cytometry: methodology and
applications in pathology. J Pathol. 1986;149:79–87.
21. Keith Wilson JW. Principles and techniques of biochemistry
and molecular biology. Cambridge: Cambridge University
Press;2010.
22. Kawazoe T, Kim HH. Tissue augmentation by white blood
cell-containing platelet-rich plasma. Cell Transplant.
2012;21:601–7.
23. Omar OM, Granéli C, Ekström K, Karlsson C, Johansson A,
Lausmaa J, et al. The stimulation of an osteogenic response by
classical monocyte activation. Biomaterials. 2011;32:8190–204.
24. Pirraco RP, Reis RL, Marques AP. Effect of monocytes/mac-
rophages on the early osteogenic differentiation of hBMSCs. J
Tissue Eng Regen Med. 2013;7:392–400.
Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF)…
25. Ekström K, Omar O, Granéli C, Wang X, Vazirisani F, Thomsen
P. Monocyte exosomes stimulate the osteogenic gene expression
of mesenchymal stem cells. PLoS One. 2013;8:e75227.
26. Perut F, Filardo G, Mariani E, Cenacchi A, Pratelli L, Devescovi
V, et al. Preparation method and growth factor content of platelet
concentrate influence the osteogenic differentiation of bone mar-
row stromal cells. Cytotherapy. 2013;15:830–9.
27. Tan KW, Chong SZ, Wong FHS, Evrard M, Tan SM-L, Keeble J,
et al. Neutrophils contribute to inflammatory lymphangiogenesis
by increasing VEGF-A bioavailability and secreting VEGF-D.
Blood. 2013;122:3666–77.
28. Schär MO, Diaz-Romero J, Kohl S, Zumstein MA, Nesic D.
Platelet-rich concentrates differentially release growth fac-
tors and induce cell migration in vitro. Clin Orthop Relat Res.
2015;473:1635–43.
29. Nami N, Feci L, Napoliello L, Giordano A, Lorenzini S,
Galeazzi M, et al. Crosstalk between platelets and PBMC: new
evidence in wound healing. Platelets. 2016;27:143–8.
30. Jurk K, Kehrel BE. Platelets: physiology and biochemistry.
Semin Thromb Hemost. 2005;31:381–92.
31. Walsh TG, Metharom P, Berndt MC. The functional role
of platelets in the regulation of angiogenesis. Platelets.
2015;26:199–211.
32. Jenne CN, Urrutia R, Kubes P. Platelets: bridging hemo-
stasis, inflammation, and immunity. Int J Lab Hematol.
2013;35:254–61.
33. Yeaman MR. Platelets: at the nexus of antimicrobial defence.
Nat Rev Microbiol. 2014;12:426–37.
34. Schmidt-Bleek K, Kwee BJ, Mooney DJ, Duda GN. Boon and
bane of inflammation in bone tissue regeneration and its link
with angiogenesis. Tissue Eng Part B Rev. 2015;21:354–64.
35. Moens S, Goveia J, Stapor PC, Cantelmo AR, Carmeliet P.
The multifaceted activity of VEGF in angiogenesis—impli-
cations for therapy responses. Cytokine Growth Factor Rev.
2014;25:473–82.
36. Lichtman MK, Otero-Vinas M, Falanga V. Transforming growth
factor beta (TGF-β) isoforms in wound healing and fibrosis.
Wound Repair Regen. 2016;215–22.
37. Kim B-C, Kim HT, Park SH, Cha J-S, Yufit T, Kim S-J, et al.
Fibroblasts from chronic wounds show altered TGF-β-signaling
and decreased TGF-? Type II receptor expression. J Cell Physiol.
2003;195:331–6.
38. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS,
Wakefield LM, et al. Transforming growth factor type beta:
rapid induction of fibrosis and angiogenesis in vivo and stimu-
lation of collagen formation in vitro. Proc Natl Acad Sci USA.
1986;83:4167–71.
39. Barbeck M, Unger RE, Booms P, Dohle E, Sader RA, Kirk-
patrick CJ, et al. Monocyte preseeding leads to an increased
implant bed vascularization of biphasic calcium phosphate bone
substitutes via vessel maturation. J Biomed Mater Res Part A.
2016;1–8.
40. Davis VL, Abukabda AB, Radio NM, Witt-Enderby PA, Claf-
shenkel WP, Cairone JV, et al. Platelet-rich preparations to
improve healing. Part II: platelet activation and enrichment, leu-
kocyte inclusion, and other selection criteria. J Oral Implantol.
2014;40:511–21.
41. Ghasemzadeh M, Hosseini E. Intravascular leukocyte migration
through platelet thrombi: directing leukocytes to sites of vascular
injury. Thromb Haemost. 2015;113:1224–35.
13
Clin Oral Invest
DOI 10.1007/s00784-017-2133-z

REVIEW

Use of platelet-rich fibrin in regenerative dentistry:

a systematic review
Richard J. Miron 1,2 & Giovanni Zucchelli 3 & Michael A. Pikos 4 & Maurice Salama 1,5,6 &
Samuel Lee 7 & Vincent Guillemette 8 & Masako Fujioka-Kobayashi 1,9,10 & Mark Bishara 11 &
Yufeng Zhang 12 & Hom-Lay Wang 2 & Fatiha Chandad 8 & Cleopatra Nacopoulos 13 &
Alain Simonpieri 14,15,16 & Alexandre Amir Aalam 17 & Pietro Felice 3 &
Gilberto Sammartino 18 & Shahram Ghanaati 19 & Maria A Hernandez 1 &
Joseph Choukroun 20

Received: 3 December 2016 / Accepted: 15 May 2017

# Springer-Verlag Berlin Heidelberg 2017

Abstract socket management, sinus lifting procedures, gingival reces-

Objectives Research across many fields of medicine now
points towards the clinical advantages of combining regener-
ative procedures with platelet-rich fibrin (PRF). This system-
atic review aimed to gather the extensive number of articles
published to date on PRF in the dental field to better under-
stand the clinical procedures where PRF may be utilized to
enhance tissue/bone formation.
Materials and methods Manuscripts were searched systemat-
ically until May 2016 and separated into the following cate-
gories: intrabony and furcation defect regeneration, extraction
sion treatment, and guided bone regeneration (GBR) includ-
ing horizontal/vertical bone augmentation procedures. Only
human randomized clinical trials were included for
assessment.
Results In total, 35 articles were selected and divided accord-
ingly (kappa = 0.94). Overall, the use of PRF has been most
investigated in periodontology for the treatment of periodontal
intrabony defects and gingival recessions where the majority
of studies have demonstrated favorable results in soft tissue
management and repair. Little to no randomized clinical trials

Electronic supplementary material The online version of this article

(doi:10.1007/s00784-017-2133-z) contains supplementary material,
which is available to authorized users.

10
* Richard J. Miron Department of Oral Surgery, Clinical Dentistry, Institute of Biomedical
richard.miron@zmk.unibe.ch Sciences, Tokushima University Graduate School, Tokushima, Japan
11
West Bowmanville Dental, Bowmanville, Ontario, Canada
12
Department of Oral Implantology, University of Wuhan, Wuhan, China
13
1 Laboratory for Research of the Musculoskeletal System, KAT Hospital,
College of Dental Medicine, Department of Periodontology, Nova
School of Medicine, National and Kapodistrian, University of Athens,
Southeastern University, Fort Lauderdale, FL, USA Athens, Greece
2
Department of Periodontics and Oral Medicine, University of Michigan, 14
Oral Surgery Department, University Federico II Naples, Naples, Italy
Ann Arbor, MI, USA
15
3 Periodontology and Implantology, Beausoleil, France
Department of Biomedical and Neuromotor Sciences, University of
16
Bologna, Bologna, Italy Periodontology and Implantology, Marseille, France
4 17
Pikos Institute, Tampa Bay, FL, USA Department of Advanced Periodontics, USC School of Dentistry, Los
5 Angeles, CA, USA
Department of Periodontology, Georgia University, Athens, GA, USA
18
6 Department of Neuroscience, Reproductive Science and
Goldstein Garber & Salama, Atlanta, GA, USA Odontostomatology, University of Naples Federico II, Naples, Italy
7
International Academy of Dental Implantology, San Diego, CA, USA 19
FORM, Frankfurt Oral Regenerative Medicine, Clinic for Maxillofacial
8 and Plastic Surgery, Johann Wolfgang Goethe University, Frankfurt am
Department of Periodontology, Laval University, Quebec City, Canada
9 Main, Germany
Department of Cranio-Maxillofacial Surgery, Inselspital, Bern University
20
Hospital, University of Bern, Bern, Switzerland Pain Clinic, Nice, France

were found for extraction socket management although PRF
has been shown to significantly decrease by tenfold dry
sockets of third molars. Very little to no data was available
directly investigating the effects of PRF on new bone forma-
tion in GBR, horizontal/vertical bone augmentation proce-
dures, treatment of peri-implantitis, and sinus lifting
procedures.
Conclusions Much investigation now supports the use of PRF
for periodontal and soft tissue repair. Despite this, there re-
mains a lack of well-conducted studies demonstrating con-
vincingly the role of PRF during hard tissue bone regenera-
tion. Future human randomized clinical studies evaluating the
use of PRF on bone formation thus remain necessary.
Clinical relevance PRF was shown to improve soft tissue
generation and limit dimensional changes post-extraction,
with little available data to date supporting its use in GBR.
Keywords Platelet-rich fibrin . Tissue regeneration . Bone
augmentation . Soft tissue regeneration
Introduction
Regenerative therapy in dentistry involves the replacement and/
or regeneration of oral tissues altered as a result of disease or
injury. One of the reported aspects complicating this endeavor
has been the complex nature of the tissues found in the oral
cavity. These include both mineralized tissues such as the cemen-
tum, alveolar bone, and dentin, as well as soft tissues connected
by ligaments (periodontal ligament), each comprising distinct
cell populations from various tissue origins (ectodermal and me-
sodermal). These cell populations reside in specialized extracel-
lular matrices organized in complex fashions [1, 2]. In the past, a
variety of regenerative procedures utilizing highly sophisticated
biomaterials were introduced to attempt their regeneration. These
included ambitious attempts with barrier membranes to perform
guided tissue/bone regeneration and the use of a variety of bone
grafting materials from human, animal and synthetic sources, as
well as bioactive growth factors such as bone morphogenetic
proteins (BMPs) and enamel matrix derivative (EMD). Other
investigators proposed that the use of three-dimensional scaffolds
fabricated from the patient’s own peripheral blood could be uti-
lized [2]. This new approach is based on the concepts that were
introduced over a decade ago consisting of a platelet concentrate
without the use of anticoagulants. Platelet-rich fibrin (PRF) was
therefore developed as an improved formulation of the previous-
ly utilized platelet-rich plasma (PRP) [3].
Unlike PRP, which requires the addition of anticoagulants
such as bovine thrombin during initial blood collection, PRF
is obtained simply by centrifugation without anticoagulants
and is therefore strictly autologous. This fibrin matrix contains
platelets and leukocytes as well as a variety of growth factors
and cytokines including transforming growth factor-beta1
Clin Oral Invest
(TGF-β1), platelet-derived growth factor (PDGF), vascular
endothelial growth factor (VEGF), interleukin (IL)-1β, IL-4,
and IL-6 [4]. Furthermore, fibrin that forms during the final
stages of the coagulation cascade, combined with cytokines
secreted by platelets, makes PRF a highly biocompatible ma-
trix especially in damaged sites where the fibrin network acts
also as a reservoir of tissue growth factors [5]. These factors
act directly on promoting the proliferation and differentiation
of osteoblasts, endothelial cells, chondrocytes, and various
sources of fibroblasts [6, 7]. Despite this, many questions
remain about the actual clinical performance of PRF.
Therefore, the purpose of the present systematic review is to
report the current state of knowledge and clinical potential of
PRF in regenerative dental therapy when compared to both
standardized controls and well-established standard regenera-
tive biomaterials from human clinical randomized trials.
Brief history of platelet concentrates
The original concept leading towards the preparation of plate-
let concentrates was that concentrated platelets and autologous
growth factors could be collected in plasma solutions that
could then be utilized in a surgical site to promote local
healing [8, 9]. It was given the popular working name
Bplatelet-rich plasma^ (PRP), introduced in the late 1990s
[10–12]. PRP is composed of over 95% platelets, a cell type
that actively secretes growth factors for initiating wound
healing and secreting factors responsible for enhancing cell
adhesion, proliferation, and migration of various cell types
[10, 13]. Around the same time period, Anitua et al. formulat-
ed a second platelet concentrate also utilizing anticoagulants
termed platelet-rich growth factor (PRGF) [14, 15].
Despite this, several factors have been shown to limit the use
of PRP and PRGF. Their preparation requires the additional use
of bovine thrombin or CaCl2 in addition to coagulation factors.
Furthermore, the preparation must be centrifuged in two separate
stages in order to increase platelet concentration without incor-
poration of leukocytes (sometimes requiring 1 h). It has further
been reported that the liquid nature of PRP also complicates its
handling and reduces its potential application since it must be
utilized in combination with other biomaterials. Lastly, the clin-
ical potential for bone regeneration with PRP is limited having a
very short release of growth factor profile [16–18]. All these
limitations have led to the emergence of a second-generation
platelet concentrate termed PRF fabricated from 100% autolo-
gous sources [19].
Advantages of PRF over PRP
PRF differs from its predecessor (PRP/PRGF) by several pa-
rameters which can be summarized as follows: the simplicity
Clin Oral Invest
of its preparation and its implementation. The time of prepa-
ration and cost of preparation are both significantly lower as
PRF does not necessitate the direct activation with additional
factors such as bovine thrombin or extrinsic anticoagulants
[3]. Because of its fibrous structure, PRF retains a larger num-
ber of cytokines and growth factors in a supportive three-
dimensional fibrin scaffold for cell migration [20]. In tissue,
PRF dissolves more slowly than PRP, forming a solid fibrin
matrix slowly remodeled in the style of a natural blood clot.
Platelets and cytokines are then effectively retained and re-
leased gradually over time [18]. The PRF scaffold allows a
continuous slow release of growth factors and cytokines over
a period of 10 days, in contrast to PRP which has been shown
to release the majority of its growth factors within the first day
[18]. Therefore, migrating cells in near proximity to PRF scaf-
folds are in an environment with fibrin and growth factors
throughout their entire growth cycle [21].
Once blood is collected (in the absence of anticoagulants),
samples must be centrifuged immediately to avoid the activa-
tion of the coagulation cascade. During centrifugation, fibrin-
ogen is concentrated to the top of the collection tube until the
circulating thrombin transforms it into a fibrin network. This
results in a fibrin clot rich in platelets, trapped between an
acellular plasma layer and erythrocytes. The solid fibrin clot
is found between the supernatant and the reddish background
formed by red blood cells. The clot may then be removed
immediately and condensed in a metal box so as to obtain a
solid covering membrane or a filling cylinder (Fig. 1). The
resulting exudate may be cut and used to hydrate graft mate-
rials if required (Fig. 1) [19, 20].
Implications of PRF in wound healing
Although leukocyte and platelet cytokines play an important
role in the PRF healing capacity, it has often been suggested
that it is the fibrin matrix supporting these elements which is
actually responsible for its therapeutic potential [20]. The keys
to tissue regeneration lie in their angiogenic potential, their
immune system control, their potential to recruit circulating
stem cells, and their ability to ensure undisturbed wound
closure/healing by epithelial tissues [20]. The angiogenic
properties of PRF may therefore be explained by the three-
dimensional structure of the fibrin matrix which holds a num-
ber of growth factors and cytokines simultaneously embedded
in the matrix including PDGF, TGF-β1, IGF, and VEGF. The
regenerative potential of these cytokines has been abundantly
studied in tissue wound healing and regeneration [4, 12,
22–33]. Furthermore, the fibrin matrix stimulates the expres-
sion of integrin avb3 which allows cells to bind to fibrin,
fibronectin, and vitronectin [33]. This cascade of events is of
utmost importance to initiate the process of angiogenesis and
thus tissue wound healing [33].
Moreover, the fibrin degradation products directly stimu-
late neutrophil migration and facilitate transmigration into the
vascular endothelium. This neutrophil activation causes secre-
tion of proteases that facilitate their penetration in the base-
ment membrane of blood vessels, in addition to their contri-
bution to degrade the fibrin clot. Neutrophils trapped within
the fibrin clot act to eliminate incoming bacteria and patho-
gens in the wound site by phagocytosis and the production of
toxic free radicals and digestive enzymes. This contributes
towards the prevention of bacterial contamination within the
surgical site [34]. PRF also contains macrophages that are
involved in the healing and repair process by playing a key
role in the transition between inflammation and wound repair
during osteogenesis [33, 34].
Methods
Development of a protocol
This review was conducted and reported according to the
PRISMA (Preferred Reporting Items for Systematic
Reviews and Meta-Analyses) [35]. A protocol including all
aspects of a systematic review methodology was developed
prior to initiation of this review. This included definition of the
focused question; a PICO (patient, intervention, comparison,
outcome) question; a defined search strategy; study inclusion
criteria; determination of outcome measures; screening
methods, data extraction, and analysis; and data synthesis.
PICO question
P: Do patients in need of clinical bone, cementum, soft
tissue, and/or PDL gain
I: Undergoing to dental treatments (i.e., guided bone/
tissue repair/regeneration or pulp repair/regeneration)
using defined non/surgical approaches combined with
the use of PRF as sole/combined biomaterial
C: Defined regenerative/reparative approaches without
the use of PRF
O: Soft and/or hard tissue reconstruction of the periodon-
tium, alveolar bone, peri-implant tissues, or tooth
structure
Defining the focused question
The following focused question was defined: BWhat indica-
tions has platelet rich fibrin (PRF) been shown effective for
tissue repair/regeneration of either soft or hard tissues in
dentistry?^
 Clin Oral Invest

Fig. 1 Fabrication of various PRF membranes from 10 mL autologous blood. Thereafter, two PRF clots were mixed with a bone grafting material
accordingly (clinical images were kindly provided by Dr. Michael A. Pikos)

Search strategy or case series were excluded if controls were not present. All
animal and in vitro studies were also excluded.
Electronic and manual literature searches were conducted in-
dependently by two authors (RJM and MFK) in several data-
Outcome measure determination
bases, including MEDLINE (OVID), EMBASE (OVID),
Cochrane Central Register of Controlled Trials (Cochrane
The primary outcome of interest was to determine the
Library), Cochrane Oral Health Group Trials Register
regenerative/reparative potential of PRF in a variety of clinical
(Cochrane Library), Web of Science (Thomson Routers),
settings utilized in dentistry. For each of the investigated clin-
and SciVerse (Elsevier). The electronic literature was searched
ical indications, different primary outcomes were considered.
for articles published up to and including May 14, 2016: com-
For studies dealing with intrabony defect regeneration, prob-
binations of several search terms and search strategies were
ing pocket depth (PPD) and clinical attachment levels (CAL)
applied to identify appropriate studies (Supplemental
were measured. For studies dealing with gingival recessions,
Tables 1–4). These include search strategies to identify the
root coverage was calculated as percentage. Studies investi-
effects of PRF on (1) intrabony defect regeneration, (2) furca-
gating the use of PRF for furcation defect regeneration quan-
tion defect regeneration, (3) management of gingival reces-
tified CAL gains as a primary outcome measure. For studies
sions, (4) guided bone regeneration and extraction socket
dealing with bone regeneration, dimensional change/density
healing, and (5) sinus floor elevation procedures. Reference
of hard tissues was compared. Similarly, sinus floor elevation
lists of review articles and of the included articles in the pres-
procedures quantified new bone formation and/or implant suc-
ent review were screened. Finally, a hand search of the
cess rates following sinus lifting procedures with/without
Journal of Clinical Periodontology, Journal of Dental
PRF. Outcomes were summarized in Supplemental
Research, Journal of Periodontal Research, Journal of
Tables 1–4 for the various clinical studies accordingly.
Periodontology, Clinical Oral Implants Research, Clinical
Implant Dentistry and Related Research, Clinical Oral
Investigations, and The International Journal of Screening method
Periodontics and Restorative Dentistry was performed from
January 2000 to May 2016. Titles and abstracts of the selected studies were independently
screened by three reviewers (RJM, MFK, and VG). The
screening was based on the question: BDoes platelet rich fibrin
Criteria for study selection and inclusion (PRF) have the ability to affect primary outcomes measured
across a variety of procedures commonly performed in
Study selection considered only articles published in English, dentistry?^ Full-text articles were obtained if the response to
describing the human clinical evaluation of PRF for the the screening question was Byes^ or Buncertain.^ The level of
above-indicated search strategies. Only human studies evalu- agreement between reviewers was determined by kappa
ating the comparative effects of PRF to an appropriate control scores. Disagreement regarding inclusion was resolved by
or to another regenerative modality in human studies were discussion between authors. For necessary missing data, the
included. All human studies evaluating PRF in a case report authors of the studies were contacted.
Clin Oral Invest
Data extraction and analysis
The following data were extracted: general characteristics (au-
thors and year of publication), defect type, number of patients,
healing period, treatment groups, primary outcome measure-
ments, and significant value. Due to the size of the study and
the number of treatment procedures compared using PRF, no
meta-analysis was performed. Instead, the data is reported in a
systematic fashion with an overview of all studies fitting the
search descriptions. Thereafter, data was extracted from the
collection of articles and summarized in separate tables and
discussed accordingly.
Results
Search outcomes
In total, the initial search strategies generated 152 articles that
were separated accordingly into intrabony, furcation, gingival
recession, guided bone regeneration (GBR)/extraction socket,
and sinus lift accordingly (Fig. 2). Of the initial searches, 45
abstracts were retained for further investigation. In total, 10
articles were excluded primarily based on their lack of con-
trols or appropriate endpoints matching our search criteria. In
total, 35 articles were kept for further evaluation. This section
aims to present viable treatment options utilizing PRF and
evaluate its performance according to published studies.
Intrabony defect regeneration with PRF
One of the main uses for PRF has been for the repair/
regeneration of periodontal intrabony defects [36–45]. To
date, 11 randomized clinical trials (RCTs) have reported the
use of PRF for intrabony defect regeneration, most often com-
paring PRF to open flap debridement alone (Table 1). Clinical
improvements were reported via PPD reductions as well as
CAL gains following regenerative periodontal therapy. All
seven studies found that the additional use of PRF increased
PPD reductions and CAL gains when compared to open flap
debridement (OFD) alone (Table 1). One study comparing the
regeneration of intrabony defects utilizing either PRF or a
bone grafting material (demineralized freeze-dried bone allo-
graft (DFDBA)) found no significant differences between
treatment groups [42]. Two studies reported the effectiveness
of PRF in combination with a bone grafting material when
compared to bone grafting material alone [36, 38]. In both
these studies, the additional use of PRF enhanced the filling
of intraosseous defects. Most recently, Panda et al. most re-
cently found that the supplemental use of PRF for intrabony
defect regeneration in combination with a barrier membrane
also led to statistically better results [39]. In summary, the
collected RCTs have demonstrated that the use of PRF leads
to statistically superior periodontal repair of intrabony defects
when compared to OFD alone and may further be combined
with regenerative biomaterials such as bone grafts or collagen
barrier membranes to further enhance periodontal regenera-
tion of intrabony defects. Despite the widespread use of PRF
demonstrating the reduction of PPD and CAL gains, it re-
mains of interest to note that no histological findings have
yet been utilized to demonstrate true histological periodontal
regeneration in human subjects. Therefore, future research to
characterize intrabony defect regeneration versus repair utiliz-
ing PRF as a biomaterial remains necessary.
Furcation defect regeneration with PRF
Similarly, PRF has also been utilized in three studies investi-
gating periodontal regeneration of class II furcation defects
(Supplemental Table 5) [46–48]. In all studies, PRF was com-
pared to OFD alone, thereby fully characterizing its regenera-
tive potential utilizing appropriate well-designed controls in
all human clinical studies. In all three studies conducted by
Sharma et al. 2011, Bajaj et al. 2013, and Pradeep et al. 2016,
the use of PRF led to a significant improvement in CAL gains
when compared to controls [46–48]. These findings report a
gain in vertical CAL of 2.33, 2.87, and 4.17 mm in test PRF
groups when compared to 1.28, 1.37, and 1.82 mm, respec-
tively, in OFD controls [46–48]. These results demonstrate the
potential for tissue repair utilizing PRF for furcation defects.
One remaining issue to address is that the results have not
confirmed the regenerative potential of PRF via histological
evaluation and therefore the process can solely be defined as
tissue Brepair.^ Furthermore, to date, no study has compared
the use of PRF to other effective regenerative materials such as
bone grafting materials or other regenerative bioactive growth
factors. In the future, its clinical performance could be better
assessed if compared to other leading regenerative agents.
Root coverage of gingival recessions with PRF
PRF has also been widely utilized as a bioactive matrix in
numerous studies for root coverage of gingival recessions
(Table 2, Fig. 3) [49–61]. Of the 13 listed studies, six studies
compared the use of coronally advanced flap (CAF) to CAF +
PRF. Of these studies, three found that PRF induced a signif-
icant increase in root coverage [49, 51, 58] whereas the other
three found no significant differences [52, 54, 60]. Of the
remaining studies, one study compared PRF to EMD and
found no differences in the reported root coverage [56]. Four
studies compared CAF + PRF to CAF + connective tissue
graft (CTF) and also found no difference in average root cov-
erage [50, 53, 55, 61]. One study compared CAF + CTG with
CAF + CTG and PRF and found a significant increase in root
coverage for the combination approach utilizing both CTG
with PRF [57]. Rajaram et al. utilized a double lateral sliding
 Clin Oral Invest

Identification
Records iden!fied through Records iden!fied through EMBASE Records iden!fied through other
PUBMED database searching database searching electronic databases
(n =140) (n = 164) (n= 116)

Records a"er duplicates removed

(n = 152)
Screening

Records screened Records excluded

(n= 152) (n = 107)

Full-text ar!cles assessed Full-text ar!cles excluded

for eligibility (n = 10)
Eligibility

(n= 45)
Included

Studies included in
qualita!ve synthesis
(n =35)

Studies included in Studies included in Studies included in Studies included in Studies included in
intrabony defects furca!on defects gingival recessions GBR sinus li"ing
(n =10) (n =3) (n =13) (n =4) (n =5)

Fig. 2 Flowchart of the screened relevant publications

bridge flap with and without PRF for the treatment of gingival
Miller class II defects and found no significant differences
between control and test groups [59]. These results seem to
hint at the fact that the use of PRF favors a slight gain in root
coverage when compared to CAF alone but does not lead to
better results when compared to EMD or to CTG.
Furthermore, several reports show that CAF in combination
with CTG leads to more width in keratinized tissue when
compared to PRF.
Interestingly, two reports commented on the added advan-
tage of PRF in pain management [55, 56]. Jankovic et al.
found in two separate studies comparing PRF to either EMD
or CTG that PRF led to lower morbidity and faster wound
healing [55, 56]. In a similar study investigating the wound
healing properties of PRF, the palatal donor site of the
epithelialized CTG was treated with PRF or a gelatin sponge
on the healing of palatal donor sites [62]. It was reported that
the PRF-enriched palatal bandage significantly accelerated
palatal wound healing and reduced the patient’s morbidity
[62].
In summary, the use of PRF for the treatment of gingival
recessions is limited. Evidence from another systematic re-
view from 2016 concluded that the additional use of PRF for
the treatment of gingival recessions did not lead to any addi-
tional benefit in root coverage or CAL (P = 0.57 and P = 0.50,
respectively) [63]. Furthermore, the reported keratinized mu-
cosa width (KMW) gain was significantly greater in the sub-
group treated with CTG when compared to PRF for studies
greater or equal to 6 months in duration [63]. The results of
that meta-analysis suggest that the use of PRF does not im-
prove the root coverage, KMW, or CAL of Miller class I and II
gingival recessions compared with other treatment modalities
including EMD or CTG but can be obtained easily at low cost
when compared to other regenerative modalities [63].
Clin Oral Invest

Table 1 Effects of PRF on

intrabony defect regeneration Author Defect Healing time Groups ΔPPD CAL gain P value
no. (months) (mm) (mm)

Thorat 32 9 OFD 3.56 2.13 ΔPPD <0.01

2011 OFD + PRF 4.56 3.69 CAL <0.01
Sharma 56 9 OFD 3.21 2.77 ΔPPD 0.006
2011 OFD + PRF 4.55 3.31 CAL n.s.
Pradeep 90 9 OFD 2.97 2.67 ΔPPD 0.002
2012 OFD + PRF 3.90 3.03 GAL n.s.
Pradeep 90 9 OFD 2.97 2.83 ΔPPD 0.018
2012 OFD + PRF 3.77 3.17 GAL n.s.
Shah 40 6 OFD + 3.70 2.97 n.s.
2015 DFDBA
OFD + PRF 3.67 2.97
Pradeep 120 9 OFD 3.01 2.96 ΔPPD <0.001
2015 OFD + PRF 4.01 4.03 CAL <0.001 both
ORF + 1% MF 3.93 3.93 treatment groups
OFD + 1% MF 4.9 4.9
+ PRF
Ajwani 40 9 OFD 1.60 1.3 PPD <0.001
2015 OFD + PRF 1.90 1.8 CAL <0.001
Elgandhy 40 6 OFD + HA 3.42 3.55 PPD <0.02
2015 OFD + HA + 3.82 3.9 CAL <0.027
PRF
Agawal 60 12 OFD + 3.60 2.61 PPD <0.05
2016 DFDBA CAL <0.05
OFD + 4.15 3.73
DFDBA +
PRF
Panda 32 9 Barrier 3.19 3.38 PPD 0.002
2016 membrane CAL = 0.001
Membrane + 3.88 4.44
PRF

PPD probing periodontal depth, CAL clinical attachment level, OFD open flap debridement, PRF platelet-rich
fibrin, DFDBA demineralized freeze-dried bone allograft, MF metformin, HA hydroxyapatite

Guided bone regeneration and extraction socket
management with PRF
One area of research that has gained tremendous popularity in
recent years is the management of dimensional changes of the
alveolar bone directly following tooth extraction [64–66].
These changes have been reported to occur within 8 weeks
following extraction [67] as a consequence of decreased blood
supply following tooth removal (periodontal ligament ab-
sence). Several advantages have been reported when filling
extraction sockets with PRF (Supplemental Table 6)
[68–71]. Hauser et al. found in a study of 23 patients that
PRF reduced dimensional changes prior to implant placement
when compared to natural socket healing [71]. Furthermore, it
was reported that raising a peri-mucosteal flap reduced the
effectiveness of PRF [71]. Girish Rao et al. found that follow-
ing third molar extractions, the filling of sockets with PRF led
to a non-significant increase in bone volume [68]. Hoaglin
et al. reported that filling third molar extraction sockets with
PRF led to a nearly tenfold decrease in osteomyelitis infec-
tions when compared to natural healing. This study was con-
ducted bilaterally in 200 patients, thus providing some of the
highest scientific evidence for the reduced rate of infection
following use of PRF [70]. Lastly, Suttapreyasri et al. found
that PRF reduced dimensional changes in premolar extraction
sites when compared to blank controls [69].
Despite the limited number of studies, the use of PRF acts
as an ideal material post-extraction by improving bone
healing/regeneration, preserving the quality and density of
the residual ridge, reducing infection, and decreasing the time
of surgery when compared to the use of a covering membrane.
These benefits are increasingly associated with a low cost of
operation and a minimal risk of infection. PRF may further be
utilized around immediate implant placement to pack gaps or
additionally to speed soft tissue wound healing (Fig. 4). There
remains however a great necessity to further evaluate dimen-
sional changes utilizing PRF in various clinical situations
using appropriately designed studies. Future clinical research
 Clin Oral Invest

Table 2 Effects of PRF on root coverage of gingival recessions

Author Study type Patient no. Healing period (months) Treatment groups Root coverage (%) P value

Aroca 2009 Split-mouth; Miller class I or II 20 6 CAF 91.5 <0.004

CAF + PRF 80.7
Jankovic 2010 Split-mouth; Miller class I or II 20 12 CAF + EMD 70.5 n.s.
CAF + PRF 72.1
Aleksic 2010 Split-mouth; Miller class I or II 19 12 CAF + CTG 88.6 n.s.
CAF + PRF 79.9
Jankovic 2012 Split-mouth; Miller class I or II 15 6 CAF + CTG 88.7 n.s.
CAF + PRF 92
Padma 2013 Split-mouth; Miller class I or II 15 1, 3, and 6 CAF 68.4 <0.0001
CAF + PRF 100
Eren 2014 Split-mouth; Miller class I or II 22 6 CAF + CTG 94.2 n.s.
CAF + PRF 92.7
Tunaliota 2015 Split-mouth; Miller class I or II 22 12 CAF + CTG 77.4 n.s.
CAF + PRF 76.6
Thamaraiselvan 2015 Split-mouth; Miller class I or II 20 3 and 6 CAF 65 n.s.
CAF + PRF 74.2
Gupta 2015 Split-mouth; Miller class I or II 26 3 and 6 CAF 86.6 n.s.
CAF + PRF 91
Keceli 2015 Split-mouth; Miller class I or II 40 3 and 6 CAF + CTG 79.9 <0.05
CAF + CTG + PRF 89.6
Dogan 2015 Split-mouth; Miller class I or II 20 6 CAF 82.1 n.s.
CAF + PRF 86.7
Rajaram 2015 Split-mouth; Miller class II 20 12 and 24 DLSBF 80 n.s.
DLSBF + PRF 78.8
Agarwal 2016 Split-mouth; Miller class I or II 30 3 and 6 CAF 33 <0.05
CAF + AM 36
CAF + PRF 56

CAF coronally advanced flap, PRF platelet-rich fibrin, EMD enamel matrix derivative, CTG connective tissue graft, DLSBF double lateral sliding bridge
flap, AM amniotic membrane

is therefore necessary. Furthermore, it remains unknown what
effect PRF may play in combination with GBR techniques.
While a collagen barrier membrane is routinely used during
such procedures, additional use or replacement with PRF may
provide further regenerative advantages when compared to
collagen barrier membranes alone. Future studies are thus
necessary to validate these potential advantages.
Sinus elevation procedures with PRF
The use of PRF for sinus elevation is relatively new with little
comparative studies or standardized protocols available.
Although the success rate of surgeries utilizing the addition
of PRF is very high, it is difficult to compare the results be-
tween various treatment methods. Three authors using PRF
alone as a graft material for sinus lift concluded that PRF
significantly promoted bone healing with bone gains of
7.52 mm [72], 10.1 mm [73], and 10.4 mm [74] between the
sinus floor and the top of the alveolar ridge. No controls were
utilized in these studies. Nevertheless, no implants were lost at
6 months, 1 year, and 6 years in their respective studies
[72–74]. Some authors further claim that the use of PRF alone
may be a valid treatment protocol for the majority of sinus lift
cases although the lack of controls and limited number of
studies have thus far limited its use [73].
Other studies compared the use of a bone grafting material
with and without the additional use of PRF [75–78]. The re-
sults show that despite the large bone gains observed with
PRF, no statistically significant differences were reported.
One reported plausible advantage of combining PRF with a
bone grafting material seems to result in a decrease in the
overall healing time and better graft material handling
[75–78]. A recent systematic review on the topic published
in 2016 by Ali et al. found that of 290 initial publications
searched, only eight met the inclusion criteria with approxi-
mately half not utilizing controls [79]. It was reported that the
identified studies showed great heterogeneity regarding surgi-
cal technique, grafting material, implant placement time, sur-
gical protocols, outcome measures, healing time for biopsy,
implant placement, and follow-up periods [79]. In summary,
although the results do not seem to confirm that PRF is better
than other biomaterials, its ease of use, combined with its
Clin Oral Invest

Fig. 3 a Multiple gingival

recessions from the canine to the
molar in the upper jaw. A frontal
view. B–D Lateral views (case
performed by Dr. Giovanni
Zucchelli). b Surgical technique:
A A flap for multiple gingival
recessions has been elevated with
a split-full-split thickness
approach. B A-PRF prepared. C
A-PRF has been applied to cover
all teeth affected by gingival
recessions. Multiple layers have
been applied. D, E Lateral view
showing the thickness of A-PRF
material applied to the root
exposures. F, G Lateral view
showing the flap coronally
advanced and covering
completely the A-PRF material. H
Frontal view showing the flap
covering in excess all gingival
recessions (case performed by Dr.
Giovanni Zucchelli). c Six
months follow-up. A Complete
root coverage with increase in
keratinized tissue height has been
achieved in all treated gingival
recessions. B–D Lateral view
showing the increase in gingival
thickness at all teeth previously
affected by gingival recessions
(case performed by Dr. Giovanni
Zucchelli)

minimal costs and high success rates, seems to illustrate that
high success rates with minimal costs can be obtained by
using PRF during sinus lifting procedures. Nevertheless,
much further research is needed to support the beneficial ef-
fect of PRF.
Discussion
In this systematic review, all randomized clinical studies using
PRF in dentistry were selected without discrepancies as com-
pared to controls or commonly utilized surgical methods. The
aim was to evaluate the current literature with respect to the
clinical indications where PRF has been investigated for
wound healing and/or tissue regeneration/repair. As was
observed in the analysis of its clinical applications, the perfor-
mance of PRF was often compared either to conventional
treatments such as OFD during intrabony/furcation defects
or to naturally healing Bblank^ defects during extraction sock-
et management. The analysis of the generated publications
revealed a great heterogeneity of results with a general lack
of conclusive evidence in large part due to the lack of study
number with appropriate controls. Therefore, only guidelines
can be drawn from the sum of these general conclusions with
an obvious need for further research.
One factor that has been frequently reported in this system-
atic literature search was the ability for PRF to stimulate re-
generation over a wide range of tissues. PRF has been shown
to quickly stimulate tissue healing by significantly increasing
the recruitment and proliferation of a variety of cells including

