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C OPYRIGHT  2013

BY

T HE J OURNAL

OF

B ONE

AND J OINT

S URGERY, I NCORPORATED

Histological Study of the Influence of Plasma Rich

in Growth Factors (PRGF) on the Healing
of Divided Achilles Tendons in Sheep
J. Andre s Ferna ndez-Sarmiento, DVM, Juan M. Domnguez, DVM, PhD, Mara M. Granados, DVM, PhD,
Juan Morgaz, DVM, PhD, Roco Navarrete, DVM, PhD, Jose M. Carrillo, DVM, PhD,

DVM, Juana Martn de las Mulas, MD, PhD,

Rafael J. Gomez-Villamandos,
DVM, PhD, Pilar Munoz-Rascon,
MD, PhD, and Ramon
Cugat, MD, PhD
Yolanda Milla n, DVM, PhD, Montserrat Garca-Balletbo,
Investigation performed at the Department of Animal Medicine and Surgery, University of Cordoba, Cordoba; the Department of Animal
Medicine and Surgery, Cardenal Herrera University, Valencia; the Department of Comparative Pathology, University of Cordoba,
Cordoba; and the Deparment of Orthopaedic Surgery and Traumatology, Hospital Quiro n, Barcelona, Spain

Background: The use of plasma rich in growth factors (PRGF) has been proposed to improve the healing of Achilles
tendon injuries, but there is debate about the effectiveness of this therapy. The objective of the present study was to
evaluate the histological effects of PRGF, which is a type of leukocyte-poor platelet-rich plasma, on tendon healing.
Methods: The Achilles tendons of twenty-eight sheep were divided surgically. The animals were randomly divided into
four groups of seven animals each. The repaired tendons in two groups received an infiltration of PRGF intraoperatively and
every week for the following three weeks under ultrasound guidance. The tendons in the other two groups received
injections with saline solution. The animals in one PRGF group and one saline solution group were killed at four weeks, and
the animals in the remaining two groups were killed at eight weeks. The Achilles tendons were examined histologically,
and the morphometry of fibroblast nuclei was calculated.
Results: The fibroblast nuclei of the PRGF-treated tendons were more elongated and more parallel to the tendon axis
than the fibroblast nuclei of the tendons in the saline solution group at eight weeks. PRGF-treated tendons showed more
packed and better oriented collagen bundles at both four and eight weeks. In addition to increased maturation of the
collagen structure, fibroblast density was significantly lower in PRGF-infiltrated tendons. PRGF-treated tendons exhibited
faster vascular regression than tendons in the control groups, as demonstrated by a lower vascular density at eight weeks.
Conclusions: PRGF was associated with histological changes consistent with an accelerated early healing process in
repaired Achilles tendons in sheep after experimental surgical disruption. PRGF-treated tendons showed improvements in
the morphometric features of fibroblast nuclei, suggesting a more advanced stage of healing. At eight weeks, histological
examination revealed more mature organization of collagen bundles, lower vascular densities, and decreased fibroblast
densities in PRGF-treated tendons than in tendons infiltrated with saline solution. These findings were consistent with a
more advanced stage of the healing process.
Clinical Implications: Based on the findings in this animal model, PRGF infiltration may improve the early healing
process of surgically repaired Achilles tendons.

njured tendons heal slowly because of less blood supply

and reduced metabolism compared with many other tissues1,2. The use of growth factors has been proposed as a

strategy to improve tendon healing3. Platelet-derived growth

factor (PDGF), transforming growth factor-b1 (TGF-b1),
vascular-endothelial growth factor (VEGF), epidermal growth

Disclosure: None of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of any
aspect of this work. None of the authors, or their institution(s), have had any financial relationship, in the thirty-six months prior to submission of this work, with
any entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. Also, no author has had any
other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The
complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.

