DR FARHAN RAZA KHAN (Orcid ID : 0000-0002-5650-6268)

Article type : Comprehensive Review

Accepted Article
Which is the most recommended medium for the storage and transport of
avulsed teeth? A systematic review

Dr Samira Adnan
BDS, FCPS
Assistant Professor, Operative Dentistry
Jinnah Sindh Medical University, Karachi
nnmst@hotmail.com

Dr Maham Muneeb Lone

BDS, FCPS
Lecturer, Operative Dentistry
Jinnah Sindh Medical University, Karachi
maham.mlone@gmail.com

Dr Farhan Raza Khan

BDS, MS, MCPS, FCPS
Associate Professor, Operative Dentistry
Aga Khan University Hospital, Karachi
farhan.raza@aku.edu

Dr Syeda Mahvash Hussain

BDS
mahvashh@gmail.com

Dr Sana Ehsan
BDS, FCPS (Operative Dentistry)
dr_ sanas@hotmail.com

Corresponding author:
Dr Farhan Raza Khan
Associate Professor & Head of Dentistry
Aga Khan University Hospital, Karachi
farhan.raza@aku.edu
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/edt.12382
This article is protected by copyright. All rights reserved.
 Running title: Recommended storage medium for avulsed teeth

Conflict of Interests: The authors (SA, MML, FRK, SMH, & SEN) confirm that there are no
Accepted Article
competing interests regarding this publication.

Authors' Contribution: SA did the literature search and drafted the manuscript, MML did data
collection. FRK contributed in the discussion, assessment of biases in the primary studies and
critically reviewed the manuscript. SMH and SE helped with initial data compilation and review of
studies. All authors have contributed substantially to the content of the paper. They have read and
approved the final manuscript.

ABSTRACT

Background/Aims: A wide variety of materials have been researched for their use as potential
storage media for avulsed teeth, but it is essential to recognize the medium most recommended for
improvement of the prognosis of avulsed teeth. The aim of this systematic review was to identify the
most recommended medium to store and transport avulsed teeth based on the survival of
periodontal ligament (PDL) cells as determined by in-vitro studies.

Methods: Only laboratory based experimental studies on PDL cells found on adult permanent teeth
were included. Data was collected using PubMed, CINAHL plus (EBSCO host) and the Cochrane
Library, along with Google Scholar and a hand search. The key terms employed were permutations
of [avulsed permanent teeth* OR dental avulsion* OR knocked out teeth*] AND [storage media* OR
transport media* OR biological transport* OR PDL cell viability* OR PDL cell survival*]. A customized
data extraction proforma was used to extract the data and to evaluate the quality and risk of bias.

Results: The initial search yielded 978 articles, but only 67 were selected. Milk was the most
recommended individual medium followed by Hank’s balanced salt solution. Among natural
products other than milk, propolis and coconut water were most frequently recommended.
Recommendations were based on maintenance of PDL cell viability followed by ease of availability,
low cost and long shelf-life.

Conclusions: Natural products are more effective in maintaining the PDL cell viability compared to
synthetic products. Some storage media recommendations were also based upon practical aspects.
Although natural products other than milk have more recommendations as a group, milk is the most
recommended storage medium individually, based not only on PDL cell viability, but also practical
considerations.

Key words: Tooth avulsion; storage media; transport media; periodontal ligaments; cell viability

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 INTRODUCTION

Tooth avulsion is defined as the complete loss of a tooth from the alveolar socket as a result of
trauma,1 which can be caused by a fall, road traffic accident, assault, sports or occupational injury.2,3
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This form of dental trauma comprises 1-11% of all traumatic injuries to the permanent dentition.4
Maxillary central incisors are the most frequently involved teeth with children aged 7-10 years
commonly affected. Avulsion can be diagnosed clinically and radiographically by the complete
absence of the tooth from its socket. An avulsed tooth experiences severed vascular and nerve
supply resulting in pulp death, especially in a mature permanent tooth with a closed apex.4-6 Avulsion
also causes tearing of the periodontal ligament as the tooth is completely displaced from the socket,
resulting in damage to the periodontal ligament (PDL) cells. These cells form part of the attachment
apparatus of the tooth and hence, subsequent successful replantation of the avulsed tooth into its
socket is greatly dependent on the presence of viable PDL cells on the root surface.7