Fig. 4 Immediate implant
placement in combination with
the use of PRF. a, b Maxillary
right cuspid fracture, extraction. c
PRF placed into fresh extraction
socket. d Dental implant
placement with PRF fragments
visible following placement
(arrow in e). f Three-week
follow-up, excellent soft tissue
wound healing. g Three-year
follow-up (case performed by Dr.
Michael A. Pikos)
endothelial cells, gingival fibroblast, chondrocytes, and oste-
oblasts, thereby heavily promoting tissue repair and angiogen-
esis at the site of injury [80, 81]. These processes are regulated
by local concentrations of cytokines and growth factors
trapped within the fibrous scaffolding, most notably derived
from autologous sources. In comparison, the growing use of
two of the main growth factors approved by the FDA are
PDGF and BMP derived from recombinant sources [82–85].
While these products sell for hundreds of dollars and are fab-
ricated in mammalian cells or bacteria, the use of PRF are
growth factors harvested purely from autologous sources via
low-cost methods. It is therefore of interest to determine the
benefit of using high supra-physiological concentrations of
growth factors in recombinant form (i.e., BMP and PDGF)
versus lower concentrations from autologous form (PRF).
Although recombinant proteins have a regenerative potential
well documented in the literature [86–88], many biological
limitations to their use (swelling and edema), coupled with
their low stability in vivo [89, 90], remain a limiting factor.
Therefore, future research should target the comparison of the
half-life and bioactivity of the growth factors found in PRF in
comparison to commercially available recombinant growth
factors.
Another interesting aspect that requires further study is to
determine the regenerative/reparative potential of PRF on soft
versus hard tissue formation. Thus far, this systematic review
seems to point to the fact that the reparative potential of PRF
favors soft tissue formation/ligament regeneration.
Periodontitis is known to be one of the most common diseases
with breakdown at the periodontium, causing destruction of
Clin Oral Invest
the cementum and periodontal ligament and intrabony defects
[91]. The use of PRF specifically for intrabony defect repair
showed significantly higher PPD reductions and CAL gains
when compared to control OFD in all seven studies.
Furthermore, a bone grafting material (DFDBA) could be
combined with PRF to further generate statistically better
CAL gains and PPD reductions [36]. Therefore, these findings
demonstrate that PRF is able to support periodontal ligament
repair as effective or potentially more effectively than com-
monly utilized biomaterials. Despite these positive findings, it
remains of interest to determine if the reparative potential of
PRF leads to true periodontal regeneration in humans.
Therefore, future human histological studies are needed.
With respect to treatment and management of gingival re-
cessions, PRF has been studied in 13 randomized human clin-
ical studies. In general, it was found that PRF had similar
advantages in root coverage of Miller class I and II defects
when compared to CTG. Noteworthy, it was however com-
monly reported that significantly higher keratinized tissue
width was found with CTG when compared to PRF. While
it is difficult to evaluate significant differences in these treat-
ment procedures due to the high success rates (generally ob-
served over 80% root coverage for all treatment groups), one
area of research that remains to be determined is precisely
under which clinical situations should one expect similar re-
sults between PRF and CTG. Since CTG procedures are as-
sociated with high patient morbidity, it may be that in the
future, such procedures could be substituted with PRF to pre-
vent high morbidity. Furthermore, the technical ability of the
clinician plays a more prominent role during CTG harvesting
Clin Oral Invest
when compared to PRF. Future studies are therefore needed to
present updated clinical guidelines.
Another reported advantage to the use of PRF is its ability
to decrease bacterial infections following surgery such as os-
teomyelitis commonly reported following third molar extrac-
tions [70]. In that study, a 9.5-fold significant decrease in
reported cases of osteomyelitis was observed in a clinical trial
with 100 patients [70]. Therefore and most likely due to the
increase in white blood cells and macrophages capable of
fighting infection, the use of PRF offers some antibacterial
defense against incoming pathogens. Furthermore, macro-
phages have been shown to be key implicators in new bone
formation both during bone modeling and remodeling, as well
as in association with bone biomaterials [92–94]. Despite this,
it remains interesting to point out that no human clinical study
to date has investigated the effects of PRF in a controlled
manner during GBR procedures, and only three studies on
extraction socket healing have investigated dimensional
changes following tooth extraction utilizing PRF
(Supplemental Table 6). A similar trend investigating sinus
lift procedures with PRF was also observed whereby a lack
of well-designed studies with appropriate controls or end-
points was commonly found. Therefore, the effect of PRF
on pure bone regeneration remains questionable and requires
more validating studies. Similarly, various reports have now
supported the use of PRF for pulp regeneration, cystic bone
defect, and papilla augmentation under various clinical indi-
cations to improve healing [95–103]. While these reports are
rare and anecdotal, future research aimed at better character-
izing the regenerative potential of PRF for various other dental
procedures remains necessary.
Very recently, a team of researchers has convincingly
shown that lower centrifugation speeds and time resulted in
higher leukocyte concentrations and release of growth factors
[104–108]. Ghanaati et al. demonstrated via histological pro-
cessing of PRF scaffolds that at higher centrifugation speeds,
the majority of leukocytes were found at the bottom of PRF
scaffolds [104]. By reducing centrifugation g-force, leuko-
cytes were more evenly distributed throughout the PRF scaf-
folds [104]. In addition, regenerative growth factors released
and gingival fibroblast activity are increased when utilizing
slower centrifugation speed and time [108]. While reports
from these studies support modifications to centrifugation pro-
tocols, the impact these may have on clinical outcomes in the
various indications highlighted throughout this review article
remains to be investigated. Future clinical study is therefore
needed.
In conclusion, this systematic review demonstrates the
widespread use of PRF in dentistry in various clinical settings.
Although this regenerative modality remains unfamiliar to
many clinicians, the evidence supporting its use has accumu-
lated over the years, demonstrating its ability to improve tissue
regeneration. The combination of PRF with regenerative
therapy has been shown to be most promising for periodontal
repair of intrabony and furcation defects, as well as soft tissue
root coverage of gingival recessions. Furthermore, evidence
from the literature suggests that PRF is able to decrease infec-
tion following tooth extraction and may further limit dimen-
sional changes following tooth loss. It was also concluded that
regeneration of bone defects (GBR procedures and sinus ele-
vation) necessitates more study with focused endpoints.
Nevertheless, its ease of use, combined with its low cost and
autologous source, makes it an ideal biomaterial worth further
investigation across a variety of surgical procedures in
dentistry.
Compliance with ethical standards
Conflict of interest Joseph Choukroun is the founder of Process of
PRF company and the developer and inventor of PRF protocol in Nice,
France. All other authors declare no conflict of interest.
Funding This work was fully funded by the Cell Biology Laboratory at
Nova Southeastern University, College of Dental Medicine.
Ethical approval No ethical approval was required for this study as
human participants or animals were not utilized in this study.
Informed consent Informed consent was not required as no human or
animal subjects were utilized in this study.
References
1. Dangaria SJ, Ito Y, Walker C, Druzinsky R, Luan X, Diekwisch
TG (2009) Extracellular matrix-mediated differentiation of peri-
odontal progenitor cells. Differentiation 78:79–90. doi:10.1016/j.
diff.2009.03.005
2. Hollander A, Macchiarini P, Gordijn B, Birchall M (2009) The
first stem cell-based tissue-engineered organ replacement: impli-
cations for regenerative medicine and society. Regen Med 4:147–
148. doi:10.2217/17460751.4.2.147
3. Choukroun J, Adda F, Schoeffler C, Vervelle A (2001) Une
opportunité en paro-implantologie: le PRF. Implantodontie 42:e62
4. Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi
J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-generation
platelet concentrate. Part II: platelet-related biologic features. Oral
Surg, Oral Med, Oral Pathol, Oral Radiol Endod 101:e45–e50.
doi:10.1016/j.tripleo.2005.07.009
5. Kang YH, Jeon SH, Park JY, Chung JH, Choung YH, Choung
HW, Kim ES, Choung PH (2011) Platelet-rich fibrin is a
bioscaffold and reservoir of growth factors for tissue regeneration.
Tissue Eng A 17:349–359. doi:10.1089/ten.TEA.2010.0327
6. He L, Lin Y, Hu X, Zhang Y, Wu H (2009) A comparative study of
platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the
effect of proliferation and differentiation of rat osteoblasts
in vitro. Oral Surg, Oral Med, Oral Pathol, Oral Radiol Endod
108:707–713. doi:10.1016/j.tripleo.2009.06.044
7. Dohan Ehrenfest DM, Diss A, Odin G, Doglioli P, Hippolyte MP,
Charrier JB (2009) In vitro effects of Choukroun’s PRF (platelet-
r i c h f i b r i n ) o n h u m a n g i n g i v a l f i br ob l a s t s , d e r m a l
prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in

primary cultures. Oral Surg, Oral Med, Oral Pathol, Oral Radiol
Endod 108:341–352. doi:10.1016/j.tripleo.2009.04.020
8. Anfossi G, Trovati M, Mularoni E, Massucco P, Calcamuggi G,
Emanuelli G (1989) Influence of propranolol on platelet aggrega-
tion and thromboxane B2 production from platelet-rich plasma
and whole blood. Prostaglandins Leukot Essent Fat Acids 36:1–7
9. Fijnheer R, Pietersz RN, de Korte D, Gouwerok CW, Dekker WJ,
Reesink HW, Roos D (1990) Platelet activation during preparation
of platelet concentrates: a comparison of the platelet-rich plasma
and the buffy coat methods. Transfusion 30:634–638
10. Jameson C (2007) Autologous platelet concentrate for the produc-
tion of platelet gel. Lab Med 38:39–42
11. Whitman DH, Berry RL, Green DM (1997) Platelet gel: an autol-
ogous alternative to fibrin glue with applications in oral and max-
illofacial surgery. J Oral Maxillofac Surg 55:1294–1299
12. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE,
Georgeff KR (1998) Platelet-rich plasma: growth factor enhance-
ment for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 85:638–646
13. Marx RE (2004) Platelet-rich plasma: evidence to support its use. J
Oral Maxillofac Surg 62:489–496
14. Anitua E (1999) Plasma rich in growth factors: preliminary results
of use in the preparation of future sites for implants. Int J Oral
Maxillofac Implants 14:529–535
15. Anitua E, Prado R, Troya M, Zalduendo M, de la Fuente M, Pino
A, Muruzabal F, Orive G (2016) Implementation of a more phys-
iological plasma rich in growth factor (PRGF) protocol: anticoag-
ulant removal and reduction in activator concentration. Platelets
27:459–466. doi:10.3109/09537104.2016.1143921
16. Lucarelli E, Beretta R, Dozza B, Tazzari PL, O’Connel SM, Ricci
F, Pierini M, Squarzoni S, Pagliaro PP, Oprita EI, Donati D (2010)
A recently developed bifacial platelet-rich fibrin matrix. Eur Cell
Mater 20:13–23
17. Saluja H, Dehane V, Mahindra U (2011) Platelet-rich fibrin: a
second generation platelet concentrate and a new friend of oral
and maxillofacial surgeons. Ann Maxillofac Surg 1:53–57. doi:
10.4103/2231-0746.83158
18. Kobayashi E, Fluckiger L, Fujioka-Kobayashi M, Sawada K,
Sculean A, Schaller B, Miron RJ (2016) Comparative release of
growth factors from PRP, PRF, and advanced-PRF. Clin Oral
Investig. doi:10.1007/s00784-016-1719-1
19. Dohan Ehrenfest DM, Del Corso M, Diss A, Mouhyi J, Charrier
JB (2010) Three-dimensional architecture and cell composition of
a Choukroun’s platelet-rich fibrin clot and membrane. J
Periodontol 81:546–555. doi:10.1902/jop.2009.090531
20. Toffler M, Toscano N, Holtzclaw D, Corso M, Dohan D (2009)
Introducing Choukroun’s platelet rich fibrin (PRF) to the recon-
structive surgery milieu. J Implant Adv Clin Dent 1:22–31
21. Tsay RC, Vo J, Burke A, Eisig SB, Lu HH, Landesberg R (2005)
Differential growth factor retention by platelet-rich plasma com-
posites. J Oral Maxillofac Surg 63:521–528. doi:10.1016/j.joms.
2004.09.012
22. Carlson NE, Roach RB Jr (2002) Platelet-rich plasma: clinical
applications in dentistry. J Am Dent Assoc 1939(133):1383–1386
23. Andrades JA, Han B, Becerra J, Sorgente N, Hall FL, Nimni ME
(1999) A recombinant human TGF-beta1 fusion protein with
collagen-binding domain promotes migration, growth, and differ-
entiation of bone marrow mesenchymal cells. Exp Cell Res 250:
485–498. doi:10.1006/excr.1999.4528
24. Lind M, Deleuran B, Thestrup-Pedersen K, Soballe K, Eriksen EF,
Bunger C (1995) Chemotaxis of human osteoblasts. Effects of
osteotropic growth factors. APMIS 103:140–146
25. Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi
J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-generation
platelet concentrate. Part III: leucocyte activation: a new feature
Clin Oral Invest
for platelet concentrates? Oral Surg, Oral Med Oral Pathol Oral
Radiol Endod 101:e51–e55. doi:10.1016/j.tripleo.2005.07.010
26. Grando Mattuella L, Poli de Figueiredo JA, Nor JE, de Araujo FB,
Medeiros Fossati AC (2007) Vascular endothelial growth factor
receptor-2 expression in the pulp of human primary and young
permanent teeth. J Endod 33:1408–1412. doi:10.1016/j.joen.
2007.08.019
27. Troost E, Hold GL, Smith MG, Chow WH, Rabkin CS, McColl
KE, El-Omar EM (2003) The role of interleukin-1beta and other
potential genetic markers as indicators of gastric cancer risk. Can J
Gastroenterol 17(Suppl B):8B–12B
28. Kishimoto T, Akira S, Narazaki M, Taga T (1995) Interleukin-6
family of cytokines and gp130. Blood 86:1243–1254
29. Kishimoto T (1989) The biology of interleukin-6. Blood 74:1–10
30. Waters JP, Pober JS, Bradley JR (2013) Tumour necrosis factor in
infectious disease. J Pathol 230:132–147. doi:10.1002/path.4187
31. Murtaugh MP, Johnson CR, Xiao Z, Scamurra RW, Zhou Y
(2009) Species specialization in cytokine biology: is interleukin-
4 central to the T(H)1-T(H)2 paradigm in swine? Dev Comp
Immunol 33:344–352. doi:10.1016/j.dci.2008.06.014
32. Li Z, Zhang Y, Sun B (2011) Current understanding of Th2 cell
differentiation and function. Protein Cell 2:604–611. doi:10.1007/
s13238-011-1083-5
33. Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C,
Dohan SL, Dohan AJ, Mouhyi J, Dohan DM (2006) Platelet-rich
fibrin (PRF): a second-generation platelet concentrate. Part IV:
clinical effects on tissue healing. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 101:e56–e60. doi:10.1016/j.tripleo.
2005.07.011
34. Clark RA (2001) Fibrin and wound healing. Ann N Y Acad Sci
936:355–367
35. Swartz MK (2011) The PRISMA statement: a guideline for sys-
tematic reviews and meta-analyses. J Pediatr Health Care 25:1–2.
doi:10.1016/j.pedhc.2010.09.006
36. Agarwal A, Gupta ND, Jain A (2016) Platelet rich fibrin combined
with decalcified freeze-dried bone allograft for the treatment of
human intrabony periodontal defects: a randomized split mouth
clinical trail. Acta Odontol Scand 74:36–43. doi:10.3109/
00016357.2015.1035672
37. Ajwani H, Shetty S, Gopalakrishnan D, Kathariya R, Kulloli A,
Dolas RS, Pradeep AR (2015) Comparative evaluation of platelet-
rich fibrin biomaterial and open flap debridement in the treatment
of two and three wall intrabony defects. J Int Oral Health 7:32–37
38. Elgendy EA, Abo Shady TE (2015) Clinical and radiographic
evaluation of nanocrystalline hydroxyapatite with or without
platelet-rich fibrin membrane in the treatment of periodontal
intrabony defects. J Indian Soc Periodontol 19:61–65. doi:10.
4103/0972-124x.148639
39. Panda S, Sankari M, Satpathy A, Jayakumar D, Mozzati M,
Mortellaro C, Gallesio G, Taschieri S, Del Fabbro M (2016)
Adjunctive effect of autologus platelet-rich fibrin to barrier mem-
brane in the treatment of periodontal intrabony defects. J
Craniofac Surg 27:691–696. doi:10.1097/scs.0000000000002524
40. Pradeep AR, Nagpal K, Karvekar S, Patnaik K, Naik SB,
Guruprasad CN (2015) Platelet-rich fibrin with 1% metformin
for the treatment of intrabony defects in chronic periodontitis: a
randomized controlled clinical trial. J Periodontol 86:729–737.
doi:10.1902/jop.2015.140646
41. Pradeep AR, Rao NS, Agarwal E, Bajaj P, Kumari M, Naik SB
(2012) Comparative evaluation of autologous platelet-rich fibrin
and platelet-rich plasma in the treatment of 3-wall intrabony de-
fects in chronic periodontitis: a randomized controlled clinical
trial. J Periodontol 83:1499–1507. doi:10.1902/jop.2012.110705
42. Shah M, Patel J, Dave D, Shah S (2015) Comparative evaluation
of platelet-rich fibrin with demineralized freeze-dried bone allo-
graft in periodontal infrabony defects: a randomized controlled
Clin Oral Invest
clinical study. J Indian Soc Periodontol 19:56–60. doi:10.4103/
0972-124x.145803
43. Thorat M, Pradeep AR, Pallavi B (2011) Clinical effect of autol-
ogous platelet-rich fibrin in the treatment of intra-bony defects: a
controlled clinical trial. J Clin Periodontol 38:925–932. doi:10.
1111/j.1600-051X.2011.01760.x
44. Pradeep AR, Bajaj P, Rao NS, Agarwal E, Naik SB (2012)
Platelet-rich fibrin combined with a porous hydroxyapatite graft
for the treatment of three-wall intrabony defects in chronic peri-
odontitis: a randomized controlled clinical trial. J Periodontol. doi:
10.1902/jop.2012.110722
45. Sharma A, Pradeep AR (2011) Treatment of 3-wall intrabony
defects in patients with chronic periodontitis with autologous
platelet-rich fibrin: a randomized controlled clinical trial. J
Periodontol 82:1705–1712. doi:10.1902/jop.2011.110075
46. Sharma A, Pradeep AR (2011) Autologous platelet-rich fibrin in
the treatment of mandibular degree II furcation defects: a random-
ized clinical trial. J Periodontol 82:1396–1403. doi:10.1902/jop.
2011.100731
47. Bajaj P, Pradeep AR, Agarwal E, Rao NS, Naik SB, Priyanka N,
Kalra N (2013) Comparative evaluation of autologous platelet-
rich fibrin and platelet-rich plasma in the treatment of mandibular
degree II furcation defects: a randomized controlled clinical trial. J
Periodontal Res. doi:10.1111/jre.12040
48. Pradeep AR, Karvekar S, Nagpal K, Patnaik K, Raju A, Singh P
(2016) Rosuvastatin 1.2 mg in situ gel combined with 1:1 mixture
of autologous platelet-rich fibrin and porous hydroxyapatite bone
graft in surgical treatment of mandibular class II furcation defects:
a randomized clinical control trial. J Periodontol 87:5–13. doi:10.
1902/jop.2015.150131
49. Agarwal SK, Jhingran R, Bains VK, Srivastava R, Madan R,
Rizvi I (2016) Patient-centered evaluation of microsurgical man-
agement of gingival recession using coronally advanced flap with
platelet-rich fibrin or amnion membrane: a comparative analysis.
Eur J Dent 10:121–133. doi:10.4103/1305-7456.175686
50. Aleksic Z, Jankovic S, Dimitrijevic B, Divnic-Resnik T,
Milinkovic I, Lekovic V (2010) The use of platelet-rich fibrin
membrane in gingival recession treatment. Srp Arh Celok Lek
138:11–18
51. Aroca S, Keglevich T, Barbieri B, Gera I, Etienne D (2009)
Clinical evaluation of a modified coronally advanced flap alone
or in combination with a platelet-rich fibrin membrane for the
treatment of adjacent multiple gingival recessions: a 6-month
study. J Periodontol 80:244–252. doi:10.1902/jop.2009.080253
52. Dogan SB, Dede FO, Balli U, Atalay EN, Durmuslar MC (2015)
Concentrated growth factor in the treatment of adjacent multiple
gingival recessions: a split-mouth randomized clinical trial. J Clin
Periodontol 42:868–875. doi:10.1111/jcpe.12444
53. Eren G, Atilla G (2014) Platelet-rich fibrin in the treatment of
localized gingival recessions: a split-mouth randomized clinical
trial. Clin Oral Investig 18:1941–1948. doi:10.1007/s00784-013-
1170-5
54. Gupta S, Banthia R, Singh P, Banthia P, Raje S, Aggarwal N
(2015) Clinical evaluation and comparison of the efficacy of
coronally advanced flap alone and in combination with platelet
rich fibrin membrane in the treatment of Miller class I and II
gingival recessions. Contemp Clin Dent 6:153–160. doi:10.
4103/0976-237x.156034
55. Jankovic S, Aleksic Z, Klokkevold P, Lekovic V, Dimitrijevic B,
Kenney EB, Camargo P (2012) Use of platelet-rich fibrin mem-
brane following treatment of gingival recession: a randomized
clinical trial. Int J Periodontics Restorative Dent 32:e41–e50
56. Jankovic S, Aleksic Z, Milinkovic I, Dimitrijevic B (2010) The
coronally advanced flap in combination with platelet-rich fibrin
(PRF) and enamel matrix derivative in the treatment of gingival
recession: a comparative study. Eur J Esthet Dent 5:260–273
57. Keceli HG, Kamak G, Erdemir EO, Evginer MS, Dolgun A
(2015) The adjunctive effect of platelet-rich fibrin to connective
tissue graft in the treatment of buccal recession defects: results of a
randomized, parallel-group controlled trial. J Periodontol 86:
1221–1230. doi:10.1902/jop.2015.150015
58. Padma R, Shilpa A, Kumar PA, Nagasri M, Kumar C, Sreedhar A
(2013) A split mouth randomized controlled study to evaluate the
adjunctive effect of platelet-rich fibrin to coronally advanced flap
in Miller’s class-I and II recession defects. J Indian Soc
Periodontol 17:631–636. doi:10.4103/0972-124x.119281
59. Rajaram V, Thyegarajan R, Balachandran A, Aari G,
Kanakamedala A (2015) Platelet rich fibrin in double lateral slid-
ing bridge flap procedure for gingival recession coverage: an
original study. J Indian Soc Periodontol 19:665–670. doi:10.
4103/0972-124x.164764
60. Thamaraiselvan M, Elavarasu S, Thangakumaran S, Gadagi JS,
Arthie T (2015) Comparative clinical evaluation of coronally ad-
vanced flap with or without platelet rich fibrin membrane in the
treatment of isolated gingival recession. J Indian Soc Periodontol
19:66–71. doi:10.4103/0972-124x.145790
61. Tunaliota M, Ozdemir H, Arabaciota T, Gurbuzer B, Pikdoken L,
Firatli E (2015) Clinical evaluation of autologous platelet-rich
fibrin in the treatment of multiple adjacent gingival recession de-
fects: a 12-month study. Int J Periodontics Restorative Dent 35:
105–114. doi:10.11607/prd.1826
62. Femminella B, Iaconi MC, Di Tullio M, Romano L, Sinjari B,
D’Arcangelo C, De Ninis P, Paolantonio M (2016) Clinical com-
parison of platelet-rich fibrin and a gelatin sponge in the manage-
ment of palatal wounds after epithelialized free gingival graft har-
vest: a randomized clinical trial. J Periodontol 87:103–113. doi:10.
1902/jop.2015.150198
63. Moraschini V, Barboza Edos S (2016) Use of platelet-rich fibrin
membrane in the treatment of gingival recession: a systematic
review and meta-analysis. J Periodontol 87:281–290. doi:10.
1902/jop.2015.150420
64. De Risi V, Clementini M, Vittorini G, Mannocci A, De Sanctis M
(2015) Alveolar ridge preservation techniques: a systematic re-
view and meta-analysis of histological and histomorphometrical
data. Clin Oral Implants Res 26:50–68. doi:10.1111/clr.12288
65. Jambhekar S, Kernen F, Bidra AS (2015) Clinical and histologic
outcomes of socket grafting after flapless tooth extraction: a sys-
tematic review of randomized controlled clinical trials. J Prosthet
Dent 113:371–382. doi:10.1016/j.prosdent.2014.12.009
66. Moraschini V, Barboza ED (2016) Quality assessment of system-
atic reviews on alveolar socket preservation. Int J Oral Maxillofac
Surg. doi:10.1016/j.ijom.2016.03.010
67. Chappuis V, Engel O, Reyes M, Shahim K, Nolte LP, Buser D
(2013) Ridge alterations post-extraction in the esthetic zone: a 3D
analysis with CBCT. J Dental Res 92:195s–201s. doi:10.1177/
0022034513506713
68. Girish Rao S, Bhat P, Nagesh KS, Rao GH, Mirle B, Kharbhari L,
Gangaprasad B (2013) Bone regeneration in extraction sockets
with autologous platelet rich fibrin gel. J Maxillofac Oral
Surgery 12:11–16. doi:10.1007/s12663-012-0370-x
69. Suttapreyasri S, Leepong N (2013) Influence of platelet-rich fibrin
on alveolar ridge preservation. J Craniofac Surg 24:1088–1094.
doi:10.1097/SCS.0b013e31828b6dc3
70. Hoaglin DR, Lines GK (2013) Prevention of localized osteitis in
mandibular third-molar sites using platelet-rich fibrin. Int J Dent
2013:875380. doi:10.1155/2013/875380
71. Hauser F, Gaydarov N, Badoud I, Vazquez L, Bernard JP,
Ammann P (2013) Clinical and histological evaluation of
postextraction platelet-rich fibrin socket filling: a prospective ran-
domized controlled study. Implant Dent 22:295–303. doi:10.
1097/ID.0b013e3182906eb3

72. Tajima N, Ohba S, Sawase T, Asahina I (2013) Evaluation of sinus
floor augmentation with simultaneous implant placement using
platelet-rich fibrin as sole grafting material. Int J Oral Maxillofac
Implants 28:77–83. doi:10.11607/jomi.2613
73. Mazor Z, Horowitz RA, Del Corso M, Prasad HS, Rohrer MD,
Dohan Ehrenfest DM (2009) Sinus floor augmentation with si-
multaneous implant placement using Choukroun’s platelet-rich
fibrin as the sole grafting material: a radiologic and histologic
study at 6 months. J Periodontol 80:2056–2064. doi:10.1902/
jop.2009.090252
74. Simonpieri A, Choukroun J, Del Corso M, Sammartino G, Dohan
Ehrenfest DM (2011) Simultaneous sinus-lift and implantation
using microthreaded implants and leukocyte- and platelet-rich fi-
brin as sole grafting material: a six-year experience. Implant Dent
20:2–12. doi:10.1097/ID.0b013e3181faa8af
75. Inchingolo F, Tatullo M, Marrelli M, Inchingolo AM, Scacco S,
Inchingolo AD, Dipalma G, Vermesan D, Abbinante A, Cagiano
R (2010) Trial with platelet-rich fibrin and Bio-Oss used as
grafting materials in the treatment of the severe maxillar bone
atrophy: clinical and radiological evaluations. Eur Rev Med
Pharmacol Sci 14:1075–1084
76. Tatullo M, Marrelli M, Cassetta M, Pacifici A, Stefanelli LV,
Scacco S, Dipalma G, Pacifici L, Inchingolo F (2012) Platelet rich
fibrin (P.R.F.) in reconstructive surgery of atrophied maxillary
bones: clinical and histological evaluations. Int J Med Sci 9:
872–880. doi:10.7150/ijms.5119
77. Zhang Y, Tangl S, Huber CD, Lin Y, Qiu L, Rausch-Fan X (2012)
Effects of Choukroun’s platelet-rich fibrin on bone regeneration in
combination with deproteinized bovine bone mineral in maxillary
sinus augmentation: a histological and histomorphometric study. J
Cranio-Maxillo-Facial Surg 40:321–328. doi:10.1016/j.jcms.
2011.04.020
78. Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C,
Dohan SL, Dohan AJ, Mouhyi J, Dohan DM (2006) Platelet-rich
fibrin (PRF): a second-generation platelet concentrate. Part V: his-
tologic evaluations of PRF effects on bone allograft maturation in
sinus lift. Oral Surg Oral Med Oral Pathol Oral Rad Endod 101:
299–303. doi:10.1016/j.tripleo.2005.07.012
79. Ali S, Bakry SA, Abd-Elhakam H (2015) Platelet-rich fibrin in
maxillary sinus augmentation: a systematic review. J Oral
Implantol 41:746–753. doi:10.1563/aaid-joi-D-14-00167
80. Roy S, Driggs J, Elgharably H, Biswas S, Findley M, Khanna S,
Gnyawali U, Bergdall VK, Sen CK (2011) Platelet-rich fibrin
matrix improves wound angiogenesis via inducing endothelial cell
proliferation. Wound Repair Regen 19:753–766. doi:10.1111/j.
1524-475X.2011.00740.x
81. Chen FM, Wu LA, Zhang M, Zhang R, Sun HH (2011) Homing of
endogenous stem/progenitor cells for in situ tissue regeneration:
promises, strategies, and translational perspectives. Biomaterials
32:3189–3209. doi:10.1016/j.biomaterials.2010.12.032
82. Steed DL, Donohoe D, Webster MW, Lindsley L (1996) Effect of
extensive debridement and treatment on the healing of diabetic
foot ulcers. Diabetic Ulcer Study Group. J Am Coll Surg 183:
61–64
83. Wieman TJ, Smiell JM, Su Y (1998) Efficacy and safety of a
topical gel formulation of recombinant human platelet-derived
growth factor-BB (becaplermin) in patients with chronic neuro-
pathic diabetic ulcers. A phase III randomized placebo-controlled
double-blind study. Diabetes Care 21:822–827
84. White AP, Vaccaro AR, Hall JA, Whang PG, Friel BC, McKee
MD (2007) Clinical applications of BMP-7/OP-1 in fractures,
nonunions and spinal fusion. Int Orthop 31:735–741. doi:10.
1007/s00264-007-0422-x
85. Miron RJ, Zhang YF (2012) Osteoinduction: a review of old con-
cepts with new standards. J Dent Res 91:736–744. doi:10.1177/
0022034511435260
Clin Oral Invest
86. Young CS, Ladd PA, Browning CF, Thompson A, Bonomo J,
Shockley K, Hart CE (2009) Release, biological potency, and
biochemical integrity of recombinant human platelet-derived
growth factor-BB (rhPDGF-BB) combined with Augment(TM)
Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP).
J Control Release 140:250–255. doi:10.1016/j.jconrel.2009.06.
030
87. Park YJ, Lee YM, Lee JY, Seol YJ, Chung CP, Lee SJ (2000)
Controlled release of platelet-derived growth factor-BB from
chondroitin sulfate-chitosan sponge for guided bone regeneration.
J Control Release 67:385–394
88. Wissink MJ, Beernink R, Poot AA, Engbers GH, Beugeling T, van
Aken WG, Feijen J (2000) Improved endothelialization of vascu-
lar grafts by local release of growth factor from heparinized colla-
gen matrices. J Control Release 64:103–114
89. Delgado JJ, Evora C, Sanchez E, Baro M, Delgado A (2006)
Validation of a method for non-invasive in vivo measurement of
growth factor release from a local delivery system in bone. J
Control Release 114:223–229
90. Oe S, Fukunaka Y, Hirose T, Yamaoka Y, Tabata Y (2003) A trial
on regeneration therapy of rat liver cirrhosis by controlled release
of hepatocyte growth factor. J Control Release 88:193–200
91. Sculean A, Gruber R, Bosshardt DD (2014) Soft tissue wound
healing around teeth and dental implants. J Clin Periodontol
41(Suppl 15):S6–22. doi:10.1111/jcpe.12206
92. Adamson R (2009) Role of macrophages in normal wound
healing: an overview. J Wound Care 18:349–351. doi:10.12968/
jowc.2009.18.8.43636
93. Miron RJ, Bosshardt DD (2016) OsteoMacs: key players around
bone biomaterials. Biomaterials 82:1–19. doi:10.1016/j.
biomaterials.2015.12.017
94. Sinder BP, Pettit AR, McCauley LK (2015) Macrophages: their
emerging roles in bone. J Bone Miner Res 30:2140–2149. doi:10.
1002/jbmr.2735
95. Subash D, Shoba K, Aman S, Bharkavi SK (2016) Revitalization
of an immature permanent mandibular molar with a necrotic pulp
using platelet-rich fibrin: a case report. J Clin Diagn Res 10:Zd21–
zd23. doi:10.7860/jcdr/2016/21793.8902
96. Rebentish PD, Umashetty G, Kaur H, Doizode T, Kaslekar M,
Chowdhury S (2016) Platelet-rich fibrin: a boon in regenerative
endodontics. Minerva Stomatol 65:385–392
97. Kim JH, Woo SM, Choi NK, Kim WJ, Kim SM, Jung JY (2017)
Effect of platelet-rich fibrin on odontoblastic differentiation in
human dental pulp cells exposed to lipopolysaccharide. J Endod
43:433–438. doi:10.1016/j.joen.2016.11.002
98. Bakhtiar H, Esmaeili S, Fakhr Tabatabayi S, Ellini MR, Nekoofar
MH, Dummer PM (2017) Second-generation platelet concentrate
(platelet-rich fibrin) as a scaffold in regenerative endodontics: a
case series. J Endod 43:401–408. doi:10.1016/j.joen.2016.10.016
99. Pradeep K, Kudva A, Narayanamoorthy V, Cariappa KM,
Saraswathi MV (2016) Platelet-rich fibrin combined with synthet-
ic nanocrystalline hydroxy apatite granules in the management of
radicular cyst. Niger J Clin Pract 19:688–691. doi:10.4103/1119-
3077.188711
100. Mirkovic S, Djurdjevic-Mirkovic T, Pugkar T (2015) Application
of concentrated growth factors in reconstruction of bone defects
after removal of large jaw cysts–the two cases report. Vojnosanit
Pregl 72:368–371
101. Meshram VS, Lambade PN, Meshram PV, Kadu A, Tiwari MS
(2015) The autologous platelet rich fibrin: a novel approach in
osseous regeneration after cystic enucleation: a pilot study.
Indian J Dent Res 26:560–564. doi:10.4103/0970-9290.176915
102. Dar M, Hakim T, Shah A, Najar L, Yaqoob G, Lanker F (2016)
Use of autologous platelet-rich fibrin in osseous regeneration after
cystic enucleation: a clinical study. J Oral Biol Craniofac Res 6:
S29–s32. doi:10.1016/j.jobcr.2016.04.004
Clin Oral Invest
103. Arunachalam LT, Merugu S, Sudhakar U (2012) A novel surgical
procedure for papilla reconstruction using platelet rich fibrin.
Contemp Clin Dent 3:467–470. doi:10.4103/0976-237x.107443
104. Ghanaati S, Booms P, Orlowska A, Kubesch A, Lorenz J,
Rutkowski J, Landes C, Sader R, Kirkpatrick C, Choukroun J
(2014) Advanced platelet-rich fibrin: a new concept for cell-
based tissue engineering by means of inflammatory cells. J Oral
Implantol 40:679–689. doi:10.1563/aaid-joi-D-14-00138
105. Choukroun J, Ghanaati S (2017) Reduction of relative centrifuga-
tion force within injectable platelet-rich-fibrin (PRF) concentrates
advances patients’ own inflammatory cells, platelets and growth
factors: the first introduction to the low speed centrifugation con-
cept. Eur J Trauma Emerg Surg. doi:10.1007/s00068-017-0767-9
106. El Bagdadi K, Kubesch A, Yu X, Al-Maawi S, Orlowska A, Dias
A, Booms P, Dohle E, Sader R, Kirkpatrick CJ, Choukroun J,
Ghanaati S (2017) Reduction of relative centrifugal forces in-
creases growth factor release within solid platelet-rich-fibrin
(PRF)-based matrices: a proof of concept of LSCC (low speed
centrifugation concept). Eur J Trauma Emerg Surg. doi:10.1007/
s00068-017-0785-7
107. Kobayashi E, Fluckiger L, Fujioka-Kobayashi M, Sawada K,
Sculean A, Schaller B, Miron RJ (2016) Comparative release of
growth factors from PRP, PRF, and advanced-PRF. Clin Oral
Investig 20:2353–2360. doi:10.1007/s00784-016-1719-1
108. Fujioka-Kobayashi M, Miron RJ, Hernandez M, Kandalam U,
Zhang Y, Choukroun J (2017) Optimized platelet-rich fibrin with
the low-speed concept: growth factor release, biocompatibility,
and cellular response. J Periodontol 88:112–121. doi:10.1902/
jop.2016.160443
 Tissue Engineering
Fo
rP
ee
Tissue Engineering Manuscript Central: http://mc.manuscriptcentral.com/ten
rR
Platelet Rich Fibrin and Soft Tissue Wound Healing: A
Systematic Review
ev

Journal: Tissue Engineering

ie
Manuscript ID Draft

Manuscript Type: Review Article - Part B

w

Date Submitted by the Author: n/a

Complete List of Authors: Miron, Richard; Nova Southeastern University, Periodontology

ON

Fujioka-Kobayashi, Masako; University of Bern, Cranio-Maxillofacial

Surgery
Bishara, Mark
zhang, yufeng; Ministry Education Key Laboratory for Oral Biomedical
Engineering School of Stomatology, Wuhan University
LY

Hernandez, Maria; Nova Southeastern University, Periodontology

Choukroun, Joseph

Growth Factors < Fundamental of Tissue Engineering (DO NOT select this
phrase; it is a header ONLY), Wound Healing < Fundamental of Tissue
Keyword:
/N

Engineering (DO NOT select this phrase; it is a header ONLY), Tissue

Engineering Applications (DO NOT select this phrase; it is a header ONLY)