J Bone Joint Surg Am. 2013;95:246-55

http://dx.doi.org/10.2106/JBJS.K.01659

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factor (EGF), basic fibroblastic growth factor (bFGF), and

insulin-like growth factor type-I (IGF-I), among others, have
been recognized to play key roles in tendon healing4,5. Although
these growth factors potentially could be used therapeutically
to accelerate the complex process of tendon healing, it seems
unlikely that a single growth factor will give a positive result
in vivo. Some authors have suggested that the interaction of
various growth factors with a natural balance of anabolic and
catabolic functions, present in the right concentration at the
right time, would be necessary to optimize the tissue environment and to favor the tendon healing process3,6.
Autologous plasma, generally called platelet-rich plasma
(PRP), is being used to treat musculoskeletal injuries. Platelets
contain a complex pool of growth factors, interleukins, and
other biologically active proteins, such as fibrinogen, fibronectin,
and vitronectin, which have prominent roles in the healing
process7. Several clinical studies have demonstrated that applications of PRP can improve the healing response after tendon
injuries8-16, but there is an intense debate about the effectiveness
of these products6,17-19.
The purpose of the present study was to evaluate the
effect of plasma rich in growth factors (PRGF), which is one
type of leukocyte-poor PRP, on tendon healing. We compared
the histological appearance of acutely surgically injured and
repaired Achilles tendons that had been treated with PRGF with
that of repaired tendons that had been infiltrated with saline
solution in sheep. Our hypothesis was that weekly infiltrations of
PRGF onto the injured tendons would accelerate the healing
response and would improve the histological properties of these
tendons.
Fig. 1

Materials and Methods

Experimental Model of Divided Achilles Tendon

he present study was approved by the Bioethical Committee on Animal

Research at our institution. We strictly adhered to the guidelines for the use
of laboratory animals proposed by the National Institutes of Health. We used
twenty-eight skeletally mature female Merino sheep weighing between 45 to
55 kg for our study. A veterinary surgeon performed general and orthopaedic
physical examinations on each animal to guarantee that all sheep were free from
lameness.
With use of sterile technique, the right hindlimb of the anesthetized
sheep was prepared for surgery. A lateral skin incision was made over the Achilles
tendon. The peritenon was incised longitudinally (Fig. 1-A). A full-thickness
transverse tendon division of the Achilles tendon was performed with use of a
scalpel, 5 cm proximal to its insertion into the calcaneus (Fig. 1-B). The tendon
was repaired with use of a three-loop pulley pattern with a nonabsorbable
synthetic monofilament (polypropylene) (Premilene USP 1; B Braun Aesculap,
Melsungen, Germany) (Fig. 1-C). The peritenon was sutured with a continuous
absorbable synthetic monofilament (polyglyconate) (Monosyn USP 3-0; B
Braun Aesculap). The wound was closed in layers and was covered with a
sterile dressing.
The repaired tendon was protected during the postoperative period
with use of a custom system that allowed the animal to bear weight on
the operatively treated hindlimb without subjecting the Achilles tendon to
20,21
macromovements
. A tarsal transarticular external skeletal fixation system was placed in the medial aspect of the limb just after skin suture and was
left in place throughout the experiment. The tarsal joint was fixed at an angle
of 140 (Fig. 1-D) and was covered with a protective bandage. An antibiotic
(benzylpenicillin [15,000 IU/kg, administered intramuscularly] plus dihy-

The surgical approach of the Achilles tendon through a lateral skin incision
allowed precise and reproducible measurements of the tenotomy area
(Fig. 1-A). The tenotomy point was 5 cm proximal to the calcaneal tuberosity. This point was measured with use of a ruler (Fig. 1-B). A three-loop
pulley pattern was used to repair the tenotomy (Fig. 1-C). The tarsal
transarticular external skeletal fixation system protected the repaired
tendon while the animal was bearing weight on the operatively treated limb.
The bars on the tibia and metatarsus were joined together with two connecting bars, forming a triangular configuration (Fig. 1-D).

drostreptomycin [15 mg/kg, administered intramuscularly]) (Shotapen LA;

Virbac Animal Health, Barcelona, Spain) was given at the time of surgery and
again after seventy-two hours. Analgesic buprenorphine 0.02 mg/kg/8 hr,
administered intramuscularly (Buprex 0.3 mg; Schering-Plough, Madrid, Spain)
was given for five days after surgery.