If an avulsed tooth is retrieved from the site of the accident, there are two possibilities in terms of
management. The ideal situation involves meticulous handling of the avulsed tooth by the patient or
an attendant (in case of a minor patient) and immediate replantation into the socket. This approach
prevents any further damage to the PDL cells and tissue repair is initiated immediately. The second
scenario, much more frequently encountered, is the tooth not being immediately replanted, but
rather brought to the dentist for replantation.8 In the latter course of management, the avulsed
tooth must not be allowed to dry and should be placed in a storage or transport medium as soon as
possible, until it can be replanted.9 This step is crucial in determining the subsequent outcome of
replantation. An appropriate storage medium ensures the viability of the PDL cells present on the
root surface of the avulsed teeth. Over the years, the composition, formulation, temperature, pH
and other aspects of many potential storage media have been the focus of research.10 A wide variety
of materials have been researched for their role as potential transport media11 in the quest for
finding the ideal medium which could maintain the viability of the PDL cells attached to the root
surface of the avulsed tooth. Without a suitable storage medium, the PDL cells are likely to
experience further damage, resulting in poor prognosis for the replanted tooth.5 Therefore, it is
essential that the choice of storage medium for avulsed teeth is based on scientific observations.

The aim of the review was to identify the most recommended storage medium that can be utilized
for storage and transport of avulsed teeth, based on PDL cell viability as determined by in-vitro
studies.

MATERIALS AND METHODS

The PICO question was: What is the most recommended medium to store and transport avulsed
teeth?

The following PICO model for systematic reviews was applied:

Population: Human PDL cells (Isolated from avulsed permanent teeth)

Intervention: Placement of PDL cells of an adult avulsed tooth in the storage medium

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 Comparison: Placement in no storage medium, a different storage medium, or the same storage
medium with variability in composition, concentration or temperature, serving as its own
comparator.
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Outcome: Survival of PDL cells in terms of viability, increase in numbers, no change in morphology or
any other positive outcome as determined by the laboratory parameters.

The protocol for the present review was registered with Prospero, (an international prospective
register for systematic reviews with registration ID: CRD42013003558). The literature search was
conducted using three major health sciences databases, PubMed (NLM), CINAHL Plus (EBSCO host)
and the Cochrane Library including publications from 1970 till March 2017. The following key terms
and their different permutations were used: [avulsed permanent teeth* OR dental avulsion*OR
knocked out teeth*] AND storage media* OR transport media* OR biological transport* OR PDL cell
viability* OR PDL cell survival*. An additional search using Google Scholar was done along with a
hand search. Grey literature was not explored. The flow chart depicting the study selection
methodology is shown as Figure 1. The selection of the studies was completed according to the
inclusion and exclusion criteria as stated in Table 1.

The studies that fulfilled the basic inclusion criteria were assessed independently by four reviewers
for their suitability. A customized data extraction form was developed to collect data from the
included studies. Any titles that were duplicated or not pertaining to the research questions were
excluded at this point, with reasons for exclusion identified on the form. The study was included if it
met the initial criteria based on three key points; a) Use of PDL cells, b) Use of some form of
transport/storage medium, and c) the description of the outcome in terms of PDL cell survival. If the
study failed to meet any one of the criteria, it was excluded from the review.

In order to assess the quality of the included studies, each item in the data extraction form was
assigned a specific value, according to the amount of information that could be obtained from the
individual study. These scores were then added and the quality of the paper was graded as fair, good
or excellent according to the maximum score obtained. Extraction of data from the selected studies
and their quality assessment was performed by four independent reviewers (SA, MML, SMH & SEN).
Each reviewer was also required to assess the data extraction form completed by other reviewers.
Any discrepancy that arose during the data extraction or when assessing the quality of the research
paper was resolved through discussion with a senior colleague (FRK) and mutual consensus
developed. In the same manner, any risk of bias in the included study was also determined. The
details of the studies included in the present systematic review are presented in Table 2.

Data analysis was performed to report the storage medium recommended by the individual studies
in terms of survival of the PDL cells (as determined under laboratory conditions using various
methods of assessment). The reviewers were not blinded regarding the authors, journals, date of
publication, or results.

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 RESULTS A total of 978 references were initially identified from the computerized search but 843
were excluded after screening the titles. A further 68 studies which included case reports and
assessment of clinical survival of teeth were excluded after further appraisal. Sixty seven studies that
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assessed the PDL cells survival were finally included and analyzed. The full text was retrieved for 63
studies, while only abstracts were available for the remaining four studies.

