Platelet-Rich Plasma Stimulates Cell
Proliferation and Enhances Matrix Gene
Expression and Synthesis in Tenocytes
From Human Rotator Cuff Tendons
With Degenerative Tears
Chris Hyunchul Jo,*1" MD, Ji Eun Kim,1" MS, Kang Sup Yoon,1 MD, and Sue Shin,* MD
Investigation performed at SMG-SNU Boramae Medical Center, Seoul National University
College of Medicine, Seoul, Korea.
Background: Platelet-rich plasma (PRP) contains various growth factors and appears to have a potential to promote tendon healing, but evidence is lacking regarding its effect on human tenocytes from rotator cuff tendons with degenerative tears.
Hypothesis: Platelet-rich plasma stimulates cell proliferation and enhances matrix gene expression and synthesis in tenocytes
isolated from human rotator cuff tendons with degenerative tears.
Study Design: Controlled laboratory study.
Methods: Tenocytes were enzymaticaliy isolated and cultured. To evaluate cell proliferation, tenocytes were cultured with 10%
(vol/vol) platelet-poor plasma (PPP), PRP .activated with calcium, and PRP activated with calcium and thrombin at platelet concentrations of 100, 200, 400, 800,1000,2000, 4000, 8000, and 16,000 X 103/|xL for 14 days. Cell number was measured at days 7
and 14. To investigate matrix gene expression and synthesis, cells were cultured with a PPP or PRP gel (10% vol/vol) at a platelet
concentration of 1000 X 103/jxLfor 14 days. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to determine the expressions of type I and 111 collagen, decorin, tenascin-C, and scleraxis, and measurements of total
collagen and glycosaminoglycan (GAG) synthesis were conducted at days 7 and 14.
Results: Platelet-rich plasma significantly increased cell proliferation at days 7 and 14 in a dose-dependent manner, and the addition of thrombin moved up the plateau of proliferation. Platelet-rich plasma significantly induced the gene expression of type I
collagen at day 7 but not at day 14, while it significantly promoted that of type III both at days 7 and 14. The ratio of type 11 I/I
collagens did not change at days 7 and 14. The expressions of decorin and scleraxis significantly increased at day 14, whereas
that of tenascin-C significantly increased at days 7 and 14. Platelet-rich plasma significantly increased total collagen synthesis at
days 7 and 14 and GAG synthesis at day 14.
Conclusion: Platelet-rich plasma promoted cell proliferation and enhanced gene expression and the synthesis of tendon matrix in
tenocytes from human rotator cuff tendons with degenerative tears.
Clinical Relevance: These findings suggest that PRP might be used as a useful biological tool for regenerative healing of rotator
cuff tears by enhancing the proliferation and matrix synthesis of tenocytes from tendons with degenerative tears.
Keywords: platelet-rich plasma; rotator cuff tear; degeneration; tenocytes; matrix; gene expression
Clinical results after rotator cuff repair have been shown
to be satisfactory regardless of the operative technique
used, which includes open surgery,18'61 mini-open surgery,so>51and arthroscopic surgery.17'64 Average satisfaction rates of 85% have been reported for open surgery
and from 84% to 95% for arthroscopic surgery.62 However,
2 important problems, namely the quality and speed of
healing, still remain to be solved despite recent advances
in mechanical fixation methods.25 Furthermore, as the
degenerative torn ends of rotator cuffs do not appear to
contribute to healing27'59 and as tendon healing is naturally slow,46'47 an additional biological strategy is required
to improve the tissue quality of torn ends and to aid the
regeneration of native tendon-to-bone insertions.25
"Address correspondence to Chris Hyunchul Jo, MD, Department of
Orthopedic Surgery, SMG-SNU Boramae Medical Center, Seoul National
University College of Medicine, 41 Boramae-gil, Dongjak-gu, 156-707
Seoul, Korea (e-mail: [email protected]).
tDepartment of Orthopedic Surgery, SMG-SNU Boramae Medical
Center, Seoul National University College of Medicine, Seoul, Korea.
* Department of Laboratory Medicine, SMG-SNU Boramae Medical
Center, Seoul National University College of Medicine, Seoul, Korea.
One or more of the authors has declared the following potential conflict
of interest or source of funding: This research was supported by the Bio &
Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (No. 2011 -0019773).
The American Journal of Sports Medicine, Vol. 40, No. 5
DOI: 10.1177/0363546512437525
©2012 The Author(s)
1035
1036 Jo et al
Platelet-rich plasma (PRP) is a platelet concentrate that
typically contains more than 1000 X 10s platelets/^L, representing a 3- to 5-fold increase as compared with whole
blood.25 Because platelets contain various growth factors
in their a-granules, PRP potentially can release these
growth factors at higher than physiological levels. As
growth factors known to be in a-granules, such as
platelet-derived growth factor (PDGF), epidermal growth
factor (EGF), transforming growth factor-beta 1 (TGFpl), msulin-like growth factor (IGF-I), vascular endothelial
growth factor (VEGF), basic fibroblast growth factor
(bFGF), and hepatocyte growth factor (HGF), are also
known to be upregulated or involved during tendon healing,11'23'38 it has been suggested that the addition of PRP
would aid tendon healing. However, basic experimental
and clinical evidence is lacking or still controversial.6'25'40
A few studies have investigated the effects of PRP on
human tenocytes.3'12'68 Anitua et al3 showed that 20%
PRP (vol/vol) stimulated tenocyte proliferation and
increased VEGF and HGF production by tenocytes, and
de Mos et al12 demonstrated that 20% PRP (vol/vol) promoted tenocyte proliferation but decreased the gene
expressions of type I and type III collagen. Recently,
Zargar Baboldashti et al68 reported that PRP diminished
the adverse effects of dexamethasone and ciprofloxacin.