Plasma Rich in Growth Factors (PRGF): Preparation

and Infiltration on Repaired Area
PRGF was prepared with use of the PRGF-Endoret system (Biotechnology
Institute [BTI], Vitoria, Spain). Disposable BTI extraction and fractioning tubes
were used in a custom-designed bench-top centrifuge. Each 5-mL extraction tube
contained 0.5 mL of sodium citrate solution (3.8%) as anticoagulant. Twenty
milliliters of blood were collected from the jugular vein of each animal and were
divided into four tubes that were centrifuged at 630 times gravity for eight minutes
22
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was collected just prior to surgery and was processed intraoperatively. The upper
layer of the centrifuged plasma was removed with use of a pipette and was
discarded. The remaining 0.5 mL of plasma fraction that lies just above but
not including the interphase zone (buffy coat) was retrieved with use of a sterile
pipette and was placed in a fractioning tube. This plasmatic fraction is the PRGF
according to the manufacturers instructions. The plasma separation procedure
was done in a laminar flow cabinet in accordance with the PRGF-Endoret system
manufacturer instructions. The 0.5 mL of PRGF from each of the four tubes were
combined to obtain a total volume of 2 mL of PRGF per animal. The platelets were
activated by adding 0.1 mL of 10% calcium chloride just prior to the injection of
PRGF in the injured tendon. The time delay between blood collection and PRGF
application was less than one hour.
The sheep were randomly divided into four groups of seven animals
each. In the sheep in the two experimental groups, 2 mL of PRGF were infiltrated
intratendinously into the divided tendon stumps with use of a 23-gauge needle
just before suturing of the peritenons. The injections were repeated every week
for the following three weeks. The repaired tendons in the other two groups
(controls) were injected with 2 mL of saline solution plus 0.1 mL of 10% calcium
chloride as described above. The infiltrations during the postoperative period
were carried out in properly sedated animals with use of sterile technique under
ultrasonographic guidance. The animals in one PRGF-treated group and one
saline solution-treated group were killed at four weeks, and the remaining animals
were killed at eight weeks.

Histological Procedure
After each animal was killed, both Achilles tendons were harvested and were
fixed in 10% neutral buffered formalin. Contralateral (non-operatively treated)
tendons were studied as normal controls. Each operatively treated tendon was
cut lengthwise on the dorsal midline with use of a scalpel blade. A 1-mm-thick
slab of tissue that included the entire repaired area was excised. This slab of
tissue also contained intact tendon fibers on either side of the repaired area. The
margin adjacent to the intact tendon fibers was yellow-inked for histological
identification. Two paraffin blocks were obtained from each operatively treated
tendon. From each block, six 4-mm-thick consecutive paraffin sections were
cut. Sections were mounted in slides, and three were stained with hematoxylin
and eosin and three were stained with Masson trichrome.
The stained sections were digitalized with use of a photomicroscope
(Axiophot; Carl-Zeiss, Oberkochen, Germany) with an attached digital camera
(DS-5M camera head; Nikon, Tokyo, Japan) coupled with a digital imaging
controller (DS-L1 camera control unit, Nikon) that allowed us to observe, focus,
and capture histological images. Slides were sampled in a systematic randomized
manner by superimposing a dotted transparent template onto each slide. Several
dots spanning the whole tissue section were marked over the glass of the slide
with use of a permanent marker. The regions of interest were chosen during
the histological examination beside a previously marked dot. In Masson
trichrome-stained sections, nine histological images per slide were captured with
a magnification of 200. In hematoxylin and eosin-stained sections, nine
histological images per slide were captured with a magnification of 200 and
nine histological images per slide were captured with a magnification of 630.
These histological images were transferred to a computer equipped with imageanalysis software (Image Pro Plus, version 6.0; MediaCybernetics, Rockville,
Maryland) to perform quantitative measurements. In order to blind the evaluation
of the histological images, all of the captures were encoded with use of an
identification number and were randomly evaluated by three independent
pathologists.

Morphology of Fibroblast Nuclei

The morphometry of fibroblast nuclei was studied with the aid of imageanalysis software on images captured at a magnification of 630 and stained
with hematoxylin and eosin. The outlines of fibroblast nuclei were traced
with use of the cursor of the image software (Fig. 2-A). The following quantitative
parameters were calculated from each nuclear profile: area (mm2), perimeter
2
(mm), major axis (mm), minor axis (mm) and roundness [(perimeter )/4 pi
area]. The nuclear aspect ratio (defined as the ratio of the minor axis to the

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major axis) was also calculated, with values approaching zero suggesting a
23
spindle shape and with the value of 1.00 representing a perfect circle . The
nuclear orientation angle was defined as the angle between the major axis of
the nucleus and the longitudinal tendon axis, with values of 0 representing a
nucleus that is perfectly aligned toward the longitudinal axis. When this value
increases, the nucleus becomes more angled until it approaches 90, where it
23
lies perpendicular to the long axis of the tendon .