For the purpose of data extraction and ease of analysis, the numerous storage media identified in
the studies were divided into broad groups according to their formulation and composition. These
groups included: Saliva and its variants, Milk and derivatives, Hank’s Balanced Salt solution and its
variants, Saline and its variants, other specialized media and Natural products other than milk. The
list of these media along with the number of studies recommending each is shown in Table 3.

The results suggest that from 1976 till 2000, most studies recommend ‘milk or its derivatives’ as the
most suitable storage medium for avulsed teeth whereas from 2001 onwards, more
recommendations are for ‘natural products other than milk’. There is a plethora of materials
included in these two broad varieties of media. The different milk products that have been
mentioned in the included studies are whole milk, skimmed milk, milk with varying fat content, baby
formula, long shelf life milk, goat milk, pro-biotic milk and chilled milk. The natural products other
than milk mentioned are saliva, soymilk, saliva officinalis, coconut water, probiotic (lactobacillus
reuteri) solution, propolis, egg white, green tea extract, aloe vera, royal jelly, pomegranate juice,
dragon’s blood sap, rice water and cranberry juice.

Overall, 22 studies exclusively recommended natural products as storage media,12-33 while two
studies recommended them in addition to Hank’s balanced salt solution (HBSS)34 and Ringer’s
lactate.35 A further three studies also recommend milk and HBSS along with different natural
products.36-38 Twenty one studies recommended milk in some form in addition to the other media
tested,36-56 while , 13 of these studies exclusively recommended milk.39,42,45-47,49-56 HBSS was found to
be the second most recommended individual medium in 16 studies.34,36-38,43,48,57-66 Out of these, four
studies57,63,65,66 exclusively recommended HBSS, while 12 studies recommended HBSS along with
other media.34,36-38,43,48,58-62,64 Specialized media was also recommended in some studies,67-73 while
saline and its variants were specifically recommended in four studies.74-77 There was only one
recommendation for saliva.78 The results are summarized in Table 3. Overall, around one-third of the
studies recommended multiple storage media rather than a single one.

The most common reason for recommendation was based on the ability of the medium to maintain
PDL cell viability, while a small number of recommendations were based on ease of availability, low
cost and long shelf-life of the storage medium. Only a few media were recommended on the basis of
actually enhancing the viability of PDL cells.23,25,26,33,70 The methods of assessing PDL cell viability was
also evaluated and Trypan Blue staining was found to be the most frequently utilized laboratory
technique to determine viable PDL cells (Figure 2).

In terms of the quality of included studies, 65 of the 67 studies were found to be in the ‘excellent’
category, as these studies encompassed all the relevant information pertaining to the study
question. The risk of bias in studies was also assessed, and is depicted in Figure 3. In regards to
randomization, there was no mention of random sequence generation in any study; while 33% of
the studies mentioned random allocation of samples.12,15,16,18,19,21,23,24,27,28,32,34,53,54,58,62,63,64,71,72,74,75
Where only abstracts could be retrieved, the risk of selective reporting was unclear. 39,40,43,44,67

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 However, no such bias could be detected in the full text articles. As the included studies were
laboratory experiments. All the parameters for risk assessments of clinical trials could not be applied
to them.
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DISCUSSION

Although there are numerous studies that have reviewed and compared media for the storage and
transfer of avulsed teeth,10,1s1,79 no systematic review determining the most recommended storage
and transport media for avulsed teeth has been reported in literature previously. The present
systematic review compared the various storage media in terms of their ability to maintain PDL cell
viability, and in some cases, to even enhance their growth. To determine the actual physical state of
the PDL cells after they had been placed in different storage media, only laboratory based studies
were selected. These studies had a relatively more stringent protocol and their outcome
measurements was relatively straight forward to determine, in comparison to clinical studies, in
which evaluation of the survival of PDL cells is based on a long follow-up period and variable
interpretation of clinical and radiographic findings in patients.