However, in these studies, tenocytes were isolated and cultured from the normal hamstring tendons of young patients
undergoing hamstring tendon release or anterior cruciate
ligament reconstruction, and as has been previously mentioned,12 tenocytes from the degenerated torn ends of rotator cuff tendons in older patients might respond to PRP
very differently from those of normal hamstring tendons
in young patients. To the best of our knowledge, no study
has been performed on the effects of PRP on tenocytes
from human rotator cuff tendons with degenerative tears.
The purpose of this study was to determine the effects of
PRP gel on the proliferation, matrix gene expression, glycosaminoglycan (GAG), and total collagen synthesis of tenocytes
from human rotator cuff tendons with degenerative tears. Our
hypothesis was that PRP gel would promote tenocyte proliferation and induce matrix gene expression and synthesis.
MATERIALS AND METHODS
Isolation and Expansion of Tenocytes From Human
Rotator Cuff Tendons
After informed consent was obtained, tendon tissues were
obtained from patients undergoing arthroscopic rotator
cuff repair for the treatment of degenerative rotator cuff
tears (n = 9). Pieces of tissue 3 X 3 mm in size were
obtained after debriding the severely frayed portion of
the lateral edge of rotator cuff tendons with a basket forceps. The study protocol was approved by the institutional
review board at our institution. All 9 patients had shoulder
pain with an insidious onset and no history of trauma.
Tendon tissue was harvested after the debridement of
severely frayed tissue from torn free ends using a basket
The American Journal of Sports Medicine
forceps. After removal of the overlying bursal or synovia!
tissue, the tendon specimens were washed twice in calcium- and magnesium-free phosphate-buffered saline
(DPBS) and finely minced. Cells were released by treating
with 0.3% collagenase II for 2 hours in low-glucose Dulbecco's
modified Eagle medium (LG DMEM) containing antibiotic
solution (100 U/mL penicillin and 100 fig/mL streptomycin)
with gentle agitation. After the same volume of DPBS was
added, undigested tissue was removed using a 100-fim nylon
sieve, and cells were collected by centrifugation, washed
twice, resuspended in LG DMEM supplemented with 10%
fetal bovine serum (FBS) and antibiotic solution (growth
medium), and plated in 100-mm tissue culture dishes at
a density of 2 to 5 X 104 cells/cm2 at 37°C in a humidified
5% C02 atmosphere. The medium was replaced twice
weekly. When cells reached 60.% to 80% confluence, they
were detached by incubation for 10 minutes with 0.25% trypsin (Welgene, Daegu, Korea), washed, and then replated at
a ratio of 1:3. Cells from the second passage to fifth passage
were used in the study.
Preparation of PRP Gel
Platelet-rich plasma (n = 9) was obtained from patients
undergoing arthroscopic rotator cuff repair for the treatment of degenerative rotator cuff tears using a plateletpheresis system with a leukoreduction set (COBE Spectra LRS
Turbo, Caridian BCT, Lakewood, Colorado) according to
a previously described standard collection program.25 The
target concentration of platelets in the final product was
1400 X 10s platelets/pJL. The system was set and primed
with saline solution and anticoagulant acid citrate dextrose as the anticoagulant according to the manufacturer's
instructions. Aliquots were taken to determine complete
blood counts. For the application experiments, platelet
counts in PRP were first adjusted with platelet-poor
plasma (PPP) to 1000 X 10splatelets/^L25 and then further
diluted or concentrated if necessary. To produce a gel from
prepared PPP or PRP, 10% calcium gluconate with or without 166.7 IU/mL thrombin (thrombin-lyophilized power of
5000 IU) (Reyon Pharmaceutical, Seoul, Korea) was added
to PPP or PRP at 1:10 (vol/vol). The dilution and gelling
procedure was performed immediately before experiments.
Throughout the experiments, cells were treated with PPP,
PRP activated with calcium (PRP-Ca), and PRP activated
with calcium and thrombin (PRP-Ca-Thr). Cells treated
with only 2% FBS were used as controls.
Assay for Tenocyte Proliferation
Cells were seeded at a density of 1 X 10s cells/cm2 in the
bottom of 24-well plates with cell culture inserts (SPL Lifesciences, Pocheon, Korea) and were allowed to attach for
24 hours in LG DMEM supplemented with 2% FBS and
antibiotic solution. Platelet-rich plasma gels (10% vol/vol)
at platelet concentrations of 100, 200, 400, 800, 1000,
2000, 4000, 8000, or 16,000 X 10s cells/ixL activated with
10% calcium gluconate with or without 166.7 IU/mL bovine
thrombin were then placed on the cell culture insert of
Vol. 40, No. 6, 2012
each well. Media were replaced every 3 days. Cell proliferation was determined using the WST colorimetric assay
(EZ-CyTox, Daeil Lab Service, Seoul, Korea) at days Y
and 14, All experiments were performed in triplicate.
Assay for Matrix Gene Expression
Cells were seeded at a density of 3 X 10s cells/cm2 in the
bottoms of 6-well plates with cell culture inserts (SPL Lifesciences) and allowed to attach for 24 hours. Platelet-poor
plasma or PRP gels (10% vol/vol) at a platelet concentration of 1000 X 103 cells/pi activated with 10% calcium gluconate with or without 166.7 IU7mL bovine thrombin were
placed on the cell culture insert of each well. Media were
replaced at 2, 7, and 9 days. Matrix gene expression was
evaluated using real-time reverse transcriptase polymerase chain reaction (RT-PCR) at days 7 and 14.
Total RNA was extracted, and reverse transcription and
amplification were performed as previously described.26
Briefly, total RNA was extracted using a Qiagen RNeasy
•mini kit (Qiagen, Hilden, Germany) and quantified using
a NanoDrop ND-100 spectrophotometer (NanoDrop, Wilmington, Delaware). First-strand complementary DNA
(cDNA) was synthesized using the Superscript III Reverse
Transcription kit (Invitrogen, Carlsbad, California).