The growing multidisciplinary field of tissue engineering aims to predictably

regenerate, enhance or replace damaged or missing tissues for a variety of
ot

conditions caused by trauma, disease and old age. One area of research
that has gained tremendous awareness in recent years is that of platelet
rich fibrin (PRF) which has been utilized across a wide variety of medical
fields for the regeneration of soft tissues. This systematic review gathered
fo

all the currently available in vitro, in vivo and clinical literature utilizing PRF
for soft tissue regeneration, augmentation and/or wound healing. In total,
164 publications met the original search criteria with a total of 48 meeting
inclusion criteria (kappa score = 94%). These studies were divided into 7
rD

in vitro, 11 in vivo and 31 clinical studies. In summary, 6 out of 7 (85.7%)

and 11 out of 11 (100%) of the in vitro and in vivo studies respectively
Abstract: demonstrated a statistically significant advantage for combining PRF to
their regenerative therapies. Out of the remaining 31 clinical studies, a
total of 8 reported the effects of PRF in a randomized clinical trial with 5
is

additional studies (13 total) reporting appropriate controls. In those clinical

studies, 9 out of the 13 studies (69.2%) demonstrated a statistically
relevant positive outcome for the primary endpoints measured. In total, 18
tri

studies (58% of clinical studies) reported positive wound healing events

associated with the use of PRF despite using controls. In total, 27 of the 31
clinical studies (87%) supported the use of PRF for soft tissue regeneration
bu

and wound healing for a variety of procedures in medicine and dentistry. In

conclusion, the results from the present systematic review highlight the
positive effects of PRF on wound healing following regenerative procedures
of soft tissues in total 43 out of 48 studies (89.6%).
tio

Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

n
 Page 1 of 37 Tissue Engineering
Fo
1
2
rP
3
4
5
6
ee
7
8
9
10
rR
11
12
13
14
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 2 of 37
Fo
1
2
rP
3
4 Platelet Rich Fibrin and Soft Tissue Wound Healing: A Systematic
5
6
Review
ee
7
8
9
10
rR
11
12
13 Richard J. Miron1*, Masako Fujioka-Kobayashi1,2,3, Mark Bishara4, Yufeng Zhang5,
14
ev
15 Maria A Hernandez1, Joseph Choukroun6
16
17
18
ie
19
1Department of Periodontology, Nova Southeastern University, Fort Lauderdale,
20
w
21
22 Florida, USA
23
24
ON

2Cranio-Maxillofacial Surgery, Bern University Hospital, Inselspital, Bern,

25
26
27 Switzerland
28
LY

29 3Department of Oral Surgery, Clinical Dentistry, Institute of Biomedical Sciences,

30
31
32 Tokushima University Graduate School, Japan
33
/N

34 4Director, Private Practice, West Bowmanville Family Dental, Ontario, Canada

35
36 5Department
37 of Oral Implantology, University of Wuhan, China
ot

38
39 6Pain Clinic, Nice, France
40
41
fo

42
43
44 * Corresponding author.
rD

45
46 Richard Miron
47
48
Nova Southeastern University
Department of Periodontology
is

49
50 Fort Lauderdale, Florida, USA
51 rmiron@nova.edu
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 3 of 37 Tissue Engineering
Fo
1
2
rP
3
Abstract:
4
5
6 The growing multidisciplinary field of tissue engineering aims to predictably
ee
7
8 regenerate, enhance or replace damaged or missing tissues for a variety of
9
10 conditions caused by trauma, disease and old age. One area of research that
rR
11 has gained tremendous awareness in recent years is that of platelet rich fibrin
12
13 (PRF) which has been utilized across a wide variety of medical fields for the
14
ev
15 regeneration of soft tissues. This systematic review gathered all the currently
16
17 available in vitro, in vivo and clinical literature utilizing PRF for soft tissue
18
ie
regeneration, augmentation and/or wound healing. In total, 164 publications met
19
20 the original search criteria with a total of 48 meeting inclusion criteria (kappa
w
21
22 score = 94%). These studies were divided into 7 in vitro, 11 in vivo and 31 clinical
23
24 studies. In summary, 6 out of 7 (85.7%) and 11 out of 11 (100%) of the in vitro
ON

25
26
and in vivo studies respectively demonstrated a statistically significant advantage
27 for combining PRF to their regenerative therapies. Out of the remaining 31
28
clinical studies, a total of 8 reported the effects of PRF in a randomized clinical
LY

29
30
31 trial with 5 additional studies (13 total) reporting appropriate controls. In those
32
33 clinical studies, 9 out of the 13 studies (69.2%) demonstrated a statistically
/N

34 relevant positive outcome for the primary endpoints measured. In total, 18

35
36 studies (58% of clinical studies) reported positive wound healing events
37
ot

38 associated with the use of PRF despite using controls. In total, 27 of the 31
39
40 clinical studies (87%) supported the use of PRF for soft tissue regeneration and
41
fo

42
wound healing for a variety of procedures in medicine and dentistry. In
43 conclusion, the results from the present systematic review highlight the positive
44
rD

45 effects of PRF on wound healing following regenerative procedures of soft

46
47 tissues in total 43 out of 48 studies (89.6%).
48
is

49
50 Keywords: Platelets, fibrin, PRP, PRF, angiogenesis, vascularization
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 4 of 37
Fo
1
2
rP
3
Introduction
4
5
6 The multidisciplinary field of tissue engineering aims to predictably repair,
ee
7
8 regenerate or restore damaged and supporting tissues including cell, tissue and
9
10 organs due to an assortment of biological conditions including congenital
rR
11
12
abnormalities, injury, disease and/or aging (1-4). During their regeneration, one
13 key aspect involves the ingrowth of a vascular source capable of supporting
14
ev
15 cellular function and future tissue development by maintaining a viable exchange
16
17 of nutrients through blood vessels (5). Although the majority of tissue engineering
18
ie
19 scaffolds are avascular by nature, it remains essential that all regenerative
20 strategies focus on the development of a vascular network in order to obtain
w
21
22 successful clinical outcomes and regeneration of either soft or hard tissues (5).
23
24 Wound healing, which is defined as the natural restorative response to
ON

25
26 tissue injury, involves a cascade of complex, orderly and elaborate events
27
28
involving many cell types guided by the release of soluble mediators and signals
LY

29 capable of influencing the homing of circulating cells to damaged tissues (6).

30
31 Typically, wound-healing events are divided into 4 overlapping phases including
32
33 hemostasis, inflammation, proliferation and remodeling (7-9). Platelets have been
/N

34
35 shown to be important cells regulating the hemostasis phase through vascular
36 obliteration and facilitating fibrin clot formation (6). They are responsible for the
37
ot

38 activation and release of important biomolecules including platelet-specific

39
40 proteins, growth factors including platelet-derived growth factor (PDGF),
41
fo

42 coagulation factors, adhesion molecules, cytokines/chemokines and angiogenic

43
factors capable of stimulating the proliferation and activation of cells involved in
44
rD

45 wound healing including fibroblasts, neutrophils, macrophages and mesenchymal

46
47 stem cells (MSCs) (10). For these reasons, the use of platelet concentrates have
48
been utilized in modern medicine for over 4 decades due to their hypothesized
is

49
50
51
impact on tissue regeneration by facilitating angiogenesis and various additional
tri

52 phases during wound healing including cell recruitment, proliferation, remodeling

53
54 and differentiation. Below we describe how tissue-engineering constructs have
bu

55
56 utilized various platelet concentrates to speed wound healing of either soft or
57
58 hard tissues.
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 5 of 37 Tissue Engineering
Fo
1
2
rP
3
Platelet Concentrates: From Platelet Rich Plasma to Platelet Rich Fibrin
4
5
6
ee
7 Autologous platelet-rich plasma (PRP) was first developed in the early 1970s and
8
9 was made popular in the 1980s (11, 12). The first generation of PRP was
10
rR
11 introduced by mixing collected blood with thrombin and excess calcium resulting
12 in activated platelets trapped within a fibrin network. Since then, different platelet
13
14 preparation protocols are now available traditionally isolated by a dual speed
ev
15
16 centrifugation process. The first spin separates red blood cells from plasma and
17
18 buffy coat. Thereafter, the platelet plug is typically separated from the platelet-
ie
19
poor plasma (PPP) in a second spin cycle generating PRP, a platelet concentrate
20
w
21 with up to 6-8 times the concentration of growth factors when compared to whole
22
23 blood (13). These platelets have been shown to secrete high levels of bioactive
24
ON

25 substances that slowly diffuse to the surrounding micro-environment facilitating

26
27
tissue regeneration (14-18). Much advancement has since been made in the
28 medical field by various groups who demonstrated that PRP could further
LY

29
30 enhance surgical wound healing of either soft or hard tissues (14, 19, 20).
31
32 Despite its widespread use, one of the reported drawbacks was the use of anti-
33
/N

34 coagulation factors delaying normal wound healing events.

35
36
37 Due to these reported limitations, further research was focused at
ot

38
39 developing a second-generation platelet concentrate without utilizing anti-
40
41 coagulation factors. As such, a platelet concentrate lacking coagulation factors,
fo

42
43
later termed platelet rich fibrin (PRF) was developed due to its anticipated
44 properties in tissue regeneration and wound healing (21-24). PRF (also termed
rD

45
46 leukocyte-PRF or L-PRF) contains more white blood cells (WBCs); necessary
47
48 cells important during the wound healing process (16, 25-29). Furthermore, since
is

49
50 WBCs including neutrophils and macrophages are one of the first cell-types
51 found in wounded sites, their role also includes to phagocytize debris, microbes
tri

52
53 and necrotic tissue thereby preventing infection. Macrophages are also key cells
54
bu

55 derived from the myeloid lineage and are considered one of the key cells
56
57 implicated in growth factor secretion during wound healing including transforming
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 6 of 37
Fo
1
2
rP
3
growth factor beta (TGF-beta), PDGF and vascular endothelial growth factor
4
5 (VEGF). These cells, together with neutrophils and platelets, are key players in
6
ee
7 wound healing and in combination with their secreted growth factors/cytokines
8
9 are capable of facilitating tissue regeneration, new blood vessel formation
10
rR
11 (angiogenesis) and prevention of infection (21-24, 27). To date, numerous
12 studies have investigated the regenerative potential of PRF in various medical
13
14 situations. The aim of this review article was to systematically characterize the
ev
15
16 potential for PRF to influence soft tissue wound healing. A systematic search was
17
18 carried out including all in vitro, in vivo and clinical studies performed on PRF to
ie
19
date dealing with soft tissue regeneration, wound healing and/or angiogenesis
20
w
21 following treatment with PRF.
22
23
24
ON

25
26
27
Methods
28 Development of a protocol
LY

29
30 A protocol including all aspects of a systematic review methodology was
31
32 developed prior to commencing the review. This included definition of the
33
/N

34 focused question; a defined search strategy; study inclusion criteria;

35 determination of outcome measures; screening methods, data extraction and
36
37 analysis; and data synthesis.
ot

38
39
40
41 Defining the focused question
fo

42
43
The following focused question was defined: ‘‘Does platelet rich fibrin (PRF)
44 affect/induce soft tissue regeneration and/or soft tissue wound healing?’’
rD

45
46
47
48 Search strategy
is

49
50 Using the MEDLINE database, the literature was searched for articles published
51 up to and including April 7th, 2016: combinations of several search terms were
tri

52
53 applied to identify appropriate studies (Table 1). Reference lists of review articles
54
bu

55 and of the included articles in the present review were screened. Finally, a hand
56
57 search of the Journal of Clinical Periodontology, the Journal of Dental Research,
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 7 of 37 Tissue Engineering
Fo
1
2
rP
3
Journal of Periodontal Research, the Journal of Periodontology, Clinical Oral
4
5 Implants Research, Clinical Implant Dentistry and Related Research, Clinical
6
ee
7 Oral Investigations and The International Journal of Periodontics and Restorative
8
9 Dentistry was performed.
10
rR
11
12 Criteria for study selection and inclusion
13
14 Study selection considered only articles published in English, describing in vitro,
ev
15
16 in vivo and human clinical studies evaluating the effect of PRF on soft tissue
17
18 wound healing. All in vitro studies were included on gingival fibroblasts,
ie
19
endothelial cells, keratinocytes and/or PDL fibroblasts. All in vivo data specifically
20
w
21 characterizing the effects of PRF on soft tissue wound healing were included. All
22
23 human studies reporting the effects of PRF were also included. Human studies
24
ON

25 were not limited to randomized clinical trials.

26
27
28 Outcome measure determination
LY

29
30 The primary outcome of interest was to determine the effect in percentage
31
32 increases that PRF is capable of inducing soft tissue regeneration and wound
33
/N

34 healing events. The outcome measures were separated into 1) in vitro studies, 2)
35 animal studies and 3) clinical studies. Since large variability in the outcomes
36
37 measured were performed by the various groups working across several fields of
ot

38
39 medicine, a meta-analysis was not considered. Outcomes were summarized in
40
41 Tables 2-4 for the various in vitro, in vivo and clinical studies according to the
fo

42
43
specific effect of PRF on soft tissue wound healing.
44
rD

45
46 Screening method
47
48 Titles and abstracts of the selected studies were independently screened by two
is

49
50 reviewers (RJM and MFK) on April 7th, 2016. The screening was based on the
51 question: ‘What effect does platelet rich fibrin (PRF) have on soft tissue
tri

52
53 regeneration and/or wound healing?’ Full text articles were obtained if the
54
bu

55 response to the screening question was ‘yes’ or ‘uncertain’. The level of

56
57 agreement between reviewers was determined by kappa scores. Disagreement
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 8 of 37
Fo
1
2
rP
3
regarding inclusion was resolved by discussion between authors. For necessary
4
5 missing data, the authors of the studies were contacted. Articles referring strictly
6
ee
7 to use in tendons, and orthopedic/bone uses were excluded if soft tissue wound
8
9 healing events were not investigated/discussed. Furthermore, review articles and
10
rR
11 clinical cases with no measurable endpoint were excluded.
12
13
14 Data extraction and analysis
ev
15
16 The following data were extracted: general characteristics (authors, year of
17
18 publication); PRF centrifugation characteristics/protocols, evaluation
ie
19
characteristics (amount of PRF utilized, volume, period, outcome measures);
20
w
21 methodological characteristics (study design, methodological quality); and
22
23 conclusions. Because of the heterogeneity of the included studies (study design,
24
ON

25 in vitro versus animal versus clinical studies, investigated parameters, materials

26
27
used, evaluation methods, outcome measures, observation periods) no mean
28 differences could be calculated, and consequently no quantitative data synthesis
LY

29
30 and meta-analysis could be performed. Instead, the data is reported in a
31
32 systematic fashion characterizing all available literature to date. Therefore, data
33
/N

34 were extracted from the reviewed articles and summarized in separate tables
35 based upon the various in vitro, in vivo and clinical studies and outcome
36
37 measures employed.
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 9 of 37 Tissue Engineering
Fo
1
2
rP
3
Results
4
5
6
ee
7 In vitro studies evaluating the effects of PRF on soft tissue regeneration
8
9 and/or wound healing
10
rR
11 The evaluation of PRF for soft tissue regeneration and/or wound healing has
12 been evaluated in 7 in vitro studies to date (Table 2). In 2008, Lundquist was one
13
14 of the first to evaluate the effects of PRF on human dermal fibroblasts (30). It was
ev
15
16 found that the proliferative effect of PRF on dermal fibroblasts was significantly
17
18 greater than fibrin sealant and recombinant PDGF-BB. Furthermore PRF induced
ie
19
rapid release of collagen 1 and sustained release and protection against
20
w
21 proteolytic degradation of endogenous fibrogenic factors important for wound
22
23 healing (30). Thereafter, Clipet et al. found that PRF induced fibroblast,
24
ON

25 keratinocyte and osteoblast cell survival, proliferation and induced osteoblast

26
27
differentiation (31). In the only in vitro report investigating the effects of PRF on
28 angiogenesis in vitro, Roy et al. found that PRF induced endothelial cell
LY

29
30 mitogenesis via extracellular signal-regulated protein kinase activation pathway
31
32 (32). In a 2nd in vitro study conducted by Lundquist et al. in 2013, PRF induced
33
/N

34 the mitogenic and migratory effect on cultured human dermal fibroblasts and they
35 further showed that fibrocytes (a cell type important for acute wound healing)
36
37 could be grown from within PRF patches further favoring wound healing and soft
ot

38
39 tissue regeneration (33). In 2014, a new protocol for PRF was introduced (termed
40
41 Advanced-PRF or A-PRF) whereby centrifugal forces were decreased and total
fo

42
43
spin times increased (34). By decreasing the rpm while increasing the
44 centrifugation time in the A-PRF group, an enhanced presence of neutrophilic
rD

45
46 granulocytes in the distal part of the clot was found contributing to monocyte
47
48 differentiation to macrophages (34), a cell responsible for inducing new bone
is

49
50 formation (35, 36). In 2015, Vahabi et al. also confirmed that PRF induced
51 gingival fibroblast proliferation at 24 hours, however found that gingival fibroblast
tri

52
53 proliferation was significantly higher in the Plasma Rich in Growth Factors
54
bu

55 (PRGF) group at 48 and 72 hours (37). Lastly, Bayer et al. discovered that PRF
56
57 contains anti-inflammatory/microbial activity by demonstrating that in human
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 10 of 37
Fo
1
2
rP
3
keratinocytes, PRF induced the expression of hBD-2, an anti-microbial agent
4
5 necessary in the treatment of chronic and infected wounds (38). In total, 6 of 7
6
ee
7 (85.6%) reported studies demonstrate a positive effect of PRF on soft tissue
8
9 regeneration in vitro.
10
rR
11
12 In vivo studies evaluating the effects of PRF on soft tissue regeneration
13
14 and/or wound healing
ev
15
16 In total, 11 studies have evaluated the effects of PRF on soft tissue wound
17
18 healing and regeneration (Table 3). In a first study, Roy et al. evaluated the
ie
19
effects of PRF after 14 days in a porcine ischemic excision wound model where
20
w
21 8-mm skin biopsies were created and filled with PRF versus control (32). It was
22
23 found that PRF significantly improved angiogenesis in chronic wounds and
24
ON

25 collagen matrix deposition (Figure 2) (32). Liu et al. found that after a 24 week
26
27
healing period, fat grafting with PRF into the ear’s auricula could be enhanced
28 with PRF as a therapeutic adjuvant to these procedures (39). Tunali et al. found
LY

29
30 that PRF centrifuged in titanium vials improved soft tissue wound healing in a
31
32 mucoperiosteal flap defect model in rabbits 30 days after implantation (40).
33
/N

34 Suzuki et al. further showed that PRF induced faster wound healing and
35 angiogenesis in the dorsal tissues of rats after 14 days (41). In another,
36
37 subcutaneous implantation model performed in mice, PRF readily integrated with
ot

38
39 surrounding tissues and was partially replaced with collagen fibers 2 weeks after
40
41 implantation (42). Soyer et al. found in a penile erethral 5 mm defect model that
fo

42
43
treatment with PRF significantly increased TGF-beta and VEGF growth factor
44 release after 24 hours (43). Furthermore, Horii et al. concluded that PRF
rD

45
46 significantly spread soft tissue healing of oral mucosititis in rats after a 14 day
47
48 healing period (Fig. 3) (44). Sun et al. demonstrated that the combination of PRF
is

49
50 with adipose derived MSCs improved the preservation of left ventricular (LV)
51 function and attenuating LV remodelling in a rat model that induced regional
tri

52
53 myocardial ischemia by left coronary artery ligation (45). In a model designed to
54
bu

55 regenerate the parotid gland following their irradiation in minipigs, both adipose-
56
57 derived stem cells and PRF significantly sped the repair of defects in
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 11 of 37 Tissue Engineering
Fo
1
2
rP
3
maxillofacial soft tissue in irradiated minipigs, and their combined use was more
4
5 effective after a 6-month healing period (46). In 2014 it was found that PRF
6
ee
7 increased type 1 collagen formation in full and split-thickness flaps and improved
8
9 skin graft take in a skin graft model performed in porcine animals (47). In 2015,
10
rR
11 adipose-derived mesenchymal stem cells embedded in PRF scaffolds promoted
12 angiogenesis, preserved heart function, and reduced left ventricular remodeling
13
14 in rat acute myocardial infarction when compared to controls (48). In total, 11 of
ev
15
16 11 studies found that PRF significantly improves soft tissue regeneration, wound
17
18 healing and/or angiogenesis in various animal models investigated.
ie
19
20
w
21
22
23 Clinical studies evaluating the effects of PRF on soft tissue regeneration
24
ON

25 and/or wound healing

26
27
In total, 31 studies investigated the effects of PRF on soft tissue wound
28 healing/regeneration in various clinical scenarios (Table 4). The use of PRF has
LY

29
30 been utilized for 20 different clinical procedures; 7 of which coming from the oral
31
32 and maxillofacial region (Table 4). In the dental field, the most commonly utilized
33
/N

34 use of PRF was for the treatment of extraction sockets (49-52), gingival
35 recessions (53-55) and palatal wound closure (56-58) with PRF being
36
37 additionally utilized for the repair of potentially malignant lesions (59),
ot

38
39 regeneration of periodontal defects (60), hyperplastic gingival tissues (61) and in
40
41 addition to periodontally accelerated osteogenic orthodontics (62). In other
fo

42
43
medical procedures, the use of PRF has been most combined for the successful
44 management of hard-to-heal leg ulcers including diabetic foot ulcers, venous leg
rD

45
46 ulcers and chronic leg ulcers (63-67). Furthermore, PRF has been investigated
47
48 for the management of hand ulcers (68), facial soft tissue defects (69),
is

49
50 laparoscopic cholecystectomy (70), in plastic surgery for the treatment of deep
51 nasolabial folds, volume-depleted midface regions, facial defects, superficial
tri

52
53 rhytids and acne scars (71), induction of dermal collagenesis (72), vaginal
54
bu

55 prolapse repair (73), urethracutaneous fistula repair (74, 75), during lipostructure
56
57 surgical procedures (76), chronic rotator cuff tears (77) and acute traumatic ear
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 12 of 37
Fo
1
2
rP
3
drum perforations (78). A total of 8 studies have reported the effects of PRF in a
4
5 randomized clinical trial (Table 4, bolded and italicized). Five non-randomized
6
ee
7 studies reported appropriately selected controls whereas 18 studies (58% of the
8
9 total listed clinical studies) reported no controls in their investigation and instead
10
rR
11 focused on the technical utilization/aspects of combining PRF during their various
12 medical procedures. In total 9 of the 13 studies utilizing appropriate controls
13
14 reported a significant positive influence combining PRF to their surgical protocols
ev
15
16 during soft tissue wound healing (Table 4). In total, 27 of the 31 studies (87%)
17
18 reported having beneficial effect for the utilization of PRF during soft tissue
ie
19
regeneration and/or soft tissue wound healing and angiogenesis in human
20
w
21 applications.
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 13 of 37 Tissue Engineering
Fo
1
2
rP
3
Discussion
4
5 Platelet concentrates, including PRP and PRF have been used for regenerative
6
ee
7 procedures in various fields of medicine including dentistry, reconstructive
8
9 surgery, plastic surgery and dermatology to delivery supernatural concentrations
10
rR
11 of autologous growth factors directly to host tissues. These growth factors have
12 been shown to be chemotactic for various cell types including monocytes,
13
14 fibroblasts, endothelial cells, stem cells, fibroblasts and osteoblasts, creating
ev
15
16 tissue micro-environments directly influencing the proliferation and differentiation
17
18 of progenitor cells (79). Furthermore, platelet concentrates are safe, reliable and
ie
19
cost-effective means to accelerate tissue healing and improving the efficiency of
20
w
21 tissue repair following injury.
22
23
24
ON

25 In the present study, we investigated specifically the regenerative potential

26
27
of soft tissue following regeneration with PRF. To date, no systematic review has
28 characterized the regenerative potential of PRF specifically for soft tissue wound
LY

29
30 healing/regeneration despite the great number of in vitro, in vivo and clinical
31
32 studies that have been reported on this topic to date. In the present systematic
33
/N

34 review, we focused specifically on soft tissues and excluded all studies where
35 PRF was utilized for bone, cartilage or tendon regeneration. In total, 48 studies
36
37 met our inclusion criteria with 31 studies being derived from human clinical
ot

38
39 studies (Table 4).
40
41
fo

42
43
While the effects of PRF were shown to enhance soft tissue regeneration
44 in all but one in vitro and in vivo studies (18 studies total), the results from the
rD

45
46 clinical studies need to be interpreted with caution. In total, 18 of the 31 clinical
47
48 studies (58%) report a beneficial effect of PRF based on the investigators
is

49
50 reported clinical experience, however in these studies, no controls were utilized
51 and the authors instead focus primary on their case reports/case series (Table 4).
tri

52
53 It may therefore be concluded that this first wave of research provides the clinical
54
bu

55 evidence that PRF seems to promote soft tissue wound healing, however, it is
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 14 of 37
Fo
1
2
rP
3
clear that future studies are needed comparing systematically the effects of PRF
4
5 in a randomized, controlled fashion across a wide-range of medical fields.
6
ee
7
8
9 Similarly, it was recently reported in a systematic review that the effects of
10
rR
11 platelet concentrates showed similar findings on bone healing/formation of
12 extraction sockets and intrabony defects (Figure 5) (80, 81). Although the results
13
14 of that meta-analysis are suggestive that platelet concentrates increase new
ev
15
16 bone formation in post-extraction sockets, the authors report that due to the
17
18 limited amount and quality of the available evidence, these results need to be
ie
19
cautiously interpreted (80). It was reported that a standardisation of the
20
w
21 experimental design was necessary for a better understanding of the true effects
22
23 of the use of platelet concentrates for enhancing post-extraction socket healing
24
ON

25 (80). Within the limits of our review article, we conclude similar findings that the
26
27
effects of PRF enhance soft tissue regeneration, however, future studies on soft
28 tissue regeneration following use of PRF need to be designed with appropriate
LY

29
30 controls and these findings need also to be interpreted with caution until further
31
32 randomized clinical trials are gathered.
33
/N

34
35 One of the reported advantages of PRF often reported was the ability for
36
37 the fibrin network containing leukocytes to resist and fight infection. Chronic non-
ot

38
39 healing wounds are a significant medical challenge and the pathogenesis of non-
40
41 healing wounds thereby requires new treatment options to improve clinical
fo

42
43
outcomes. One of the main factors to date hypothesized to further speed the
44 wound healing properties of PRF in comparison to PRP is the fact it contains
rD

45
46 higher levels of WBCs that favor the continuous release of growth factors.
47
48 Recently we demonstrated that both PRF, and the new formulation of PRF
is

49
50 termed advanced-PRF were both able to release significantly higher levels of
51 growth factors when compared to PRP over a 10-day period (82). Furthermore,
tri

52
53 macrophages have been shown to be key players during tissue regeneration,
54
bu

55 wound healing and prevention of infection (27, 35, 36). Furthermore, they contain
56
57 antimicrobial effects capable of reducing bacterial contamination following
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 15 of 37 Tissue Engineering
Fo
1
2
rP
3
surgeries (27). This finding was best exemplified in the healing of third molar
4
5 extraction sockets (50). It was reported that infection (osteomyelitis) is commonly
6
ee
7 reported in 9.5% of wisdom tooth removal and when a PRF plug was inserted
8
9 following extraction, this was significantly reduced to 1% of cases (50). Despite
10
rR
11 these reported findings, very little is yet known what the antibacterial properties
12 of PRF are as very little/few studies have investigated this phenomenon (83).
13
14
ev
15
16 Furthermore, to date very little is known regarding the effects of the fibrin
17
18 architecture and leukocyte content from these products as both these
ie
19
components are too often neglected as contributing factors in the tissue
20
w
21 regenerative potential of PRF. The presence of leukocytes has a great impact on
22
23 the biology of wound healing (16, 29), not only due to their additional release of
24
ON

25 growth factors and their implications in antibacterial immune defense, but also
26
27
because they are key regulators controlling the wound healing environment
28 through local factor regulation. Future basic research should focus specifically on
LY

29
30 the contribution of these cells in specific cell knock-down/knock-in systems to
31
32 determine the functional roles of each cell in the wound-healing process when
33
/N

34 PRF is utilized. For instance, it has been reported that addition of activated
35 macrophage to wounds in aging mice and human accelerated healing time (27).
36
37 Thus, in theory, the concept of developing newer modified protocols of PRF to
ot

38
39 further increase the number of WBCs would in principle, increase wound repair.
40
41 Nevertheless, a better understanding of the individual roles of the various cells
fo

42
43
found in PRF could prove to be an important finding for the development of these
44 technologies leading to modern changes to their protocols further increasing their
rD

45
46 regenerative potential.
47
48
is

49
50 Conclusion
51 In summary, 2 main findings can be drawn from the present systematic review: 1)
tri

52
53 the currently available literature supports soft tissue regeneration following soft
54
bu

55 tissue regenerative procedures utilizing PRF; and 2) there is a lack of appropriate

56
57 controls to the majority of studies drawing conclusive evidence that PRF is able
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 16 of 37
Fo
1
2
rP
3
to further as most of the clinical studies to date thus far highlight the use of PRF
4
5 in case series experiments or retrospective analysis without comparative results
6
ee
7 to appropriate controls. Therefore, it is imperative that the next wave of research
8
9 utilizing PRF as an adjunct to soft tissue regenerative therapies design
10
rR
11 appropriate studies with necessary controls to further evaluate the regenerative
12 potential of PRF for soft tissue wound healing.
13
14
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 17 of 37 Tissue Engineering
Fo
1
2
rP
3
References
4
5
6 1. Coury, A.J. Expediting the transition from replacement medicine to tissue
ee
7
8 engineering. Regenerative biomaterials 3, 111, 2016.
9 2. Dai, R., Wang, Z., Samanipour, R., Koo, K.I., andKim, K. Adipose-Derived
10 Stem Cells for Tissue Engineering and Regenerative Medicine Applications.
rR
11 Stem cells international 2016, 6737345, 2016.
12 3. Rouwkema, J., andKhademhosseini, A. Vascularization and Angiogenesis in
13
14
Tissue Engineering: Beyond Creating Static Networks. Trends Biotechnol 2016.
4. Zhu, W., Ma, X., Gou, M., Mei, D., Zhang, K., andChen, S. 3D printing of
ev
15
16 functional biomaterials for tissue engineering. Current opinion in biotechnology
17 40, 103, 2016.
18 5. Upputuri, P.K., Sivasubramanian, K., Mark, C.S., andPramanik, M. Recent
ie
19
developments in vascular imaging techniques in tissue engineering and
20
regenerative medicine. BioMed research international 2015, 783983, 2015.
w
21
22 6. Guo, S., andDipietro, L.A. Factors affecting wound healing. J Dent Res 89,
23 219, 2010.
24 7. Gosain, A., andDiPietro, L.A. Aging and wound healing. World journal of
ON

25 surgery 28, 321, 2004.

26
27
8. Eming, S.A., Brachvogel, B., Odorisio, T., andKoch, M. Regulation of
28 angiogenesis: wound healing as a model. Progress in histochemistry and
cytochemistry 42, 115, 2007.
LY

29
30 9. Eming, S.A., Kaufmann, J., Lohrer, R., andKrieg, T. [Chronic wounds. Novel
31 approaches in research and therapy]. Der Hautarzt; Zeitschrift fur Dermatologie,
32
Venerologie, und verwandte Gebiete 58, 939, 2007.
33
/N

34 10. Nurden, A.T. Platelets, inflammation and tissue regeneration. Thrombosis

35 and haemostasis 105 Suppl 1, S13, 2011.
36 11. Heyns Adu, P., Eldor, A., Yarom, R., andMarx, G. Zinc-induced platelet
37 aggregation is mediated by the fibrinogen receptor and is not accompanied by
ot

38 release or by thromboxane synthesis. Blood 66, 213, 1985.

39
40
12. Marx, R.E., Carlson, E.R., Eichstaedt, R.M., Schimmele, S.R., Strauss, J.E.,
41 andGeorgeff, K.R. Platelet-rich plasma: Growth factor enhancement for bone
fo

42 grafts. Oral surgery, oral medicine, oral pathology, oral radiology, and
43 endodontics 85, 638, 1998.
44 13. Peerbooms, J.C., van Laar, W., Faber, F., Schuller, H.M., van der Hoeven,
rD

45
H., andGosens, T. Use of platelet rich plasma to treat plantar fasciitis: design of a
46
47 multi centre randomized controlled trial. BMC Musculoskeletal Disorders 11, 69,
48 2010.
is

49 14. Rozman, P., andBolta, Z. Use of platelet growth factors in treating wounds
50 and soft-tissue injuries. Acta dermatovenerologica Alpina, Pannonica, et
51 Adriatica 16, 156, 2007.
tri

52
53
15. Alsousou, J., Thompson, M., Hulley, P., Noble, A., andWillett, K. The biology
54 of platelet-rich plasma and its application in trauma and orthopaedic surgery: a
bu

55 review of the literature. The Journal of bone and joint surgery British volume 91,
56 987, 2009.
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 18 of 37
Fo
1
2
rP
3
16. Davis, V.L., Abukabda, A.B., Radio, N.M., Witt-Enderby, P.A., Clafshenkel,
4
5 W.P., Cairone, J.V., andRutkowski, J.L. Platelet-rich preparations to improve
6 healing. Part I: workable options for every size practice. The Journal of oral
ee
7 implantology 40, 500, 2014.
8 17. De Pascale, M.R., Sommese, L., Casamassimi, A., andNapoli, C. Platelet
9 derivatives in regenerative medicine: an update. Transfusion medicine reviews
10
rR
11
29, 52, 2015.
12 18. Grambart, S.T. Sports medicine and platelet-rich plasma: nonsurgical
13 therapy. Clinics in podiatric medicine and surgery 32, 99, 2015.
14 19. Whitman, D.H., Berry, R.L., andGreen, D.M. Platelet gel: an autologous
ev
15 alternative to fibrin glue with applications in oral and maxillofacial surgery.
16
Journal of oral and maxillofacial surgery : official journal of the American
17
18 Association of Oral and Maxillofacial Surgeons 55, 1294, 1997.
ie
19 20. Borzini, P., Mazzucco, L., Giampaolo, A., andHassan, H.J. Platelet gel - the
20 Italian way: a call for procedure standardization and quality control. Transfusion
w
21 medicine (Oxford, England) 16, 303, 2006.
22 21. Choukroun, J., Diss, A., Simonpieri, A., Girard, M.O., Schoeffler, C., Dohan,
23
24 S.L., Dohan, A.J., Mouhyi, J., andDohan, D.M. Platelet-rich fibrin (PRF): a
ON

25 second-generation platelet concentrate. Part IV: clinical effects on tissue healing.

26 Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 101,
27 e56, 2006.
28 22. Dohan, D.M., Choukroun, J., Diss, A., Dohan, S.L., Dohan, A.J., Mouhyi, J.,
LY

29
30
andGogly, B. Platelet-rich fibrin (PRF): a second-generation platelet concentrate.
31 Part I: technological concepts and evolution. Oral surgery, oral medicine, oral
32 pathology, oral radiology, and endodontics 101, e37, 2006.
33 23. Dohan, D.M., Choukroun, J., Diss, A., Dohan, S.L., Dohan, A.J., Mouhyi, J.,
/N

34 andGogly, B. Platelet-rich fibrin (PRF): a second-generation platelet concentrate.

35 Part II: platelet-related biologic features. Oral surgery, oral medicine, oral
36
37 pathology, oral radiology, and endodontics 101, e45, 2006.
ot

38 24. Dohan, D.M., Choukroun, J., Diss, A., Dohan, S.L., Dohan, A.J., Mouhyi, J.,
39 andGogly, B. Platelet-rich fibrin (PRF): a second-generation platelet concentrate.
40 Part III: leucocyte activation: a new feature for platelet concentrates? Oral
41 surgery, oral medicine, oral pathology, oral radiology, and endodontics 101, e51,
fo

42
43
2006.
44 25. Martin, P., andLeibovich, S.J. Inflammatory cells during wound repair: the
rD

45 good, the bad and the ugly. Trends in cell biology 15, 599, 2005.
46 26. Tsirogianni, A.K., Moutsopoulos, N.M., andMoutsopoulos, H.M. Wound
47 healing: immunological aspects. Injury 37 Suppl 1, S5, 2006.
48 27. Adamson, R. Role of macrophages in normal wound healing: an overview.
is

49
50 Journal of wound care 18, 349, 2009.
51 28. Davis, V.L., Abukabda, A.B., Radio, N.M., Witt-Enderby, P.A., Clafshenkel,
tri

52 W.P., Cairone, J.V., andRutkowski, J.L. Platelet-rich preparations to improve

53 healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other
54 selection criteria. The Journal of oral implantology 40, 511, 2014.
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 19 of 37 Tissue Engineering
Fo
1
2
rP
3
29. Ghasemzadeh, M., andHosseini, E. Intravascular leukocyte migration
4
5 through platelet thrombi: directing leukocytes to sites of vascular injury.
6 Thrombosis and haemostasis 113, 1224, 2015.
ee
7 30. Lundquist, R., Dziegiel, M.H., andAgren, M.S. Bioactivity and stability of
8 endogenous fibrogenic factors in platelet-rich fibrin. Wound repair and
9 regeneration : official publication of the Wound Healing Society [and] the
10
rR
11
European Tissue Repair Society 16, 356, 2008.
12 31. Clipet, F., Tricot, S., Alno, N., Massot, M., Solhi, H., Cathelineau, G., Perez,
13 F., De Mello, G., andPellen-Mussi, P. In vitro effects of Choukroun's platelet-rich
14 fibrin conditioned medium on 3 different cell lines implicated in dental
ev
15 implantology. Implant dentistry 21, 51, 2012.
16
32. Roy, S., Driggs, J., Elgharably, H., Biswas, S., Findley, M., Khanna, S.,
17
18 Gnyawali, U., Bergdall, V.K., andSen, C.K. Platelet-rich fibrin matrix improves
ie
19 wound angiogenesis via inducing endothelial cell proliferation. Wound repair and
20 regeneration : official publication of the Wound Healing Society [and] the
w
21 European Tissue Repair Society 19, 753, 2011.
22 33. Lundquist, R., Holmstrom, K., Clausen, C., Jorgensen, B., andKarlsmark, T.
23
24 Characteristics of an autologous leukocyte and platelet-rich fibrin patch intended
ON

25 for the treatment of recalcitrant wounds. Wound repair and regeneration : official
26 publication of the Wound Healing Society [and] the European Tissue Repair
27 Society 21, 66, 2013.
28 34. Ghanaati, S., Booms, P., Orlowska, A., Kubesch, A., Lorenz, J., Rutkowski,
LY

29
30
J., Landes, C., Sader, R., Kirkpatrick, C., andChoukroun, J. Advanced platelet-
31 rich fibrin: a new concept for cell-based tissue engineering by means of
32 inflammatory cells. The Journal of oral implantology 40, 679, 2014.
33 35. Miron, R.J., andBosshardt, D.D. OsteoMacs: Key players around bone
/N

34 biomaterials. Biomaterials 82, 1, 2016.