The present systematic review demonstrated that the most recommended transport medium for
avulsed teeth is milk. Milk has a unique combination of nutrients, capable of maintaining PDL cell
viability, and with its physiological pH of 6.5-7.2, it can be considered as the best storage medium in
most situations.80 The PDL cells have been shown to survive for 2-6 hours when immersed in milk.81
Chilled milk or milk with a lower fat content is considered a better choice for storing avulsed teeth,
but there are some impediments in using milk as a storage medium. The antigens present in milk
could interfere with the process of reattachment of PDL cells when the tooth is replanted.7
Moreover, the milk needs to be fresh and refrigerated.20 Sour milk should not be used as it harmful
to the PDL cells.82 Recent studies26,28,30,32 have favored other natural media (other than milk) for
storage of avulsed teeth, , but the low cost, presence of nutrients and ease of availability make milk
a more practical choice than any other medium.

HBSS is a specially designed storage medium containing essential nutrients.80 Although, it is not
easily available in most parts of the world, it is marketed in some countries as ‘Save-a-tooth’ [Save-
A-Tooth, PA, USA]. It has shown to maintain PDL cell viability and has demonstrated clonogenic and
mitogenic capacity of the PDL fibroblast, with replenishment of the lost metabolites. This ability has
been documented to be sustained for up to 48 hours. It has been recommended that the avulsed
tooth be placed in HBSS for 30 minutes before replantation into the socket, regardless of which
storage medium the tooth was placed in prior to this procedure.83,84

Among the natural products other than milk, six studies recommended propolis,12,13,27,32-34 and five
studies proposed coconut water as storage media for transporting avulsed teeth.15,16,18,26,35 There
were four recommendations for green tea extract.17,23,30,35 The rest of the media from this category
were recommended in either only one or two studies. Although natural products other than milk
had the largest number of studies recommending them as a group, when the single most
recommended medium was explored, the number of recommendations for any specific medium was

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 greatly diluted. No consensus was found for on a storage medium as being the best or ideal.
Contrary to the fact that saliva is biological and easy available, saliva as a storage or transport
medium for avulsed teeth was only recommended in one study, which recommended placing the
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avulsed tooth in saliva for not more than 30 minutes.78 This is because the presence of enzymes and
bacteria present in saliva exert harmful effects on the PDL cells.39

Propolis is a natural product with anti-inflammatory properties and it is also known to maintain or
enhance the PDL cell viability. Various formulations of propolis have been studied.12,13,32,33 However,
no consideration during its recommendation has been given to the ease of its availability or the cost.

The recommendation for coconut water is based on the maintenance of PDL cell viability, cost
effectiveness and ease of availability. Coconut water may be easily accessible in some parts of the
world,56 and this would make it a viable storage medium in case of an avulsion for some geographic
locations. However, it cannot be recommended universally due to the practical limitations. Even if
ease of availability is considered, the primary factor in recommending a storage medium, this may
not hold true universally for every part of the world. This holds true for milk also, where some
countries may not have pasteurized milk readily available. Therefore, a practical approach would be
to identify a list of recommended media that can be used in a variety of locations and situations.

There was a wide variety of methods in the studies included in the present systematic review that
were utilized for the assessment of these PDL cells. Therefore, it was difficult to directly compare
studies with one another. The most common method reported for assessing the viability of the PDL
cells was the Trypan blue exclusion or staining test. Trypan blue solution in the strength of 0.4%, is
routinely used as a cell stain to assess cell viability using the dye exclusion test. This test is based on
the concept that viable cells do not take dye, and it is only taken up by dead cells which become
permeable.18 It is considered one of the most accurate methods to detect the remaining viable cells.
The cells can also be counted using hemocytometer in the same instance. A disadvantage of using
Trypan blue is that the dye is cytotoxic to some degree, and also stains the background which can
result in inaccuracy when counting the cells.53 Another method that was used to determine PDL cell
viability was MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) which is
basically a colorimetric test determining the metabolic activity of cells. This tetrazolium-based assay
is useful because of its rapid results, objectivity, and ease of manipulation as well as immediate
identification of viable PDL cells.85 The results of MTT assay are comparable to the histological
examination of the PDL cells in vitro.86