Briefly, first-strand cDNA was synthesized from cellular
mRNAs (1 jxg) by heating a mixture (1 p,g mRNA, 1 |xL Oligo(dT)20 [50 uM], 1 pi dNTP [10 mM], and up to 10 pi
DW) to 65°C for 5 minutes, cooling on ice for 1 minute,
and then adding a mixture containing 2 |xL 10 X RT buffer,
4 pi MgCl2 (25 mM), 2 pi DTT (0.1 M), 1 pL RNaseOut
(40 U/mL), and 1 |xL Superscript III Reverse Transcriptase
(200 U/mL) (Invitrogen). The reaction mixture was held at
50°C for 50 minutes to promote cDNA synthesis, and the
reaction was terminated by heating to 85°C for 5 minutes
and then snap cooling at 0°C for 1 minute. Finally,
RNase H (1 pL, 2 U/mL) was added and incubated at
37°C for 20 minutes to remove RNA strands from
RNA-cDNA hybrids. Synthesized cDNA was either stored
at -20°C or used immediately for real-time RT-PCR.
To perform real-time RT-PCR utilizing a LightCycler
480 (Roche Applied Science, Mannheim, Germany), TaqMan Gene Expression Assays (Applied Biosystems, Foster
City, California) were used as a probe/primer set specified
for type I collagen (assay ID: Hs00164004_ml), type III collagen (assay ID: Hs00943809_ml), scleraxis (assay ID:
Hs03054634_gl), decorin (assay ID: Hs00266491_ml),
tenascin-C (assay ID: 1115665_ml), and GAPDH (assay
ID: Hs99999905_ml). The PCRs were performed in a final
volume of 20 pi containing 10 p,L 2X LightCyclerH 480
Probes Master (FastStart Taq DNA polymerase, reaction
buffer, dNTP mix [with dUTP instead of dTTP], and
6.4 mM MgCl2) (Roche Applied Science), 1 pL TaqMan
Gene Expression Assay (Applied Biosystems), 5 jxL cDNA
as the template, and 4 pL H2O using the following program: 95°C for 10 minutes, 40 cycles at 95°C for 10 seconds, and 60°C for 1 minute, followed by 72°C for 4
seconds, and a final cooling at 40°C for 30 seconds. Experiments were performed in triplicate, and averaged values
PRP in Rotator Cuff Tendons
1037
were calculated for normalized expression levels. During
PCR amplification, amplified product amounts were monitored by continuous measurement of fluorescence. Gene
expressions were normalized versus GAPDH as follows:
the cycle number at which the transcript of each gene
was detectable (threshold cycle, Ct) was normalized
against the Ct of GAPDH, which is referred to as ACt.
Gene expressions relative to GAPDH are expressed as
2
, Where ACt = CT gene of interest ~ CT GAPDH-
Assay for Total Collagen and GAG Synthesis
To assay total collagen and GAG synthesis, cells were
seeded at a density of 3 X 10s cells/cm2 in the bottom of
6-well plates with cell culture inserts (SPL Lifesciences)
and allowed to attach for 24 hours. A PRP gel (10% vol/
'vol) with a platelet concentration of 1000 X 10s cells/pi
activated with 10% calcium gluconate with or without
166.7 lU/mL bovine thrombin was added to cell culture
inserts in wells. All assays were performed in culture
supernatants and in triplicate at days 7 and 14.
Total soluble collagen was measured in culture supernatants using the Sircol assay (Biocolor, Newtownabbey,
Northern Ireland) according to the manufacturer's instructions. Briefly, 900 pL of Sirius Red reagent was added to
100 pi of supernatant and mixed for 30 minutes at room
temperature. The collagen-dye complex was precipitated
by centrifugation at 12,000 rpm for 10 minutes and washed
with 750 pi of ice-cold Acid-Salt Wash Reagent provided
with the kit (Biocolor). Wash reagent was used to remove
unbound dye from the surface of pellets and the inside surface of microcentrifuge tubes. After centrifugation at
12,000 rpm for 10 minutes, supernatants were discarded,
and pellets were dissolved in the acidic solution provided.
Absorbance was measured at 555 nm. The calibration
curve was set up on the basis of a collagen standard provided by the manufacturer.
Glycosaminoglycan amount was measured using the
Blyscan 1,9-dimethylmethylene blue (DMMB) assay kit
(Biocolor) according to the manufacturer's instructions.
Briefly, Blyscan dye reagent (500 pi) was added to supernatants (500 pi) and the kit standard and mixed for 30
minutes at room temperature. Insoluble pellets of sulfated
GAG were precipitated by centrifugation at 12,000 rpm for
10 minutes. Bound dye was released with the dissociation
reagent (500 pi), and the absorbance was measured at
656 run. The calibration curve was set up on the basis of
a collagen standard provided by the manufacturer.
Statistical Analysis
All data values were tested for normality using the
Shapiro-Wilk test and expressed as means and standard
deviations. The significances of differences were determined using the independent t test and 1-way analysis of
variance. For post hoc analysis, the Dunnett test was
used for comparison with controls, whereas the Tukey
test was used for comparisons of PPP-, PRP-Ca-, and
The American Journal of Sports Medicine
1038 Jo et al
FBS
PPP
100
1,000
2,000.
4,000
200
8,000
400
800
16,000 (x103 platelets/jil)
Figure 1. Tenocytes from human rotator cuff tendons with degenerative tears. Cells were cultured for 14 days with a platelet-rich
plasma gel (10% vol/vol) at platelet concentrations of 100, 200, 400, 800, 1000, 2000, 4000, 8000, and 16,000 X 103 cells/|xL
Platelet-rich plasma was activated with 10% calcium gluconate and placed on a cell culture insert in each well. Note that cell
proliferation increased with platelet concentration and plateaued at around 4000 X 10s cells/jLL No definite morphological
change was observed during culture. FBS, fetal bovine serum; PPP, platelet-poor plasma.