35 36. Sinder, B.P., Pettit, A.R., andMcCauley, L.K. Macrophages: Their Emerging
36
37 Roles in Bone. Journal of bone and mineral research : the official journal of the
ot

38 American Society for Bone and Mineral Research 30, 2140, 2015.
39 37. Vahabi, S., Vaziri, S., Torshabi, M., andRezaei Esfahrood, Z. Effects of
40 Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and
41 Viability of Human Gingival Fibroblasts. Journal of dentistry (Tehran, Iran) 12,
fo

42
43
504, 2015.
44 38. Bayer, A., Lammel, J., Rademacher, F., Gross, J., Siggelkow, M., Lippross,
rD

45 S., Kluter, T., Varoga, D., Tohidnezhad, M., Pufe, T., Cremer, J., Glaser, R.,
46 andHarder, J. Platelet-released growth factors induce the antimicrobial peptide
47 human beta-defensin-2 in primary keratinocytes. Experimental dermatology 2016.
48 39. Liu, B., Tan, X.Y., Liu, Y.P., Xu, X.F., Li, L., Xu, H.Y., An, R., andChen, F.M.
is

49
50 The adjuvant use of stromal vascular fraction and platelet-rich fibrin for
51 autologous adipose tissue transplantation. Tissue engineering Part C, Methods
tri

52 19, 1, 2013.
53 40. Tunali, M., Ozdemir, H., Kucukodaci, Z., Akman, S., andFiratli, E. In vivo
54 evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet
bu

55
56
concentrate. The British journal of oral & maxillofacial surgery 51, 438, 2013.
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 20 of 37
Fo
1
2
rP
3
41. Suzuki, S., Morimoto, N., andIkada, Y. Gelatin gel as a carrier of platelet-
4
5 derived growth factors. Journal of biomaterials applications 28, 595, 2013.
6 42. Li, Q., Pan, S., Dangaria, S.J., Gopinathan, G., Kolokythas, A., Chu, S.,
ee
7 Geng, Y., Zhou, Y., andLuan, X. Platelet-rich fibrin promotes periodontal
8 regeneration and enhances alveolar bone augmentation. BioMed research
9 international 2013, 638043, 2013.
10
rR
11
43. Soyer, T., Ayva, S., Boybeyi, O., Aslan, M.K., andCakmak, M. The effect of
12 platelet rich fibrin on growth factor levels in urethral repair. Journal of pediatric
13 surgery 48, 2545, 2013.
14 44. Horii, K., Kanayama, T., Miyamoto, H., Kohgo, T., Tsuchimochi, T.,
ev
15 Shigetomi, T., andYokoi, M. Platelet-rich fibrin has a healing effect on
16
chemotherapy-induced mucositis in hamsters. Oral surgery, oral medicine, oral
17
18 pathology and oral radiology 117, 445, 2014.
ie
19 45. Sun, C.K., Zhen, Y.Y., Leu, S., Tsai, T.H., Chang, L.T., Sheu, J.J., Chen,
20 Y.L., Chua, S., Chai, H.T., Lu, H.I., Chang, H.W., Lee, F.Y., andYip, H.K. Direct
w
21 implantation versus platelet-rich fibrin-embedded adipose-derived mesenchymal
22 stem cells in treating rat acute myocardial infarction. International journal of
23
24 cardiology 173, 410, 2014.
ON

25 46. Chen, Y., Niu, Z., Xue, Y., Yuan, F., Fu, Y., andBai, N. Improvement in the
26 repair of defects in maxillofacial soft tissue in irradiated minipigs by a mixture of
27 adipose-derived stem cells and platelet-rich fibrin. The British journal of oral &
28 maxillofacial surgery 52, 740, 2014.
LY

29
30
47. Reksodiputro, M., Widodo, D., Bashiruddin, J., Siregar, N., andMalik, S.
31 PRFM enhance wound healing process in skin graft. Facial plastic surgery : FPS
32 30, 670, 2014.
33 48. Chen, Y.L., Sun, C.K., Tsai, T.H., Chang, L.T., Leu, S., Zhen, Y.Y., Sheu,
/N

34 J.J., Chua, S., Yeh, K.H., Lu, H.I., Chang, H.W., Lee, F.Y., andYip, H.K. Adipose-
35 derived mesenchymal stem cells embedded in platelet-rich fibrin scaffolds
36
37 promote angiogenesis, preserve heart function, and reduce left ventricular
ot

38 remodeling in rat acute myocardial infarction. American journal of translational

39 research 7, 781, 2015.
40 49. Sammartino, G., Dohan Ehrenfest, D.M., Carile, F., Tia, M., andBucci, P.
41 Prevention of hemorrhagic complications after dental extractions into open heart
fo

42
43
surgery patients under anticoagulant therapy: the use of leukocyte- and platelet-
44 rich fibrin. The Journal of oral implantology 37, 681, 2011.
rD

45 50. Hoaglin, D.R., andLines, G.K. Prevention of localized osteitis in mandibular

46 third-molar sites using platelet-rich fibrin. International journal of dentistry 2013,
47 875380, 2013.
48 51. Suttapreyasri, S., andLeepong, N. Influence of platelet-rich fibrin on alveolar
is

49
50 ridge preservation. The Journal of craniofacial surgery 24, 1088, 2013.
51 52. Yelamali, T., andSaikrishna, D. Role of platelet rich fibrin and platelet rich
tri

52 plasma in wound healing of extracted third molar sockets: a comparative study.

53 Journal of maxillofacial and oral surgery 14, 410, 2015.
54 53. Anilkumar, K., Geetha, A., Umasudhakar, Ramakrishnan, T., Vijayalakshmi,
bu

55
56
R., andPameela, E. Platelet-rich-fibrin: A novel root coverage approach. Journal
57 of Indian Society of Periodontology 13, 50, 2009.
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 21 of 37 Tissue Engineering
Fo
1
2
rP
3
54. Jankovic, S., Aleksic, Z., Klokkevold, P., Lekovic, V., Dimitrijevic, B., Kenney,
4
5 E.B., andCamargo, P. Use of platelet-rich fibrin membrane following treatment of
6 gingival recession: a randomized clinical trial. The International journal of
ee
7 periodontics & restorative dentistry 32, e41, 2012.
8 55. Eren, G., Tervahartiala, T., Sorsa, T., andAtilla, G. Cytokine (interleukin-
9 1beta) and MMP levels in gingival crevicular fluid after use of platelet-rich fibrin or
10
rR
11
connective tissue graft in the treatment of localized gingival recessions. Journal
12 of periodontal research 2015.
13 56. Jain, V., Triveni, M.G., Kumar, A.B., andMehta, D.S. Role of platelet-rich-
14 fibrin in enhancing palatal wound healing after free graft. Contemporary clinical
ev
15 dentistry 3, S240, 2012.
16
57. Kulkarni, M.R., Thomas, B.S., Varghese, J.M., andBhat, G.S. Platelet-rich
17
18 fibrin as an adjunct to palatal wound healing after harvesting a free gingival graft:
ie
19 A case series. Journal of Indian Society of Periodontology 18, 399, 2014.
20 58. Femminella, B., Iaconi, M.C., Di Tullio, M., Romano, L., Sinjari, B.,
w
21 D'Arcangelo, C., De Ninis, P., andPaolantonio, M. Clinical Comparison of
22 Platelet-Rich Fibrin and a Gelatin Sponge in the Management of Palatal Wounds
23
24 After Epithelialized Free Gingival Graft Harvest: A Randomized Clinical Trial.
ON

25 Journal of periodontology 87, 103, 2016.

26 59. Pathak, H., Mohanty, S., Urs, A.B., andDabas, J. Treatment of Oral Mucosal
27 Lesions by Scalpel Excision and Platelet-Rich Fibrin Membrane Grafting: A
28 Review of 26 Sites. Journal of oral and maxillofacial surgery : official journal of
LY

29
30
the American Association of Oral and Maxillofacial Surgeons 73, 1865, 2015.
31 60. Ajwani, H., Shetty, S., Gopalakrishnan, D., Kathariya, R., Kulloli, A., Dolas,
32 R.S., andPradeep, A.R. Comparative evaluation of platelet-rich fibrin biomaterial
33 and open flap debridement in the treatment of two and three wall intrabony
/N

34 defects. Journal of international oral health : JIOH 7, 32, 2015.

35 61. di Lauro, A.E., Abbate, D., Dell'Angelo, B., Iannaccone, G.A., Scotto, F.,
36
37 andSammartino, G. Soft tissue regeneration using leukocyte-platelet rich fibrin
ot

38 after exeresis of hyperplastic gingival lesions: two case reports. Journal of

39 medical case reports 9, 252, 2015.
40 62. Munoz, F., Jimenez, C., Espinoza, D., Vervelle, A., Beugnet, J., andHaidar,
41 Z. Use of leukocyte and platelet-rich fibrin (L-PRF) in periodontally accelerated
fo

42
43
osteogenic orthodontics (PAOO): Clinical effects on edema and pain. Journal of
44 clinical and experimental dentistry 8, e119, 2016.
rD

45 63. Danielsen, P., Jorgensen, B., Karlsmark, T., Jorgensen, L.N., andAgren, M.S.
46 Effect of topical autologous platelet-rich fibrin versus no intervention on
47 epithelialization of donor sites and meshed split-thickness skin autografts: a
48 randomized clinical trial. Plastic and reconstructive surgery 122, 1431, 2008.
is

49
50 64. O'Connell, S.M., Impeduglia, T., Hessler, K., Wang, X.J., Carroll, R.J.,
51 andDardik, H. Autologous platelet-rich fibrin matrix as cell therapy in the healing
tri

52 of chronic lower-extremity ulcers. Wound repair and regeneration : official

53 publication of the Wound Healing Society [and] the European Tissue Repair
54 Society 16, 749, 2008.
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 22 of 37
Fo
1
2
rP
3
65. Steenvoorde, P., van Doorn, L.P., Naves, C., andOskam, J. Use of
4
5 autologous platelet-rich fibrin on hard-to-heal wounds. Journal of wound care 17,
6 60, 2008.
ee
7 66. Jorgensen, B., Karlsmark, T., Vogensen, H., Haase, L., andLundquist, R. A
8 pilot study to evaluate the safety and clinical performance of Leucopatch, an
9 autologous, additive-free, platelet-rich fibrin for the treatment of recalcitrant
10
rR
11
chronic wounds. The international journal of lower extremity wounds 10, 218,
12 2011.
13 67. Londahl, M., Tarnow, L., Karlsmark, T., Lundquist, R., Nielsen, A.M.,
14 Michelsen, M., Nilsson, A., Zakrzewski, M., andJorgensen, B. Use of an
ev
15 autologous leucocyte and platelet-rich fibrin patch on hard-to-heal DFUs: a pilot
16
study. Journal of wound care 24, 172, 2015.
17
18 68. Chignon-Sicard, B., Georgiou, C.A., Fontas, E., David, S., Dumas, P., Ihrai,
ie
19 T., andLebreton, E. Efficacy of leukocyte- and platelet-rich fibrin in wound
20 healing: a randomized controlled clinical trial. Plastic and reconstructive surgery
w
21 130, 819e, 2012.
22 69. Desai, C.B., Mahindra, U.R., Kini, Y.K., andBakshi, M.K. Use of Platelet-Rich
23
24 Fibrin over Skin Wounds: Modified Secondary Intention Healing. Journal of
ON

25 cutaneous and aesthetic surgery 6, 35, 2013.

26 70. Danielsen, P.L., Agren, M.S., andJorgensen, L.N. Platelet-rich fibrin versus
27 albumin in surgical wound repair: a randomized trial with paired design. Annals of
28 surgery 251, 825, 2010.
LY

29
30
71. Sclafani, A.P. Safety, efficacy, and utility of platelet-rich fibrin matrix in facial
31 plastic surgery. Archives of facial plastic surgery 13, 247, 2011.
32 72. Sclafani, A.P., andMcCormick, S.A. Induction of dermal collagenesis,
33 angiogenesis, and adipogenesis in human skin by injection of platelet-rich fibrin
/N

34 matrix. Archives of facial plastic surgery 14, 132, 2012.

35 73. Gorlero, F., Glorio, M., Lorenzi, P., Bruno-Franco, M., andMazzei, C. New
36
37 approach in vaginal prolapse repair: mini-invasive surgery associated with
ot

38 application of platelet-rich fibrin. International urogynecology journal 23, 715,

39 2012.
40 74. Soyer, T., Cakmak, M., Aslan, M.K., Senyucel, M.F., andKisa, U. Use of
41 autologous platelet rich fibrin in urethracutaneous fistula repair: preliminary report.
fo

42
43
International wound journal 10, 345, 2013.
44 75. Guinot, A., Arnaud, A., Azzis, O., Habonimana, E., Jasienski, S.,
rD

45 andFremond, B. Preliminary experience with the use of an autologous platelet-

46 rich fibrin membrane for urethroplasty coverage in distal hypospadias surgery.
47 Journal of pediatric urology 10, 300, 2014.
48 76. Braccini, F., Chignon-Sicard, B., Volpei, C., andChoukroun, J. Modern
is

49
50 lipostructure: the use of platelet rich fibrin (PRF). Revue de laryngologie -
51 otologie - rhinologie 134, 231, 2013.
tri

52 77. Zumstein, M.A., Rumian, A., Lesbats, V., Schaer, M., andBoileau, P.
53 Increased vascularization during early healing after biologic augmentation in
54 repair of chronic rotator cuff tears using autologous leukocyte- and platelet-rich
bu

55
56
fibrin (L-PRF): a prospective randomized controlled pilot trial. Journal of shoulder
57 and elbow surgery / American Shoulder and Elbow Surgeons [et al] 23, 3, 2014.
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 23 of 37 Tissue Engineering
Fo
1
2
rP
3
78. Habesoglu, M., Oysu, C., Sahin, S., Sahin-Yilmaz, A., Korkmaz, D., Tosun,
4
5 A., andKaraaslan, A. Platelet-rich fibrin plays a role on healing of acute-traumatic
6 ear drum perforation. The Journal of craniofacial surgery 25, 2056, 2014.
ee
7 79. Sclafani, A.P., Romo, T., 3rd, Ukrainsky, G., McCormick, S.A., Litner, J.,
8 Kevy, S.V., andJacobson, M.S. Modulation of wound response and soft tissue
9 ingrowth in synthetic and allogeneic implants with platelet concentrate. Archives
10
rR
11
of facial plastic surgery 7, 163, 2005.
12 80. Del Fabbro, M., Corbella, S., Taschieri, S., Francetti, L., andWeinstein, R.
13 Autologous platelet concentrate for post-extraction socket healing: a systematic
14 review. European journal of oral implantology 7, 333, 2014.
ev
15 81. Anuroopa, P., Patil, P., Kumar, R.V., andKripal, K. Role and Efficacy of L-
16
PRFmatrix in the Regeneration of Periodontal Defect: A New Perspective.
17
18 Journal of clinical and diagnostic research : JCDR 8, Zd03, 2014.
ie
19 82. Kobayashi, E., Fluckiger, L., Fujioka-Kobayashi, M., Sawada, K., Sculean, A.,
20 Schaller, B., andMiron, R.J. Comparative release of growth factors from PRP,
w
21 PRF, and advanced-PRF. Clinical oral investigations 2016.
22 83. Cieslik-Bielecka, A., Dohan Ehrenfest, D.M., Lubkowska, A., andBielecki, T.
23
24 Microbicidal properties of Leukocyte- and Platelet-Rich Plasma/Fibrin (L-PRP/L-
ON

25 PRF): new perspectives. Journal of biological regulators and homeostatic agents

26 26, 43s, 2012.
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 24 of 37
Fo
1
2
rP
3
Table 1: Search terms used to identify the relevant studies
4
5
6 Search Terms
ee
7 "Platelet Rich Fibrin" OR "PRF" OR "Platelet-Rich Fibrin" OR "Leukocyte Platelet
8 Rich Fibrin" OR "Leukocyte Platelet-Rich Fibrin" OR "LPRF" OR "L-PRF" OR
9
"Advanced Platelet Rich Fibrin" OR "Advanced PRF" OR "A-PRF" OR "APRF"
10
rR
11 AND
12 "Soft Tissue Regeneration" OR "Soft Tissue Wound Regeneration" OR "Soft
13 Tissue Wound-Healing" OR "Wound Healing" OR "Wound-Healing" OR "Soft
14 Tissue Augmentation" OR "Angiogenesis"
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
Fo
Page 25 of 37 Tissue Engineering

1 rP
2
3
4 ee
5
6
7 rR
Table 2: In vitro studies evaluating the effects of PRF on soft tissue regeneration and/or wound healing

ev
8 Centrifugation
Author Year Cell Type Findings/Conclusions
9 Protocol
10 Human PRF induced higher fibroblast proliferation when compared to fibrin sealant
11
12
13
14
Lundquist
et al.
2008 dermal
iew
fibroblasts
400 g for 10 min

1100 g for 6 min

(with trisodium
and recombinant PDGF. Furthermore, PRF protected against proteolytic
degradation of endogenous fibrogenic factors important for wound healing.

ON
15 Endothelial PRF induced endothelial cell mitogenesis via extracellular signal-regulated
Roy et al. 2011 citrate) followed
16 cells protein kinase activation pathway.
17 by 4500 g for 25
18 min (with CaCl2)
19
20
21
Clipet et
al.
2012
Osteoblast,
Keratinocyte
s, Fibroblasts
400 g for 12 min
LY
Soluble growth factors from PRF induced cell viability and proliferation and
osteoblast differentiation.
22
23
24
Lundquist
et al.
2013
Human
dermal
fibroblasts
3000 g for 8 min
followed by 2 min
at 3000 g
/N
Fibrocytes (an important cell for acute wound healing) was grown from
within PRF patches implicating their role in wound healing and soft tissue
regeneration.

ot
25
26 Introduction of Advanced-PRF (A-PRF): By decreasing the rpm/g-force while
27 Ghanaati increasing the centrifugation time in A-PRF, an enhanced presence of
2014 PRF clots 150 g for 14 min

fo
28 et al. neutrophilic granulocytes and macrophages, cells implicated in wound
29 healing.
Vahabi et Gingival PRGF induced significantly higher gingival fibroblast proliferation at 48 and

rD
30
2015 400 g for 12 min
31 al. fibroblasts 72 hours when compared to PRF.
32 Primary 2000 g for 10 min
33 Bayer et PRF contains some anti-inflammatory/microbial effects in human
2016 keratinocyte followed by 2000
34
35
36
al.
s g for 10 min
ist
keratinocytes through the expression of an antimicrobial peptide hBD-2.

37
38
39 rib
40
41
ut
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo Tissue Engineering Page 26 of 37

1 rP
2
3
4 ee
5
6
7 rR
Table 3: In vivo studies evaluating the effects of PRF on soft tissue regeneration and/or wound healing

ev
8 Healing Centrifugation
Author Year Model Defect type Findings/Conclusions
9 Period Protocol
10 1100 g for 6 min
11
12
13
14
Roy et al. 2011
Porcine
ischemic
iew
excisional
wound model
8-mm
disposable
punch biopsies
14 days
(with trisodium
citrate) followed
by 4500 g for 25
min (with CaCl2)
PRF improved wound angiogenesis in chronic
wounds and collagen matrix deposition.

ON
15
Rabbit soft
16 3, 5, 10, PRF induced the formation of new bone with new
17 Tunali et tissue healing Mucoperiosteal 3500 rpm for 15
2012 15, 30 connective tissue in a rabbit model of wound
18 al. in the oral flaps min
days healing within 30 days.
19
20
21
cavity
Rabbit fat
grafting in
Subcutaneous LY
4, 12,

/N
22 injections into 3000 rpm for 10 The efficacy of adipose tissue implantation can be
Liu et al. 2013 plastic and 24
23 the ear’s min enhanced by using PRF as a therapeutic adjuvant.
reconstructive weeks
24 auricula
surgery

ot
25
Subcutaneous Subcutaneous injection of growth factors extracted
26 Subcutaneous
27 Suzuki et implantation on 3000 rpm for 10 from PRF incorporated into a gelatin gel was found
2013 injection in 14 days

fo
28 al. the dorsal min to be more effective in acceleration of wound
rats
29 tissues of rats healing than the commonly used PRP.
Subcutaneous Subcutaneous PRF readily integrated with surrounding tissues

rD
30
7, 14 2100 rpm (400g)
31 Li et al. 2013 injections in implantation and was partially replaced with collagen fibers 2
32 days for 12 min
nude mice under the cutis weeks after implantation.
33 5 mm vertical
34
35
36
Soyer et
al.
2013
Rat penile
erethral repair
incision in the
penile urethra
24
hours
2400 rpm for 12
min
ist
Use of PRF after urethral repair increased early
TGF-β-R and VEGF expressions in urethral tissues

37
38
39
Horii et
al.
2014
Oral mucositis
induced in
Intraperitoneal
injection of 5-
fluorouracil
5, 9 and
14 days
3000 rpm (400g)
for 10 min rib
The PRF group exhibited significant improvements
in the size and histologic features of the ulcer and in

ut
hamsters themyeloperoxidase activity.
40 followed by
41
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo
Page 27 of 37 Tissue Engineering

1 rP
2
3
4 ee
5
6
7 rR light scratching
of the cheek
pouch
8
9
10 ev
Male rat
Regional
myocardial The combination of PRF with adipose derived MSCs

iew
Sun et al. 2014 myocardial ischemia by left 42 days 600 g for 5 min improved the preservation of left ventricular (LV)
11
12 infarctions coronary artery function and attenuating LV remodeling.
13 ligation
14 Maxillofacial
Right parotid Both adipose-derived stem cells and PRF facilitated
soft tissue

ON
15
Chen et gland 6 3000 rpm (400g) the repair of defects in maxillofacial soft tissue in
16 2014 defects in
al. irradiation (20 months for 10 min irradiated minipigs, and their combined use was
17 irradiated
Gy) most effective.
18 minipigs
19
20
21 Reksodip
2014
Porcine facial
plastic and
full-thickness
(FTSG) and
split-thickness
LY
14, 30
1500 g for 15
min, 1800 g for
PRF in FTSG and STSG increased type 1 collagen
formation. PRF in addition to STSG gave the best
22
23
24
utro et al. reconstructive
surgery
(STSG) skin
grafts
days

/N
60 min skin graft take.

ot
25 Acute
26 myocardial
Adipose-derived mesenchymal stem cells
27 Ventricular infarction
Chen et embedded in PRF scaffolds promoted angiogenesis,
2015 remodeling in induction 6 weeks 400 g for 10 min

fo
28
29 al. preserved heart function, and improved left
rats through left
ventricular remodeling.

rD
30 coronary artery
31
ligation
32
33
34
35
36 ist
37
38
39 rib
40
41
ut
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo Tissue Engineering Page 28 of 37

1 rP
2
3
4 ee
5
6
7 rR
Table 4: Clinical studies evaluating the effects of PRF on soft tissue regeneration and/or wound healing

ev
8 Healing Centrifugation
Author Year Model Defect type Findings/Conclusions
9 Period Protocol
10 Arterial leg ulcers,
11
12
13
14
Steenvoor
de et al.
2008 iew
12 patients,
hard-to-heal
wounds
diabetic foot
ulcers and
postoperative
wound infections
Varied
consider
ably
3000 rpm (400g)
for 10 min
PRF achieved full healing or a significant reduction in
wound diameter with no adverse effects.

ON
15
16 (no controls)
17 Randomized
18 observation of
19
20
21 Danielsen
20 patients,
epithelialization
of donor sites and
meshed split- LY 3000 rpm
Epithelial coverage of donor wounds did not differ
significantly between platelet-rich fibrin and

/N
22 2008 chronic leg 5, 8 days (400g) for 10 control on day 5 (43.5 percent versus 34.4 percent,
et al. thickness skin
23 ulcer min p = 0.65) or day 8 (76.6 percent versus 94.8
autografts in
24 percent, p = 0.17).
chronic ulcers vs

ot
25
26 skin autografts
27 with/out PRF

fo
28 7 mm of clinical
29 attachment loss
Anilkumar 1 patient, root 2700 rpm for 12 Complete coverage was achieved six months after the
2008 on labial surface 1 month

rD
30
et al. coverage min procedure, with excellent tissue contour and color.
31 of anterior teeth
32 (no controls)
33 12 patients, 17 venous leg Weekly Complete closure was achieved in 66.7% in PRF
34
35
36
O'Connell
et al.
2008 chronic lower-
extremity ulcers
ulcers (VLU) (no
controls)
visits for
12 weeks
3000 g for 10
min
ist
group demonstrating VLU versus 44% in non-treated
VLU group.
37
38
39
Danielsen
et al.
2010
51 patients,
laparoscopic
Randomized, PRF
versus albumin
10 days
3000 rpm
(400g) for 10 rib
PRF did not improve wound strength significantly
compared with albumin but suppressed
subcutaneous collagen synthesis and deposition

ut
cholecystectomy min
40 during early repair of surgical wounds in humans.
41
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo
Page 29 of 37 Tissue Engineering

1 rP
2
3
4 ee
5
6
7 rR PRF used for
treatment of deep
nasolabial folds,
9.9

ev
8 months Full patient biocompatibility. No patients reported
50 patients, volume-depleted 1100 rpm for 6
9 Sclafani 2011 (range, 3- any swelling lasting more than 5 days. Most patients
plastic surgery midface regions, min
10 30 were satisfied with treatment.

iew
superficial rhytids,
11 months).
12 and acne scars (no
13 controls)
14 50 patients, 168 defects,
extractions (or extractions with The proposed protocol with PRF is a reliable

ON
15
16 Sammartin avulsions) for patients on anti- therapeutic option to avoid significant bleeding after
2011 9 hours 400 g 18 min
17 o et al. the prevention of coagulant dental extractions without the suspension of
18 hemorrhagic therapies (no anticoagulant therapy in heart surgery patients.
19
20
21
complications

4 patients,
controls)
PRF injected into
the deep dermis
LY
22
23
24 Sclafani et
induction of
Dermal
and immediate
subdermis of the /N 1100 rpm for 6
Injection of PRF into the deep dermis and subdermis
of the skin stimulates a number of positive cellular

ot
25 2011 Collagenesis, upper arms 10 weeks
al. min changes including increases in collagen production
26 Angiogenesis followed by 5mm
and angiogenesis.
27 and full thickness
adipogenesis biopsy collection

fo
28
29 (no controls)

rD
30 16 lower
31
15 patients, extremity chronic Authors conclude that PRF is easy to prepare and
32 Jorgensen 3000 rpm for 12
2011 recalcitrant wounds of varying 6 weeks apply in the clinics, is safe, and may be a clinically
33 et al. min

ist
34 chronic wounds etiology (no effective treatment of recalcitrant chronic wounds.
35 controls)
36 Surgery for
37
38
39
Gorlego et
al.
2012
10 patients,

repair
prolapse with
vaginal prolapse recurrence (stage
II or higher) (no
1, 6, 12,
18, 24
months
3000 rpm for 12
min rib
The use of PRF for site-specific prolapse repair is
associated with a good functional outcome because
of the healing and mechanical properties of PRF
40
41
controls)
ut
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo Tissue Engineering Page 30 of 37

1 rP
2
3
4 ee
5
6
7 Jankovic rR
2012
15 patients,
Miller Class I or PRF or connective
6 month
3000 rpm
(400g) for 10
No difference could be found between PRF and CTG
groups in gingival recession therapy, except for a
greater gain in keratinized tissue width obtained

ev
8 et al. II gingival tissue graft (CTG)
min in the CTG group and enhanced wound healing
9 recessions
associated with the PRF group.
10

iew
Use of PRF in a 3-
11
year-old boy
12 1 patient, Following treatment with PRF, no urethral fistula
13 following 1, 3
Soyer et al. 2012 urethracutaneou 400 g for 10 min was detected. Therapy with PRF improved clinical
14 hypospadias months
s fistula repair outcomes in this case report.
repair (no

ON
15
16 controls)
17 Post-operative
18 hand wounds 1, 2, 7,

LY
19 Chignon- 2700rpm PRF application on fresh postoperative hand
68 patients, (PRF vs reference 14, 21,
20 Sicard et 2012 (400g) for 12 wounds showed a median improvement of 5 days
wound healing care with 28, 60
21 al. min faster wound healing in comparison to control.
petroleum jelly days
22
23
24 1 patient, palatal
mesh)
48 year old male,
7, 14, 21, /N
3000 rpm for 10
Patient showed a delayed wound healing after

ot
25 Jain et al. 2012 palatal wound (no subepithelial connective tissue graft harvestation,
wound healing 28 days min
26 control) which was re-treated successfully with PRF.
27 By offering a matricial support to angiogenesis and
fat tissue
232 patients

fo
by stimulating the proliferation of pre-adipocytes,
28
29 Braccini et extracted from 2, 4, 6, 8 3400 rpm for 10
2013 during the PRF demonstrated a beneficial role on the
al. inner side of knees months min

rD
30 lipostructure cicatrization and the consolidation of an adipocyte
31 (no control)
graft.
32
33 200 defects, PRF

ist
34 placed in half, 1% of cases with PRF were infected versus 9.5% in
100 patients, 3rd
35 Hoaglin et reevaluated for 7-10 2700 rpm for 10- control untreated sites. PRF may be utilized to
2013 molar
36 al. localized osteitis days 12 min significantly reduce osteomyelitis following 3rd

rib
extractions
37 (control = no molar extractions.
38 therapy)
39 1 patient, facial 30 year old 2, 6 3000 rpm for 10 Innovative technique of enhancement of skin wound
40
41
Desai et al. 2013
soft tissue defect motorcycle weeks min
ut
healing by local application of PRF
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo
Page 31 of 37 Tissue Engineering

1 rP
2
3
4 ee
5
6
7 rR 20 patients,
accident
extraction
PRF did not influence alveolar ridge preservation
nor enhanced bone formation in the extraction
8
9
10
Suttaprey
asri et al.
2013
ev
defect wound
fill
sockets, split
mouth, PRF vs
blood clot
1, 2, 4, 6,
8 weeks
3000 rpm for 10
min
socket. The use of PRF revealed some effectiveness
by accelerating soft-tissue healing during the first
11
12
13
14
Guinot et
al.
2014
iew
33 patients,
urethroplasty
coverage in
Urethroplasties
performed using
Duplay's
8 months
(range,
6–18
3000 rpm for 10
min
4 weeks.
PRF seemed to be a safe and efficient covering
technique and was reported an additional approach
to coverage for hypospadias surgery, and may help to

ON
15 distal technique (no reduce the incidence of postoperative complications
months)
16 hypospadias controls) when coverage healthy tissue is not available.
17 20 consecutive
18 patients, repair arthroscopic 3000 rpm Arthroscopic rotator cuff repair with the
19
20
21
Zumstein
et al.
2014 of chronic
rotator cuff
tears
treatment +/-
PRF LY 6, 12
weeks
(400g) for 10
min
application of L-PRF is technically feasible and
yielded higher early vascularization.
22
23
24 Kulkarni et
2014
18 patients,
healing of free
Palatal wound
healing with PRF 7, 14, 21 /N 3000 rpm for 10
PRF as a dressing is an effective method of enhancing
the healing of the palatal donor site and consequently

ot
25 al. gingival graft (control without days min
reducing post-operative morbidity.
26 donor sites PRF)
27 PRF versus Here we found that PRF is a biomaterial that
32 patients with

fo
28 nothing was used quickens the healing of ear drum which is
29 Habesoglu acute traumatic 2700 rpm for 12
2014 for the repair of 1 month autogenous and simply prepared. In the study
et al. ear drum min

rD
30
ear drum group, the rate of ear drum closure was 64.3% and
31 perforations
32 perforation in the control group it was 22.2% (P<0.05).
33 PRF was applied every
39 patients, hard
34
35
36
Löndahl et
al.
2015 to heal diabetic
foot ulcers (DFU)
weekly to DFU for
up to 20 weeks
(no control)
week up
to 20
weeks
3000 g for 10
min
ist
PRF was well-tolerated, easy to use and had potential
in the armamentarium of the DFU treatment.

37
38
39
Pathak et
al.
2015
26 patients, oral
mucosal lesions
following
PRF over healing
areas of
potentially
7, 15, 30,
and 60
3000 rpm for 10
min
rib
The results of the present study suggest that PRF
membrane is a successful coverage agent that aids in
40
41
excisions malignant lesions
days
ut
the healing of superficial oral mucosal wounds.