The present systematic review attempted to answer the question of the most recommended storage
medium using in-vitro studies. Parameters indicating a good prognosis for the replanted avulsed
tooth such as the lack of replacement resorption, external invasive resorption, external cervical
resorption, pain and swelling can only be assessed clinically and over long follow-up periods.
Therefore, the clinical application of the investigated storage media can only be established after
their use in clinical trials and/or field situations. It is difficult to conduct clinical trials evaluating the
storage media most suited to storing avulsed teeth as avulsion occurs without warning, and the
traumatized patient may or may not be able to recover the avulsed tooth from the site of the
accident, to bring to the clinic to be replanted.87 Also, such time may be extremely variable. Hence to
standardize the inclusion criteria dictated by the dry time of the PDL cells would be very difficult.
Thus, identification of a the most recommended storage medium for avulsed teeth in the context of

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 clinical success/ survival warrants a separate systematic review, focusing exclusively on the clinical
trials and/ or case reports where clinical parameters for the survival of a replanted avulsed tooth
(after being kept in storage/ transport media) would be analyzed. This could be considered the
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primary shortcoming of the present review, in that no studies assessing the clinical survival of teeth
were included. This was mainly because the parameters of assessment of clinical success were
different to those of laboratory based studies, and this would make the interpretation of data
meaningless. Moreover, the majority of the studies in the present review did not mention the
methods adopted by their authors to eliminate biases such as random allocation or blinding (Figure
3). The inferences of the investigation would be more expressive had the methods to control such
biases been applied.

Ideally, the most recommended storage medium should be able to not only maintain PDL cell
viability but also enhance it. However, this enhancement of PDL cell viability was only observed in a
few studies.23,25,26,33,41,70,72 The actual number of cells that remained viable after being placed in the
storage medium could not be compared in the selected studies because of the great variability in the
methodology of studies. This technical heterogeneity made a generalized assumption next to
impossible. The ‘best’ medium encompassing all the ideal characteristics may yet to be identified,
but this systematic review has managed to identify the most recommended storage media that can
be used to store or transport avulsed teeth.

CONCLUSIONS
Compared to synthetic products, natural products may be more effective in maintaining the PDL cell
viability. Due to the wide variety of storage media assessed using variable methodologies; the
included studies could not be compared in all aspects for the qualities of the storage media.
However, the literature suggests milk as the most viable option in terms of PDL cell viability, ease of
availability and cost effectiveness followed by HBSS. Among the natural products other than milk,
propolis and coconut water were frequently recommended.

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Accepted Article
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Legends to Tables

Table 1: Criteria for inclusion, exclusion, intervention recorded and features of PDL cells assessed

Table 2: Included studies, transport media assessed, method of PDL viability assessment and the
recommended medium

Table 3: Recommendations for storage media groups as reported in the included studies

Legends to Figures

Figure 1: Study flow diagram

Figure 2: Laboratory methods used to assess the periodontal ligament cell survival (n=67)

Figure 3: Overall risk of bias for the included studies

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 Table 1: Criteria for inclusion, exclusion, intervention recorded and features of PDL cells
assessed
Accepted Article
Inclusion criteria: Laboratory based experimental studies on human adult
PDL cells

Exclusion criteria: Studies on primary teeth or fractured teeth

Animal based studies

Publications in any other language besides English

Case reports, literature reviews

Studies assessing clinical survival of teeth

Details of intervention Number of storage media included in selected study

Type of storage media

Time for which PDL cells placed in storage medium

Temperature at which PDL cells stored in storage medium

Method of assessment

Recommended medium and reason for recommendation

Features of PDL cells Viability (maintained, enhanced)

assessed:
Mitotic ability

Expression of growth factors

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 Table 2: Included studies, transport media assessed, method of PDL viability assessment and the
recommended medium
Accepted Article
Method of Recommende
Author(s), ref # Media assessed
assessment d media

Kristerson et Tissue culture media, Liquid nitrogen Not mentioned Tissue culture
al.67 media, Liquid
nitrogen

Blomlof et al. 44 Milk, Saliva, EMEM Trypan blue Whole milk

staining/ exclusion

Blomlof et al. 39 Saliva, Milk, Tap water, Saline 3-H urdine Milk, Saline
leakage test

Patil et al.40 Milk, Saline Number counted Milk, Saline

by
hemocytometer

Alacam et al.58 HBSS, Custodial solution, Sterile saline LDH HBSS,

measurements Custodial
solution

Olson et al.41 Milk, Gatorade, Save-a-Tooth, Save-a- MTS assay Milk, Save-A-
Tooth + PDGF, EMEM Tooth with
PGDF