PRP-Ca-Thr-treated cells. P < .05 was considered to be
statistically significant.
RESULTS
Characteristics of Rotator Cuff Tears and PRP
The average age of the 9 patients from whom tendons were
harvested was 57.8 ± 11.9 years (range, 39-69 years), and
there were 4 men and 5 women. Average anteroposterior
size and mediolateral retraction were 31.6 ± 18.2 mm
and 16.2 ± 14.4 mm, respectively.
The average age of the 9 patients from whom PEP was
prepared was 52.7 ± 19.2 years (range, 23-69 years). Platelet, red blood cell (REG), and white blood cell (WBC) counts
were determined using a fully automated analyzer (XE2100, Sysmex, Kobe, Japan). Platelet counts increased
from 199.00 ± 36.93 (X 103 platelets/pi) in whole blood
to 956.22 ± 55.12 in PRP, a 4.9-fold increase from baseline
(P < .001). Mean RBC and WBC counts reduced from
4.48 ± 0.31 and 6.11 ± 1.56 in whole blood to 0.15 ±
0.06 and 0.01 ± 0.01 in PRP, respectively (P < .001). The
average concentration of fibrinogen in PRP was 197.05 ±
10.55 mg/dL.
Effect of PRP on the Proliferation of Tenocytes From
Tendons With Degenerative Tears
Platelet-rich plasma activated with calcium and PRP-CaThr significantly stimulated the proliferation of tenocytes
in a dose-dependent manner in comparison with the control, whereas PPP did not (Figure 1). No definite morphological change was observed during culture. The addition
of thrombin for activation showed a tendency of advancing
the platelet concentration for a cell proliferation plateau
(Figure 2). At day 7, PRP-Ca with a platelet concentration
of 4000 X 103/|xL showed the greatest proliferation by
4.98-fold compared with the control (Figure 2A). However, cell proliferation plateaued statistically at concentrations over 2000 X 103/n,L. For PRP-Ca-Thr, the
greatest proliferation was observed at a concentration of
8000 X 10s/|xL, an increase of 4.44-fold compared with
the control. A proliferation plateau was reached at a concentration of 400 X 108/|xL. The activation of PRP with
calcium and thrombin further promoted proliferation at
the lower concentrations of 100, 200, and 400 X 103/|xL.
At day 14, the highest proliferation was observed at a concentration of 8000 cells X 103/pJL for PRP-Ca and at
16,000 cells X 10s/jiL for PRP-Ca-Thr, which represented
increases of 5.70- and 5.41-fold, respectively (Figure 2B).
At concentrations of 4000 and 2000 X 103/fi,L, proliferation plateaued for PRP-Ca and PRP-Ca-Thr, respectively. The addition of thrombin further enhanced
proliferations at the lower concentrations of 100, 200,
400, and 800 X 103/|xL.
Effect of PRP on Matrix Gene Expression of Tenocytes
From Tendons With Degenerative Tears
Platelet-rich plasma activated with calcium and PRP-CaThr significantly induced the gene expression of type I collagen compared with the control at day 7, whereas PPP did
not (Figure 3). Platelet-rich plasma activated with calcium
and PRP-Ca-Thr upregulated the gene expression of type I
collagen by 2.32- and 3.58-fold, respectively (P = .034 and
P < .001, respectively). At day 14, although PRP-Ca and
PRP-Ca-Thr increased the gene expression of type I
Vol. 40, No. 5, 2012
PRP in Rotator Cuff Tendons
1039
18 i
16 4
'
• Control
•E §10
?? S
• PPP
iPRPw/Ca
• PRPw/Ca-Thr
i-l
li 6 1
1
cc
Control PPP
100
200
400
800
1,000 2,000 4,000 8,000 16,000
(x10> platelets/pi)
4 2
0
Day 7
Control PPP
100
200
400
800
1,000 2,000 4,000
8,000 16,000
(x10>platelets/iil)
Figure 2. Relative cell proliferation measured using a WST
colorimetric assay (EZ-CyTox assay, Daeil Lab Service,
Seoul, Korea). Cells were cultured for 14 days with a platelet-rich plasma (PRP) gel (10% vol/vol) at platelet concentrations of 100, 200, 400, 800, 1000, 2000, 4000, 8000, and
16,000 X 103 cells/p.L Platelet-rich plasma was activated
with calcium only (PRP-Ca) or with calcium plus thrombin
(PRP-Ca-Thr). (A) At day 7, cell proliferation increased in
a dose-dependent manner. The greatest proliferation was
4.98-fold at a concentration of 4000 cells X 103/|xL for
PRP-Ca and 4.44-fold at a concentration of 8000 cells X
103/|xL for PRP-Ca-Thr. The addition of thrombin further
enhanced cell proliferation at lower concentrations of 100,
200, and 400 cells X 103/(j,L compared with cells activated
with calcium only, resulting in moving up the proliferation plateau from 2000 cells X 103/(jU- for PRP-Ca (solid line) to 400
cells X 103/|xl_ for PRP-Ca-Thr (dotted line). (B) At day 14,
cell proliferation also increased in a dose-dependent manner.
The greatest proliferation was 5.70-fold at a concentration of
8000 cells X 103/fjLl_ for PRP-Ca and 5.41 -fold at a concentration of 8000 cells X 103/|xLfor PRP-Ca-Thr. Thrombin further
stimulated cell proliferation at lower concentrations of 100,
200, 400, and 800 cells X 103/|xL and advanced the cell proliferation plateau from a concentration of 4000 cells x 1 o3/|j,L
for PRP-Ca (solid line) to 2000 cells X 103/|xLfor PRP-Ca-Thr
(dotted line).
collagen compared with the control, no statistically significant difference was found because of a large variation
between samples.