42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
Fo Tissue Engineering Page 32 of 37

1 rP
2
3
4 ee
5
6
7 Ajwani et rR
2015
20 patients, split
mouth, 2- and 3-
open-flap
debridement
(OFD) +/- PRF 9 months
3000 rpm (400g) Adjunctive use of PRF with OFD significantly

ev
8 al. Wall Intrabony for 10 min improves defect fill when compared to OFD alone.
(control OFD
9 Defects
alone)
10

iew
20 patients, split Soft tissue healing
11
Yelamali et mouth, 3rd following 3000 rpm for 10 PRF is significantly better in promoting soft tissue
12 2015 1 week
13 al. molar extraction, PRF vs min healing when compared to PRP.
14 extractions PRP
2 patients, PRF led to rapid and good healing of soft tissues and

ON
15
exeresis of lesions 1, 3, 7,
16 di Lauro et exeresis of 2700 rpm for 12 the authors suggest that the use of PRF can be
2015 and application of 14, 30
17 al. hyperplastic min applied to cover wounds after exeresis of oral
PRF (no control) days
18 gingival lesions neoformations such as hyperplastic gingival lesions.
19
20
21 Eren et al. 2015
14 patients,
gingival
Treatment with
connective tissue
graft or PRF
LY 1, 6
months
3000 rpm for 10
min
PRF resulted in earlier vessel formation and tissue
maturation compared to CTG.
22
23
24
recession
40 patients with
(control = CTG)
/N
ot
25 one site of Palatal wounds The PRF-enriched palatal bandage significantly
Femminell 1, 2, 3, 4 3000 rpm for 10
26 2016 Miller Class I or covered with PRF accelerates palatal wound healing and reduces
a et al. weeks min
27 II gingival vs palatal sponge patient morbidity.
recession

fo
28
29 Paraffin
2000g for 10 min PRF induced hBD-2 (implicated in wound healing)

rD
30 Bilateral gluteal Embedding and
31 Bayer et al. 2016 10 days followed by expression when applied to experimentally
wounds mRNA extraction
32 2000g for 10 min generated skin wounds.
33 (no controls)

ist
34 11 patients,
A Wilcko’s
35 periodontally Combination with traditional bone grafts, PRF
Muñoz et modified PAOO 1, 2, 4, 8, 3000 rpm for 10
36 2016 accelerated potentially accelerates wound healing and reduces

rib
al. technique with L- 10 days min
37 osteogenic post-surgical pain, inflammation, infection.
PRF (no controls)
38 orthodontics
39
40
41
ut
42
43
io
n
44
45
46 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
47
48
 Page 33 of 37 Tissue Engineering
Fo
1
2
rP
3
Figure Legends:
4
5
6 Figure 1: Flow chart of the screened relevant publications
ee
7
8 Figure 2: Treatment of porcine ischemic wound with PRFM. Four Ischemic wounds
9 (solid circles) were developed on the back of pigs as described in Methods. Six additional
10
rR
11
non-ischemic wounds were developed (open circles). Arrows indicate direction of blood
12 flow in flaps containing ischemic wounds. The upper left and lower right ischemic
13 wounds were treated with PRFM at the time of wounding. Representative digital images
14 of excisional wounds treated or not with PRFM on days 0 and 4 post-wounding. Adapted
ev
15 with permission from (Roy et al., 2011)
16
17
18 Figure 3: Macroscopic aspects of the cheek pouches of hamsters injected with 5-
ie
19 fluorouracil. The control group (A-C), the fibrin group (D-F), and the PRF group (G-I)
20 are shown. Notice the significantly faster wound healing associated with the PRF group
w
21 (Horii et al., 2014).
22
23
24
Figure 4: Illustration of IVIS study and anatomical and pathologicalfindings on day 42
ON

25 after AMI induction (n = 8). A. Serial assessmentsof living imaging by In Vivo Imaging
26 System (IVIS) after acute myocardial infarction (AMI). B. The anatomical findings
27 showed the cross-section areaof the heart at the papillary muscle (blue arrows) among the
28 four groups.The left ventricular (LV) chamber size was highest in AMI group, lowest in
LY

29
sham-control group, and notably higher in AMI + platelet-rich fibrin (PRF) group than in
30
31 AMI + PRF + adipose-derived mesenchymal stem cell (ADMSC);Conversely, the infarct
32 size showed an apposite pattern of LV chamber size.The black arrows indicated PRF
33 scaffold tissue in AMI + PRF group and ADMSC-embedded PRF scaffold (AMI + PRF
/N

34 + ADMSC) (i.e., engineered ADMSC grafts) group, respectively.Scale bars in right lower
35 corners represent 5 mm. C. *vs. other groups with differentsymbols (*, †, ‡, §), p < 0.001.
36
37 Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison
ot

38 post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Adapted with
39 permission from (Chen et al., 2015).
40
41 Figure 5: Clinical diagram of intrabony defect regeneration with PRF. Notice the initial
fo

42
43
lesions and soft tissue recessions that have successfully been regenerated following
44 application with PRF. Adapted with permission from (Anuroopa et al., 2014).
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 34 of 37
Fo
1
2
rP
3
4
5
6
ee
7
8
9
10
rR
11
12
13
14
ev
15
16
17
18
ie
19
20
w
21
22 Figure 1: Flow chart of the screened relevant publications
23 127x55mm (300 x 300 DPI)
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 35 of 37 Tissue Engineering
Fo
1
2
rP
3
4
5
6
ee
7
8
9
10
rR
11
12
13
14
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
Figure 2: Treatment of porcine ischemic wound with PRFM. Four Ischemic wounds (solid circles) were
47
developed on the back of pigs as described in Methods. Six additional non-ischemic wounds were developed
48 (open circles). Arrows indicate direction of blood flow in flaps containing ischemic wounds. The upper left
is

49 and lower right ischemic wounds were treated with PRFM at the time of wounding. Representative digital
50 images of excisional wounds treated or not with PRFM on days 0 and 4 post-wounding. Adapted with
51 permission from (Roy et al., 2011)
tri

52 101x166mm (300 x 300 DPI)

53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 36 of 37
Fo
1
2
rP
3
4
5
6
ee
7
8
9
10
rR
11
12
13
14
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32 Figure 3: Macroscopic aspects of the cheek pouches of hamsters injected with 5-fluorouracil. The control
group (A-C), the fibrin group (D-F), and the PRF group (G-I) are shown. Notice the significantly faster
33
/N

wound healing associated with the PRF group (Horii et al., 2014).
34 101x74mm (300 x 300 DPI)
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
47
48
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Page 37 of 37 Tissue Engineering
Fo
1
2
rP
3
4
5
6
ee
7
8
9
10
rR
11
12
13
14
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37 Figure 4: Illustration of IVIS study and anatomical and pathologicalfindings on day 42 after AMI induction (n
ot

38 = 8). A. Serial assessmentsof living imaging by In Vivo Imaging System (IVIS) after acute myocardial
39 infarction (AMI). B. The anatomical findings showed the cross-section areaof the heart at the papillary
40 muscle (blue arrows) among the four groups.The left ventricular (LV) chamber size was highest in AMI
41 group, lowest in sham-control group, and notably higher in AMI + platelet-rich fibrin (PRF) group than in
fo

AMI + PRF + adipose-derived mesenchymal stem cell (ADMSC);Conversely, the infarct size showed an
42 apposite pattern of LV chamber size.The black arrows indicated PRF scaffold tissue in AMI + PRF group and
43 ADMSC-embedded PRF scaffold (AMI + PRF + ADMSC) (i.e., engineered ADMSC grafts) group,
44 respectively.Scale bars in right lower corners represent 5 mm. C. *vs. other groups with differentsymbols
rD

45 (*, †, ‡, §), p < 0.001. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple
46 comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Adapted with permission
47 from (Chen et al., 2015).
48 101x91mm (300 x 300 DPI)
is

49
50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
 Tissue Engineering Page 38 of 37
Fo
1
2
rP
3
4
5
6
ee
7
8
9
10
rR
11
12
13
14
ev
15
16
17
18
ie
19
20
w
21
22
23
24
ON

25
26
27
28
LY

29
30
31
32
33
/N

34
35
36
37
ot

38
39
40
41
fo

42
43
44
rD

45
46
Figure 5: Clinical diagram of intrabony defect regeneration with PRF. Notice the initial lesions and soft tissue
47
recessions that have successfully been regenerated following application with PRF. Adapted with permission
48 from (Anuroopa et al., 2014).
is

49 203x279mm (300 x 300 DPI)

50
51
tri

52
53
54
bu

55
56
57
58
tio

59
60
Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801
n
LITERATURE REVIEW

Two Neglected Biologic Risk Factors in Bone Grafting and

Implantology: High Low-Density Lipoprotein Cholesterol
and Low Serum Vitamin D
Joseph Choukroun1*
Georges Khoury, DDS2
Fouad Khoury, MD, PhD3
Philippe Russe, DDS4
Tiziano Testori, MD5
Yataro Komiyama, DDS, PhD6
Gilberto Sammartino, MD, PhD7
Patrick Palacci, DDS8
Mustafa Tunali, DDS, PhD9
Elisa Choukroun10

Following a failure of a bone graft or an implant placement, the hypothesis of a biological abnormality is rarely
considered as a possible cause. A systematic search of peer-reviewed literature for dyslipidemia or vitamin D
deficiency may explain this lack of consideration. Excess low-density lipoprotein cholesterol (dyslipidemia) is
responsible for a slower bone metabolism or lower dental implant osseointegration. In addition, vitamin D is a
key factor for linking innate and adaptive immunity. Both of these factors are compromised under the
conditions of vitamin D deficiency. Therefore, vitamin D deficiency slows implant osseointegration and increases
the risk of graft infection. Vitamin D is also involved in immune function and therefore allergic reactions.

Key Words: cholesterol, LDL cholesterol, vitamin D, failures, implants, bone grafts, infections, immune
defense, osseointegration

INTRODUCTION levels should be more systematically investigated.

T
Good cholesterol (high-density lipoprotein [HDL]) and
he search for a biological anomaly
bad cholesterol (low-density lipoprotein [LDL]) need
labeled as a risk factor before oral
to be included in this investigation because both
surgery is limited to disease states such
could have a negative effect on bone growth and
as diabetes. However, it seems in recent osseointegration (high LDL or low HDL). Vitamin D is
years that cholesterol and vitamin D one the most important hormones involved in bone
growth. In addition, vitamin D also plays a role in
1
Pain Clinic, Nice, France. reducing the effects of inflammation and helps
2
University Paris VII, Paris, France.
3
Munster University, Olsberg, Germany. improve the body’s natural immune reactions.
4
Private practice, Reims, France.
5
Galeazzi Institute, Universita di Milano, Milan, Italy.
6
Branemark Osseointegraion Center, Tokyo, Japan.
7
University Frederico 2, Napoli, Italy.
DYSLIPIDEMIA
8
Branemark Osseointegration Center, Marseille, France. LDL cholesterol and bone metabolism
9
Haidarpasa Hospital, Istanbul, Turkey.
10
University of Nice, France.
* Corresponding author, e-mail: joseph.choukroun@free.fr Cholesterol is transported in the plasma predomi-
DOI: 10.1563/AAID-JOI-D-13-00062 nantly as cholesteryl esters associated with lipopro-

110 Vol. XL /No. One /2014


teins. There are 2 types of lipoproteins: LDL (bad)
and HDL (good).
In addition to cardiovascular diseases, there is
evidence that high levels of cholesterol and
triglycerides cause alterations in bone tissue.
Krieger1 demonstrated an increase in the number
of osteoclasts, the inhibition of osteoblastic activity,
and a decreased bone remodeling in hyperlipidemic
rats. According to Luegmayr et al,2 elevated levels
of cholesterol may lead to an imbalance in the
bone-remodeling process, a reduction of bone mass
by increasing the activity, and a differentiation of
osteoclasts. Furthermore, recent studies have point-
ed out possible links between periodontal infection
and an increased risk for cardiovascular disease.3,4
An increase of circulating levels of oxidized LDL
induces alveolar bone loss5,6 and is associated with
the severity of the local inflammatory response to
bacteria as well as the susceptibility to periodontal
disease in diabetic patients.7
Osteoblasts can bind, internalize, and then
metabolize HDL3/LDL cholesterol and are capable
of selectively taking up cholesteryl esters from these
lipoproteins.8 The bone releases enzymes that are
involved in the oxidation of LDL. Therefore, it is
possible that the oxidized LDL accumulated in the
bone could induce subsequent deleterious cellular
effects on bone density. Hyperlipidemia causes a
reduction of bone density in vivo due to the
inhibition of osteoblast differentiation by bioactive
lipids.9,10
Indeed, the Demer group showed that oxidized
LDL caused an inhibition of the alkaline phospha-
tase activity and also mineralization,11 which are
markers of osteoblast differentiation. In addition, it
has recently been shown that oxidized LDL also
induces cell death by apoptosis of osteoblastic
cells.12
Hirasawa et al8 confirmed that atherogenic
conditions (high LDL levels) caused the death of
osteoblasts.
What is the role of HDL? Various antioxidants
carried by HDL may interrupt the cascade of events
leading to the oxidation of LDL.13 Another impor-
tant property of HDL is its ability to inhibit cell
death induced by oxidized LDL. In particular, it has
been reported that HDL inhibits the apoptosis of
monocytic cells by inducing cholesterol efflux and
thus preventing the accumulation of cholesterol
Choukroun et al
caused by the presence of oxidized LDL.14 HDL
should be considered as a bone cell protector.
Low vitamin D incidence on osseointegration and
bone grafts
The most important related compound of vitamin D
is vitamin D3 (cholecalciferol). Vitamin D is a steroid
hormone that is acquired via diet or synthesized in
the skin from cholesterol when sun (ultraviolet light)
exposure is adequate. Cholesterol is converted to
pre–vitamin D3 and then isomerized to vitamin D3.
After binding to vitamin D–binding carrier protein,
vitamin D3 is transported to the liver, where it is
enzymatically hydroxylated by CYP27A1, generating
25-hydroxyvitamin D3 (calcidiol, or 25OHD3).15
In the bone, vitamin D stimulates the activity of
osteoclasts and increases the production of extra-
cellular matrix proteins by osteoblasts. Vitamin D
also stimulates intestinal calcium absorption and
inhibits the synthesis and secretion of parathyroid
hormone.16–19 Vitamin D deficiency can result from
inadequate dietary intake together with the insuf-
ficient exposure to sunlight. Vitamin D deficiency in
these patients is associated with a catabolic bone
turnover and its main consequence, the occurrence
of osteoporotic fractures.20 Deficiency has also been
linked to impaired fracture healing in clinical
practice and in patients suffering a fracture.21,22
These results support the role of the steroid
hormone in controlling bone regeneration. As
osseointegration of dental implants also depends
on bone regeneration, peri-implant bone formation
was shown to be reduced in vitamin D–deficient
rats.23 Surprisingly, only limited preclinical data on
the effect of vitamin D supplementation on bone
regeneration are available.24
Overall, these studies suggest that vitamin D
supplementation has beneficial effects on bone
turnover in patients with vitamin D deficiency,
which might also hold true for bone regeneration.
In a recent study, Dvorak et al25 indicated that
vitamin D deficiency has a negative impact on
cortical peri-implant bone formation in ovariecto-
mized rats, which can be compensated by a vitamin
D–rich diet.
Vitamin D deficiency has been also implicated in
various diseases such as diabetes, high blood
pressure, cardiovascular diseases, and many can-
cers.19 It has also been implicated in several allergic
disorders and immune system dysregulation. Our
Journal of Oral Implantology 111
Two Neglected Biologic Risk Factors in Bone Grafting and Implantology
TABLE 1
Lipoprotein values*
Cholesterol total ,2 g/L
Triglycerides ,2 g/L
LDL cholesterol Men ,1.6 g/L Women ,1.5 g/L
HDL cholesterol .0.35 g/L
*HDL indicates high-density lipoprotein; LDL, low-density
lipoprotein.
understanding of vitamin D metabolism and
biological effects has grown exponentially in recent
years, and it has become clear that vitamin D has
extensive immunomodulatory effects. It is now
known that cells from the immune system contain
all the characteristics needed to convert 25-hydrox-
yvitamin D to active 1,25-dihydroxyvitamin D during
a bacterial infection.26,27 Liu et al28 showed that
stimulation of TLR 2/1 engages a vitamin D–
dependent intracellular circuit that results in the
expression of antimicrobial peptides, such as
defensins and cathelicidins, which enhances the
microbicidal capability of the monocytes. These
peptides have a broad range of actions against
microorganisms, including bacteria, fungi, and
viruses. It is interesting to observe that sera from
African American individuals, who are known to
have substantially lower serum vitamin D levels
than whites, were inefficient in inducing genetic
expression of cathelicidin.27 This article published
by Liu et al28 triggered in recent years a very large
scientific interest related to vitamin D, with more
than 1500 articles published in 2012 and more than
2500 already in 2013. The nasal carriage in
Staphylococcus aureus is a major risk for infections
with the bacterium.29 The vitamin D level is directly
connected to this infection risk; Olsen suggests that
vitamin D can improve the antibacterial immune
response and thereby prevent S aureus colonization
and carriage and subsequent disease.30,31
Flynn showed that vitamin D levels ,20 ng/mL
have a significant impact on length of stay, organ
dysfunction, and infection rates.32 Frieri and Val-
luri33 showed that there was also a positive
correlation with allergy subtypes and sinus infec-
tions with low vitamin D. In one of the early
observational studies that pointed toward a con-
nection between vitamin D and respiratory infec-
tions, Ginde et al34 found an inverse relationship
between serum 25(OH)D concentrations and the
112 Vol. XL /No. One /2014
incidence of upper respiratory infections. Ginde et
al35 also found that vitamin D deficiency is
associated with a high prevalence of severe
infections in the hospital emergency department.
Vitamin D has since been studied in several
clinical trials to characterize its role in respiratory
infections.36 In 2010, Sabetta et al37 conducted a
prospective cohort study showing that serum
25(OH)D concentrations of 38 ng/mL or greater
were associated with a 2-fold decrease in the
number of upper respiratory infections.
Serum levels
Serum level values are shown in Table 1 (lipopro-
tein) and Table 2 (vitamin D).
DISCUSSION
Vitamin D also plays a predominant role in allergy.
Insufficiency of vitamin D seems to be connected to
the onset of atopy and food allergies.38 The
hypothesis is that vitamin D could have a central
role in these pathological situations and that it may
represent a novel preventive and/or therapeutic
strategy. Numerous data are published on the
relationship between vitamin D and asthma and
allergies.39,40 These results may indicate that the
timing of the intervention of vitamin D levels may
be a factor in subsequent allergic diseases. An
alternative explanation is that different absolute
amounts of vitamin D have alternate physiologic
effects on allergic pathogenesis. Furthermore,
although beyond the scope of this review, vitamin
D may also affect the body’s susceptibility and
response to infectious organisms, a major trigger of
wheezing at a young age.39,40 In conclusion, the
vitamin D serum level plays a predominant role in
bone metabolism, sensibility to infections, and
many allergy symptoms. We advance the hypoth-
esis that allergic patients are often deficient in
vitamin D.
The result of high LDL is a reduction of bone
metabolism, inhibition of phosphatase alkaline, and
TABLE 2
Serum 25(OH) vitamin D levels
Deficiency ,10 ng/mL
Insufficiency 10–30 ng/mL
Optimal .30 ng/mL

an increase of the fat part in the bone. The result is a
lower osseointegration and slower bone growth.
Branemark said in 1985, during an international
meeting, ‘‘When I see a yellow bone, I cancel the
transplant!’’ At that time, it was only a clinical
observation. We now understand why. We have
been focused on the high cholesterol risk in bone
grafts for more than 10 years, and now we can
explain why we had more failures in these cases.
Daily needs of vitamin D are 4000 IU, and the
lack of sufficient vitamin D in one’s diet is very
common: An estimated 1 billion people worldwide
are vitamin D deficient. Forty percent to 100% of US
and European elderly men and women still living in
the community (not in nursing homes) are deficient
in vitamin D.20 Stoker et al41 found that about two-
thirds of preoperative spinal fusion patients were
vitamin D insufficient. About 50% of these insuffi-
cient patients were deficient (less than 15 ng/mL). In
France, a review of patients in the Besançon
hospital made by Malpica et al42 revealed that
91% of patients were insufficient. In this insufficient
population, 40% were deficient (less than 10 ng/
mL). In the Suvimax French Study, the authors
found 79% of patients insufficient (women patients,
average 47 years old; range, 35–60 years). We can
conclude that most of the population is insufficient.
The medical consensus in 2012 is that ‘‘the
population over the age of 65 years old has to be
supplemented without any lab control.’’43 Recently,
Maier et al44 asked: Is there an epidemic vitamin D
deficiency in German orthopedic patients? Among
preoperative orthopedic patients, Maier et al found
that 85% were insufficient and 60% were deficient.
There is also clearly a close relationship between
statins, cholesterol, and vitamin D. It is interesting to
note that cholesterol and vitamin D have the same
precursor, namely, 7-dehydrocholesterol.
We have a similarity to the clinical benefits of
vitamin D in that it has been suggested that statins
might somehow be analogues of vitamin D.45 We
know that statins have a beneficial effect in the
reduction of infective or inflammatory episodes,21 in
a pattern similar to vitamin D. Statins increase the
blood level of vitamin D as 25-hydroxyvitamin D
and also the activated hormone 1,25-dihydroxyvi-
tamin D.46,47
The supplementation of vitamin D in a daily diet
also decreases the LDL cholesterol.48 The link
between osteoporosis and the metabolic syndrome
Choukroun et al
could influence the therapeutic approach in both
disorders and vitamin D. Supplementation may play
an important role in prevention of these severe
conditions.
CONCLUSION
We suggest exploring vitamin D serum level
(prescription: 25OH vitamin D ¼ D2 þ D3) and LDL
Cholesterol (prescription: cholesterol total þ LDL þ
HDL cholesterol) systematically in patients who are
diabetic, allergic, with hypertension, and previously
in a difficult case of implants and/or bone grafting.
This exploration is largely indicated in the case of a
failure of bone graft or implant placement. Correct-
ing these detected anomalies is obviously recom-
mended. Further multicentric studies may be
helpful for finding a correlation between implant
facilities and vitamin D or/and cholesterol dosage.
ABBREVIATIONS
HDL: high-density lipoprotein
LDL: low-density lipoprotein
Vitamin D3: cholecalciferol
REFERENCES
1. Krieger M. The best of cholesterol, the worst of cholester-
ols: a tale of two receptors. Proc Natl Acad Sci USA. 1998;95:4077–
4080.
2. Luegmayr E, Glantschnig H, Wesolowski GA, et al.
Osteoclast formation, survival and morphology are highly depen-
dent on exogenous cholesterol/lipoproteins. Cell Death Differ. 2004;
11(suppl 1):S108–S118.
3. Blaizot A, Vergnes JN, Nuwwareh S, Amar J, Sixou M.
Periodontal diseases and cardiovascular events: meta-analysis of
observational studies. Int Dent J. 2009;59:197–209.
4. Tonetti MS. Periodontitis and risk for atherosclerosis: an
update on intervention trials. J Clin Periodontol. 2009;36(suppl 10):
15–19.
5. Tomofuji T, Ekuni D, Azuma T, et al. Involvement of toll-like
receptor 2 and 4 in association between dyslipidemia and
osteoclast differentiation in apolipoprotein E deficient rat peri-
odontium. Lipids Health Dis. 2013;12(1):1.
6. Fentoğlu O, Köroğlu BK, Kara Y, et al. Serum lipoprotein-
associated phospholipase A2 and C-reactive protein levels in
association with periodontal disease and hyperlipidemia. J Perio-
dontol. 2011;82:350–359.
7. Bastos AS, Graves DT, Loureiro AP, et al. Lipid peroxidation
is associated with the severity of periodontal disease and local
inflammatory markers in patients with type 2 diabetes. J Clin
Endocrinol Metab. 2012;97:E1353–E1362.
8. Hirasawa H, Tanaka S, Sakai A, et al. ApoE gene deficiency
enhances the reduction of bone formation induced by a high-fat
diet through the stimulation of p53-mediated apoptosis in
osteoblastic cells. Bone Miner Res. 2007;22:1020–1030.
Journal of Oral Implantology 113
Two Neglected Biologic Risk Factors in Bone Grafting and Implantology
9. Brodeur MR, Brissette L, Falstrault L, Luangrath V, Moreau
R. Scavenger receptor of class B expressed by osteoblastic cells are
implicated in the uptake of cholesteryl ester and estradiol from LDL
and HDL3. J Bone Miner Res. 2008;23:326–337.
10. Brodeur MR. Implication des Lipoprotéines dans le métab-
olisme normal et pathologique du tissu osseux [PhD thesis].
Montreal, Canada: University Montreal Canada; 2009.
11. Parhami F, Morrow AD, Balucan J, et al. Lipid oxidation
products have opposite effects on calcifying vascular cell and bone
cell differentiation: a possible explanation for the paradox of
arterial calcification in osteoporotic patients. Arterioscler Thromb
Vasc Biol. 1997;17:680–687.
12. Klein BY, Rojansky N, Ben-Yehuda A, Abou-Atta I, Abedat S,
Friedman G. Cell death in cultured human Saos2 osteoblasts
exposed to low-density lipoprotein. Cell Biochem. 2003;90:42–58.
13. Navab M, Ananthramaiah GM, Reddy ST, et al. The
oxidation hypothesis of atherogenesis: the role of oxidized
phospholipids and HDL. Lipid Res. 2004;45:993–1007.
14. Jiang P, Yan PK, Chen JX, et al. High density lipoprotein 3
inhibits oxidized low density lipoprotein-induced apoptosis via
promoting cholesterol effiux in RAW264.7 cells. Acta Pharmacol Sin.
2006;27:151–157.
15. Lehmann B. The vitamin D3 pathway in human skin and its
role for regulation of biological processes. Photochem Photobiol.
2005;81:1246–1251.
16. DeLuca HF. The vitamin D story: a collaborative effort of
basic science and clinical medicine. FASEB J. 1988;2:224–236.
17. Bouillon R, Okamura WH, Norman AW. Structure-function
relationships in the vitamin D endocrine system. Endocr Rev. 1995;
16:200–257.
18. Christakos S, Dhawan P, Liu Y, Peng X, Porta A. New
insights into the mechanisms of vitamin D action. J Cell Biochem.
2003;88:695–705.
19. Dusso AS, Brown AJ, Slatopolsky E. Vitamin D. Am J Physiol
Renal Physiol. 2005;289:F8–F28.
20. Holick M. Vitamin D deficiency. N Engl J Med. 2007;357:
266–281.
21. Brinker MR, O’Connor DP, Monla YT, Earthman TP.
Metabolic and endocrine abnormalities in patients with nonunions.
J Orthop Trauma. 2007;21:557–570.
22. Alkalay D, Shany S, Dekel S. Serum and bone vitamin D
metabolites in elective patients and patients after fracture. J Bone
Joint Surg. 1989;71:85–87.
23. Kelly J, Lin A, Wang CJ, Park S, Nishimura I. Vitamin D and
bone physiology: demonstration of vitamin D deficiency in an
implant osseointegration rat model. J Prosthodont. 2009;18:473–
478.
24. Delgado-Martinez AD, Martinez ME, Carrascal MT, Rodri-
guez-Avial M, Munuera L. Effect of 25-OH-vitamin D on fracture
healing in elderly rats. J Orthop Res. 1998;16:650–653.
25. Dvorak G, Fügl A, Watzek G, Tangl S, Pokorny P, Gruber R.
Impact of dietary vitamin D on osseointegration in the ovariecto-
mized rat. Clin Oral Implants Res. 2012;23:1308–1313.
26. Hewison M. Vitamin D and immune function: autocrine,
paracrine or endocrine? Scand J Clin Lab Invest Suppl. 2012;243:92–
102.
27. Hansdottir S, Monick MM. Vitamin D effects on lung
immunity and respiratory diseases. Vitam Horm. 2011;86:217–237.
28. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of
a vitamin D-mediated human antimicrobial response. Science. 2006;
311:1770–1773.
29. Bode LG, Kluytmans JA, Wertheim HF, et al. Preventing
114 Vol. XL /No. One /2014
surgical-site infections in nasal carriers of Staphylococcus aureus. N
Engl J Med. 2010;362:9–17.
30. Olsen K, Falch BM, Danielsen K, et al. Staphylococcus aureus
nasal carriage is associated with serum 25-hydroxyvitamin D levels,
gender and smoking status. Eur J Clin Microbiol Infect Dis. 2012;31:
465–473.
31. Grimnes G, Jorde R, Simonsen GS, Furberg AS. Staphylo-
coccus aureus nasal carriage is associated with serum 25-
hydroxyvitamin D levels, gender and smoking status. The Tromsø
Staph and Skin Study. Eur J Clin Microbiol Infect Dis. 2012;31:465–
473.
32. Flynn L, Zimmerman LH, McNorton K, et al. Effects of
vitamin D deficiency in critically ill surgical patients. Am J Surg.
2012;203:379–382.
33. Frieri M, Valluri A. Vitamin D deficiency as a risk factor for
allergic disorders and immune mechanisms. Allergy Asthma Proc.
2011;32:438–444.
34. Ginde AA, Liu MC, Camargo CA Jr. Demographic differ-
ences and trends of vitamin D insufficiency in the US population,
1988–2004. Arch Intern Med. 2009;169:626–632.
35. Ginde AA, Camargo CA Jr, Shapiro NI. Vitamin D
insufficiency and sepsis severity in emergency department patients
with suspected infection. Acad Emerg Med. 2011;18:551–554.
36. Youssef DA, Miller CW, El-Abbassi AM, et al. Antimicrobial
implications of vitamin D. Dermatoendocrinol. 2011;3:220–229.
37. Sabetta JR, De Petrillo P, Cipriani RJ, Smardin J, Burns LA,
Landry ML. Serum 25-hydroxyvitamin D and the incidence of acute
viral respiratory tract infections in healthy adults. PLoS One. 2010;5:
e11088.
38. Searing DA, Leung DYM. Vitamin D in atopic dermatitis,
asthma and allergic diseases. Immunol Allergy Clin North Am. 2010;
30:397–409.
39. Reinholz M, Ruzicka T, Schauber J. Vitamin D and its role in
allergic disease. Clin Exp Allergy. 2012;42:817–826.
40. Bozzetto S, Carraro S, Giordano G, Boner A, Baraldi E.
Asthma, allergy and respiratory infections: the vitamin D hypoth-
esis. Allergy. 2012;67:10–17.
41. Stoker GE, Buchowski JM, Bridwell KH, Lenke LG, Riew KD,
Zebala LP. Preoperative vitamin D status of adults undergoing
surgical spinal fusion. Spine (Phila Pa 1976). 2013;38:507–515.
42. Malpica J, Dumoulin G, Bardet R, Wendling D. Évaluation du
statut vitaminique D des patients du CHU de Besançon. Besançon,
France: Société de Médecine de Franche-Comté; 2011.
43. Benhamou CL, Souberbielle JC, Cortet B, Fardellone P,
Gauvain JB, Thomas T. La vitamine D chez l’adulte: recommenda-
tions du GRIO. Presse Med. 2011;40:673–682.
44. Maier GS, Jakobs P, Roth KE, Kurth AA, Maus U. Is there an
epidemic vitamin D deficiency in German orthopaedic patients?
Clin Orthop Relat Res. 2013;471:3029–3035.
45. Grimes DS. Are statins analogues of vitamin D? Lancet.
2006;368:83–86.
46. Perez-Castrillon JL, Vega G, Abad L, et al. Effects of
atorvastatin on vitamin D levels in patients with ischaemic heart
disease. Am J Card. 2007;99:903–905.
47. Yavuz B, Ertugrul DT, Cil H, et al. Increased levels of 25-
hydroxyvitamin D and 1,25-dihydroxyvitamin D after rosuvastatin
treatment: a novel pleiotropic effect of statins? Cardiovasc Drugs
Ther. 2009;23:295–299.
48. Major GC, Alarie F, Doré J, Phouttama S, Tremblay A.
Supplementation with calcium þ vitamin D enhances the beneficial
effect of weight loss on plasma lipid and lipoprotein concentra-
tions. Am J Clin Nutr. 2007;85:54–59.
Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2016, Article ID 5319718, 7 pages
http://dx.doi.org/10.1155/2016/5319718

Research Article
Is Low Serum Vitamin D Associated with
Early Dental Implant Failure? A Retrospective Evaluation
on 1625 Implants Placed in 822 Patients

Francesco Mangano,1 Carmen Mortellaro,2 Natale Mangano,3 and Carlo Mangano4

1
Department of Surgical and Morphological Science, Dental School, University of Insubria, 21100 Varese, Italy
2
Department of Health Sciences, University of Eastern Piedmont, 28100 Novara, Italy
3
Division of Endocrinology and Metabolism, Moriggia Pelascini Hospital, 22015 Gravedona ed Uniti, Italy
4
Department of Dental Sciences, University Vita Salute San Raffaele, 20132 Milan, Italy

Correspondence should be addressed to Francesco Mangano; francescomangano1@mclink.net

Received 1 June 2016; Revised 22 July 2016; Accepted 30 August 2016

Academic Editor: Marcos Minicucci

Copyright © 2016 Francesco Mangano et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Aim. To investigate whether there is a correlation between early dental implant failure and low serum levels of vitamin D. Methods.
All patients treated with dental implants in a single centre, in the period 2003–2015, were considered for enrollment in this study.
The main outcome was early implant failure. The influence of patient-related variables on implant survival was calculated using the
Chi-square test. Results. 822 patients treated with 1625 implants were selected for this study; 27 early failures (3.2%) were recorded.
There was no link between gender, age, smoking, history of periodontitis, and an increased incidence of early failures. Statistical
analysis reported 9 early failures (2.2%) in patients with serum levels of vitamin D > 30 ng/mL, 16 early failures (3.9%) in patients
with levels between 10 and 30 ng/mL, and 2 early failures (9.0%) in patients with levels <10 ng/mL. Although there was an increasing
trend in the incidence of early implant failures with the worsening of vitamin D deficiency, the difference between these 3 groups
was not statistically significant (𝑃 = 0.15). Conclusions. This study failed in proving an effective link between low serum levels of
vitamin D and an increased risk of early implant failure. Further studies are needed to investigate this topic.

1. Introduction (surgical technique, loading conditions, and time), and others

Dental implants are now a reliable solution for the func-
tional and esthetic rehabilitation of partially and completely
edentulous patients; this has been demonstrated by long-term
clinical trials, with survival rates of greater than 95% [1–3].
In order to achieve long-term survival, osseointegration
of the dental implant needs to occur; that is, a direct
connection must be established between the bone and the
implant surface, without the interposition of fibrous tissue
[4]; once established, this close bond must be maintained
over time, resulting in a clinically asymptomatic fixation of
the implant under functional load [5]. Osseointegration is a
complex phenomenon and depends on many factors; some
are related to the implant (material, macroscopic design, and
implant surface), others to the surgical-prosthetic protocol
to the patient (quantity/quality of bone at the receiving site
and the host response) [4, 5].
Although survival rates of dental implants are now
high, there still remains a seemingly unavoidable number of
failures: either cases in which correctly placed implants do
not integrate with the bone or cases of peri-implant tissue
infection [6, 7]. To be specific, failure to osseointegrate and
peri-implantitis are the most frequent causes of early implant
failure [3, 6, 7]. Such events occur during the early stages of
healing (within 2-3 months of implantation) and therefore
before the implant is functionally loaded with the prosthetic
restoration; these failures are unevenly distributed within the
general population and tend to occur in some subjects in
particular. In these individuals multiple or repeated failures
over time are possible [6, 7]. Early failures occur even when
2 Mediators of Inflammation

optimal materials are used, surgical protocols are strictly dental implants [9, 24–35]: almost all these studies have been
followed, and the quantity/quality of bone at the recipient site done on animal models [24–32] and very few on humans [33–
is sufficient [6–8]. All these observations would suggest the 35]. The purpose of this retrospective study was therefore to
existence of specific patient-related risk factors; this prompts investigate any possible correlation between low blood levels
an investigation into the regulatory mechanisms controlling of vitamin D and early implant failure (failure occurring in
bone metabolism, bone remodelling, and bone turnover [9, the four months prior to the full restoration of the implant,
10]. because of a lack of osseointegration or because of infection).
Vitamin D plays a fundamental role in bone metabolism
[11–13]. It is a fat-soluble vitamin which promotes the absorp-
tion of calcium in the intestine and regulates calcium and 2. Materials and Methods
phosphate homoeostasis in the tissues and it is a fundamental
2.1. Data Collection. All patients who had been treated with
element in the mineralization of bones and teeth [11–13].
Morse-taper connection dental implants (Leone! Implant
It also acts as a hormone and is vital for the health of the
System, Florence, Italy) [3, 8] inserted to support fixed
blood vessels and the brain [14, 15]. It has been demonstrated
prosthetic restorations in one single dental centre (Grave-
that vitamin D plays a crucial role in the health of the
dona, Como, Italy), in the period between January 2003 and
cardiovascular tract [16], the immune system [17], and the
December 2015, were evaluated for possible enrollment into
respiratory tract [18, 19].
this retrospective study. Patients were enrolled into the study
Vitamin D in an inactive form (cholecalciferol or vita-
if they were over 18 years of age, had good oral and general
min D3) is ingested or produced in the skin on exposure
health, and had not undergone bone regenerative therapy
to sunlight [11, 12]. This inactive form undergoes double
prior to implant placement. The exclusion criteria were
hydroxylation in the liver and the kidneys and is thereby
incomplete medical records, the presence of specific systemic
transformed into its active form, known as either calcitriol
diseases (uncontrolled diabetes mellitus, immunodeficient
or 1,25-dihydroxyvitamin D3 [11, 12]. This active form exerts
states, and bleeding disorders), and the abuse of alcohol and
its action on various tissues by binding to the vitamin D
drugs; patients undergoing radiotherapy and chemotherapy
receptors and regulating the transcription of specific target
and those who were pregnant were also excluded. All the data
genes [12–23].
used for the study were obtained from the medical records
Serum levels of vitamin D in the 25(OH)D form are the
of the patients enrolled. The patient data was evaluated; this
most accurate way of determining vitamin D status: a subject
included gender (male or female), age at time of surgery,
with <10 ng/mL is considered to be vitamin D deficient;
history of chronic periodontal disease, smoking habits, and
one with 10–30 ng/mL is considered to have low levels of
serum vitamin D levels. Vitamin D levels were taken from
vitamin D. The optimal blood level of vitamin D is a value
blood tests, which had been requested two weeks prior
>30 ng/mL [12, 13]. Vitamin D deficiency is high in the
to surgery. The medical records also contained a range of
general population [12]: in Italy, for example, it is estimated
information as regards the implant or implants, that is, their
that about 80% of people can be deficient, particularly in the
site (maxilla or mandible), location (incisor, canine, premolar,
northern regions where exposure to the sun is lower [20]. This
and molar), the length and diameter of the implant, the type
deficiency increases with age and encompasses the majority
of prosthetic restoration, and the loading conditions. Lastly,
of the elderly population of Italy who are not taking vitamin
the medical records contained all the information on any
D supplements [20]. Until a few years ago, the guidelines
implant failure. These included their cause (lack of osseoin-
estimated that the daily intake of vitamin D required to
tegration in the absence of infection, infection of the peri-
maintain adequate blood levels was 200 IU (5 mcg) in adults
implant tissues or peri-implantitis, or implant failure due
aged between 19 and 50, 400 IU (10 mcg) in adults aged
to progressive bone loss caused by to prosthetic overload).
between 51 and 69, and at least 600 IU (15 mcg) in those over
It also included their classification: early failure, occurring
70 [12, 13]. These guidelines have now been revised upwards
in the early healing period, that is, the four months after
and it is currently believed that the amount of vitamin D
implant placement, prior to the placement of restoration and
which should be taken daily is 2000 IU (50 mcg) and up to
loading, or late failure, occurring after loading. There were
4000 (100 mcg) in the case of, for example, pregnant women
also details of any possible biological complications (peri-
[12, 13].
implant mucositis and peri-implantitis) and/or prosthetic
There is now substantial literature on the negative effects
complications (mechanical and/or technical).
of low levels of vitamin D, especially in severely compro-
mised patients: vitamin D deficiency seems to be associated
with increased mortality, cardiovascular events, and reduced 2.2. Insertion Protocol for the Implants. All implants were
functioning of the immune and musculoskeletal systems [15– inserted under the same strict protocol by the same specialist
19, 21–23]. On the other hand, normalizing levels of vitamin D (C. M.) who had 25 years’ experience in implant dentistry,
can lead to substantial benefits for critically ill patients, with in the period between January 2003 and December 2015.
effects on the muscles, the respiratory system, the heart, and The implants were inserted after raising a full thickness
the immune system [18, 21, 23]. mucoperiosteal flap; the implant site preparation and implant
Despite the importance of vitamin D and its effects on placement were performed in compliance with modern
bone metabolism [11, 12] few studies have, to date, investi- surgical protocols and in accordance with the manufacturer’s
gated the effects of its depletion on the osseointegration of instructions. After placement, cover screw was positioned
Mediators of Inflammation
and the implants were submerged. Immediately after posi-
tioning, patients were prescribed antibiotic coverage with
2 g of amoxicillin (or 600 mg of clindamycin in patients
allergic to penicillins) for 6 days. Postoperative pain was
controlled with nimesulide 100 mg daily for 2 days. Patients
were given detailed instructions on oral hygiene and were
prescribed chlorhexidine 0.12% mouth rinse twice a day for
6 days. The patients were recalled for suture removal after
10 days. The implants were left to heal submerged for a total
period of 4 months, to allow undisturbed healing and achieve
osseointegration. After 4 months of undisturbed healing, the
patient was recalled for the implant to be uncovered. The
cover screw was replaced with the healing abutment, and
sutures were positioned. Two weeks later, the sutures were
removed and an impression was taken for the manufacture
of the temporary restoration. The temporary restoration was
maintained in situ for 3 months, in order to monitor the
response of the implant, as well as the peri-implant tissues,
to masticatory load; at the end of this period, the temporary
restoration was replaced with the final restoration. The final
restorations were metal porcelain, cemented with a zinc
oxide-eugenol cement. A periapical radiograph was taken
to check on the sealing of the restoration. All patients were
included in a follow-up protocol with an annual check-up at
one of the scheduled professional oral hygiene sessions.
2.3. Primary Outcome. Early implant failure, occurring
within 4 months after implant placement and therefore prior
to placement of the prosthetic restoration and the functional
load of the implant, was the primary outcome studied. Early
implant failures were divided into two different categories: (a)
early failures due to lack of osseointegration and subsequent
implant mobility, in the absence of clinical signs of infection;
(b) early failures due to infection of the bone tissue around
the implant, with inflammation (peri-implantitis) of peri-
implant tissues and the presence of fistula, pain, swelling,
pus and/or exudate, pocket depth >6 mm with bleeding, and
marginal bone resorption >2.5 mm.
2.4. Statistical Analysis. All the data retrieved from the
individual medical records were recorded on a generic
spreadsheet (Excel!, Microsoft Office, Redmond, MA, USA)
which was used for the descriptive, qualitative, and quan-
titative analyses. The mean, standard deviation, median,
and confidence intervals were calculated for the quantitative
variables (e.g., patients’ age and vitamin D levels in serum).
A patient-based technique was used to calculate implant
survival. In this analysis, the “event” was implant failure: thus
in patients receiving more than one implant, the occurrence
of even a single implant failure led to the patient being
classified as a “failure.” The influence of different variables
on implant survival was taken into consideration: gender
(male or female), age at time of surgery (three age groups
were examined: <40, 40–60 years, and >60 years), smoking
habits (regardless of the actual number of cigarettes smoked),
a history of chronic periodontitis [36], and serum levels of
vitamin D. In the analysis of serum levels of vitamin D,
three classes of patients were considered: severely deficient
3
patients (serum vitamin D <10 ng/mL), patients with low
levels (serum vitamin D between 10 and 30 ng/mL), and
patients with adequate levels (serum vitamin D >30 ng/mL).
The influence of each of these variables on implant survival
was calculated using the Chi square test. The significance
level was set at 0.05. The overall implant survival, the survival
within the different groups, and the analysis of the influence
of the different variables on survival were all made using
dedicated statistical analysis software (SPSS 17.0!, SPSS Inc.,
Chicago, IL, USA).
3. Results
Of the 915 patients originally evaluated for enrollment in
this study, 93 presented with conditions corresponding to
the exclusion criteria and were therefore excluded from the
assessment. By contrast, 822 patients (mean age 57.3 ± 14.2
years; median age 58; range 18–90; and 95% CI, 56.3–58.2),
receiving 1625 implants, did not have any of the condi-
tions contained in the exclusion criteria and were therefore
enrolled into this retrospective study. The distribution of
patients by groups, with relative incidence of failures, was
reported in Table 1. In total, 27 early failures were recorded
(19 due to failure of osseointegration and 8 due to peri-
implant tissue infection), with an overall incidence of 3.2%.
No differences were observed in the incidence of early failures
between males and females (𝑃 = 0.97) nor according to age at
time of surgery (𝑃 = 0.98). Although the percentage of early
failures in smokers was slightly higher than that detected in
nonsmokers, there was no statistically significant difference
(𝑃 = 0.56) between these two groups of patients. The same
was true for patients with a history of periodontal disease;
they displayed a slightly higher incidence of early failures than
patients who had not been affected by periodontitis, but this
difference was not significant (𝑃 = 0.73). The average serum
level of vitamin D in the general population was 29.9 ng/mL
(±12.1; median 29; range 5–73; and 95% CI, 29.1–30.7). In
patients in whom early implant failure occurred, the average
serum level of vitamin D was 25.5 (±13.2; median 24; range
8–55; and 95% CI, 20.6–30.4). Statistical analysis reported
a rather low incidence of early failures (2.2%) in patients
with blood vitamin D levels >30 ng/mL. The incidence of
early failure was almost double in patients with insufficient
serum levels of vitamin D (10–30 ng/mL) and became even
higher (9.0%) in patients with serious vitamin D deficiency.
Although the statistical analysis revealed a trend toward
an increased incidence of failure in patients with severe
vitamin D deficiency, the analysis did not reveal a statistically
significant difference (𝑃 = 0.15) in the incidence of early
implant failure in these three groups of patients. Similar
results (𝑃 = 0.14) were obtained comparing the incidence
of failures in the group of severely deficient patients (2/22:
9.0%) with the incidence of failures in all other patients
(25/800: 3.1%). Finally, the statistical analysis did not reveal
a significant difference (𝑃 = 0.13) when comparing the
incidence of failures in the group of patients with serum
vitamin D levels >30 ng/mL (9/394: 2.2%) with the incidence
of failures in all other patients (18/428: 4.2%). The details of
early failure were reported in Table 2.
4 Mediators of Inflammation

Table 1: Distribution of patients (by gender, age at surgery, smoking habit, history of periodontal disease, and vitamin D levels in serum),
related failures, survival rate within groups, and differences between groups (Chi square test).