Histological Study of Collagen Fibers

The histological appearance of the collagen fibers was studied on images captured at a magnification of 200 and stained with Masson trichrome. A 5-point
semiquantitative grading scale was designed to evaluate the packing and orientation of collagen fibers (see Appendix). This semiquantitative grading scale
was a modification based on the grading scales described in three different
24-26
publications
.

Fibroblast Density
The fibroblast density was determined with use of a counting frame that
was superimposed onto each histological image; these images were captured at a
magnification of 630 and were stained with hematoxylin and eosin (Fig. 2-B).
The number of fibroblasts per area was calculated according to the formula:
fibroblasts/mm2 = N/AF , where N was the number of fibroblasts counted
within the counting frame, and AF was the area of the counting frames (in our
study, AF = 0.004 mm2).

Vascular Density
Vascular density was quantified with use of histological images that were captured at a magnification of 200 and stained with hematoxylin and eosin. The
number of blood vessels in each image area was divided by the area of the
histological image captured at a magnification of 200 (in our study, 0.142 mm2).

Evaluation of the Inflammatory Cell Infiltration

The infiltration of inflammatory cells was analyzed with use of images captured
at a magnification of 200 and stained with hematoxylin and eosin. As the
different inflammatory cell subtypes were not quantified, the inflammatory cell
infiltration was defined on the basis of the density of the main inflammatory cell
subtype observed in the histological sections (lymphocytes). The density of
inflammatory cell infiltration was characterized with use of a 5-point semiquantitative grading scale. A value of 0 was assigned if inflammatory process
was absent, a value of 1 was assigned when there was slight inflammatory cell
infiltration, a value of 2 was assigned when there was moderate inflammatory
cell infiltration, a value of 3 was assigned when there was strong inflammatory
cell infiltration, and a value of 4 was assigned where there was severe inflammatory cell infiltration homogeneously spread in the entire field of view.

Statistical Analysis
Descriptive statistics for each study group (including the mean, standard deviation, and frequencies for categorical data) were calculated. The nonparametric
Kruskal-Wallis test and the Mann-Whitney U test were used to analyze the significance between groups for quantitative parameters (morphology of fibroblast
nuclei, fibroblast density, and vascular density). Categorical data (packing of
collagen fibers, orientation of collagen fibers, and inflammatory cell infiltration)
were analyzed with use of the chi-square test to determine if the infiltration with
PRGF or saline solution yielded significant differences in the frequency distribution between study groups. Values were presented as mean and the standard
deviation (SD). The level of significance was set at p < 0.05.

Source of Funding
This study was supported by the Garca-Cugat Foundation for Biomedical
Research and the Ministry of Health of the Spanish Government. One of the
authors (J.A.F.-S.) was the recipient of a grant from the Ministry of Education
and Science of Spanish Government (AP2006-00150). The authors do not have

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Fig. 2

Representative images demonstrating computer analysis of the histopathological captures. The nuclear profiles of the fibroblasts were traced manually with
use of the cursor of the image software that had been loaded onto a digital tablet, and morphometric parameters of the fibroblast nuclei were calculated
(Fig. 2-A). To determine the fibroblast density, a four-squares frame was superimposed onto each image, and the nuclei (asterisks) that were included
inside each square without touching the exclusion borders (red borders) were counted. The number of fibroblast nuclei was divided between the total area
of the four squares to obtain the fibroblast density (Fig. 2-B).

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Fig. 3

The main morphometric parameters of fibroblast nuclei (the nuclear aspect ratio [Fig. 3-A] and the nuclear orientation angle [Fig. 3-B]) were
significantly different between the PRGF group and the saline solution group at eight weeks. Fibroblast density was significantly different between
the PRGF and saline solution groups at both four and eight weeks (Fig. 3-C). There was a significant decrease in vascularization in the PRGF-treated
tendons at eight weeks (Fig. 3-D). Asterisks indicate a significant difference between the PRGF and saline solution groups (p < 0.05). In all experimental
groups, n = 7.
any financial interest or other relationship with any commercial company related to this study.