The gene expression of type III collagen was significantly induced by PRP-Ca and PRP-Ca-Thr compared
with the control at days 7 and 14, whereas PPP did not
(Figure 4). At day 7, PRP-Ca and PRP-Ca-Thr upregulated
the gene expression of type III collagen by 3.57- and
Day 14
Figure 3. Gene expression level of type I collagen measured
by real-time reverse transcriptase polymerase chain reaction.
Relative quantifications were calculated by dividing the
mRNA expression level in cells treated with platelet-rich
plasma (PRP) by that in control cells. At day 7, PRP activated
with calcium (PRP-Ca) and PRP activated with calcium and
thrombin (PRP-Ca-Thr), but not platelet-poor plasma (PPP),
significantly upregulated type I collagen expression by
2.32- and 3.58-fold, respectively (P = .034 and P < .001,
respectively). At day 14, although PPP, PRP-Ca, and PRPCa-Thr also enhanced expression by 3.00-, 5.97-, and
7.40-fold, respectively, there were no statistical significances. P values were calculated as compared with the control.
i Control
i PPP
i PRP w/ Ca
• PRP w/ Ca-Thr
Day 7
Day 14
Figure 4, Gene expression level of type III collagen measured
by real-time reverse transcriptase polymerase chain reaction.
Relative quantifications were calculated by dividing the mRNA
expression level in cells treated with platelet-rich plasma (PRP)
by that in control cells. At day 7, PRP activated with calcium
(PRP-Ca) and PRP activated with calcium and thrombin
(PRP-Ca-Thr), but not platelet-poor plasma (PPP), significantly
upregulated type 111 collagen expression by 3.57- and 5.23fold, respectively (all P < .001). At day 14, PRP-Ca and
PRP-Ca-Thr increased the gene expression by 3.24- and
3.93-fold, respectively (P = .003 and P < .001, respectively).
P values were calculated as compared with the control.
5.23-fold, respectively (all P < .001), and at day 14, these
gene expressions were upregulated by 3.24- and 3.93-fold,
respectively (P = .003 and P < .001, respectively).
The American Journal of Sports Medicine
1040 Jo et al
'*'
1
P=.004
10 -
™01
T
9 -
• Control
• PPP
• PRP w/ Ca
• PRP w/ Ca-Thr
if"
11::
II
„
• Control
• PPP
• PRP w/ Ca
• PRP w/Ca-Thr
"ULl
o\
Day 7
Day 14
Day 7
Figures. The ratio of the gene expression of type III to I collagen. Both at days 7 and 14, platelet-rich plasma (PRP) activated with calcium and PRP activated with calcium and
thrombin did not significantly change the ratio of type 11 I/I collagen expression level compared with the control. PPP,
platelet-poor plasma.
Day 14
Figure 7, Gene expression level of tenascin-C measured
using real-time reverse transcriptase polymerase chain reaction. Relative quantifications were calculated by dividing the
mRNA expression level in cells treated with platelet-rich
plasma (PRP) by that in control cells. The gene expression
of tenascin-C was significantly upregulated with PRP activated with calcium and thrombin only at days 7 and 14 in
comparison with the control by 6.20- and 5.28-fold at days
7 and 14, respectively (P < .001 and P = .004, respectively).
P values were calculated as compared with the control. PPP,
platelet-poor plasma.
• Control
• PPP
P=.035
» PRP w/ Ca
• PRP w/ Ca-Thr
P=.033
i Control
Day 7
• PPP
• PRPw/Ca
• PRP w/Ca-Thr
Day 14
Figure 6. Gene expression level of decorin measured using
real-time reverse transcriptase polymerase chain reaction. Relative quantifications were calculated by dividing the mRNA
expression level in cells treated with platelet-rich plasma
(PRP) by that in control cells. Platelet-poor plasma (PPP),
PRP activated with calcium, and PRP activated with calcium
and thrombin significantly upregulated the gene expression of
decorin at day 14 by 1.58-, 1.86-, and 1.73-fold, respectively
(P = .016, P < .001, and P = .003, respectively). P values
were calculated as compared with the control.
Platelet-poor plasma, PRP-Ca, and PRP-Ca-Thr did not
significantly elevate the ratio of type III/I collagen expression at days 7 and 14 compared with, the control (Figure 5).
Platelet-poor plasma, PRP-Ca, and PRP-Ca-Thr significantly upregulated the gene expression of decorin compared with the control at day 14 but not at day 7 (Figure
6). At day 14, PPP, PRP-Ca, and PRP-Ca-Thr upregulated
the expression of decorin by 1.58-, 1.86-, and 1.73-fold,
respectively (P = .016, P < .001, andP = .003, respectively).
For tenascin-C expression, PRP-Ca-Thr significantly
enhanced by 6.20- and 5.28-fold in comparison with the
controls at days 7 and 14, respectively (P < .001 and
P = .004, respectively), whereas PPP and PRP-Ca did not
Day 7
Day 14
Figure 8. Gene expression level of scleraxis measured using
real-time reverse transcriptase polymerase chain reaction.
Relative quantifications were calculated by dividing the
mRNA expression level in cells treated with platelet-rich
plasma (PRP) by that in control cells. At day 7, there was
no significant difference between the 4 groups. Platelet-rich
plasma activated with calcium (PRP-Ca) and PRP activated
with calcium and thrombin (PRP-Ca-Thr), but not plateletpoor plasma (PPP), significantly increased the gene expression of scleraxis by 2.52- and 2.51-fold with PRP-Ca and
PRP-Ca-Thr at day 14, respectively (P = .033 and P = .035,
respectively). P values were calculated as compared with
the control.
(Figure 7). Platelet-rich plasma activated with calcium
and PRP-Ca-Thr significantly increased the gene expression of scleraxis at day 14 by 2.52- and 2.51-fold with
PRP-Ca and PRP-Ca-Thr, respectively (P = .033 and
P = .035, respectively), whereas PPP did not (Figure 8).