Number of patients Early failures Incidence of early failures 𝑃 value∗

Gender
Males 424 14 3.3%
0.97
Females 398 13 3.2%
Age at surgery
<40 years 92 3 3.2%
40–60 years 380 12 3.1% 0.98
>60 years 350 12 3.4%
Smoking habit
Yes 261 10 3.8%
0.56
No 561 17 3.0%
History of periodontal disease
Yes 279 10 3.5%
0.73
No 543 17 3.1%
Vitamin D level in serum
<10 ng/mL 22 2 9.0%
10–30 ng/mL 406 16 3.9% 0.15
>30 ng/mL 394 9 2.2%
Statistically significant difference 𝑃 < 0.05.
∗
𝑃 value = Chi square test.

4. Discussion evaluated the effect of the topical application of vitamin D

A relatively small number of experimental studies has
attempted to investigate the effects of vitamin D on the
osseointegration of dental implants [24–32]. The majority of
these studies would appear to indicate a positive effect of
vitamin D on osseointegration, but it is not yet entirely clear
whether supplementation would promote the healing of peri-
implant bone tissue clinically [24, 33–35].
A recent review of the literature on animal studies
has shown that vitamin D supplementation can stimulate
new bone formation and increase the contact between the
bone and the surface of titanium implants [24]. Specifically,
Kelly et al. demonstrated that vitamin D deficiency could
significantly compromise the establishment of osseointegra-
tion of Ti6Al4V implants in rats [25]. Similar results were
reported by Dvorak et al. [26]. In an experimental study on
ovariectomized rats, the authors demonstrated that vitamin
D deficiency could impair the formation of peri-implant
bone; the normalization of blood levels via supplementation
of vitamin D stimulated new bone formation [26]. Similar
results were reported by Zhou et al., who found an increase
in osseointegration in osteoporotic rats given vitamin D
supplements [27], and Wu et al., who demonstrated an
increase in the percentage of contact between bone and
implant in diabetic rats given vitamin D supplements [28].
Finally, Liu et al. reported that the administration of vitamin
D could increase the fixation of dental implants in mice
suffering from chronic kidney disease [29].
A further possibility for study, in order to understand
the effects of the administration of vitamin D on bone
healing of the peri-implant tissues, is that of coating the
implant surface with vitamin D [30–32]. Salomó-Coll et al.
to the surface of implants inserted in postextraction sockets
in dogs, with histological and histomorphometric analyses
of tissues removed at 12 weeks [30]. Topical application
of vitamin D increased the percentage of bone to implant
contact of 10% [30]. Similarly promising results were reported
by Cho et al. in a histological and histomorphometric study
on rabbits, where the coating of anodized implant surfaces
with a solution of poly(D,L-lactide-co-glycolide) PLGA and
1𝛼,25-dihydroxyvitamin D3 (1𝛼,25-(OH)2D3) stimulated the
apposition of new bone on fixtures [31]. Finally, in a further
experimental work in rabbits, implants with a surface coated
in 1,25-(OH)2D3 have shown an improved tendency to
osseointegrate compared to noncoated implants; however,
this difference was not statistically significant [32].
Unfortunately, very few clinical studies have so far inves-
tigated the effects of vitamin D deficiency on osseointegration
and on bone regeneration in dentistry [33–35]. This is
probably due to the fact that there are many factors which
can determine the success or failure of dental implants; the
attention of clinicians has been mostly focused on drawing
up surgical and prosthetic protocols and identifying new
materials and implant surfaces to improve osseointegration,
rather than on the analysis of patient-related risk factors
[6–9]. In a recent clinical work, Alvim-Pereira et al. found
no relationship between polymorphism of the vitamin D
receptor and implant failure [33]. In a randomized, con-
trolled, double-blind study, Schulze-Späte et al. investigated
the effects of supplementation with a combination of vitamin
D3 (5000 IU) and calcium (600 mg) on the formation of
new bone following maxillary sinus lift [34]. Ten patients
were assigned to the test group and given vitamin D and
calcium; ten other patients were assigned to the control
Mediators of Inflammation 5

Table 2: Details of all early implant failures.

History of
Patient Vitamin D level Position of the
Gender Age at surgery Smoking habit periodontal Type of failure
in serum failed implant
disease
1 M 66 Yes No 20 ng/mL FO #24
2 M 55 No No 35 ng/mL FO #25
3 F 42 No No 55 ng/mL FO #14
4 M 38 No Yes 48 ng/mL FO #15
5 F 57 Yes Yes 45 ng/mL PI #15
6 F 76 No Yes 24 ng/mL FO #26
7 M 45 Yes No 55 ng/mL FO #17
8 M 56 No No 10 ng/mL PI #17
9 F 58 No No 8 ng/mL FO #26
10 M 62 Yes Yes 22 ng/mL FO #25
11 M 45 No No 12 ng/mL FO #13
12 M 44 No No 28 ng/mL FO #21
13 F 38 No No 25 ng/mL PI #16
14 M 69 Yes Yes 24 ng/mL PI #24
15 F 74 No No 25 ng/mL FO #26
16 M 70 Yes No 18 ng/mL FO #27
17 M 69 No No 33 ng/mL FO #46
18 F 58 Yes No 32 ng/mL FO #46
19 F 46 Yes Yes 12 ng/mL PI #35
20 M 52 No Yes 9 ng/mL PI #33
21 F 43 No Yes 29 ng/mL FO #46
22 F 26 No No 33 ng/mL FO #37
23 F 68 Yes No 12 ng/mL PI #42
24 M 78 Yes No 15 ng/mL FO #35
25 M 65 No No 22 ng/mL FO #44
26 F 66 No Yes 20 ng/mL FO #36
27 F 70 No Yes 18 ng/mL PI #45

group and received only calcium [34]. Six to eight months
after surgery for bone regeneration, bone samples were
taken for histological analysis during implant placement
[34]. Although supplementation with vitamin D3 would have
increased the serum levels of vitamin D with potentially
positive effects on bone remodelling at the cellular level, no
statistically significant difference was demonstrated between
the two groups at the histological level [34].
The results of our study would appear to suggest that a
severe deficiency of vitamin D in the blood might be related
to an increase in the incidence of early implant failure. In fact,
the incidence of early implant failure was rather low (2.2%)
in patients with normalized levels of vitamin D in the blood
(>30 ng/mL), rose to almost double (3.9%) in patients with
insufficient serum levels (10–30 ng/mL), and were rather high
(9.0%) in patients characterized by severe deficiency states.
However, despite the tendency to an increased incidence of
early failure in patients characterized by deficiency states, the
differences between the three groups of patients were not
statistically significant (𝑃 = 0.15).
Our study also confirms that the serum values of vitamin
D in the local population are rather low: we found that
the proportion of patients with insufficient levels was 49.4%
and that the percentage with a severe deficiency was 2.7%.
The percentage of patients with adequate levels was 47.9%.
This is not surprising, as most of the patients treated came
from northern Italy and southern Switzerland, regions where
exposure to sunlight is somewhat reduced for long periods
of the year. In the light of this, the administration of vitamin
D in the weeks prior to placement of a dental implant
could be useful, particularly in patients with severe deficiency
states; in these patients, vitamin D supplementation should
be maintained for the whole life, in order to guarantee a good
remodelling of the bone around the implant.
This study has the distinction of being one of the first
clinical studies carried out on a large number of patients
to investigate the possibility of an association between low
blood levels of vitamin D and the incidence of early failure
in implantology. By restricting the analysis to early failures,
occurring in the first period of healing and therefore prior
6 Mediators of Inflammation
to placement of the prosthetic restoration, we were able to
focus our research and avoid a range of factors (linked to
the restoration itself and the prosthetic load) which could
have confused the issue in the study. It is, in fact, well known
that implant survival and thus osseointegration depend on a
large number of factors (related to the surgical and prosthetic
protocol, the materials used, and lastly the patient) [4, 5]
and it can be difficult to identify which of them might be
determining the success or failure of the treatment [7]. In
order to avoid this and to limit the confounding factors, the
same materials were used for all the patients in this study (the
same implant system for all the patients) [1, 3, 8]. In addition,
the same surgical protocol was used, involving submerged
healing in the absence of prosthetic loading [3]. Thus the
only possible confounding factors were the different quantity
and quality of bone at the implant receiving sites and the
patients’ responses: these are unavoidable factors. However,
some of those categories of patients most at risk of implant
failure (patients undergoing bone regeneration to create the
conditions for the positioning of the implant fixtures or those
with particular medical conditions which might increase the
risk of treatment failure) were excluded in the present study.
This study has limits. It is a retrospective work, in which
the number of patients having a severe deficiency of vitamin
D in the blood was low (only 22); thus the presence of even
just one less failure in this group would have led to quite
different results. It is possible that some residual confounding
may have biased the association between vitamin D and
implant failures that we observed. For instance, this study did
not investigate the influence of other patient-related factors
(e.g., the bone quality) which can affect implant survival
in the period immediately following implant placement. In
addition, if subjects with low levels of vitamin D were also
likely to receive more than 1 implant, their risk of being
classified as “failures” may increase. However, no patient
in this study experienced more than 1 failure, and the
probability of implant failure was not higher (1.5% versus
2.1%) in presence of another implant. Therefore, randomized,
controlled clinical trials are needed to confirm the presence
of an association between low serum levels of vitamin D and
an increase in the incidence of early failure in implantology.
It would be appropriate to assess whether supplementation of
vitamin D in the weeks before the operation could lead to a
reduction in early failures, whether due to lack of osseointe-
gration or implant infection. Further scientific studies with
an appropriate design and a more rigorous statistical analysis
will therefore be required in order to thoroughly investigate
this issue.
5. Conclusions
Until now, very few studies, and those mainly on animal
models, have involved assessing the influence of blood
levels of vitamin D levels on the osseointegration of dental
implants. Although most of these studies have shown that
the administration of vitamin D can improve the healing
of the peri-implant bone tissue, it is not yet clear whether
vitamin D supplements can promote the osseointegration of
dental implants. Our retrospective clinical study aimed to
investigate if there is a link between low levels of vitamin D
in the blood and an increased risk of early implant failures.
Although the incidence of early implant failures was higher in
patients with low serum levels of vitamin D, our study failed
in proving an effective link between low levels of vitamin D
in the blood and an increased risk of early implant failure.
Further higher level studies (prospective controlled trials or,
even better, randomized controlled clinical trials) with a more
rigorous statistical analysis are therefore needed to investigate
this issue. If an association between low serum levels of
vitamin D and higher risk of early implant failure could be
demonstrated, the clinician could give a set dose of vitamin
D in the weeks before surgery, in order to normalize serum
levels and obtain a positive effect on the healing process.
Competing Interests
The authors declare no conflict of interests for the present
study.
Acknowledgments
The authors are grateful to Giovanni Veronesi, Ph.D., Depart-
ment of Biostatistics, University of Varese, Italy, for help with
writing this manuscript.
References
[1] C. Mangano, F. Iaculli, A. Piattelli, and F. Mangano, “Fixed
restorations supported by Morse-taper connection implants:
a retrospective clinical study with 10–20 years of follow-up,”
Clinical Oral Implants Research, vol. 26, no. 10, pp. 1229–1236,
2015.
[2] S. T. Becker, B. E. Beck-Broichsitter, C. M. Rossmann, E.
Behrens, A. Jochens, and J. Wiltfang, “Long-term survival of
straumann dental implants with tps surfaces: a retrospective
study with a follow-up of 12 to 23 years,” Clinical Implant
Dentistry and Related Research, vol. 18, no. 3, pp. 480–488, 2016.
[3] F. Mangano, A. Macchi, A. Caprioglio, R. L. Sammons, A.
Piattelli, and C. Mangano, “Survival and complication rates
of fixed restorations supported by locking-taper implants: a
prospective study with 1 to 10 years of follow-up,” Journal of
Prosthodontics, vol. 23, no. 6, pp. 434–444, 2014.
[4] R. Trindade, T. Albrektsson, and A. Wennerberg, “Current
concepts for the biological basis of dental implants: foreign
body equilibrium and osseointegration dynamics,” Oral and
Maxillofacial Surgery Clinics of North America, vol. 27, no. 2, pp.
175–183, 2015.
[5] A. Jemat, M. J. Ghazali, M. Razali, and Y. Otsuka, “Surface
modifications and their effects on titanium dental implants,”
BioMed Research International, vol. 2015, Article ID 791725, 11
pages, 2015.
[6] B. R. Chrcanovic, J. Kisch, T. Albrektsson, and A. Wennerberg,
“Factors influencing early dental implant failures,” Journal of
Dental Research, vol. 95, no. 9, pp. 995–1002, 2016.
[7] A. Monje, L. Aranda, K. T. Diaz et al., “Impact of maintenance
therapy for the prevention of peri-implant diseases: a systematic
review and meta-analysis,” Journal of Dental Research, vol. 95,
no. 4, pp. 372–379, 2016.
Mediators of Inflammation
[8] C. Mangano, F. Mangano, J. A. Shibli, M. Ricci, R. L. Sammons,
and M. Figliuzzi, “Morse taper connection implants supporting
‘planned’ maxillary and mandibular bar-retained overdentures:
a 5-year prospective multicenter study,” Clinical Oral Implants
Research, vol. 22, no. 10, pp. 1117–1124, 2011.
[9] J. Choukroun, G. Khoury, F. Khoury et al., “Two neglected
biologic risk factors in bone grafting and implantology: high
low-density lipoprotein cholesterol and low serum vitamin D,”
Journal of Oral Implantology, vol. 40, no. 1, pp. 110–114, 2014.
[10] L. F. Cooper, “Systemic effectors of alveolar bone mass and
implications in dental therapy,” Periodontology 2000, vol. 23, no.
1, pp. 103–109, 2000.
[11] J. A. MacLaughlin, R. R. Anderson, and M. F. Holick, “Spectral
character of sunlight modulates photosynthesis of previtamin
D3 and its photoisomers in human skin,” Science, vol. 216, no.
4549, pp. 1001–1003, 1982.
[12] M. F. Holick, “High prevalence of vitamin D inadequacy and
implications for health,” Mayo Clinic Proceedings, vol. 81, no. 3,
pp. 353–373, 2006.
[13] C. M. Weaver, C. M. Gordon, K. F. Janz et al., “The National
Osteoporosis Foundation’s position statement on peak bone
mass development and lifestyle factors: a systematic review and
implementation recommendations,” Osteoporosis International,
vol. 27, no. 4, pp. 1281–1386, 2016.
[14] P. P. D. Santos, B. P. M. Rafacho, A. D. F. Gonçalves et al.,
“Vitamin D induces increased systolic arterial pressure via
vascular reactivity and mechanical properties,” PLoS ONE, vol.
9, no. 6, Article ID e98895, 2014.
[15] J. T. Keeney and D. A. Butterfield, “Vitamin D deficiency and
Alzheimer disease: common links,” Neurobiology of Disease, vol.
84, pp. 84–98, 2015.
[16] D. Papandreou and Z.-T.-N. Hamid, “The role of vitamin D
in diabetes and cardiovascular disease: an updated review of
the literature,” Disease Markers, vol. 2015, Article ID 580474, 15
pages, 2015.
[17] B. Prietl, G. Treiber, T. R. Pieber, and K. Amrein, “Vitamin D
and immune function,” Nutrients, vol. 5, no. 7, pp. 2502–2521,
2013.
[18] D. R. Thickett, T. Moromizato, A. A. Litonjua et al., “Associa-
tion between prehospital vitamin D status and incident acute
respiratory failure in critically ill patients: a retrospective cohort
study,” BMJ Open Respiratory Research, vol. 2, no. 1, Article ID
e000074, 2015.
[19] C. Schnedl, H. Dobnig, S. A. Quraishi, J. D. McNally, and K.
Amrein, “Native and active vitamin D in intensive care: who
and how we treat is crucially important,” American Journal of
Respiratory and Critical Care Medicine, vol. 190, no. 10, pp. 1193–
1194, 2014.
[20] G. Isaia, R. Giorgino, G. B. Rini, M. Bevilacqua, D. Maugeri, and
S. Adami, “Prevalence of hypovitaminosis D in elderly women
in Italy: clinical consequences and risk factors,” Osteoporosis
International, vol. 14, no. 7, pp. 577–582, 2003.
[21] K. Amrein, K. B. Christopher, and J. D. McNally, “Understand-
ing vitamin D deficiency in intensive care patients,” Intensive
Care Medicine, vol. 41, no. 11, pp. 1961–1964, 2015.
[22] K. Amrein, C. Schnedl, A. Holl et al., “Effect of high-dose
vitamin D3 on hospital length of stay in critically ill patients with
vitamin D deficiency: the VITdAL-ICU randomized clinical
trial,” The Journal of the American Medical Association, vol. 312,
no. 15, pp. 1520–1530, 2014.
[23] D. N. Gumieiro, B. P. Murino Rafacho, B. L. Buzati Pereira et al.,
“Vitamin D serum levels are associated with handgrip strength
7
but not with muscle mass or length of hospital stay after hip
fracture,” Nutrition, vol. 31, no. 7-8, pp. 931–934, 2015.
[24] F. Javed, H. Malmstrom, S. V. Kellesarian, A. A. Al-Kheraif, F.
Vohra, and G. E. Romanos, “Efficacy of vitamin D3 supplemen-
tation on osseointegration of implants,” Implant Dentistry, vol.
25, no. 2, pp. 281–287, 2016.
[25] J. Kelly, A. Lin, C. J. Wang, S. Park, and I. Nishimura, “Vitamin D
and bone physiology: demonstration of vitamin D deficiency in
an implant osseointegration rat model,” Journal of Prosthodon-
tics, vol. 18, no. 6, pp. 473–478, 2009.
[26] G. Dvorak, A. Fügl, G. Watzek, S. Tangl, P. Pokorny, and R.
Gruber, “Impact of dietary vitamin D on osseointegration in the
ovariectomized rat,” Clinical Oral Implants Research, vol. 23, no.
11, pp. 1308–1313, 2012.
[27] C. Zhou, Y. Li, X. Wang, X. Shui, and J. Hu, “1,25Dihydroxy
vitamin D3 improves titanium implant osseointegration in
osteoporotic rats,” Oral Surgery, Oral Medicine, Oral Pathology
and Oral Radiology, vol. 114, no. 5, pp. S174–S178, 2012.
[28] Y.-Y. Wu, T. Yu, X.-Y. Yang et al., “Vitamin D3 and insulin com-
bined treatment promotes titanium implant osseointegration in
diabetes mellitus rats,” Bone, vol. 52, no. 1, pp. 1–8, 2013.
[29] W. Liu, S. Zhang, D. Zhao et al., “Vitamin D supplementation
enhances the fixation of titanium implants in chronic kidney
disease mice,” PLoS ONE, vol. 9, no. 4, Article ID e95689, 2014.
[30] O. Salomó-Coll, J. E. Maté-Sánchez de Val, M. P. Ramı́rez-
Fernandez, F. Hernández-Alfaro, J. Gargallo-Albiol, and J. L.
Calvo-Guirado, “Topical applications of vitamin D on implant
surface for bone-to-implant contact enhance: a pilot study in
dogs part II,” Clinical Oral Implants Research, vol. 27, no. 7, pp.
896–903, 2016.
[31] Y. J. Cho, S. J. Heo, J. Y. Koak, S. K. Kim, S. J. Lee, and L. H. Lee,
“Promotion of osseointegration of anodized titanium implants
with a 1a,25-dihydroxyvitamin D3 submicron particle coating,”
The International Journal of Oral and Maxillofacial Implants, vol.
26, no. 6, pp. 1225–1232, 2011.
[32] Y. Naito, R. Jimbo, M. S. Bryington et al., “The influence
of 1𝛼.25-dihydroxyvitamin D3 coating on implant osseointe-
gration in the rabbit tibia,” Journal of Oral and Maxillofacial
Research, vol. 5, no. 3, article e3, 2014.
[33] F. Alvim-Pereira, C. C. Montes, G. Thomé, M. Olandoski, and
P. C. Trevilatto, “Analysis of association of clinical aspects and
vitamin D receptor gene polymorphism with dental implant
loss,” Clinical Oral Implants Research, vol. 19, no. 8, pp. 786–795,
2008.
[34] U. Schulze-Späte, T. Dietrich, C. Wu, K. Wang, H. Hasturk, and
S. Dibart, “Systemic vitamin D supplementation and local bone
formation after maxillary sinus augmentation—a randomized,
double-blind, placebo-controlled clinical investigation,” Clini-
cal Oral Implants Research, vol. 27, no. 6, pp. 701–706, 2016.
[35] G. Bryce and N. MacBeth, “Vitamin D deficiency as a suspected
causative factor in the failure of an immediately placed dental
implant: a case report,” Journal of the Royal Naval Medical
Service, vol. 100, no. 3, pp. 328–332, 2014.
[36] M. S. Zangrando, C. A. Damante, A. C. Sant’ana, M. L. R.
De Rezende, S. L. Greghi, and L. Chambrone, “Long-term
evaluation of periodontal parameters and implant outcomes
in periodontally compromised patients: a systematic review,”
Journal of Periodontology, vol. 86, no. 2, pp. 201–221, 2015.
 MEDIATORS of

INFLAMMATION

The Scientific Gastroenterology Journal of

World Journal
Hindawi Publishing Corporation
http://www.hindawi.com
Research and Practice Hindawi Publishing Corporation
Diabetes Research
Hindawi Publishing Corporation
Disease Markers
Hindawi Publishing Corporation
http://www.hindawi.com Volume 201
Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Journal of International Journal of

Immunology Research
Hindawi Publishing Corporation
Endocrinology
Hindawi Publishing Corporation
http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 2014

Submit your manuscripts at

http://www.hindawi.com

PPAR Research
Hindawi Publishing Corporation
http://www.hindawi.com
BioMed
Research International
Hindawi Publishing Corporation
Volume 2014 http://www.hindawi.com Volume 2014

Journal of
Obesity

Evidence-Based
Journal of Stem Cells Complementary and Journal of
Ophthalmology
Hindawi Publishing Corporation
International
Hindawi Publishing Corporation
Alternative Medicine
Hindawi Publishing Corporation Hindawi Publishing Corporation
Oncology
Hindawi Publishing Corporation
http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 201

Parkinson’s
Disease

Computational and
Mathematical Methods
in Medicine
Hindawi Publishing Corporation
http://www.hindawi.com
Behavioural
Neurology
AIDS
Research and Treatment
Oxidative Medicine and
Cellular Longevity
Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation
Volume 201 http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 2014
 BLOOD DRAWING
Equipment

1. 2. 3.

Stick the bandage on your hand Tighten the tourniquet Apply alcohol and tap

Int. Ext.
4. 5.

Cephalic
Basilic

Palpate with the index Spot the best vein Place the guide with a pen

Introduce the
6. 7. needle with a
straightforward
movement :
LO LO facing up
- Bevel
- Angle of 15-30°

Hold the needle (bevel facing you) Pull the skin at 10cm Apply the bandage

8. 9. End of the drawing

- Remove the tourniquet
LO - Prepare your finger above the LO
bandage without pressing
- Withdraw the needle with a fast movement
- Press on the puncture point for hemostasis
- Fold the patient arm for 1 minute
Connect the tubes - Retract the needle and throw it in the dedicated container
 A BLOOD DRAWING AT MY DENTIST ?
You will soon have a surgery in our dental office.
Your dentist may have to perform a blood drawing the day of
the surgery.

Why ? Your blood will be taken just before the surgery and immediately
centrifuged. Your dentist will retrieve a small concentrate (a fibrin clot,
very rich in growth factors).

What for ? These clots will protect your wounds, promote healing,
reduce the post-operative pain, decrease the risk of infections and
accelerate the bone regeneration.

Are we drawing a lot of blood ? No, about the same amount as for
blood tests. There is no more tiredness to fear after the surgery.

Are they contraindications ? NO ! All patients can benefit from this

technique. There are no contraindications. On the contrary, health
problems can make this procedure an essential contribution to a good
healing.

Nothing can replace your blood !

There are biomaterials (human, animal or synthetic), that may be added
to your blood. But only your blood can bring you as efficiently and
naturally all the growth factors necessary to your healing. Moreover,
there are no risk of rejection to fear, as it’s your own blood.

This natural technique requires the discomfort of a blood drawing but

will greatly facilitate your healing and help your body to grow bone and
gum in resorbed sites.

The whole practice is available to answer your questions

 https://doi.org/10.5125/jkaoms.2019.45.6.332
ORIGINAL ARTICLE pISSN 2234-7550 · eISSN 2234-5930

Socket preservation using eggshell-derived nanohydroxyapatite with

platelet-rich fibrin as a barrier membrane: a new technique
Vivekanand Sabanna Kattimani1, Krishna Prasad Lingamaneni1, Girija Easwaradas Kreedapathi2,
Kiran Kumar Kattappagari3
Departments of 1Oral and Maxillofacial Surgery and 3Oral and Maxillofacial Pathology, Sibar Institute of Dental Sciences, Guntur,
2
Department of Physics, Periyar University Salem, Salem, India

Abstract (J Korean Assoc Oral Maxillofac Surg 2019;45:332-342)

Objectives: Socket grafting is vital to prevent bone resorption after tooth extraction. Several techniques to prevent resorption have been described,
and various bone graft substitutes have been developed and used with varying success. We conducted this pilot study to evaluate the performance of
nanohydroxyapatite (nHA) derived from chicken eggshells in socket preservation.
Materials and Methods: This was a prospective, single center, outcome assessor-blinded evaluation of 23 sockets (11 patients) grafted with nHA
and covered with platelet-rich fibrin (PRF) membrane as a barrier. Bone width and radiographic bone density were measured using digital radiographs
at 1, 12, and 24 weeks post-procedure. Postoperative histomorphometric and micro-computed tomography (CT) evaluation were performed. The study
protocol was approved by the Institutional Ethics Committee.
Results: All patients had uneventful wound healing without graft material displacement or leaching despite partial exposure of the grafted socket. Tis-
sue re-epithelialized with thick gingival biotype (>3 mm). Width of the bone was maintained and radiographic density increased significantly with a
trabecular pattern (73.91% of sockets) within 12 weeks. Histomorphometric analysis showed 56.52% Grade 3 bone formation and micro-CT analysis
revealed newly formed bone with interconnecting trabeculae.
Conclusion: Use of a PRF membrane with nHA resulted in good bone regeneration in sockets. Use of a PRF membrane prevents periosteal-releasing
incisions for primary closure, thereby facilitating the preservation of keratinized mucosa and gingival architecture. This technique, which uses eggshell-
derived nHA and PRF membrane from the patient’s own blood, is innovative and is free of disease transfer risks. nHA is a promising economic bone
graft substitute for bone regeneration and reconstruction because of the abundant availability of eggshell waste as a raw material.

Key words: Bone, Tooth extraction, Socket grafting, Wound healing, Bone regeneration
[paper submitted 2018. 12. 15 / revised 1st 2019. 4. 13, 2nd 2019. 5. 1 / accepted 2019. 5. 12]

I. Introduction review of dimensional changes of the alveolar bone following

Socket grafting is vital to prevent bone resorption1. Remod-
eling of alveolar bone is a continuous process in physiology
and pathology, but alveolar bone is essential for the retention
of teeth. Extraction of teeth leads to an edentulous space and
loss of alveolar bone, function, and aesthetics2-6. A systemic
Vivekanand Sabanna Kattimani
Department of Oral and Maxillofacial Surgery, Sibar Institute of Dental
Sciences, Takkellapadu, Guntur, Andra Pradesh 522509, India
TEL: +91-863-2292249 FAX: +91-863-2292139
E-mail: drvivekanandsk@gmail.com
ORCID: https://orcid.org/0000-0002-9812-7207
This is an open-access article distributed under the terms of the Creative
CC
extraction showed a mean reduction of 3.8 mm in width and
1.24 mm of height in the first six months after extraction7.
Replacement of missing teeth is essential, but without the
support of adequate healthy alveolar bone, teeth replacement
is difficult8. Socket preservation attempts are therefore made
to maintain bone to enable patients to have implant supported
prosthesis in the future without the requirement for additional
surgical procedures for augmentation1.
Socket preservation has been performed using different
materials with varying success rates1,9-12. Materials used for
grafting include autogenous bone, allograft, xenograft, and
alloplasts, among others1,9. Various membranes have been
used for wound closure, including polytetrafluoroethylene

Commons Attribution Non-Commercial License (http://creativecommons.org/

licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and bovine and porcine collagen matrices1. Graft materials
and reproduction in any medium, provided the original work is properly cited.
Copyright © 2019 The Korean Association of Oral and Maxillofacial Surgeons. All enhance bone formation by promoting osteoconduction, os-
rights reserved.