Results
ll animals reached the end of the study without incident.
Morphometric data on fibroblast nuclei are shown in
Table I. Fibrocyte nuclei in normal Achilles tendons had a
spindle shape, as demonstrated by a low value for the nuclear
aspect ratio (0.07 0.01) (see Appendix). The PRGF-treated
tendons showed lower values of the nuclear aspect ratio than
did the saline solution-treated tendons at both four and eight
weeks. Nuclei were significantly more elongated in PRGFtreated tendons than in saline solution-treated tendons at
eight weeks (p = 0.008) (Fig. 3-A). Fibrocyte nuclei in normal
tendons were highly aligned with respect to the longitudinal
tendon axis (mean nuclear orientation angle, 2.7 0.9). Fibroblast nuclei tended to be more parallel to the longitudinal

axis in PRGF-treated tendons than in tendons infiltrated with

saline solution. This orientation was reflected by a lower value for
the nuclear orientation angle, which was significantly lower
at eight weeks compared with the saline solution group (p =
0.016) (Fig. 3-B). Other morphometric parameters of fibroblast
nuclei (major axis, minor axis, roundness, area, and perimeter)
showed a more normal appearance in the PRGF groups than in
the saline solution groups, with significant differences between
the groups in terms of the major axis, roundness, and perimeter
at eight weeks (Table I).
Normal tendons were characterized by highly packaged
and perfectly aligned collagen bundles (see Appendix). At four
weeks after surgery, the mean value for packing of collagen
fibers was significantly greater in the PRGF group than in the
saline solution group (3.82 0.51 versus 3.26 0.56; p <
0.001). Similarly, at eight weeks after surgery, the PRGF group
also had a significantly higher value for packing of collagen fibers

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TABLE I Quantitative Parameters (Morphometric Data on Fibroblast Nuclei, Fibroblast Density, and Vascular Density) in Normal,
Saline Solution, and PRGF Groups*
4 Weeks
Normal

Saline Solution
(N = 7)

PRGF
(N = 7)

0.07 0.01
2.7 0.9

0.39 0.02
16.0 2.8

0.36 0.02
12.3 1.1

31.11 4.16
1.93 0.28
7.44 0.84
44.80 11.92
62.35 7.64

11.53 0.98
4.06 0.33
1.71 0.05
36.53 6.05
27.32 2.22

Fibroblast density
(fibroblasts/mm2)

228 98

Vascular density
(vessels/mm2)

0.47 0.66

Morphometric data
Nuclear aspect ratio
Nuclear orientation
angle (deg)
Major axis (mm)
Minor axis (mm)
Roundness
Area (mm2)
Perimeter (mm)

8 Weeks
P
Value

Saline Solution
(N = 7)

PRGF
(N = 7)

P
Value

0.056
0.095

0.32 0.02
12.2 1.7

0.26 0.01
9.2 0.9

0.008
0.016

12.37 0.41
4.05 0.16
1.76 0.06
38.95 2.55
28.79 0.90

0.222
1.000
0.222
0.548
0.421

13.00 0.33
3.69 0.22
2.02 0.11
36.55 2.54
29.79 0.76

14.55 0.56
3.40 0.07
2.34 0.07
37.55 2.01
32.41 1.05

0.008
0.056
0.008
0.690
0.016

2699 156

2153 247

0.008

2612 130

1905 110

0.008

13.95 5.26

10.88 3.92

0.421

11.62 1.37

6.39 1.50

0.008

*The values are presented as the mean and the standard deviation. Significant difference between the saline solution and PRGF groups.

in comparison with the saline solution group (4.16 0.49 versus

3.70 0.55; p < 0.001) (Table II). The orientation of collagen
fibers was significantly better in the PRGF group in comparison with the saline solution group at both four weeks
(3.64 0.63 versus 3.20 0.70; p < 0.001) and eight weeks
after surgery (4.21 0.51 versus 3.63 0.62; p < 0.001) (Table