Vol. 40, No. 6, 2012
PEP in Rotator Cuff Tendons 1041
Control
PPP
PRP w/ Ca
PRPw/Ca-Thr
Day 7
Day 14
Figure 9. Total collagen synthesis measured using the Sjrcol
assay (Biocolor, Newtownabbey, Northern Ireland). Total collagen synthesis significantly increased with platelet-poor
plasma (PPP), platelet-rich plasma activated with calcium
(PRP-Ca), and PRP activated with calcium and thrombin
(PRP-Ca-Thr) at days 7 and 14. At day 7, total collagen production increased by 2.81-, 1.84-, and 1.94-fold with PPP,
PRP-Ca, and PRP-Ca-Thr, respectively (P < .001, P = .007,
and P = .003, respectively) when compared with the control.
At day 14, total collagen production also increased by 1.94-,
1.47-, and 1.53-fold with PPP, PRP-Ca, and PRP-Ca-Thr,
respectively (P < .001, P = .026, and P = .011, respectively).
P values were calculated as compared with the control.
Effect of PRP on Total Collagen and GAG Synthesis
Platelet-poor plasma, PEP-Ca, and PRP-Ca-Thr significantly increased total collagen production compared with
the control at days 7 and 14 (Figure 9). At day 7, total collagen production was increased by 2.81-, 1.84-, and 1.94fold with PPP, PRP-Ca, and PRP-Ca-Thr, respectively
(P < .001, P = .007, and P = .003, respectively) when compared with the control. At day 14, total collagen production
also increased by 1.94-, 1.47-, and 1.53-fold with PPP, PRPCa, and PRP-Ca-Thr, respectively (P < .001, P = .026, and
P = .011, respectively) when compared with the control.
Glycosaminoglycan synthesis was significantly enhanced
with PRP-Ca and PRP-Ca-Thr at day 14, whereas PPP
showed no significant effects (Figure 10). At day 7, GAG
synthesis was not significantly different with PPP, PRPCa, or PRP-Ca-Thr treatment. At day 14, GAG synthesis
significantly increased by 1.93- and 1.88-fold with PRP-Ca
and PRP-Ca-Thr compared with the control, respectively
(P = .015 and P = .022, respectively).
DISCUSSION
The results of the present study demonstrate that PRPactivated calcium with or without thrombin significantly
stimulated the proliferation of tenocytes from human rotator cuff tendons with degenerative tears in a dose-dependent manner. Platelet-rich plasma—activated calcium
with or without thrombin also significantly upregulated
the gene expressions of type I and III collagen but did
Control
PPP
PRPw/Ca
PRP w/ Ca-Thr
Day 7
Day 14
Figure 10. Glycosaminoglycan (GAG) synthesis measured
using the Blyscan assay (Biocolor, Newtownabbey, Northern
Ireland). At day 7, there was no significant difference
between the 4 groups. At day 14, GAG synthesis significantly
increased by 1.93- and 1.88-fold with platelet-rich plasma
(PRP) activated with calcium and PRP activated with calcium
and thrombin, respectively (P = .015 and P = .022, respectively). P values were calculated as compared with the control. PPP, platelet-poor plasma.
not significantly influence the ratio of type III/I collagen
expression level. Platelet-rich plasma significantly induced
the gene expressions of decorin, a representative proteoglycan of tendon, and of tenascin-C, a representative glycoprotein of tendon.41'42 In addition, PRP significantly
upregulated the gene expression of scleraxis, a tendon-specific marker.49'54 Furthermore, the syntheses of total collagen and GAG were also significantly enhanced with PRP.
However, gene expression levels and the amount of matrix
molecules synthesized varied considerably by culture time
and activation method. Meanwhile, PPP neither stimulated the proliferation of tenocytes nor induced the expression or synthesis of tendon matrix except for decorin at day
14 and total collagen, suggesting the importance of bioactive materials released from granules in the platelet. The
addition of thrombin for platelet activation significantly
accelerated cell proliferation especially at lower concentrations compared with the addition of calcium only and demonstrated a tendency of lowering the platelet concentration
for reaching the proliferation plateau. Meanwhile, it did
not significantly affect matrix gene expression and synthesis except for tenascin-C. Considering that final cell proliferation level is more influenced by the proliferation rate in
the earlier culture period than in the later period, and that
matrix gene expression and synthesis were not significantly affected except for tenascin-C, these results would
be because of the initial burst release of growth factors
with the addition of thrombin.34'85 Taken together, the
results of the present study suggest that PRP has positive
effects on the proliferation, matrix gene expression, and
synthesis of tenocytes from degenerated torn tendons and
that the degrees of these effects are dependent on the
kind of matrix molecules, culture period, and the concentration and activation status of PRP. These results suggest
that PRP might be applied usefully in the treatment of
1042 Jo et al
degenerative tendinopathy, whereas its application strategy, such as the concentration, activation method, timing,
and application numbers of PRP should be further
investigated.
In this study, we investigated the effects of PRP on
tenocytes from human rotator cuff tendons with degenerative tears. The in vitro effects of PRP on tenocytes have
been reported previously by a few authors.3'4'12'48'56'60'68
Three of these studies described the effects of PRP on
tenocyte proliferation and/or matrix synthesis.3'4'12 Anitua et al3'4 reported that 20% PRP releasate (vol/vol)
increased cell proliferation and the production of VEGF
and HGP, and de Mos et al12 demonstrated that PRP
releasate increased cell number and total collagen levels
but decreased the gene expressions of type I and III collagen without affecting the HI/I ratio. However, both studies were performed using tenocytes isolated from the
hamstring tendons of healthy young donors3 or children
aged 13 to 15 years.12 It has been reported that tendons
from different sites have different structures, compositions, cell phenotypes, and metabolic characteristics.1'14
Furthermore, the behaviors of tenocytes are known to
depend on donor age, anatomic site or origin, and status;
that is, whether they are obtained from intact, injured, or
degenerated tendons.5'19'29'39'67 A number of authors have
demonstrated that tendons or fibroblasts from intact and
torn tendons16 or ligaments8 also behave differently and
they have suggested that intact tendon tissue is not
appropriate for studying cellular tendon degeneration.16'39 Taken together, it appears that the previous
studies based on the use of tenocytes from the hamstrings
of young, healthy donors might not reflect the biology of
tenocytes from rotator cuff tendons with degenerative
tears in older patients or the responses of tenocytes to
PRP. We consider that the effects of PRP on any specific
disease or injury should be investigated using tissue or
cells from appropriate diseased or injured sites, and
thus, we suggest that the results of this study provide
useful information about potential effects of PRP on the
repair of torn rotator cuffs.