332
 Socket preservation using nHA and PRF

teoinduction, and osteogenesis13-16. However, no graft mate-
rial to date is considered ideal. Recently, nanohydroxyapatite
derived from chicken eggshell (nHA) was developed17-19 and
used as an innovative graft material for bone regeneration
purposes20-22, but none of these previous studies evaluated the
performance of nHA and a platelet-rich fibrin (PRF) mem-
brane in socket preservation20-23.
We therefore performed this pilot study to evaluate the
histomorphometric and radiographic characteristics of bone
regeneration when nHA and a PRF membrane were used for
alveolar bone preservation and enhancement in the maxilla
and mandible of humans to enable implant-supported restora-
tion.
II. Materials and Methods
1. Study design
A total of 11 (eight male and three female) patients be-
tween the ages of 15 to 45 years who presented to the out-
patient Department of Oral and Maxillofacial Surgery, Sibar
Institute of Dental Sciences (Guntur, India) for extraction
and willing to participate in the study protocol were enrolled
during the period from June 2017 to June 2018. The Institu-
tional Ethics Committee of Sibar Institute of Dental Sciences
approved the study protocol (IEC-16/09/2014) and this study
was registered with the Clinical Trials Registry-India (CTRI).
Patients and patients’ relatives were informed about the study

A B C D E
Exposure 75 KV 4.00 mA 19.96 s Exposure 84 KV 5.00 mA 19.96 s
Dose 349 mGy.cm2 Dose 596 mGy.cm2
Axial Thickness=630 !m Axial Thickness=630 !m
Panoramic Thickness=990 !m Panoramic Thickness=10.0 mm
Oblique Thickness=90 !m Spacing=899 !m Oblique Thickness=90 !m Spacing=899 !m

F G

Fig. 1. A. Bone defect immediately after extraction with curette in place. B. Bone graft material placed till crestal level in the mandibular left
first molar socket. C. Wound closure using platelet-rich fibrin membrane and figure of eight suture. D. Well healed wound after two weeks. E.
Well-formed bone observed during exposure for implant bed preparation. F. Cone-beam computed tomography pre-extraction view with
roots and bone defect marked in red circles. G. Bone formation (red circles) assessed before implant placement.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J Korean Assoc
Oral Maxillofac Surg 2019

333
J Korean Assoc Oral Maxillofac Surg 2019;45:332-342
protocol before enrollment and written informed consent
was obtained. This was a prospective, single center, outcome
assessor-blinded pilot study.
1) Sample size calculation
The sample size required for this pilot study was 10 pa-
tients but we recruited 11 patients involving one or more
sockets per patient depending on the clinical scenario. Sam-
ple size was calculated with 80% power and a 5% alpha error
based on a two-sided test. Total of 23 extraction sockets were
assessed among the 11 patients.
2) Inclusion and exclusion criteria for patient selection
Patients with grossly decayed teeth, unrestorable teeth,
those willing to undergo implant restoration, and those read-
ily available for periodic recall were enrolled. Pregnant
patients, patients with gross tooth mobility due to complete
bone loss (more than 3/4 of root length), those in a vulnerable
category or with a systemic illness (diabetes, thyroid, hy-
pertension, etc.), and medically compromised patients were
excluded from the study protocol to eliminate bias.
3) Materials
(1) Nanohydroxyapatite
nHA granules used for grafting were synthesized from
natural calcium precursors obtained from chicken eggshell
waste and produced using a rapid microwave processing
technique17,18. The nHA obtained in this manner is typical
flower-like nanostructured HA19. It is composed of leaf-like
flakes extending radially from the center with a width of 100
to 200 nm and length of 0.5 to 1 μm. nHA contains Ca, P, O, C,
and Mg18,19. Incorporation of Mg ions into the HA structure is
important for the development of artificial bones19.
(2) Platelet-rich fibrin membrane
PRF membrane was prepared using autologous blood under
standard aseptic precautions. Intravenous blood (5 mL) was
drawn from the antecubital region using a sterile disposable
syringe. Blood was centrifuged using a tabletop centrifuge
for 10 minutes at 3,000 rpm immediately after collection. The
resultant product comprised acellular plasma as the top layer,
a PRF clot in the middle, and red blood cells at the bottom.
Successful preparation of PRF depends on speedy blood col-
lection and immediate centrifugation before clotting begins.
PRF component was separated and molded into a thin sheet
of approximately 1×1 cm using a sterile, wet piece of gauze.
334
2. Operative procedure
Extraction of the tooth or tooth root was performed atrau-
matically under local anesthesia. If necessary, curettage was
performed (Fig. 1. A) and irrigation was done with Betadine
and saline. Socket compression was not performed to prevent
the collapse of bony walls. After hemostasis, nHA grafting
was performed to the crestal level in all 23 sockets.(Fig. 1. B)
PRF membrane was transferred over the grafted socket and
tucked between buccal and lingual/palatal gingiva and stabi-
lized using 3-0 black silk suture (figure of eight). The suture
passed through the membrane to prevent slippage. PRF mem-
brane prevented leaching of materials and periosteal-releasing
incisions for primary closure.(Fig. 1. C) Postoperatively, all
patients were administered antibiotics and analgesics for five
days as a standard of care. Patients were recalled after 8 days
for suture removal.
Implant bed was prepared using a trephine bur (3×10 mm)
followed by a standard sequential drilling protocol for im-
plant placement 24 weeks after grafting. Depending on initial
stability, implants were loaded immediately or in a delayed
manner using NobelParallel conical connection root form im-
plants made by Nobel Biocare (Kloten, Sweden).
3. Clinical and radiographic evaluation
Wound healing was assessed prospectively.(Fig. 1. D) Pa-
tients were followed-up for 24 weeks until implant insertion.
(Fig. 1. E) Preoperative cone-beam computed tomography
(CBCT) revealed bony defects with roots.(Fig. 1. F) Bone
width was measured using bone calipers and radiographic
bone density was measured using digital intraoral peri-apical
radiograph at week 1, 12, and 24 postoperatively. CBCT
was obtained for planning and assessment before implant
placement.(Fig. 1. G) Digora software (Digora for Windows
2.8.107.458 Network Client, Soredex Orion Corporation,
Finland) was used to assess follow-up density changes and
bone formation characteristics.(Fig. 2) Radiographic evalu-
ation criteria were modified and adapted for assessment24,25.
Gridlines in digital IOPA were used as reference lines to
assess bone height postoperatively for available bone for
implant size selection. The assessor was blinded for the time
duration of follow-up during radiographic evaluation.
1) Micro-CT evaluation
During implant placement, a trephine biopsy (taken using
a 3×10-mm trephine bur) harvested from the grafted site was
 Socket preservation using nHA and PRF

subjected to histomorphometric and micro-CT evaluation.
(Fig. 3) Micro-CT analysis was carried out using a Phoe-
nixV/tome/Xs machine (GE Sensing & Inspection Technolo-
gies GmbH, Wunstorf, Germany).(Fig. 4. A-C) A series of
microfocus CT images were acquired as x-ray images while
progressively rotating the sample step by step through a
360º rotation at increments less than 1º per step.(Fig. 4. D)
These projections were used to reconstruct volumetric data.
The multiline configuration of the flat panel covered with a
movable shield reduced scattering artifacts. A temperature-
stabilized DXR500L detector (General Electric, Boston, MA,
USA) with 3,072 pixels provided a voxel size of 1.6 µm for
assessment and allowed scanning of objects with voxel sizes
<2 µm.
2) Histomorphometric evaluation
After micro-CT analysis, sections were decalcified using
5% EDTA. After processing, specimens were embedded in a
paraffin wax block and sliced with a microtome to a thickness
of 5 µm for H&E staining.(Fig. 5) The core was cut trans-

Fig. 2. Intra-oral periapical radiographic

images for assessment of bone forma-
tion and density using Digora software.
Vivekanand Sabanna Kattimani et al: Soc-
ket preservation using eggshell-derived nano-
hydroxyapatite with platelet-rich fibrin as a barrier
membrane: a new technique. J Korean Assoc Oral
Maxillofac Surg 2019

A C D

Fig. 3. A. Thick tissue biotype observed after removal of the screw-retained abutment of an immediate-loaded implant following suture
removal after 1 week. B. Trephined bone (3×10 mm size) harvested from the left mandibular first molar site 24 weeks after grafting (during
implant bed preparation) for micro-computed tomography (CT) and histomorphometric analysis. C, D. Micro-CT sections of trephine bone
showing intervening trabeculae.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J Korean Assoc
Oral Maxillofac Surg 2019

335
J Korean Assoc Oral Maxillofac Surg 2019;45:332-342

A B

C D

Fig. 4. A-C. Micro-computed tomography (CT) assessment of trephined bone using PhoenixV/tome/Xs machine image analyzing software.
D. Schematic presentation of micro-CT image acquisition process for analysis using the PhoenixV/tome/Xs machine.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J Korean Assoc
Oral Maxillofac Surg 2019

10-mm bone 5-mm trephine bone

A B
Fig. 5. A. Microphotograph of two (10
mm and 5 mm) trephine bone speci-
mens showing intervening trabeculae
with a rim of osteoblasts (arrows). B.
Osteoid and fibrous connective tissue
with plump lacunae with prominent os-
teocytes was visible (arrows). A, B. H&E
staining (×10).
Vivekanand Sabanna Kattimani et al: Soc-
ket preservation using eggshell-derived nano-
hydroxyapatite with platelet-rich fibrin as a barrier
membrane: a new technique. J Korean Assoc Oral
Maxillofac Surg 2019

versely and interpreted using image analysis software by two
oral and maxillofacial pathologists blinded to the procedure.
Histology analysis criteria were modified and adopted for
evaluation as follows: Grade 0, no bone formation; Grade 1,
minimal bone formation; Grade 2, moderate bone formation;
Grade 3, abundant bone formation; and Grade 4, exuberant
bone formation at the grafted site26.
4. Statistical analysis
Results were tabulated using Microsoft Excel 2010 (Micro-
soft, Redmond, WA, USA). Data were summarized as means
and percentages and analyzed by dependent t-tests using
IBM SPSS Statistics software (ver. 20.0; IBM, Armonk, NY,
USA). Kappa correlation was calculated to assess the degree
of observer agreement for radiological assessment.
III. Results
1. Clinical observations
All patients (23 sockets) had uneventful wound healing.
No graft material displacement or leaching was observed,
even though the graft material was partially exposed. Tissue

336
 Socket preservation using nHA and PRF

re-epithelialized entirely within 2 weeks with a thick gingival
biotype (>3 mm). Radiographic analysis showed increased
density with a trabecular pattern in 73.91% of sockets while
in the remaining 26.09% of sockets, a ground glass appear-
ance was observed.(Fig. 6) After 24 weeks, bone fill with
well-preserved ridges was observed. Socket wall width was
measured at the crestal bone level and was consistent from
week 4 to week 24.(Tables 1-3) Graft particles were incorpo-
rated into the generated bony tissue and the augmented site
was clinically indistinguishable from the original neighboring
bony tissue. Many of these sites had successfully placed im-
plants.
2. Histomorphometric and micro-CT observations

Micro-CT analysis revealed newly formed bone with in-

Ground glass terconnecting trabeculae and H&E staining revealed thin tra-
26.09%
beculae of woven bone. Plenty of cellular osteoid in the form
of trabeculae with intervening loose relatively dense cellular
fibrous tissue was observed. Trabeculae showed a rich anas-
tomosing pattern with numerous enlarged lacunae containing
prominent osteocytes. Numerous bundles of collagen fibers
were also observed. Osteoid showed eosinophilic areas sur-
Trabecular rounded by a rim of osteoblasts.(Fig. 5) The graft material
73.91% was in apposition with the osteoid. No osteoclasts or inflam-
Fig. 6. Pattern of bone regeneration and distribution of these pat- matory cells were observed. Grade 2 bone formation was ob-
terns. served in 13.04% of grafted sockets, Grade 3 bone formation
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived
nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J in 56.52% of grafted sockets, and Grade 4 bone formation in
Korean Assoc Oral Maxillofac Surg 2019 8.70% of grafted sockets.(Fig. 7)

Table 1. Socket preservation, changes in alveolar width at different time intervals, pattern of bone regeneration, and histopathology grad-
ing 24 weeks after bone grafting
Alveolar width (mm) Mean density after Grade of bone
Tooth No. (FDI) of Pattern of bone
No. formation based on
socket preservation Initial Final 1 wk 12 wk 24 wk regeneration
histopathology1
1 37 13 12 60.60 40.32 76.34 Trabecular 3
2 36 14 13 63.27 55.50 88.80 Trabecular 3
3 11 14 14 54.34 34.43 70.46 Ground glass 3
4 12 13 13 50.12 45.10 65.27 Ground glass 4
5 13 15 14 53.24 40.23 58.89 Ground glass 2
6 14 14 13 50.32 43.12 65.13 Ground glass 4
7 15 13 12 44.45 34.10 67.73 Trabecular 3
8 23 15 14 54.43 45.20 73.49 Trabecular 2
9 24 14 14 50.32 40.12 75.21 Trabecular 3
10 33 13 13 45.56 34.34 58.12 Ground glass 3
11 35 13 12.5 56.75 45.65 69.15 Ground glass 2
12 44 13 12.5 45.43 34.43 72.14 Trabecular 3
13 45 14 13 45.21 32.54 73.16 Trabecular 3
14 36 14.5 14.5 50.68 45.00 69.76 Trabecular 3
15 21 14 13.5 54.45 34.80 68.23 Trabecular 3
16 36 14.5 14 50.89 29.01 75.20 Trabecular 3
17 36 15 14 59.43 36.03 76.08 Trabecular 3
18 36 14 14 66.34 35.04 79.05 Trabecular -
19 38 15 14.5 63.12 60.06 80.03 Trabecular -
20 14 14 13.5 64.34 58.09 76.83 Trabecular -
21 15 13 13 44.65 35.07 69.01 Trabecular 3
22 47 15 14 56.87 43.05 70.01 Trabecular -
23 48 16 15 68.54 46.03 74.01 Trabecular -
(FDI: Fédération Dentaire Internationale; FDI World Dental Federation)
1
Histologic analysis graded on the following 5-point scale: 1) Grade 0, no bone formation; 2) Grade 1, minimal bone formation; 3) Grade 2,
moderate bone formation; 4) Grade 3, abundant bone formation; 5) Grade 4, exuberant bone formation.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J Korean Assoc
Oral Maxillofac Surg 2019

337
J Korean Assoc Oral Maxillofac Surg 2019;45:332-342

Table 2. Comparison of initial and final alveolar width (mm) scores by dependent t-test
Time points Mean SD Mean Diff. SD Diff. % of change Paired t P -value
Initial 14.04 0.86
Final 13.48 0.80 0.57 0.43 4.02 6.2395 0.0001*
(SD: standard deviation, Diff.: difference)
*P <0.05.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J Korean Assoc
Oral Maxillofac Surg 2019

Table 3. Comparison of density scores 1 week, 12 weeks, and 24 weeks by dependent t-test
Time points Mean SD Mean Diff. SD Diff. % of change Paired t P -value
1st wk 54.49 7.35
12th wk 41.19 8.26 13.31 7.17 24.42 8.9052 0.0001*
1st wk 54.49 7.35
24th wk 71.83 6.74 –17.34 6.32 –31.81 –13.1497 0.0001*
12th wk 41.19 8.26
24th wk 71.83 6.74 –30.65 8.29 –74.41 –17.7222 0.0001*
(SD: standard deviation, Diff.: difference)
*P <0.05.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J Korean Assoc
Oral Maxillofac Surg 2019

IV. Discussion 60.0 56.52

Histological grading of bone (%)

50.0
1. General aspects
40.0
Socket preservation is essential for maintaining adequate
30.0
alveolar bone height and width and enhancing bone regen-
eration. Alveolar bone is required to support the growth of 20.0
13.04
thick soft tissue. Various materials and techniques have been
8.70
10.0
used for socket preservation1,27. Selection of a socket pres-
0.00
ervation technique and graft material remains the choice of 0.0
the clinician. Many factors such as the pattern of resorption Grade 1 Grade 2 Grade 3 Grade 4

(resorbable or non-resorbable), graft material origin (bovine,
synthetic, coral, eggshell), material availability, economic
factors, and understanding of the evidence for regeneration
influence the choice of graft material14-16.
The disadvantages associated with harvesting autogenous
grafts have prompted research into novel materials for graft-
ing14. An increasing number of synthetic alternatives are
becoming available16. Improvements in technology have
facilitated the synthesis of hydroxyapatite graft particles that
are osteoinductive28-30.
Synthetic HA is a calcium phosphate material that can
have different densities, structures, and surface chemistries
depending on its exact composition. These characteristics are
responsible for the bone bonding properties, turnover rate,
and longevity of the material in situ 18. The efficacy of vari-
ous HA materials in long-term ridge preservation has been
proven. However, HA used in socket preservation should
Fig. 7. Histological grade of bone regeneration and distribution of
these grades.
Vivekanand Sabanna Kattimani et al: Socket preservation using eggshell-derived
nanohydroxyapatite with platelet-rich fibrin as a barrier membrane: a new technique. J
Korean Assoc Oral Maxillofac Surg 2019
preserve existing bone and modulate early bone regenera-
tion to facilitate implant osseointegration31,32. Synthetic nHA
does not require a donor site, and raw material is available
in unlimited quantities26,33. Control of particle size and inter-
particulate spaces to mimic those of natural bone is possible
during manufacturing17-19.
2. Nanohydroxyapatite derived from chicken eggshell
We used nHA derived from chicken eggshell in the pres-
ent study. Nanosynthetic HA has shown promising results for
socket preservation, and our findings further support the util-

338

ity of nanosynthetic HA for this purpose34. Histology sections
from superficial to deeper cuts showed a consistent increase
in newly formed bone with a decrease in the connective tis-
sue area fraction. Histomorphometric findings indicated mod-
erate to abundant bone formation within 24 weeks. Moreover,
lower amounts of residual graft particles were observed,
similar to the study of Goetz et al.35. Goetz et al.35 used Nano-
Bone synthetic material for socket filling and reported nearly
complete ossification with small residues of material after 4
months follow-up. NanoBone was osteoconductive based on
its ability to promote bony ingrowth and initiate integration
with newly formed bone. Thus total incorporation of gener-
ated bony tissue and mineral particles was achieved34,35. New
tissue formation in the healed socket was observed in ap-
proximately 75% of all examined specimens. Ultimately, the
graft material should induce new bone formation34. New bone
formation was evident in all samples and augmented sockets
had an adequate morphology and density.
Preliminary experimental studies have also evaluated nano-
sized ceramics as a promising class of graft substitute materi-
als because of their osteo-integrative properties36. In these
studies, nHA paste was used with or without an additional
barrier membrane36. This paste promoted wound healing in
the extraction sockets. Nanoparticles can hypothetically re-
duce the inflammatory response and support active osseous
organization36,37.
The nHA used in this study meets the characteristics of an
ideal graft material noted by Gross31 in 1997. The material
is available in granular/nanopowder form, which is easy to
handle, and is not prone to infection. In this study, the grafted
area was exposed to the oral cavity because we did not use
primary closure or a barrier membrane other than a PRF
membrane to prevent initial leaching of the material until it
became cohesive with a blood clot. nHA is hydrophilic and
sticks to the socket wall and settles down in the blood soon
after filling. There is no possibility of disease transfer because
the material is derived from chicken eggshell. Histology
clearly showed the development of bone reminiscent of na-
tive bone. Mobility of the tooth has been shown to be main-
tained when this material is grafted into large cystic defects23,
and it also supports implant prostheses successfully. Similar
findings have been reported for synthetic grafts38 with clini-
cally successful implants observed after five years38. Further-
more, bone density and volume have been shown to increase
over time38,39. As noted by Boyne40, synthetics act as a natural
barrier for epithelial downgrowth into the socket. The ability
to maintain the height and width of alveolar bone and soft
Socket preservation using nHA and PRF
tissue anatomy has distinct functional and esthetic advantag-
es33,40. We observed a significant increase in density from the
week 1 to week 12 (P <0.05). Minimal bone loss (0.57±0.43
mm) was observed over 24 weeks compared to baseline,
consistent with the literature1. Alveolar bone preservation
and replacement therapy can be achieved using nHA without
a collagen barrier membrane. nHA is therefore a promising
graft material that can be produced in an environmentally-
friendly manner17,19.
3. Socket preservation technique and barrier membrane
We used nHA for socket preservation23. No cases of leach-
ing or displacement of the graft material were observed and
no periosteal-releasing incisions occurred. Use of an inva-
sive method is recommended against at this point as any
procedure that requires primary intention healing with the
advancement of flaps can result in an increased inflamma-
tory response and a decrease in vestibular depth, creating an
unaesthetic scar41. The study by Fickl et al.42 showed that flap
elevation results in the loss of more bone. This is because
rupture of the periosteum and connective tissue insertion con-
sequently reduces blood flow, causing lysis of osteocytes and
necrosis of mineralized tissue42. This necrotic bone will grad-
ually be removed through resorption by osteoclasts present
in the periosteum43,44. We overcame this problem of bone loss
by not elevating the flap. Elevation also negatively affects the
esthetics of the ridge and papilla by altering the mucogingival
line position2. We used PRF membrane to cover the graft ma-
terial during the initial period; we consider this an essential
step for successful grafting with the existing attached gingiva
and future success with implant restoration.
Many current socket preservation techniques use a barrier
membrane for primary closure, but there are often difficulties
in adoption of the membrane, and a second surgical proce-
dure is required to remove the membrane45. In general, mem-
brane use is time-consuming and expensive45-47. Murphy45,46
reported exposure of non-resorbable membrane in 87% of
cases, leading to infection, swelling, sloughing, and reces-
sion. We did not encounter these complications using PRF
membrane to cover the graft material. Synthetic grafts do
not require a second surgical procedure, nor do they elicit an
inflammatory reaction, which is usually seen with a barrier
membrane47. Formation of dense lamellar bone at the grafted
area has been observed20. Socket grafting eliminates or reduc-
es the incidence of dry socket33; consistent with this, no cases
of dry socket were encountered in our study. Moreover, inva-
339
J Korean Assoc Oral Maxillofac Surg 2019;45:332-342
sive procedures like guided bone regeneration and sinus floor
elevation are not needed when socket grafting is adopted and
planned properly before extraction41.
The PRF membrane in our study acted as a cover for graft
material and as a barrier membrane for guided bone regen-
eration. The effect of PRF as a barrier membrane for bone
formation and soft tissue healing may be limited, but PRF
and nHA may interact additively or synergistically. The use-
fulness of PRF in the maxillofacial area is expanding48. Our
study design did not allow us to distinguish between the ef-
fects of nHA and PRF, although histomorphometry analysis
at the bottom of the socket and middle of the socket showed
similar bone formation characteristics as that in the crestal
area. In previous studies, nHA alone enhanced bone forma-
tion in cystic defects23,49. Even after disintegration of the PRF
membrane, material leaching was not observed. Alveolar
bone loss occurs if socket preservation techniques are not
employed1. We therefore did not include a control group
in this preliminary study as this study is part of an ongoing
study protocol to delineate the histomorphometric and micro-
CT characteristics of bone formation in relation to radiogra-
phy; previous studies have used only radiography (IOPA and
CBCT/CT scan) for assessment of cystic defect grafting23,49.
Grafting prevents hematoma and infection. Manipulation
of graft material is easy because of the hydrophilic nature of
nHA. It can be used as putty when mixed with a drop of sa-
line or native blood or serum from the PRF test-tube. Its ease
of use, absence of disease transfer risk, ready availability in
large quantities, and environment-friendly production make
nHA an economic graft substitute material for enhancement
of bone regeneration and reconstruction. Application of nHA
in grafted sockets supports implant restoration, indicating os-
seointegration.
4. Future research work
Advances in tissue engineering techniques are likely to
provide novel biomaterials for everyday clinical use41. Long-
term follow-up data are mandatory to evaluate the presence
of grafted particles that could eventually interfere with the
longevity of the implant. We are currently performing a study
to further elucidate the histochemical and morphometric
nature of the bone formed, as well as assess the resorption
kinetics of nHA. The results of the current pilot study suggest
that multicenter randomized controlled clinical trials with
long-term implant restoration follow-up are warranted. Vari-
ous grafting materials are available for purchase. Cost varies
340
according to the manufacturer and depends on many factors
such as the raw material source and processing technique in-
volved14,16,31,32. Comparison of the costs of grafting is beyond
the scope of this study.
Limitations of this pilot study are our small sample size
and the fact that socket morphology, width, and presence of
buccal/lingual cortical walls were not taken into account in
our analyses.
V. Conclusion
Sockets preserved with nHA showed good bone regenera-
tion. Use of nHA and a PRF membrane as a barrier can assist
the healing process and provide better bone quality while pre-
serving alveolar bone. Micro-CT provides good insight into
bone healing and bone quality in three dimensions compared
to conventional histopathology. The technique described
here does not require primary closure, thereby facilitating the
preservation of keratinized mucosa and gingival architecture.
nHA derived from chicken eggshell is a promising novel
bone graft substitute.
ORCID
Vivekanand Sabanna Kattimani, https://orcid.org/0000-
0002-9812-7207
Krishna Prasad Lingamaneni, https://orcid.org/0000-0002-
0098-2900
Girija Easwaradas Kreedapathi, https://orcid.org/0000-
0002-2529-0062
Kiran Kumar Kattappagari, https://orcid.org/0000-0001-
6614-6617
Authors’ Contributions
V.S.K. designed the study and K.P.L. mentored the study.
V.S.K. collected data and wrote the manuscript. K.K.K.
and G.E.K. performed the review and assisted for the study.
K.K.K. participated in the study assessment and helped to
draft the histological assessment part of the manuscript. All
authors read and approved the final manuscript.
Acknowledgements
We would like to thank our colleagues in the Department
of Oral and Maxillofacial Radiology and Oral and Maxil-
lofacial Pathology for their support during execution of this

study protocol as well as the Department of Physics, Periyar
University, Salem for their collaboration. The Department
of Sciences and Technology, Government of India provided
funding for synthesis of nHA under the Waste Management
Technology Development and Transfer division. We would
also like to thank Prof. Krishnan Balasubramanian and Mr.
Harikrishnan Ravichandran of the Center for Nondestructive
Evaluation, Indian Institute of Technology Madras, Chennai,
Tamil Nadu for the micro-CT evaluation of the specimens.
Finally, we would like to thank Dr. Javali for his guidance
with statistical analysis.
This study was supported by Department of Science and
Technology, Government of India (grant No. DST/TSG/
WM/2015/576/G). The study protocol is registered with the
Clinical Trials Registry-India (CTRI; CTRI/2014/12/005340).
Ethics Approval and Consent to Participate
The study protocol was approved by the Institutional Eth-
ics Committee of Sibar Institute of Dental Sciences (IEC-
16/09/2014), and the written informed consent was obtained
from all patients and patients’ relatives.
Conflict of Interest
No potential conflict of interest relevant to this article was
reported.
References
1. Atieh MA, Alsabeeha NH, Payne AG, Duncan W, Faggion CM,
Esposito M. Interventions for replacing missing teeth: alveolar
ridge preservation techniques for dental implant site development.
Cochrane Database Syst Rev 2015;(5):CD010176.
2. Camargo PM, Lekovic V, Weinlaender M, Klokkevold PR, Kenney
EB, Dimitrijevic B, et al. Influence of bioactive glass on changes in
alveolar process dimensions after exodontia. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2000;90:581-6.
3. Iasella JM, Greenwell H, Miller RL, Hill M, Drisko C, Bohra AA,
et al. Ridge preservation with freeze-dried bone allograft and a col-
lagen membrane compared to extraction alone for implant site de-
velopment: a clinical and histologic study in humans. J Periodontol
2003;74:990-9.
4. Lekovic V, Kenney EB, Weinlaender M, Han T, Klokkevold P,
Nedic M, et al. A bone regenerative approach to alveolar ridge
maintenance following tooth extraction. Report of 10 cases. J Peri-
odontol 1997;68:563-70.
5. Lekovic V, Camargo PM, Klokkevold PR, Weinlaender M, Ken-
ney EB, Dimitrijevic B, et al. Preservation of alveolar bone in
extraction sockets using bioabsorbable membranes. J Periodontol
1998;69:1044-9.
6. Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing
and soft tissue contour changes following single-tooth extraction:
a clinical and radiographic 12-month prospective study. Int J Peri-
Socket preservation using nHA and PRF
odontics Restorative Dent 2003;23:313-23.
7. Tan WL, Wong TL, Wong MC, Lang NP. A systematic review of
post-extractional alveolar hard and soft tissue dimensional changes
in humans. Clin Oral Implants Res 2012;23 Suppl 5:1-21.
8. John V, De Poi R, Blanchard S. Socket preservation as a precursor
of future implant placement: review of the literature and case re-
ports. Compend Contin Educ Dent 2007;28:646-53; quiz 654, 671.
9. Ten Heggeler JM, Slot DE, Van der Weijden GA. Effect of socket
preservation therapies following tooth extraction in non-molar
regions in humans: a systematic review. Clin Oral Implants Res
2011;22:779-88.
10. Hämmerle CH, Araújo MG, Simion M; Osteology Consensus
Group 2011. Evidence-based knowledge on the biology and treat-
ment of extraction sockets. Clin Oral Implants Res 2012;23 Suppl
5:80-2.
11. Vignoletti F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M.
Surgical protocols for ridge preservation after tooth extraction. A
systematic review. Clin Oral Implants Res 2012;23 Suppl 5:22-38.
12. Vittorini Orgeas G, Clementini M, De Risi V, de Sanctis M. Surgi-
cal techniques for alveolar socket preservation: a systematic re-
view. Int J Oral Maxillofac Implants 2013;28:1049-61.
13. Damien CJ, Parsons JR. Bone graft and bone graft substitutes: a
review of current technology and applications. J Appl Biomater
1991;2:187-208.
14. Habal MB. Different forms of bone grafts. In: Habal MB, Reddi
AH, Mitchell J, eds. Bone grafts and bone substitutes. Philadelphia:
W.B. Saunders; 1992:6-8.
15. Hench LL. Bioceramics: from concept to clinic. J Am Ceram Soc
1991;74:1487-510.
16. Greenwald AS, Boden SD, Goldberg VM, Khan Y, Laurencin CT,
Rosier RN; American Academy of Orthopaedic Surgeons. The
Committee on Biological Implants. Bone-graft substitutes: facts,
fictions, and applications. J Bone Joint Surg Am 2001;83 Suppl 2
Pt 2:98-103.
17. SureshKumar G, Thamizhavel A, Girija EK. Microwave conver-
sion of eggshells into flower-like hydroxyapatite nanostructure for
biomedical applications. Mater Lett 2012;76:198-200.
18. SureshKumar G, Thamizhavel A, Yokogawa Y, NarayanaKalkura S,
Girija EK. Synthesis, characterization and in vitro studies of zinc
and carbonate co-substituted nano-hydroxyapatite for biomedical
applications. Mater Chem Phys 2012;134:1127-35.
19. SureshKumar G, Girija EK. Flower-like hydroxyapatite nanostruc-
ture obtained from eggshell: a candidate for biomedical applica-
tions. Ceram Int 2013;39:8293-9.
20. Park JW, Bae SR, Suh JY, Lee DH, Kim SH, Kim H, et al. Evalua-
tion of bone healing with eggshell-derived bone graft substitutes in
rat calvaria: a pilot study. J Biomed Mater Res A 2008;87:203-14.
21. Kim SH, Kim W, Cho JH, Oh NS, Lee MH, Lee SJ. Comparison
of bone formation in rabbits using hydroxyapatite and β-tricalcium
phosphate scaffolds fabricated from egg shells. Adv Mater Res
2008;47-50:999-1002.
22. Lee SW, Kim SG, Balázsi C, Chae WS, Lee HO. Comparative
study of hydroxyapatite from eggshells and synthetic hydroxyapa-
tite for bone regeneration. Oral Surg Oral Med Oral Pathol Oral
Radiol 2012;113:348-55.
23. Kattimani V, Lingamaneni KP, Chakravarthi PS, Kumar TS, Sid-
dharthan A. Eggshell-derived hydroxyapatite: a new era in bone
regeneration. J Craniofac Surg 2016;27:112-7.
24. Kattimani VS, Chakravarthi SP, Neelima Devi KN, Sridhar MS,
Prasad LK. Comparative evaluation of bovine derived hydroxy-
apatite and synthetic hydroxyapatite graft in bone regeneration of
human maxillary cystic defects: a clinico-radiological study. Indian
J Dent Res 2014;25:594-601.
25. Kattimani VS, Bajantai NV, Sriram SK, Sriram RR, Rao VK, De-
sai PD. Observer strategy and radiographic classification of healing
after grafting of cystic defects in maxilla: a radiological appraisal. J
Contemp Dent Pract 2013;14:227-32.
341
J Korean Assoc Oral Maxillofac Surg 2019;45:332-342
26. Brownfield LA, Weltman RL. Ridge preservation with or without
an osteoinductive allograft: a clinical, radiographic, micro-com-
puted tomography, and histologic study evaluating dimensional
changes and new bone formation of the alveolar ridge. J Periodon-
tol 2012;83:581-9.
27. Morjaria KR, Wilson R, Palmer RM. Bone healing after tooth ex-
traction with or without an intervention: a systematic review of ran-
domized controlled trials. Clin Implant Dent Relat Res 2014;16:1-
20.
28. Ripamonti U. Osteoinduction in porous hydroxyapatite implanted
in heterotopic sites of different animal models. Biomaterials
1996;17:31-5.
29. Kasaj A, Willershausen B, Reichert C, Röhrig B, Smeets R,
Schmidt M. Ability of nanocrystalline hydroxyapatite paste to
promote human periodontal ligament cell proliferation. J Oral Sci
2008;50:279-85.
30. Gosain AK, Song L, Riordan P, Amarante MT, Nagy PG, Wilson
CR, et al. A 1-year study of osteoinduction in hydroxyapatite-
derived biomaterials in an adult sheep model: part I. Plast Reconstr
Surg 2002;109:619-30.
31. Gross JS. Bone grafting materials for dental applications: a practi-
cal guide. Compend Contin Educ Dent 1997;18:1013-8, 1020-2,
1024, passim; quiz.
32. Darby I. Periodontal materials. Aust Dent J 2011;56 Suppl 1:107-
18.
33. Ashman A. Postextraction ridge preservation using a synthetic al-
loplast. Implant Dent 2000;9:168-76.
34. Gholami GA, Najafi B, Mashhadiabbas F, Goetz W, Najafi S.
Clinical, histologic and histomorphometric evaluation of socket
preservation using a synthetic nanocrystalline hydroxyapatite in
comparison with a bovine xenograft: a randomized clinical trial.
Clin Oral Implants Res 2012;23:1198-204.
35. Götz W, Gerber T, Michel B, Lossdörfer S, Henkel KO, Heine-
mann F. Immunohistochemical characterization of nanocrystalline
hydroxyapatite silica gel (NanoBone(r)) osteogenesis: a study on
biopsies from human jaws. Clin Oral Implants Res 2008;19:1016-
26.
36. Chris Arts JJ, Verdonschot N, Schreurs BW, Buma P. The use of a
bioresorbable nano-crystalline hydroxyapatite paste in acetabular
bone impaction grafting. Biomaterials 2006;27:1110-8.
37. Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R. En-
hanced functions of osteoblasts on nanophase ceramics. Biomateri-
als 2000;21:1803-10.
38. Froum S, Orlowski W. Ridge preservation utilizing an alloplast
prior to implant placement--clinical and histological case reports.
342
Pract Periodontics Aesthet Dent 2000;12:393-402; quiz 404.
39. Ashman A, LoPinto J, Rosenlicht J. Ridge augmentation for imme-
diate postextraction implants: eight year retrospective study. Pract
Periodontics Aesthet Dent 1995;7:85-94; quiz 95.
40. Boyne PJ. Use of HTR in tooth extraction sockets to maintain
alveolar ridge height and increase concentration of alveolar bone
matrix. Gen Dent 1995;43:470-3.
41. Pagni G, Pellegrini G, Giannobile WV, Rasperini G. Postextraction
alveolar ridge preservation: biological basis and treatments. Int J
Dent 2012;2012:151030.
42. Fickl S, Zuhr O, Wachtel H, Bolz W, Huerzeler MB. Hard tissue
alterations after socket preservation: an experimental study in the
beagle dog. Clin Oral Implants Res 2008;19:1111-8.
43. Araújo MG, Lindhe J. Dimensional ridge alterations following
tooth extraction. An experimental study in the dog. J Clin Peri-
odontol 2005;32:212-8.
44. Aimetti M, Romano F, Griga FB, Godio L. Clinical and histologic
healing of human extraction sockets filled with calcium sulfate. Int
J Oral Maxillofac Implants 2009;24:902-9.
45. Murphy KG. Postoperative healing complications associated with
Gore-Tex periodontal material. Part II. effect of complications on
regeneration. Int J Periodontics Restorative Dent 1995;15:548-61.
46. Murphy KG. Postoperative healing complications associated with
Gore-Tex periodontal material. Part I. incidence and characteriza-
tion. Int J Periodontics Restorative Dent 1995;15:363-75.
47. De Sanctis M, Zucchelli G, Clauser C. Bacterial colonization of
bioabsorbable barrier material and periodontal regeneration. J Peri-
odontol 1996;67:1193-200.
48. Miron RJ, Choukroun J. Platelet rich fibrin in regenerative dentist-
ry: biological background and clinical indications. Hoboken: John
Wiley & Sons; 2017.
49. Kattimani VS, Lingamaneni KP. Natural bioceramics: our experi-
ence with changing perspectives in the reconstruction of maxillofa-
cial skeleton. J Korean Assoc Oral Maxillofac Surg 2019;45:34-42.
How to cite this article: Kattimani VS, Lingamaneni KP, Kreed-
apathi GE, Kattappagari KK. Socket preservation using eggshell-
derived nanohydroxyapatite with platelet-rich fibrin as a barrier
membrane: a new technique. J Korean Assoc Oral Maxillofac Surg
2019;45:332-342. https://doi.org/10.5125/jkaoms.2019.45.6.332
 Platelet Rich Fibrin Course
Dr Joseph CHOUKROUN, MD Dr Elisa CHOUKROUN, DDS
joseph@a-prf.com elisa@a-prf.com

1. Concept
Principle : blood centrifugation without anticoagulants.
PRF was launched in 2000 by J. Choukroun (Choukroun J. et al. 2001)
PRF contains platelets, but also a high concentration of fibrin and leucocytes.
PRF is often called L-PRF (Leucocytes - PRF) for its firm formulation by J. Choukroun in 2001.
Nowadays, new modifications in centrifugation speed, G-force and time have led to the
development of a more bioactive PRF now called A-PRF (Advanced - PRF) (= LSCC for Low
Speed Centrifugation Concept, 2014).
Fibrin is the main component of PRF. Discoveries from basic science researches show that :
- Fibrin is a provisional extra cellular matric entirely derived from autogenous sources
- Fibrine contains a supra –physiological concentration of human growth factors
- Fibrine releases slowly and continuously cytokines for up to and over 10 days.

2. Indications
The use of PRF is indicated in all types of oral surgeries :
- Socket preservation : PRF is used as a tissue matrix. Fill the socket as much as you
can (compress the plugs with a gauze or a compactor). The socket has to be left
open, no primary closure is necessary. Sutures are done only to maintain PRF in
the socket. There is no need to wait more than 3 months to place the implant.
In most cases, no additional biomaterial is needed.
- Soft tissue management : PRF is used as a tissue matrix. The best technique is the
tunnel technique with a full flap. Same rule applies : Use as much PRF as you can.
- Bone graft : Sticky bone with S-PRF.
Otherwise, cut the membranes (1 or 2) into small pieces and mix them with the
biomaterial. Hydrate this mix with the PRF exudate from the PRF box.
- Sinus lift :
.Protection of the Schneiderian membrane : 2 membranes placed first
.PRF mixed with the biomaterial
.Treatment of Schneiderian membrane perforations
.Closure of the osteotomy window
.PRF can also be used alone, only with a simultaneous implant placement
- Guided Bone Regeneration : PRF can be used as a barrier membrane to cover the
augmented bone and to improve the vascularization of both hard and soft tissues.
It’s idealy placed especially when no re-entry is planned.
3. Preparation/Protocols
PRF ou L - PRF (1st protocol of 2001) : 2700 tr/min & 12 min

A - PRF + (clots, membranes, plugs) : Red tubes :

1. Draw your tubes (fast filling)
2. Put them in the centrifuge, in a balanced way (use the colors to help you)
3. Launch the program A - PRF + : 1300 tr/min & 14 min
4. At the end of the spinning : Remove the tubes from the machine, remove the caps
& Put the tubes in the tube-holder.

Wait 5 minutes if the clots are not completely solid.

Don’t leave the tubes in the centrifuge, and neither the clots in the tubes.

Remove the clot from the tube and peel of the red clot, to keep only the fibrin.
- For a clot : Use like this
- For a membrane : Place the clot in the PRF-BoX and put the tray over
- For a plug : Place the clot in the dedicated white cylinder and press it with the piston

S - PRF : (Sticky bone & large membranes) : Green tubes :

1. Draw your green tubes (fast filling) + 1 red tube
2. Put them in the centrifuge, in a balanced way (use the colors to help you)
3. Launch the program A - PRF + : 1300 tr/min & 14 min
4. At the end of the spinning : Remove the tubes from the machine, remove the caps
& Put the tubes in the tube-holder.
5. With a 2mL seringue, take the liquid from the green tubes
6. Apply this liquid on your biomaterial (sticky bone) or alone in a container (large membrane)
7. To accelerate the clotting : Add 1 clot on your sticky bone or 1 membrane on your large
membrane (From the red rube)

To have more liquid, draw more green tubes.

Add more liquid to yout sticky bone if you have a large graft
or if you want a denser sticky bone
If you want to graft first and then « congeal » it on site :
- Cut 1 or 2 membranes in small pieces in the cup
- Add your biomaterial
- Add few drops of the PRF-BoX exsudate
- Apply this mix on the site to graft
- Congeal the mix with drops of S-PRF
Limits of this protocol :
- If you do the blood drawing at the beginning of the surgery (A PRF for membranes), you’ll
need to do a second drawing at the time of the graft (S PRF for the liquid)
- Or do the drawing for both A PRF (red) & S PRF (green) at the moment of the graft.

i - PRF : Green tubes : Liquid for 15-20 min.

Women : i PRF settings : 700 tr/min (60 g) & 3 min
Men : i PRF M settings : 700 tr/min (200 g) & 4 mn

i - PRF + (Aesthetics & Orthopedics) : Purple tubes : Liquid for 20 min.