II). Most histological fields of view of PRGF-treated tendons

showed a regular and dense formation of collagen bundles, with
fibers slightly angled to the longitudinal tendon axis at four weeks
after surgery. Conversely, most histological views of the saline
solution-treated tendons at four weeks showed only a moderate
packing of collagen fibers, with disordered orientation (Fig. 4). At

Fig. 4

Photomicrographs showing the histopathological appearance of the collagen structure. Collagen fibers were more packed in PRGF-treated tendons at both
four and eight weeks. Similarly, collagen fibers showed a better orientation in the PRGF group than in the saline solution group. The arrows indicate the
longitudinal tendon axis orientation (200, Masson trichrome; bar = 100 mm).

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TABLE II Packing of Collagen Fibers, Orientation of Collagen Fibers, and Inflammatory Cell Infiltration in Saline Solution
and PRGF-Treated Groups
4 Weeks
Normal

Saline
Solution

PRGF

Packing of collagen fibers

Percentage of specimens
receiving each grade
Grade 4
Grade 3
Grade 2
Grade 1
Grade 0
Mean grade*

100%

40

31.9%
62.2%
5.9%

3.26 0.56

5.6%
70.7%
23.7%

3.82 0.51

Orientation of collagen fibers

Percentage of specimens
receiving each grade
Grade 4
Grade 3
Grade 2
Grade 1
Grade 0
Mean grade*

100%

40

0.4%
34.1%
51.5%
13.0%
1.1%
3.20 0.70

3.7%
60.7%
31.1%
4.4%

3.64 0.63

Inflammatory cell infiltration

Percentage of specimens
receiving each score
Severe (4 points)
Strong (3 points)
Moderate (2 points)
Slight (1 point)
Absent (0 points)
Mean score* (points)

100%
00

4.4%
13.7%
36.3%
35.9%
9.6%
1.67 0.98

3.0%
7.8%
24.1%
34.8%
30.4%
1.18 1.04

8 Weeks
P Value

Saline
Solution

PRGF

P Value

<0.001

4.4%
61.1%
34.1%
0.4%

3.70 0.55

21.1%
73.7%
5.2%

4.16 0.49

<0.001

<0.001

2.6%
63.3%
28.9%
5.2%

3.63 0.62

25.9%
69.3%
4.8%

4.21 0.51

<0.001

<0.001

2.2%
11.1%
32.2%
46.7%
7.8%
1.53 0.87

0.4%
2.2%
13.7%
37.0%
46.7%
0.73 0.81

<0.001

*The values are given as the mean and the standard deviation.

eight weeks, 21.1% of histological captures in PRGF-treated

tendons showed a homogeneous formation of tightly packed
collagen bundles, with 25.9% of captures showing collagen
fibers that were parallel to the longitudinal tendon axis.
Histological captures in the saline solution group exhibited
a more delayed stage of collagen maturation at this time
(Fig. 4).
Fibroblast density was relatively low in normal tendons
(mean, 228 98 fibroblasts/mm 2) (see Appendix), although
this parameter was considerably elevated in all of the tendons that had been injected with either PRGF or saline
solution (Table II). PRGF-treated tendons showed significantly lower fibroblast density than did saline solutiontreated tendons at both four weeks (p = 0.008) and eight
weeks (p = 0.008) (Fig. 3-C). Histological captures of PRGFtreated tendons showed lower fibroblast density along
with an increased maturation of the collagen structure
(Fig. 5-A).

Vascularization was barely seen in normal tendons. In

contrast, histological evaluation revealed an intense vascular response in all experimental tendons (Fig. 5-B). PRGFtreated tendons exhibited faster vascular regression than saline
solution-treated tendons, as demonstrated by a significantly
lower vascularization in the PRGF group at eight weeks (p =
0.008) (Table II; Fig. 3-D).
The main inflammatory cell subtype observed in all
of the experimental groups was lymphocytes. The inflammatory cell infiltration was significantly lower in the
PRGF-treated group than in the saline solution group at both
four weeks (1.18 1.04 versus 1.67 0.98; p < 0.001) and eight
weeks (0.73 0.81 versus 1.53 0.87; p < 0.001) (Table II).
At eight weeks, most histological captures in the saline
solution group (92.2%) exhibited at least a slight degree
of inflammation, whereas in 46.7% of captures in the
PRGF group the inflammatory cell infiltration was absent
(Fig. 5-C).