Harkacz et al.42 Tap water, Saliva, Skimmed milk (Oak Bio-rad model Skim milk
farm's brand), 2% Milk, Whole milk, 0.5 2550 EIA plate (Oaks farm’s
% Milk, 1% Milk, Gatorade reader brand)

Lekic et al.78 Saliva, Milk, HBSS, α-MEM Clonogenic Saliva

capacity

Ashkenazi et Culture media, α-MEM, Milk, HBSS, Trypan blue HBSS, Milk
al.43 Viaspan, Conditioned medium staining/ exclusion

Marino et al.45 Regular pasteurized milk, Long shelf life Cell proliferation Long shelf life
milk, Save-A-Tooth, MEM, Tap water assay milk

Ashkenazi et HBSS, Culture medium, α-MEM, Viaspan Trypan blue HBSS, Culture
al.59 staining/ exclusion media

Ashkenazi et Viaspan, HBSS, α-MEM, α-MEM with Trypan blue HBSS, alpha
al.60 15% Fetal Calf serum, Antibiotics staining/exclusion MEM
(Penicillin 100 unit/ ml, Gentamicin 50
µg/ ml, Fungizone 0.3 µg/ml)

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 Pearson et al.46 Whole milk, Reconstituted powdered Cell proliferation Enfamil (baby
milk, Evaporated milk, Similac, Enafamil assay formula milk)

Vajrabhaya et HBSS, Conditioned medium Staining with Culture media

Accepted Article
al.68 neutral red dye,
observed with
spectro-
photometer

Sigalas et al.61 Culture medium, Milk, HBSS, Soft Wear Trypan blue HBSS, Culture
contact lens solution, Opti Free contact staining/ exclusion media
lens solution, Solo Care contact lens
solution, Gatorade, Tap water

Martin et al.12 HBSS, Milk, Saline, Propolis 50%, Propolis Number counted Propolis 20%,
100% by
hemocytometer

Al- Nazhan et Bausch and Lomb (Renu) contact lens Morphology using Renu, Optifree
al.69 solution, Ciba Vision (Titmus) contact SEM
lens solution, Alcon (Opti-free) contact
lens solution, EMEM

Ozan et al.13 Propolis 10%, Propolis 20%, Long- shelf Trypan blue Propolis 10%,
life light milk with lower fat content, staining/ exclusion
HBSS, DMEM

Ozan et al.14 Saliva officinalis (4%, 2.5%, 1.5%, 0.5%) Trypan blue Saliva
solution, HBSS, PBS, Tap water staining/ exclusion officinalis

Gopikrishna et Coconut water, HBSS, Milk Trypan blue Coconut water

al.15 staining/ exclusion (fresh)

Gopikrishna et Coconut milk, Propolis, HBSS, Milk Number counted Coconut milk
al.16 by (fresh)
hemocytometer

Ozan et al.57 PBS containing 4.0% M. rubra juice, PBS Trypan blue HBSS
containing 2.5% M. rubra juice, PBS staining/ exclusion
containing 1.5% M. rubra juice, PBS
containing 0.5% M. rubra juice, HBSS,
PBS, Tap water

Cehreli et al.70 Casein phosphopeptide- amorphous Iodide orange Casein

calcium phosphate paste at 10-3, 10-4, 10- staining phosphopepti
6
, 10-8, 10-12 dilutions de-
amorphous
calcium 10-12
dilution

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 Moreira-Neto et Still mineral water, Coconut water, Neutral red assay Whole milk
al.47 Saline, Coconut water with Sodium
Bicarbonate, Milk
Accepted Article
Caglar et al.48 HBSS, Saline, Probiotic solution Trypan blue HBSS,
(Lactobacillus reuteri solution), Milk staining/ exclusion Probiotic
solution, Milk

Souza et al.49 Sterile HBSS, Non-sterile HBSS, Save-a- Trypan blue Skim milk
Tooth, Skimmed milk, MEM, Water staining/ exclusion (Oaks farm
brands)

de Souza et al.65 Recently prepared HBSS, HBSS stored for MTT assay HBSS (recently
6 months, HBSS stored for 12 months, prepared)
Save-a-Tooth’s system HBSS, MEM,
Water

Souza et al.50 Skimmed milk, Whole milk, HBSS, MTT assay Skimmed milk,
Natural coconut water, Industrialized Whole milk
coconut water Save-a-Tooth's system
HBSS, Tap water, MEM