Women : i PRF + settings : 700 tr/min (60 g) & 5 min
Men : i PRF + M settings : 700 tr/min (200 g) & 6 mn

5. Biological conditions
LDL Cholesterol
The cholesterol metabolism mainly occurs into osteoblasts.
LDL cholesterol is an oxidant and induces cell apoptosis.
A high level of LDL cholesterol deteriorates the bone health.
LDL cholesterol testing is highly recommanded : it has to be < 1,4 g/L.
Lab prescription : LDL Cholesterol + Cholesterol total
Vitamin D
Vitamin D is a major hormon, necessary to the bone homeostasis and to an efficent
immune response. Many failures and peri-implantitis should be linked to this metabolic
desease.
Lab testing is also recommanded : it has to be > 50ng/mL for a surgery.
Lab prescription : 25 OH - vit D2 + D3
Supplementation : - Starts the day of the pre-op consultation : 2000 UI/day
- Adapt to the patient serum level after lab test results :
If level > 30ng/mL : 2000 UI/day
If level is between 20 & 30ng/mL : 4000 UI/day
If level is between 10 & 20ng/mL : 6000 UI/day
If level < 10ng/mL : 10 000 UI/day
Metronidazole (pure powder)
The use of metronidazole helps to reduce the contamination of the bone graft during
surgery. Metronidazole is the most efficient molecule against anaerobe bacteria. The
powder (pure) is mixed to the biomaterial before addind the i-PRF, A-PRF or PRF
exudate. Only few milligrams are necessary, around 50mg (1 cap).

6. Pressure & tension

Pressure on a tissue leads to a lack of angiogenesis. This essential rule was published
by Mammoto in 2009. All healing failures are linked to a deficit of vascularization.

Tension
Tension free flap closure is highly recommended. However the tension is also
coming from the vestibule (speaking, smilling, coughing, chewing…). With high
tension, the periosteum becomes ischemic and the bone resorbtion becomes
obligatory.
Solutions :
- Decrease the tension of the flap at the maximum.
- Suture with apical mattress : deep U suture in the vestibule.
- Always check the tension at the end of the surgery: pull lips and cheeks
- If tension persist (flap mobilité despite apical mattress) : Incision in the vestibule.

1cm

Vestibule

Apical mattress

Pressure
The pressure is applied by the flap and will induce ischemia of the periosteum.
Solutions :
- Screw tenting
- Apical mattress
- Titanium mesh
- Cortical bone plate

Pressure free implant placement

When placed with a high torque, implants can cause a high pressure on the bone, ,
especially on the cortex, leading to an ischemia of this bone and its resorbtion.
The best is to over-drill the cortical bone on 2mm depth or to perform subcrestal
implant placement, to free this bone from any pressure.

The grafted bone, less elastic, is even more fragile. Any implant placement will
increase the stress applied on the bone. Strongly reduced insertion torque is highly
recommended. Immediate loading should be avoided.

Sutures
When a flap is raised, the re attachment of the periosteum needs ew weeks to be
sufficient. The sutures have to stay least 3-4 weeks. The use of a monofilament is
highly recommended The easiest solution is to use a resorbable monofilament.

7. Pain management
Analgesia
Pain is coming from the inflammation.
In order to reduce inflammation, cortison is the most efficient molecule.
Its biologic hal life is around 3 days.
One dose is enough to reduce the inflammation :
Prednisolone : 1mg/kg de Prednisolone or Dexamethasone : 0,20 mg/kg

Only one dose is sufficient is most of the cases. If necessary, a second dose can be given
after 3 days. Pain killers as Ibuprofen or Paracetamol should be associated.

Anesthesia
To improve a weak anesthesia (local acidity), it’s possible to increase the pH of the
gum with Sodium Bicarbonate 1,4% : 5-10mL injected in the area of the anesthesia,
after the injection of anesthetics.
Allergia
Allergic symptoms are coming from the release of histamine from the macrophages
after allergens exposure.
It’s easy to prevent any allergic reaction by blocking the histamine release before the
surgery : Use of antihistaminic drugs as Hydroxyzine : Atarax®
1 tablet of 25 mg/day/3 days before the surgery, at bedtime.

Sedation
Use of antihistaminics (ex:Hydroxyzine) : Atarax®
About 1mg/kg for an adult, to start 2h before the surgery.
In case of an high anxiety, give the same quantity the night before.
Do not drive.

Saliva
Atropine Sulfate : 0,25 mg : IM, IV, sub-cutaneous or mouth floor
If injected on mouth floor : inject fews drops of anesthetics before because atropine
injection can be painful in this site
 biomedicines
Article
Evidence for Contamination of Silica Microparticles
in Advanced Platelet-Rich Fibrin Matrices Prepared
Using Silica-Coated Plastic Tubes
Tetsuhiro Tsujino 1 , Akira Takahashi 2 , Sadahiro Yamaguchi 3 , Taisuke Watanabe 4 ,
Kazushige Isobe 5 , Yutaka Kitamura 6 , Takaaki Tanaka 7 , Koh Nakata 8 and
Tomoyuki Kawase 9, *
1 Private Practice, Hiroshima 732-0066, Japan; tetsudds@gmail.com
2 Private Practice, Kawasaki 213-0033, Kanagawa, Japan; atakahashihdc@ybb.ne.jp
3 Private Practice, Ohta 373-0808, Japan; y-sada@mwd.biglobe.ne.jp
4 Division of Anatomy and Cell Biology of the Hard Tissue, Institute of Medicine and Dentistry,
Niigata University, Niigata 951-8514, Japan; watatai@mui.biglobe.ne.jp
5 Tokyo Plastic Dental Society, Kita-ku, Tokyo 114-0002, Japan; kaz-iso@tc4.so-net.ne.jp
6 Department of Oral and Maxillofacial Surgery, Matsumoto Dental University, Shiojiri 399-0781, Japan;
shinshu-osic@mbn.nifty.com
7 Department of Materials Science and Technology, Niigata University, Niigata 950-2181, Japan;
tctanaka@eng.niigata-u.ac.jp
8 Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, Niigata 951-8520,
Japan; radical@med.niigata-u.ac.jp
9 Division of Oral Bioengineering, Institute of Medicine and Dentistry, Niigata University, Niigata 951-8514, Japan
* Correspondence: kawase@dent.niigata-u.ac.jp; Tel.: +81-25-262-7559
!"#!$%&'(!
Received: 20 May 2019; Accepted: 18 June 2019; Published: 19 June 2019 !"#$%&'

Abstract: Platelet-rich fibrin (PRF) therapy has been widely applied in regenerative dentistry,
and PRF preparation has been optimized to efficiently form fibrin clots using plain glass tubes.
Currently, a shortage of commercially available glass tubes has forced PRF users to utilize silica-coated
plastic tubes. However, most plastic tubes are approved by regulatory authorities only for diagnostic
use and remain to be approved for PRF therapy. To clarify this issue, we quantified silica microparticles
incorporated into the PRF matrix. Blood samples were collected into three di↵erent brands
of silica-containing plastic tubes and were immediately centrifuged following the protocol for
advanced-PRF (A-PRF). Advanced-PRF-like matrices were examined using a scanning electron
microscope (SEM), and silica microparticles were quantified using a spectrophotometer. Each brand
used silica microparticles of specific size and appearance. Regardless of tube brands and individual
donors, significant, but not accidental, levels of silica microparticles were found to be incorporated
into the A-PRF-like matrix, which will be consequently incorporated into the implantation sites.
Presently, from the increasing data for cytotoxicity of amorphous silica, we cannot exclude the
possibility that such A-PRF-like matrices negatively influence tissue regeneration through induction
of inflammation. Further investigation should be performed to clarify such potential risks.

Keywords: advanced platelet-rich fibrin; amorphous silica; coating; blood collection tube; health hazard

1. Introduction
Similar to platelet-rich plasma (PRP), platelet-rich fibrin (PRF) has been demonstrated to be
e↵ective in tissue repair/regeneration in various fields of regenerative medicine [1–5]. The major
advantages of PRF over PRP are that it does not necessitate superior operator skills, anti-coagulants,

Biomedicines 2019, 7, 45; doi:10.3390/biomedicines7020045 www.mdpi.com/journal/biomedicines

Biomedicines 2019, 7, 45 2 of 11

or coagulation factors, implying that fewer sections of the PRF preparation protocol can be biased.
Thus, the use of standardized blood-collection tubes and centrifuges under the same centrifugal
conditions reproducibly prepare similar-quality PRF from the same individuals at the same time points.
Therefore, PRF has been increasingly used around the world.
However, PRF users have recently faced a serious problem where, except for some brands
specialized for PRF preparation, major medical equipment manufacturers have discontinued the
production of plain glass tubes for blood collection [6]. Therefore, some PRF users have had to purchase
PRF-specific glass tubes, even though conventional, non-approved glass tubes are less expensive
than approved plastic tubes and can be obtained routinely from non-authorized distributors. If such
glass tubes are not available, many PRF users would use silica-coated plastic tubes specific for blood
coagulation, which were originally produced for diagnostic use, without a careful quality check. In fact,
many manufacturers caution PRF users against the use of silica-coated plastic tubes, including plastic
tubes containing silica-coated film, for the preparation of PRF for regenerative therapy because these
products have not been approved for PRF preparation by regulatory agencies, unlike blood-transfusion
bags in Japan and probably all other countries. Nonetheless, medical and dental clinicians in Japan
still have the right to use their discretion while choosing blood collection tubes [6]. This regulatory
matter is discussed in detail in the Discussion section.
Upon contact with the glass surface, coagulation factor XII in the plasma can be activated to
consequently produce a fibrin clot through the activation of the intrinsic coagulation cascade [7].
Since the plastic surface does not have the potential to stimulate coagulation, PRF cannot be prepared in
plastic tubes by the well-known centrifugation-based protocol. We have recently developed a method
using a synthetic collagen-like protein to indirectly stimulate coagulation via activation of platelets and
reproducibly prepare a PRF-like matrix [8]. In addition, in a preclinical animal study, we demonstrated
that the combination of PRF and particles made of the collagen-like protein could be used as a safe and
promising bone substitute in bone regenerative therapy.
However, such hybrid plastic tubes are not yet available commercially, and therefore many PRF
users employ existing products as replacements of the original plain glass tubes. Among the existing
products, the plastic tubes for blood coagulation meet their requirements [9]. Although detailed
specifications seem to vary for individual products, they commonly contain silica microparticles inside
the tubes because of the glass-like property of silica. However, many PRF users rarely pay attention
to the possible health hazards. It has been reported that silica microparticles, depending on their
amorphousness and size, can be harmful to our body [10,11]. As these products are generally used for
diagnostic purposes only, to date, manufacturers have not disclosed the information regarding either
specifications or ease of detachment of silica microparticles.
To evaluate the quality of the PRF prepared using silica-coated plastic tubes, we characterized the
morphology of the silica microparticles and examined their possible contamination in the resulting
PRF matrix.

2. Experimental Section

2.1. Preparation of Advanced-PRF (A-PRF) Matrices

After obtaining individual written informed consent, blood samples were collected, without
anticoagulants, from six non-smoking healthy male volunteers aged 30–60 years. The study design
and consent forms for all procedures (project identification code: 2297) were approved by the ethics
committee for human participants at the Niigata University School of Medicine (Niigata, Japan) on
14 October 2015, in accordance with the Helsinki Declaration of 1964, as revised in 2013.
Fresh blood samples (approximately 9.0 mL) from each donor were collected into two brands
of evacuated silica-coated plastic tubes, Vacuette® serum clot activator tubes (Greiner Bio-One
GmbH, Kremsmünster, Austria) and Neotube® celite tubes (NP-PS0909; NIPRO Corp, Osaka, Japan)
or evacuated tubes containing silica-coated film, Venoject II® clot activator film-containing tubes
Biomedicines 2019, 7, 45 3 of 11

(VP-P100K; TERUMO Corp, Tokyo, Japan) as a variation of silica-coated tubes. The specifications of
these tube brands are summarized in Table 1. Blood was immediately centrifuged at 200⇥ g for 14 min
(A-PRF protocol) using a Duo centrifuge (Process, Nice, France) at ambient temperature (20–22 C).

Table 1. Characteristics of plastic tubes used in this study.

Neotube Vacuette Venoject II

Material of tube plastic (PET) plastic (PET) plastic (PET)
Silica types amorphous not disclosed not disclosed
Object coated inner-wall surface inner-wall surface film
Additives not disclosed not disclosed thrombin

Quality checks were carried out on individual blood samples by performing platelet and other
blood cell counts using a pocH 100iV automated hematology analyzer (Sysmex, Kobe, Japan).

2.2. Detachment of Silica Microparticles from the Inner Walls of Tubes or Films
Disinfectant, 70% ethanol (2 mL) was added to each tube which were then tapped several times
and sonicated for 60 s in an ultrasonic cleaner (Citizen, Tokyo, Japan). The suspension of detached
silica microparticles was transferred into plastic dishes and air-dried on a clean bench.

2.3. Scanning Electron Microscope (SEM) Examination

Dried silica microparticles were attached to carbon tape and directly examined under a scanning
electron microscope (SEM) (TM-1000, Hitachi, Tokyo, Japan) with an accelerating voltage of 15 kV [12,13].

2.4. Spectrophotometric Quantification of Detached Silica Microparticles

Pure water (2 mL) was added to each tube and silica microparticles were detached thoroughly
as described above. Then, the concentrations of silica suspensions (100 µL) were determined using a
spectrophotometer (PiCOSCOPE; Ushio Inc., Tokyo, Japan). In a preliminary study, we confirmed that
KOH did not significantly influence either the silica microparticles or the spectrophotometric determination.

2.5. Enzymatic Degradation of A-PRF Matrices to Release Silica Microparticles

A-PRF clots were gently compressed with a compression device (JMR, Niigata, Japan) [14],
cut into 6–8 pieces, washed with Phosphate Bu↵ered Saline (PBS) three times and degraded in 0.1%
Trypsin + 1.06 mM Ethylenediaminetetraacetic acid (EDTA) (Life Technologies, Carlsbad, CA, USA)
at 37 C overnight until almost complete degradation occurred. Fibrin and cell-derived debris were
further lysed in 1 M KOH for 1 h. The released silica microparticles were washed three times with
pure water to remove colored elements, such as hemoglobin, and resuspended in pure water (2 mL).
The concentrations of the silica microparticles were determined using a spectrophotometer.

2.6. Statistical Analysis

The data were expressed as mean ± standard deviation (SD). For two-group comparisons, statistical
analyses were conducted to compare the mean values by Student t-test (SigmaPlot 12.5; Systat Software,
Inc., San Jose, CA, USA). Di↵erences with p values < 0.05 were considered significant.

3. Results
The macroscopic appearance of the silica-coated plastic tube (Neotube) pre- and post-centrifugation
is shown in Figure 1. Before centrifugation, the tube wall appears homogenously hazy overall but
some spots were dense. In contrast, after centrifugation, the tube wall turned out to be clear.
Biomedicines 2019, 7, 45 4 of 11
Biomedicines 2019, 7, x FOR PEER REVIEW 4 of 11

Figure 1. Macroscopic appearance of the silica-coated plastic tube (Neotube) pre- (a) and
Figure 1. Macroscopic appearance of the silica-coated plastic tube (Neotube) pre- (a) and post-
post-centrifugation (b).
centrifugation (b).

The
The components
components on on the
the hazy
hazy wall
wall of of pre-centrifugation
pre-centrifugation tubestubes were
were examined
examined using
using SEM.
SEM.
Microscopic observations of silica microparticles contained in Neotube, Vacuette and
Microscopic observations of silica microparticles contained in Neotube, Vacuette and Venoject II tubes are Venoject II tubes
are
shownshown in Figure
in Figure 2. After
2. After vigorously
vigorously washing
washing thetheinside
insideofofthe
thetubes
tubeswith
with 70%
70% ethanol,
ethanol, the
the resulting
resulting
suspensions
suspensions were were dried
dried on
on plastic
plastic dishes
dishes andand examined
examined with with SEM.
SEM. According
According to to manufacturers’
manufacturers’
information,
information, the theinner
innerwalls
wallsororthethe plastic
plastic filmfilm
areare coated
coated withwith silica.
silica. However,
However, theirand
their size size and varied
shape shape
varied with individual
with individual tube brands.
tube brands. Silica microparticles
Silica microparticles contained contained in theIIVenoject
in the Venoject II tubes
tubes were were
composed
composed
mainly of mainly of small pebble-like
small pebble-like crushed microparticles,
crushed microparticles, while those while
of those
Neotubeof Neotube were diverse
were diverse and
and larger.
larger.
Neotube Neotube tubes contained
tubes contained pieces ofpieces
brokenofhoneycomb
broken honeycomb
structuresstructures and needle-shape
and needle-shape structures
structures in addition
in
to addition to small pebble-like
small pebble-like microparticles.
microparticles. Vacuette tubes Vacuette
weretubes were composed
composed of both pebble-like,
of both pebble-like, crushed
crushed microparticles and pieces of short cylinder-like structures. In general, microparticles
microparticles and pieces of short cylinder-like structures. In general, microparticles contained in Venoject contained
in Venoject
II tubes wereII the
tubes were the
smallest amongsmallest among
the silica the silica microparticles
microparticles examined. examined.
Biomedicines 2019,
Biomedicines 7, x45FOR PEER REVIEW
2019, 7, 55of
of 11
11

Figure 2. Scanning electron microscopy (SEM) observations of silica microparticles contained in (a) Neotube tubes (b) Vacuette tubes and (c) Venoject II tubes at low
(Upper)2.and
Figure high magnification
Scanning (Lower).(SEM) observations of silica microparticles contained in (a) Neotube tubes (b) Vacuette tubes and (c) Venoject II tubes at
electron microscopy
low (Upper) and high magnification (Lower).
Biomedicines 2019, 7, 45 6 of 11
Biomedicines 2019, 7, x FOR PEER REVIEW 6 of 11

A-PRF-like clots
A-PRF-like clots formed
formedininindividual
individualplastic
plastictubes
tubesafter
after centrifugation
centrifugation andand
thethe balance
balance of
of A-
A-PRF-like matrices and red thrombi are shown in Figure 3. Compared with
PRF-like matrices and red thrombi are shown in Figure 3. Compared with other PRF derivativesother PRF derivatives
prepared by
prepared byhigh-speed
high-speedcentrifugation,
centrifugation,A-PRF
A-PRFclotsclots
prepared by low-speed
prepared centrifugation
by low-speed are usually
centrifugation are
attached with a longer red thrombus than that of high-speed centrifugation.
usually attached with a longer red thrombus than that of high-speed centrifugation. However, when However, when
silica-containing tubes
silica-containing tubes that
that efficiently
efficiently facilitate
facilitate coagulation
coagulation are
are used,
used, the
the red
red thrombus
thrombus became
became longer
longer
and larger
and larger than
than glass
glass tubes.
tubes.

Figure 3. (a) Advanced platelet-rich fibrin (A-PRF)-like clots formed in individual plastic tubes after
Figure 3. (a) Advanced platelet-rich fibrin (A-PRF)-like clots formed in individual plastic tubes after
centrifugation and (b) the balance of the A-PRF-like matrix and red thrombus in 100 mm dishes.
centrifugation and (b) the balance of the A-PRF-like matrix and red thrombus in 100 mm dishes.
SEM observations
SEM observations of
of silica
silica microparticles
microparticles embedded
embedded in in and
and attached
attached to
to individual
individual A-PRF-like
A-PRF-like
matrices are shown in Figure 4. In all the matrices, silica microparticles were found.
matrices are shown in Figure 4. In all the matrices, silica microparticles were found. While silica While silica
microparticles were
microparticles werealmost
almosthomogenously
homogenouslydistributed
distributedin in
thethe
A-PRF matrix
A-PRF prepared
matrix by Neotube,
prepared they
by Neotube,
werewere
they localized in several
localized regions
in several in theincases
regions of Vacuette
the cases and Venoject
of Vacuette II.
and Venoject II.
Biomedicines 2019, 7, 45 7 of 11
Biomedicines 2019, 7, x FOR PEER REVIEW 7 of 11

Figure 4. SEM observations of silica microparticles embedded in and attached to individual A-PRF-like matrices prepared by (a) Neotube, (b) Vacuette and (c) Venoject II.
Figure 4. SEM observations of silica microparticles embedded in and attached to individual A-PRF-like matrices prepared by (a) Neotube, (b) Vacuette and (c)
Venoject II.
Biomedicines 2019, 7, 45 8 of 11

Biomedicines 2019, 7, x FOR PEER REVIEW

The contents and percentages of silica microparticles contained in A-PRF-like matrices8 versus of 11
whole
contents of pre-use tubes are shown in Figure 5. We quantified the contents of silica
The contents and percentages of silica microparticles contained in A-PRF-like matrices versus
microparticles
using
whole acontents
spectrophotometer by modification
of pre-use tubes are shown inofFigure
our previous method that
5. We quantified was applied
the contents to platelet
of silica
counts [15]. In a preliminary study, we confirmed a strong correlation between absorbance
microparticles using a spectrophotometer by modification of our previous method that was applied values and
dilutions of silica suspensions as good standard curves (Figure 5a). Based on this data,
to platelet counts [15]. In a preliminary study, we confirmed a strong correlation between absorbance the percentages
of silicaand
values microparticles entrapped
dilutions of silica and retained
suspensions in A-PRF-like
as good standard curves matrices varied
(Figure 5a). Basedwith individual
on this data, tube
brands. The highest
the percentages recovery
of silica (approximately
microparticles entrapped30.9%) was observed
and retained in the A-PRF-like
in A-PRF-like matrix
matrices varied withprepared
by Neotube.
individual Inbrands.
tube contrast,
Thethe recovery
highest in the
recovery cases of Vacuette
(approximately 30.9%)and
was Venoject II were
observed in 5.2% and 10.6%,
the A-PRF-like
matrix prepared by Neotube. In contrast, the
respectively, and much lower than that of Neotube. recovery in the cases of Vacuette and Venoject II were
5.2% and 10.6%, respectively, and much lower than that of Neotube.

Figure (a)Representative
5. (a)
Figure 5. Representative standard
standard curves
curves for silica
for silica contents
contents and
and (b) (b) contents
contents and percentages
and percentages of of
silica microparticlescontained
silica microparticles containedin in A-PRF-like
A-PRF-like matrices
matrices versus
versus wholewhole contents
contents of pre-use
of pre-use tubes. n tubes.
= 8. n = 8.

4.4. Discussion
Discussion
According
According to tothe
themanufacturers’
manufacturers’ information
information [16],[16], the silica
the silica microparticles
microparticles used for used for coating
coating the the
Neotubes
Neotubes are are celite,
celite,which
whichareare amorphous
amorphous silica.
silica. As for Astheforsilica
the silica microparticles
microparticles used inusedother in other brands,
brands,
we
wespeculate
speculate thatthatamorphous
amorphous silica
silica is probably
is probably used, used,
based based
on the ongeneral
the general
consensusconsensus
regarding regarding
the the
health
health hazards
hazards of ofcrystalline
crystalline silica
silica [11,17]
[11,17] and/or
and/or the material
the material safetysafety dataprovided
data sheet sheet provided by the other
by the other
manufacturers [18,19].However,
manufacturers [18,19]. However, wewe could
could notnot
reach reach a conclusion
a conclusion basedbased
on thison this information.
information.
The specific gravity of amorphous silica (2.15–2.30) is much higherred
The specific gravity of amorphous silica (2.15–2.30) is much higher than than blood
red cells
blood cells
(approximately 1.10); therefore, silica microparticles can be precipitated immediately after
(approximately 1.10); therefore, silica microparticles can be precipitated immediately after centrifugation
centrifugation under citrated conditions [20,21]. However, it is plausible that without anti-coagulants,
under citrated conditions [20,21]. However, it is plausible that without anti-coagulants, coagulation to
coagulation to form a fibrin clot is triggered immediately by contact with silica microparticles and
form a fibrin clot is triggered immediately by contact with silica microparticles and silica microparticles
silica microparticles are entrapped within the fibrin clot. Our findings expectedly supported this
are entrapped
scenario; however, within the fibrin
for many PRF users clot.who Ourdo findings
not expectexpectedly
contamination supported this scenario;
of silica components in thehowever,
for
PRF many
matrix,PRF users
this maywhoprovedotonot beexpect contamination
a surprise. Although the of recovery
silica components
rate may be in influenced
the PRF matrix,by thethis may
prove
particletosize
be aandsurprise. Although
other factors, the recovery rate
silica contamination may be influenced
was observed by the
in all the plastic tubesparticle
tested.size
Theseand other
factors,
findingssilica
showed contamination was observed
that the PRF matrix preparedin byall the plastic tubes
silica-containing tested.
plastic tubesThese findings
undoubtedly showed that
carries
the
thePRF
silicamatrix prepared
microparticles bythe
into silica-containing
implantation sites. plastic
Thesetubes undoubtedly
microparticles cancarries the silica
be released microparticles
like growth
factors as fibrin clots are degraded by plasmin or other non-specific
into the implantation sites. These microparticles can be released like growth factors as endogenous proteases and may clots are
fibrin
influence the surrounding tissues and cells.
degraded by plasmin or other non-specific endogenous proteases and may influence the surrounding
Despite contamination of silica microparticles in the resulting A-PRF-like matrices, the amounts
tissues and cells.
varied with tube brands. The surface area of the silica coating film is substantially smaller than those
Despite contamination of silica microparticles in the resulting A-PRF-like matrices, the amounts
on the inner wall of the tube. Thus, it is obvious that the mass of the silica microparticles in Venoject
varied with tube brands. The surface area of the silica coating film is substantially smaller than those
II tubes is much lower than that in other tube brands (Vacuette and Neotube) and consequently the
on
mass of silicawall
the inner of the tube.
microparticles Thus, in
included it is obvious that
A-PRF-like matrixtheinmass of the
Venoject silica must
II tubes microparticles
be lower than in Venoject
IIthose
tubes is much lower than that in other tube brands (Vacuette and Neotube)
of other brand tubes. In addition to the mass of silica microparticles, the silica entrapment and and consequently the
mass of silica
retention microparticles
processes are primarily included
thoughtin to A-PRF-like
be influencedmatrixby other in factors,
Venoject II tubes
such must beand
as appearance lower than
those
the sizeofof
other brand tubes. Furthermore,
the microparticles. In addition to thefactors
these mass of may silica microparticles,
influence the recoverythe silicaextraction
during entrapment and
retention processes are primarily thought to be influenced by other factors, such as appearance and the
size of the microparticles. Furthermore, these factors may influence the recovery during extraction and
Biomedicines 2019, 7, 45 9 of 11

light transparency during spectrophotometric determination. It is plausible that as the microparticles

become smaller, the entrapment and retention become more difficult. Therefore, we think that the
reduction in recovery may not be necessarily related to or caused by the reduction in entrapment
and/or retention.
On the other hand, even though amorphous silica is less toxic than crystalline silica and is actually
used in a variety of products, including food and toothpaste, recent investigations have raised the
possibility that amorphous silica can cause inflammation or cytotoxicity [22–26]. It is further described
that particle size, which is usually correlated to the surface area, is more important than dose in
exerting such negative e↵ects. Since the cytotoxic and inflammatory e↵ects of amorphous silica are
thought to be mainly due to the reactive oxygen generated on the silica surface, a larger surface area
per given mass is more reactive and has a higher potential to generate reactive oxygen species [22,23].
In addition, as described in the case of the complex interaction of crystalline silica [10], at present,
we cannot exclude the possible interactions of amorphous silica at non-toxic levels with unidentified
factors to exert unexpected health hazards.
In Japan, a new regulatory framework for regenerative medicine, including PRP/PRF therapy,
was established in 2014, and individual hospitals and clinics are required to pass inspection by the
regulatory agencies prior to treatment of their patients with PRP/PRF [6]. Nonetheless, medical and
dental clinicians in Japan still have the right to choose the blood-collection tubes on the basis of their
discretion for the treatment of patients [6]. Thus, the use of silica-coated plastic tubes is not prohibited
for PRF preparation. Even though individual countries have individual regulatory frameworks for
medical treatment, similar situations regarding PRF therapy have been found in several other countries.
However, from a medical but not commercial point of view, we need to survey those individual
situations at the global level and propose universal standards of medical devices, besides preparation
of protocols, for patient-oriented PRF therapy through international collaboration.
The major advantages of PRF are low cost and high safety. Introducing possible silica hazards to
this balance seems risky and may hamper the advances in PRF therapy. O’Connell forthrightly raised
his concern over its use in PRF preparation in his article [27] and his concerns about safety have not yet
been eradicated. To date, many e↵orts have been made to clarify the hazards of amorphous silica at
cellular and molecular levels [10,23,24,26]. Until the safety of amorphous silica for implantation use is
assured by international or national regulatory authorities, we should only use the conventional types
of plain glass tubes for PRF preparation for the benefit of patients.

5. Conclusions
The most appropriate therapeutic methods are generally selected based on the balance between
risk and benefit. PRF therapy alone is superior to other therapies using medicines or bone substitutes
in terms of cost and safety. Thus, it can be confirmed that PRF therapy is advantageous based on
the balance between low risk and low benefit. Until the possible health hazards of amorphous silica
microparticles contaminated in PRF matrices cannot be ruled out by strong evidence, PRF therapy using
silica-containing tubes has no convincing advantages over other costly therapies and only increases
the risk of complications.

Author Contributions: Conceptualization, T.T. (Tetsuhiro Tsujino) and T.K.; methodology, T.T. (Tetsuhiro Tsujino),
T.T. (Takaaki Tanaka) and T.K.; validation, T.T. (Tetsuhiro Tsujino), A.T. and T.K.; formal analysis, T.T. (Takaaki
Tanaka); investigation, T.T. (Tetsuhiro Tsujino), A.T., S.Y., T.W., K.I., Y.K., T.T. (Takaaki Tanaka) and T.K.; data
curation, K.N.; writing—original draft preparation, T.T. (Tetsuhiro Tsujino) and T.K.; writing—review and
editing, T.T. (Tetsuhiro Tsujino), K.I., T.T. (Takaaki Tanaka) and T.K.; visualization, T.K.; supervision, K.N.; project
administration, T.W. and T.K.; funding acquisition, T.K.
Funding: This study was financially supported in part by Japan Society for the Promotion of Science Grants-in-Aid
for Scientific Research (JSPS KAKENHI, Grant Number 18K09595).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Biomedicines 2019, 7, 45 10 of 11

References
1. Barbon, S.; Stocco, E.; Macchi, V.; Contran, M.; Grandi, F.; Borean, A.; Parnigotto, P.P.; Porzionato, A.;
De Caro, R. Platelet-Rich Fibrin Sca↵olds for Cartilage and Tendon Regenerative Medicine: From Bench to
Bedside. Int. J. Mol. Sci. 2019, 20, 1701. [CrossRef] [PubMed]
2. Kawase, T. Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine:
basic principles and concepts underlying recent advances. Odontology 2015, 103, 126–135. [CrossRef]
[PubMed]
3. Miron, R.J.; Fujioka-Kobayashi, M.; Bishara, M.; Zhang, Y.; Hernandez, M.; Choukroun, J. Platelet-Rich Fibrin
and Soft Tissue Wound Healing: A Systematic Review. Tissue Eng. Part B Rev. 2017, 23, 83–99. [CrossRef]
[PubMed]
4. Yu, P.; Zhai, Z.; Jin, X.; Yang, X.; Qi, Z. Clinical Application of Platelet-Rich Fibrin in Plastic and Reconstructive
Surgery: A Systematic Review. Aesthet. Plast. Surg. 2018, 42, 511–519. [CrossRef] [PubMed]
5. Zumstein, M.A.; Berger, S.; Schober, M.; Boileau, P.; Ny↵eler, R.W.; Horn, M.; Dahinden, C.A. Leukocyte- and
platelet-rich fibrin (L-PRF) for long-term delivery of growth factor in rotator cu↵ repair: Review, preliminary
results and future directions. Curr. Pharm. Biotechnol. 2012, 13, 1196–1206. [CrossRef] [PubMed]
6. Kawase, T.; Okuda, K. Comprehensive Quality Control of the Regenerative Therapy Using Platelet
Concentrates: The Current Situation and Prospects in Japan. BioMed Res. Int. 2018, 2018, 6389157.
[CrossRef] [PubMed]
7. Margolis, J. Glass surface and blood coagulation. Nature 1956, 178, 805–806. [CrossRef] [PubMed]
8. Tsukioka, T.; Hiratsuka, T.; Nakamura, M.; Watanabe, T.; Kitamura, Y.; Isobe, K.; Okudera, T.; Okudera, H.;
Azuma, A.; Uematsu, K.; et al. An on-site preparable, novel bone-grafting complex consisting of human
platelet-rich fibrin and porous particles made of a recombinant collagen-like protein. J. Biomed. Mater. Res.
Part B Appl. Biomater. 2018, 107B, 1420–1430. [CrossRef]
9. Dohan Ehrenfest, D.M.; Del Corso, M.; Diss, A.; Mouhyi, J.; Charrier, J.B. Three-dimensional architecture and
cell composition of a Choukroun’s platelet-rich fibrin clot and membrane. J. Periodontol. 2010, 81, 546–555.
[CrossRef]
10. Brown, T. Silica exposure, smoking, silicosis and lung cancer—Complex interactions. Occup. Med.
(Oxford, England) 2009, 59, 89–95. [CrossRef]
11. Steenland, K.; Ward, E. Silica: A lung carcinogen. CA Cancer J. Clin. 2014, 64, 63–69. [CrossRef] [PubMed]
12. Isobe, K.; Suzuki, M.; Watanabe, T.; Kitamura, Y.; Suzuki, T.; Kawabata, H.; Nakamura, M.; Okudera, T.;
Okudera, H.; Uematsu, K.; et al. Platelet-rich fibrin prepared from stored whole-blood samples. Int. J.
Implant Dent. 2017, 3, 6. [CrossRef] [PubMed]
13. Kawabata, H.; Isobe, K.; Watanabe, T.; Okudera, T.; Nakamura, M.; Suzuki, M.; Ryu, J.; Kitamura, Y.;
Okudera, H.; Okuda, K.; et al. Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole
Blood Samples after Extended Storage. Biomedicines 2017, 5, 57. [CrossRef] [PubMed]
14. Kobayashi, M.; Kawase, T.; Horimizu, M.; Okuda, K.; Wol↵, L.F.; Yoshie, H. A proposed protocol for the
standardized preparation of PRF membranes for clinical use. Biologicals 2012, 40, 323–329. [CrossRef]
[PubMed]
15. Kitamura, Y.; Suzuki, M.; Tsukioka, T.; Isobe, K.; Tsujino, T.; Watanabe, T.; Watanabe, T.; Okudera, H.;
Nakata, K.; Tanaka, T.; et al. Spectrophotometric determination of platelet counts in platelet-rich plasma.
Int. J. Implant Dent. 2018, 4, 29. [CrossRef]
16. NIPRO Information for Medical Personnel. Available online: http://med.nipro.co.jp/med_eq_category_
detail?id=a1U1000000b1000533AEAQ (accessed on 13 May 2019).
17. Madl, A.K.; Donovan, E.P.; Ga↵ney, S.H.; McKinley, M.A.; Moody, E.C.; Henshaw, J.L.; Paustenbach, D.J.
State-of-the-science review of the occupational health hazards of crystalline silica in abrasive blasting
operations and related requirements for respiratory protection. J. Toxicol. Environ. Health Part B Crit. Rev.
2008, 11, 548–608. [CrossRef] [PubMed]
18. Becton Dickinson Vacutainer Brand SST Tubes Containing Silica and Gel, Material Safety Data Sheet.
Available online: http://catalog.bd.com/ecat/msds/d01/vs60313.pdf (accessed on 13 May 2019).
19. Mannello, F.; Tanus-Santos, J.E.; Meschiari, C.A.; Tonti, G.A. Differences in both matrix metalloproteinase
9 concentration and zymographic profile between plasma and serum with clot activators are due to the presence
of amorphous silica or silicate salts in blood collection devices. Anal. Biochem. 2008, 374, 56–63. [CrossRef]
Biomedicines 2019, 7, 45 11 of 11

20. Cement Concrete & Aggregates Australia. Amorphous Silica Properties, Characterisation and Uses.
Available online: https://www.ccaa.com.au/imis_prod/documents/TECH_NOTE_79_-_Amorphous_Silica.
pdf (accessed on 12 May 2019).
21. Ashworth, C.T.; Adams, G. Blood specific gravity studies: Relationship of specific gravity of whole
blood to specific gravity of plasma, red blood cell count, hematocrit, and hemoglobin as indicators of
hemoconcentration. J. Lab. Clin. Med. 1941, 26, 1934–1939.
22. Barnes, C.A.; Elsaesser, A.; Arkusz, J.; Smok, A.; Palus, J.; Lesniak, A.; Salvati, A.; Hanrahan, J.P.; Jong, W.H.;
Dziubaltowska, E.; et al. Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity.
Nano Lett. 2008, 8, 3069–3074. [CrossRef]
23. Kaewamatawong, T.; Kawamura, N.; Okajima, M.; Sawada, M.; Morita, T.; Shimada, A. Acute pulmonary
toxicity caused by exposure to colloidal silica: particle size dependent pathological changes in mice.
Toxicol. Pathol. 2005, 33, 743–749. [CrossRef]
24. Kusaka, T.; Nakayama, M.; Nakamura, K.; Ishimiya, M.; Furusawa, E.; Ogasawara, K. E↵ect of silica particle
size on macrophage inflammatory responses. PLoS ONE 2014, 9, e92634. [CrossRef] [PubMed]
25. Merget, R.; Bauer, T.; Kupper, H.U.; Philippou, S.; Bauer, H.D.; Breitstadt, R.; Bruening, T. Health hazards
due to the inhalation of amorphous silica. Arch. Toxicol. 2002, 75, 625–634. [CrossRef] [PubMed]
26. Sandberg, W.J.; Lag, M.; Holme, J.A.; Friede, B.; Gualtieri, M.; Kruszewski, M.; Schwarze, P.E.; Skuland, T.;
Refsnes, M. Comparison of non-crystalline silica nanoparticles in IL-1beta release from macrophages.
Part. Fibre Toxicol. 2012, 9, 32. [CrossRef] [PubMed]
27. O’Connell, S.M. Safety issues associated with platelet-rich fibrin method. Oral Surg. Oral Med. Oral Pathol.
Oral Radiol. Endod. 2007, 103, 587. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).