Previous studies on the effects of PRP on tenocytes suffer
from a lack of standardization or from inadequate characterization of PRP preparations, which result in interstudy
differences in platelet concentrations, activations, and
WBC contamination levels. Furthermore, these factors
could affect the concentrations and the release kinetics of
growth factors, which make it difficult to compare study
results and could explain contradictory results. Accordingly,
in the present study, we used a plateletpheresis system to
prepare PRP,25 which, we believe, provides the most consistent and reproducible PRP with minimal WBC contamination. Furthermore, we agree with Zimmermann et al69
that this type of preparation provides the only practical
means of elucidating the platelet-dependent and leucocyteindependent mechanisms underlying the clinical effects of
PRP.
The results of this study show that PRP stimulated
tenocyte proliferation in a dose-dependent manner. Moreover, PRP was found to have no inhibitory effect on cell
proliferation at levels of more than 50-fold over the
The American Journal of Sports Medicine
physiological level. A number of studies have addressed
the effect of PRP concentration on the proliferation of
osteoblasts, periodontal cells, or mesenchymal stem
cells.21'22'24'33'87'53 Some authors have reported that PRP
at higher concentrations did not further promote or even
suppressed proliferation.10'20'24'28 On the other hand,
only a few studies have investigated the effect of different
concentrations of PRP on the proliferation of tendon fibreblasts.5'12 We agree with Anitua et al5 that the biological
effects of PRGF may depend on platelet concentration
and on the anatomic origins of cells. In the current study,
we tested a variety of platelet concentrations from 100 to
16,000 cells X 103/jiL and examined 2 different activation
methods, that is, calcium and calcium plus thrombin. Considering that physiological platelet concentrations range
from 150 to 300 cells X 108/[juL,13 the concentrations used
in the study correspond to approximately 0.5- to more
than 50-fold over the physiological levels, which would
seem to be sufficient for simulating clinical situations. In
addition, considering that the in vivo half-life of platelets
is about 7 days,13 some authors who reported negative
effects for PRP on cell proliferation may have investigated
too short a period, 3 to 7 days, to examine the effects of
PRP on cell proliferation.10'20'24'28 Our results support this
suggestion, as cell proliferation plateaued at a lower concentration, 400 cells X 108/(j,L at day 7, whereas at day 14, cell
proliferation was highest at a concentration of 2000 cells X
103/|xL.
Several studies have examined matrix molecular
changes in rotator cuff tendons with chronic tendinopathy.31'41'43"45'65 In one study of ruptured supraspinatus
tendons, a significant reduction in total collagen content
and an increase in the expression of type I and II collagen
with an increase in type III/I ratio were found.45 However,
relatively little is known about changes in proteoglycans
and glycoprotein.31'43'44'55 In the case of proteoglycan,
a generalized increase in the amounts of hyaluronan and
sulfated GAG has been reported,44 but little is known of
other proteoglycans.41 Lo et al31 reported a significant
increase in aggrecan and a significant decrease of decorin
in ruptured rotator cuff tendons. Glycoproteins such as
tenascin-C and fibronectin are known to be increased in
ruptured tendons.43'55 On the other hand, the results of
the present study show that PRP has some reversing or
recovering effects, at least in part, to the above changes
in chronic tendinopathy and tendon rupture on tenocytes
from human rotator cuff tendons with degenerative tears;
that is, in the present study, PRP significantly increased
the gene expression of type I collagen but maintained the
type III/I ratio, stimulated total collagen synthesis, and
enhanced the gene expression of decorin. These results,
along with the observed increased cell proliferation and
the increased expression of scleraxis, a tendon-specific
marker,50 suggest that PRP could reverse matrix molecular changes caused by tendon degeneration.
Type III collagen is present in normal tendons (less than
10% of total collagen) and is weaker than type I collagen. It
is characterized by a small fibril in a woven pattern that is
deficient in cross-hiking.58 Type III collagen is synthesized
as a repair response to tissue injury, especially in adhesive
^
Vol. 40, No. S, 2012
or scar tissue, in a larger amount up to 20% to
30%.15'86'63'66 This increase of type III collagen, especially
relative to type I collagen, could inhibit the growth of collagen fibrils, lead to adhesion and contracture formation,2''7'65
and may result in the tendon being less resistant to stress
and thus at increased risk of rupture.9'32'45 Some authors
have reported that larger amounts of type III collagen production in the supraspinatus tendon are closely associated
with shoulder contracture57 and that the maintenance of
type HI collagen expression level is important for avoiding
excessive scar formation in injured rotator cuff tendons,
which suggests that type HI collagen production in injured
rotator cuff tendons is associated with scar and adhesion formation rather than healing.65 In this respect, the results of
the present study regarding increased type I collagen expression without an increase in type HI/I ratio suggest that PRP
has a positive influence on regeneration rather than on healing via scarring on tenocytes from rotator cuff tendons with
degenerative tears.
One limitation of the present study is that no control
tenocytes from normal healthy tendons were included.
Practically, it is difficult to obtain normal tendons
from age-matched patients without a rotator cuff tear.
However, other studies have used cells from normal tendons, and thus, some comparison is possible. Considering
batch-to-batch variation, especially in the gene expression
of type I collagen, further studies considering the control
and chronicity of tears, such as size, would be necessary.