Mousavi et al.74 Standard ORS, HBSS, Tap water Trypan blue Oral
staining/ exclusion Rehydration
Solution (ORS)

Subramaniam et Oral rehydration salt-liquid (ORS-L) Number counted Oral

al.75 by Rehydration
hemocytometer Salt Liquid
(100%)

Rajendran et HBSS, Milk, Ricetral Trypan blue Ricetral, HBSS

al.62 staining/ exclusion

Hwang et al.17 HBSS, Tap water, Milk, Green tea Number counted Green tea
extract, Commercial green tea by extract
hemocytometer

Gjertsen et al.33 Propolis 25%, Propolis 50%, Propolis Apoptosis assay Propolis 50%,
100%, 0.4% Ethanol/HBSS, cell culture
medium (DMEM/10% PBS)

Silva et al.36 Long-shelf-life coconut water, Long- MTS assay Soymilk, HBSS,
shelf-life whole milk, Long-shelf-life Milk
soymilk, Gatorade, Egg white, HBSS,
DMEM, Distilled water

Moazami et al.37 Soymilk, HBSS, Powdered baby formula Trypan blue HBSS, Soymilk,
milk, DMEM, Tap water staining/ exclusion Powdered
baby formula

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 milk

Chen et al.71 Epigallocatechin-3-gallate, HBSS, Milk Trypan blue Epigallocatech

staining/ exclusion in-3-gallate
Accepted Article
Mahal et al.34 HBSS, Egg albumen, Propolis Number counted HBSS, Egg
by albumin,
hemocytometer Propolis

Silva et al.38 Soy milk, Coconut water, HBSS, Whole Multiparametric Soy milk,
milk assay HBSS, Milk

Sanghavi et al.18 ORS, Coconut water, Propolis 50% Trypan blue Coconut water
staining/ exclusion (fresh)

Eskandarian et ORS 25%, ORS 50%, ORS 100%, DMEM MTT assay ORS 50%,
al.76

Badakhsh et al.19 Aloe vera (10%, 30%, 50%, 100%- diluted MTT assay Egg white
with DMEM), Egg white, DMEM

Tavassoli-Hojjati Pomegranate juice (1%, 2.5%, 5%, 7.5%), Neutral red assay Pomegranate
et al.20 1% HBSS, Tap water juice 7.5%

Hiremath et al.72 Platelet rich fibrin(PRF) with platelet Number counted PRF with PRP
poor plasma (PRP), Platelet poor plasma by
hemocytometer

Ebenezar et al.63 De-ionized water, De-ionized water with Number counted HBSS with L-
L-DOPA, HBSS, HBSS with L-DOPA by DOPA
hemocytometer

Sharma et al.21 Milk, Aloe vera, Egg white Trypan blue Aloe vera
staining/ exclusion

Bharath et al.35 HBSS, Ringer lactate solution, Tender Use of optical Ringer lactate,
coconut water, Green tea extract microscope and Tender
optical coconut
calorimeter water, Green
tea extract

Sricholpech et HBSS, Royal jelly, UHT low fat milk MTT assay Royal jelly
al.22

Subramaniam et Oral hydration salt-liquid (ORS-L), HBSS, Trypan blue ORS-liquid,

al.64 Milk staining/ exclusion HBSS

Choi et al.51 Milk Microassay data Whole milk

analysis, Reactive
oxygen species,

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 Western blot
analysis

Ghasempour et Green tea extract, HBSS, Water Trypan blue Green tea
Accepted Article
al.23 staining/ exclusion extract

Caglar et al.24 Probiotic yogurt, HBSS, Saline, Milk Trypan blue Probiotic
staining/ exclusion solution

Jabarifar et al.77 HBSS, Tap water, ORS 25%, ORS 50%, MTT assay, ORS (100%)
ORS 100% apoptosis assay

Martins et al.25 10% Dragon's blood sap, Phosphate Trypan blue Dragon's
buffered saline, DMEM, Whole milk, exclusion test and blood sap
Water MTT assay

Souza et al.26 Skim milk, Natural coconut water, Whole MTT assay Natural
milk, Freshly prepared HBSS, Save-a- coconut water
Tooth, Industrialized coconut water, Tap
water, MEM