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Fig. 5

Photomicrographs used for the histopathological study of fibroblast density

(Fig. 5-A), vascular density (Fig. 5-B), and inflammatory cell infiltration (Fig.
5-C). The arrows indicate blood vessels (200, hematoxylin and eosin; bar =
100 mm).

Discussion
latelet-rich plasma is being used with increasing frequency
in sports medicine to try to accelerate healing and to allow
an earlier return to sports18,19. Despite this interest, there have
been only two clinical studies that have evaluated the effectiveness
of PRP in the treatment of ruptures of the Achilles tendon in

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humans: a case-controlled study that showed a faster functional

recovery in association with the use of PRP9, and a randomized
controlled trial that showed no effect16. However, several animal
studies have shown promising results in association with the use
of PRP for the treatment of tendon lesions27-35.
The ratio of the minor axis to the major axis of the
nucleus (nuclear aspect ratio) and the angle between the major
axis of the nucleus and the longitudinal tendon axis (nuclear
orientation angle) provide information about fibroblast nuclei shape and alignment along the longitudinal tendon axis,
respectively. In our study, measurement of the nuclear aspect
ratio demonstrated that the PRGF-treated tendons had a
more elongated nucleus than did the saline solution-treated
tendons, suggesting a more mature histological appearance in
the PRGF-treated tendons. We observed that fibroblasts in
the PRGF-treated groups were more parallel to the longitudinal tendon axis than those in the saline solution groups, as
demonstrated by a lower value of the nuclear orientation
angle.
Early inflammatory and proliferative phases are defined
by a dramatic increase in the fibroblast population. Approximately three or four weeks after the traumatic event, the fibroblast density reaches its peak and then decreases progressively
during the remodeling and maturation phases36,37. The neovascularization is prominent during early stages of tendon healing
but then progressively disappears27. In our study, the PRGFtreated tendons had lower fibroblast density and a faster decrease
in neovascularization compared with the tendons that had been
infiltrated with saline solution, suggesting that PRGF was associated with histological changes consistent with accelerated
early healing.
The collagen structure is closely related to the biomechanical properties of tendons36. Packing and orientation of
collagen bundles are major features in the collagen structure,
and they have been evaluated in several studies24-26. We observed an improvement in both the packing and the orientation of collagen bundles in PRGF-treated tendons, showing
a more mature collagen structure compared with saline solutiontreated tendons.
Platelets modulate inflammation by secreting chemokines that control chemotaxis of leukocytes and macrophages
in injured tissues17. The features of the inflammatory response
may determine the success of tendon repair17. In our study,
PRGF-treated tendons showed lower inflammatory cell infiltration than did saline solution-treated tendons at both four
and eight weeks. No research has been conducted to investigate
the exact effect of PRP on the inflammatory response after
acute tendon injury. Further investigations are needed to
clarify the role of PRP in the modulation of the inflammatory
response during the healing process.
Platelet-rich plasma is a general term that encompasses
many different autologous plasma products38, even though they
are obtained with use of different protocols that may result in
different concentrations of platelets, leukocytes, and growth
factors and even though these products differ both qualitatively
and quantitatively and show different biologic effects38,39. The

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PRGF-Endoret system produces a volume of autologous plasma