In addition, not all cells isolated from torn degenerative
rotator cuff tendons would be tenocytes. There may be
other cells such as endothelial cells, inflammatory cells,
and so on. However, we harvested small pieces of tendon
after removing overlaying synovium and then finely
minced them. During the procedure, any suspicious tissue
other than tendon was removed. So the majority of cells
used in the study would be tenocytes. Another limitation
is that we did not normalize the measurements of total
collagen and GAG synthesis with DNA or total protein.
Nonetheless, it does not seem to significantly affect the
results, as we performed the assays in subconfluent densities, which did not allow cells to proliferate significantly
further because of contact inhibition. Another limitation
is that we used allogenic PRP, whereas autologous PRP
is usually used in clinical practice. This was because of
the time for obtaining an adequate number of tenocytes,
which usually takes several weeks. Nevertheless, we consider that allogenic and autologous PRP are likely to be
similar in an in vitro setting, as it is unlikely that an
immune reaction affected our experimental result considering that platelet concentrates can be intravenously
infused without ABO blood matching. Another possible
limitation is that we used PRP gels without further
replacement, as we simulated a single PRP application.
However, no consensus has been reached regarding the
method of PRP use for the treatment of rotator cuff disease. We believe that multiple replacement of PRP with
growth medium might exaggerate the effects of PRP and
would not be a good starting point. Based on this study,
additional studies are required simulating different clinical situations.
PRP in Rotator Cuff Tendons
1043
In conclusion, this study demonstrates that PRP promotes the proliferation of tenocytes from human rotator
cuff tendons with degenerative tears and that it enhances
the gene expression and the synthesis of tendon matrix.
The results of this study indicate that PRP might offer
a useful biological strategy for promoting the regeneration
of rotator cuff tears.
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Axial Load-Bearing Capacity of an
Osteochondral Autograft Stabilized
With a Resorbable Osteoconductive
Bone Cement Compared With a
Press-Fit Graft in a Bovine Model
Marc-Olivier Kiss,*1 DMV, MD; Annie Levasseur,* MSc, Yvan Petit,** PhD,
and Patrick Lavigne,*t§ MD, PhD
Investigation performed at the Universite de Montreal, Montreal, Canada
Background: Osteochondral autografts in mosaicplasty are inserted in a press-fit fashion, and hence, patients are kept nonweightbearing for up to 2 months after surgery to allow bone healing and prevent complications. Very little has been published
regarding alternative fixation techniques of those grafts.
Hypothesis: Osteochondral autografts stabilized with a resorbable osteoconductive bone cement would have a greater loadbearing capacity than standard press-fit grafts.
Study Design: Controlled laboratory study.
Methods: Biomechanical testing was conducted on 8 pairs of cadaveric bovine distal femurs. For the first 4 pairs, 6 single osteochondral autografts were inserted in a press-fit fashion on one femur. On the contralateral femur, 6 grafts were stabilized with
a calcium triglyceride osteoconductive bone cement. For the 4 remaining pairs of femurs, 4 groups of 3 adjacent press-fit grafts
were inserted on one femur, whereas on the contralateral femur, grafts were cemented. After a maturation period of 48 hours,
axial loading was applied on all single grafts and on the middle graft of each 3-in-a-row series.
Results: For the single-graft configuration, median loads required to sink the press-fit and cemented grafts by 2 and 3 mm were
281.87 N versus 345.56 N (P = .015) and 336.29 N versus 454.08 N (P = .018), respectively. For the 3-in-a-row configuration,
median loads required to sink the press-fit and cemented grafts by 2 and 3 mm were 260.31 N versus 353.47 N (P = .035)
and 384.83 N versus 455.68 N (P = .029), respectively.
Conclusion: Fixation of Osteochondral grafts using bone cement appears to improve immediate stability over the original mosaicplasty technique for both single- and multiple-graft configurations.
Clinical Relevance: Achieving greater primary stability of Osteochondral grafts could potentially accelerate postoperative recovery, allowing early weightbearing and physical therapy.
Keywords: articular cartilage; knee; autografting; transplantation; bone cement
Focal cartilage defects are a frequent joint injury. Fortunately, different management options can be offered to
patients presenting with. such, a condition. Mosaicplasty,
first described by Matsusue et al24 and later popularized
by Hangody et al,15 is one of the reconstructive procedures
available. This technique, which relies on press-fit insertion of cylindrical Osteochondral autografts, is usually recommended for cartilage lesions of up to 4 cm2.16 According
to published case series, good to excellent clinical results
are achieved in 69% to 96% of patients, depending on the
injury and lesion location.7'11'13'16'23'31. Mosaicplasty is
technically challenging for surgeons, and various factors
will dictate the end results, such as preserving graft chondrocytes' viability as well as re-establishing and maintaining joint congruency until definitive bony integration of the
graft.3'10'19'28'84 Histological analyses from in vivo animal
§Address correspondence to Patrick Lavigne, MD, PhD, Universite de
Montreal, Faculty of Medicine, Orthopaedic Division, 5345 #320 Assomption Boulevard, Montreal, Quebec, H1T4B3 Canada (e-mail: lavigne
[email protected]).
Imaging and Orthopedic Research Laboratory (LIO), Research Center, Hopital du Sacre-Coeur de Montreal, Montreal, Canada.
tOrthopaedic Division, Faculty of Medicine, Universite de Montreal,
Montreal, Canada.
*Department of Mechanical Engineering, Ecole de Technologie
Superieure, Montreal, Canada.
One or more of the authors has declared the following potential conflict of interest or source of funding: A research grant of Canadian $3000
was obtained from FREOM (Montreal Foundation for Research and Education in Orthopedics). The cement used in this study was provided free
of charge by a local representative of the Doctors Research Group.
The American Journal of Sports Medicine, Vol. 40, No. 5
DO1: 10.1177/0363546512438382
©2012 The Author(s)
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