Ulusoy et al.52 UHT long-shelf-life lactose-free cow milk, MTT assay Goat milk
UHT long-shelf-life whole cow milk, UHT
long-shelf-life skimmed cow milk, UHT
long-shelf-life soy milk, UHT long-shelf-
life goat milk, UHT long-shelf-life follow
on milk with probiotic, 20% Propolis, Egg
white, Tap water, EMM

Prueksakorn et HBSS, UHT processed milk, Thai propolis Trypan blue 2.5mg/ ml
al.27 extract (0.25mg/ml, 0.5mg/ml, 1mg/ml, staining/ exclusion Thai propolis
2.5mg/ml, 5mg/ml, 10mg/ml), PBS extract

Saini et al.53 Coconut milk, Probiotic milk, HBSS Trypan blue Probiotic milk
staining/ exclusion

Saluja et al.54 Low fat (3%) milk, Medium fat (3.5%) Trypan blue Light milk with
milk, High fat (4%) milk staining/ exclusion low fat
content

Sharma et al.28 Regular pasteurized milk, Rice water, Egg Trypan blue Rice water
white staining/ exclusion

Anegundi et al.29 Cranberry juice, Pomegranate juice, Neutral red assay Cranberry
Plum, Guava, Green tea extract juice

Fulzele et al.66 HBSS, Aloevera gel, Packaged drinking Trypan blue HBSS
water staining/ exclusion

Hedge et al.55 0.9% Saline, Chilled homogenous milk, Trypan blue Chilled milk

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 GC tooth mousse plus staining/ exclusion

Lee et al.73 Save-a-Tooth, EMT toothsaver Quantifying ATP EMT

(Dentosafe), HBSS, Water with luminescent toothsaver
Accepted Article
dye

Adeli et al.30 HBSS, Pasteurized long-life whole milk, MTT assay Green tea
Hypotonic sucrose solution, Green tea extract
extract, Green tea extract with sucrose,
DMEM, Distilled water

Reis et al.31 Soya milk, Whole milk, HBSS, DMEM XTT assay, Neutral Soya milk
red uptake test,
Sulphorhodamine
test (SRB)

Babaji et al.32 HBSS, Propolis, Aloe vera, Pomegranate Trypan blue Propolis (50%
juice staining/ exclusion in 0.4%
ethanol)

de Souza et al.56 Skimmed milk, Whole milk, Recently MTT assay Skimmed milk
prepared HBSS, Save-a-Tooth system’s
HBSS, Natural coconut water, Propolis,
Egg white

Legend: EMEM- Eagle’s Minimum Essential Medium, HBSS- Hank’s Balanced Salt Solution, LDH-
Lactate dehydrogenase, PDGF- Platelet Derived Growth Factor, α-MEM- Alpha Minimum Essential
Medium, DMEM- Dulbecco’s Modified Eagle’s Medium, PBS- Physiologically Buffered Saline, ORS-
Oral Rehydration Solution, UHT- Ultra Heat Treatment, EMM- Eagle’s Maintenance Medium, MTT-3-
[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay

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 Table 3: Recommendations for storage media groups as reported in the included studies
Accepted Article
Storage medium and their derivatives Number of studies

Saliva and derivates 1

Milk and derivates 13

HBBS and variants 4

Saline and variants 4

Specialized culture media 7

Natural products other than milk 22

HBBS AND milk derivatives 3

Saline AND milk derivative 2

HBSS AND specialized culture media 5

Milk AND HBSS AND natural products other than milk 3

HBSS AND natural products other than milk 1

Ringer’s Lactate AND natural products other than milk 1

HBSS AND saline variants 1

*HBSS stands for Hank’s balanced salt solution

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Accepted Article

Figure 1: Study flow diagram

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 Method used to assess PDL survival
Accepted Article
Trypan blue staining

20
MTT Assay
27

Number counted by
Heamcytometer
Neutral Red Assay

3
Other methods

8
9

Figure 2: Laboratory methods used to assess the periodontal ligament cell survival (n=67)

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 Random sequence allocation

(selection bias) 67% 33%

Accepted Article
Allocation concealment

(selection bias) 100%

Blinding of outcome
assessor
98.5%
(detection bias)

Selective reporting

(reporting bias) 7% 93%

n=67 studies 0%___________25%_____________50%___________75%_________100%

high risk of bias unclear risk of bias low risk of bias

Figure 3: Overall risk of bias for the included studies

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