having a platelet concentration above baseline, free of leukocytes
and erythrocytes, elaborated by a one-step centrifugation process
and using sodium citrate as anticoagulant and calcium chloride as
platelet activator39.
Schepull et al.16 and de Vos et al.11,40 reported that the use
of PRP did not demonstrate any effect on the healing of human
Achilles tendon in clinical studies, but the methodology that
they followed for obtaining the autologous platelet concentrate
was different from the PRGF methodology that we used in our
experimental study. Schepull et al.16 used a PRP preparation
obtained by double centrifugation, which yielded a product
with an extremely high concentration of platelets (tenfold
above baseline). Previous studies demonstrated that high concentrations of some growth factors, such as TGF-b, could be
deleterious for tendon healing as TGF-b drives fibrogenesis,
potentially stimulates the development of scar tissue, and is
associated with the onset of fibrosis22. de Vos et al.11,40 used a
PRP product obtained by means of a methodology that collects a high concentration of leukocytes. PRGF is a product
free from leukocytes, avoiding the proinflammatory effects of
the proteases and cytokines contained in white blood cells41.
While some studies have involved only a single application of PRP11,16,41, we used weekly applications of PRGF
during the following three weeks after surgery on the basis
of a previous study in sheep by Anitua et al.22. While PRGF
was injected into intact tendons without any induced injury
in the study by Anitua et al.22, a complete division of the
Achilles tendon was surgically induced in the present study.
Such intense damage triggers a strong proliferative healing
response in the injured area36,37. Anitua et al. reported that the
injection of PRGF in intact tendons increased cell density and
promoted neovascularization after four weeks of weekly administration in their experimental model22. Our findings suggested that when PRGF was injected into injured tendons, the
histological response was different. We observed a decrease in
fibroblast density in PRGF-treated tendons, which may be
explained by an acceleration in the healing process induced by
the PRGF injections. As the healing process advances, a progressive decrease in cell density occurs in the repaired area and
the maturation phase of the healing process begins36,37. Thus,
PRGF-treated tendons showed a histologically more advanced
stage of tendon healing than did saline solution controls at the
same postoperative time.
The present study had several limitations. One limitation
was that we did not perform any biomechanical testing of the
Achilles tendon to assess whether the histological differences
that were observed between the PRGF and saline solution
groups were mechanically relevant. The main objective of the
present study was to histologically evaluate the effect of PRGF
on tendon healing. In light of these promising results, future
studies are warranted to clarify whether histological improvements in tendon healing are related to superior biomechanical
properties. Another limitation of the present study is related to
the formulation of PRGF in sheep. The composition of PRGF in
humans recently was described by Anitua et al.42, but we are

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aware of no studies on the concentration of platelets, leukocytes,

erythrocytes, or growth factors in the PRGF of sheep. Another
concern is that multiple weekly injections were performed in
this study and that these injections may elicit an inflammatory
response because of the disruption of the peritenon. We considered this unavoidable damage to be the same in both the
PRGF groups and the saline solution groups. Hence, differences
observed between experimental groups should be attributed to
the injected product itself.
In conclusion, we found that PRGF was associated with
histological changes consistent with accelerated early healing
process in tendons after acute injury and repair of Achilles
tendons in sheep. PRGF-treated tendons showed better orientation and a more elongated shape of fibroblast nuclei, suggesting
a more advanced stage of healing compared with saline solutiontreated tendons. Histological examination of the repaired areas
showed a more mature organization of collagen bundles, less
inflammatory cell infiltration, faster vascular regression, and
lower fibroblast density in PRGF-treated tendons than in saline
solution-treated controls.
Appendix
A table showing the semiquantitative grading scale for
the assessment of collagen fibers and photomicrographs
showing the normal histological appearance of the Achilles
tendon in sheep are available with the online version of this
article as a data supplement at jbjs.org. n

J. Andres Fernandez-Sarmiento, DVM

Juan M. Domnguez, DVM, PhD
Mara M. Granados, DVM, PhD
Juan Morgaz, DVM, PhD
Roco Navarrete, DVM, PhD

Rafael J. Gomez-Villamandos,
DVM, PhD
DVM
Pilar Munoz-Rascon,
Department of Animal Medicine and Surgery,

University of Cordoba,
Campus de Rabanales,

Ctra. Madrid Km 396, 14014, Cordoba,

Spain.
E-mail address for J.A. Fernandez-Sarmiento: v12fesaj@uco.es
Jose M. Carrillo, DVM, PhD
Department of Animal Medicine and Surgery,
Cardenal Herrera University,
Edificio Seminario s/n, 46113,
Moncada, Valencia, Spain
Juana Martn de las Mulas, MD, PhD
Yolanda Millan, DVM, PhD
Department of Comparative Pathology,

University of Cordoba,
Campus de Rabanales,

Ctra. Madrid Km 396, 14014, Cordoba,

Spain
MD, PhD
Montserrat Garca-Balletbo,
Cugat, MD, PhD
Ramon
Department of Orthopaedic Surgery and Traumatology,
Plac
Hospital Quiron,
xa dAlfonso Comin,
5-7, 08023, Barcelona, Spain

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