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Platelet-Rich Plasma in the Management of

Articular Cartilage Pathology: A Systematic
Review
ARTICLE in CLINICAL JOURNAL OF SPORT MEDICINE: OFFICIAL JOURNAL OF THE CANADIAN ACADEMY OF SPORT
MEDICINE NOVEMBER 2013
Impact Factor: 2.27 DOI: 10.1097/01.jsm.0000432855.85143.e5 Source: PubMed

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Supplementation With Platelet-Rich Plasma Improves the

In Vitro Formation of Tissue-Engineered Cartilage With
Enhanced Mechanical Properties
Massimo Petrera, M.D., J. N. Amritha De Croos, Ph.D., Jonathan Iu, B.Sc.,
Mark Hurtig, D.V.M., M.V.Sc., Rita A. Kandel, M.D., F.R.C.P.C., and
John S. Theodoropoulos, M.D., F.R.C.S.C., M.Sc.

Purpose: This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and
biomechanical properties of tissue-engineered cartilage. Methods: Chondrocytes isolated from bovine metacarpalphalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]).
Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the
concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from
a mature cow. After 2 weeks, samples (n 8) were analyzed histologically, biochemically, and biomechanically. Data were
analyzed using the Wilcoxon test (signicance, P < .05). Results: Chondrocytes cultured in 20% PRP formed thicker
cartilage tissue (1.6  0.2 mm) than did cells grown in 20% FBS (0.7  0.008 mm; P .002) and 20% PPP (0.8  0.2 mm;
P .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1  3.6 kPa versus
15.6  1.5 kPa (P .0002) for 20% PPP and 20.4  3.5 kPa (P .007) for 20% FBS. Glycosaminoglycan (GAG) content
was increased in tissues formed in 20% PRP (176  18.8 mg GAG/mg) compared with those grown in 20% FBS (112 
10.6 mg GAG/mg; P .01) or 20% PPP (131.5  14.8 mg GAG/mg; P .11). Hydroxyproline content was similar whether
the media was supplemented with 20% PRP (8.7  0.9 mg/mg), 20% FBS (7.6  0.9 mg/mg; P .37), or 20% PPP (6.4 
1 mg/mg; P .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9  3.5 mg/mg), 20% FBS (9.3 
2.5 mg/mg; P .99), or 20% PPP (7.2  1.3 mg/mg; P .78). Immunostained samples showed prevalence of type II collagen
in tissues formed in the presence of 20% PRP. Conclusions: The presence of PRP in the culture media enhances the
in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. Clinical Relevance: Understanding the in vitro effects of PRP on tissueengineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.

From the University of Toronto Orthopaedic Sports Medicine (M.P., J.S.T.),

Mount Sinai Hospital and Womens College Hospital, Toronto; Department of
Pathology and Laboratory Medicine (J.N.A.D., J.I., R.A.K.), University of
Toronto, BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital,
Toronto; and Department of Biomedical Sciences (M.H.), University of
Guelph, Ontario, Canada.
The authors report the following potential conict of interest or source of
funding in relation to this article: M.H. serves on the board of, and has
received an operating grant from, Canadian Arthritis Network, and has
received payment for educational seminars/laboratories from International
Cartilage Repair Society; J.S.T. has received payment for consultancy from
Zimmer and Smith & Nephew Canada.
Received October 4, 2012; accepted July 9, 2013.
Address correspondence to Massimo Petrera, M.D., University of Toronto
Orthopaedic Sports Medicine, Mount Sinai Hospital, 600 University Avenue,
Ste 476C, Toronto, ON M5G 1X5, Canada. E-mail: massimopetrera@gmail.
com
2013 by the Arthroscopy Association of North America
0749-8063/12655/$36.00
http://dx.doi.org/10.1016/j.arthro.2013.07.259

he goal of tissue engineering in cartilage repair

strategies is to create biomechanically superior
hyaline cartilage that can integrate with host tissue.
One challenge of this approach is the dedifferentiation
of repair cells to a broblast-like phenotype and the
difculty of producing tissue constructs with cells that
exhibit an articular cartilage phenotype.1,2 Studies have
shown that in appropriate culture conditions, cell
phenotype can be modulated, leading to the generation
of cells suitable for cartilage repair.2-4 However, in the
absence of bioactive factors or mechanical stimulation,
this repair tissue has reduced extracellular matrix
(ECM) and inferior mechanical properties compared
with hyaline cartilage.5-7 Certain culture conditions can
lead to improved ECM composition and mechanical
properties.5,7-9 In vitro, growth factors such as transforming growth factoreb1 (TGF-b1), basic broblast
growth factor (FGF), platelet-derived growth factor,

Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 29, No 10 (October), 2013: pp 1685-1692

1685

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M. PETRERA ET AL.

insulin growth factor (IGF), and bone morphogenetic

protein (BMP) have shown the ability to regulate
articular cartilage homeostasis through proanabolic and
anticatabolic actions. In particular, TGF-b1, BMP-2 and
BMP-7, FGF-18, and IGF-1 stimulate chondrocyte
proliferation and synthesis of ECM, whereas TGF-b1,
BMP-2, and FGF-2 decrease the catabolic activity of
interleukin-1 and matrix metalloproteinases.10-14
Platelet-rich plasma (PRP) is a concentrate of platelets
obtained through centrifugation of whole blood that
represents an excellent source of autologous growth
factors. The high concentration of growth factors
released with the degranulation of the alpha granules of
activated platelets promotes regeneration of tissues that
possess low healing capacity. The potential of PRP to
accelerate the healing process in fractures, cartilage
damage, chronic tendinopathies, and ligament and
muscle injuries has resulted in several applications in
orthopaedic surgery.15-20 The use of PRP in the treatment of cartilage injury stems from the hypothesis that
the high concentration of growth factors released by
platelets could potentially accelerate chondrocyte
proliferation and ECM production while at the same
time downregulating catabolic pathways. Clinical applications range from the treatment of focal chondral
lesions to the treatment of diffuse cartilage loss, such as
osteoarthritis.18-20
The use of PRP to enhance cartilage repair has also
been investigated with variable results. Using a rabbit
model, Sun et al.21 showed that osteochondral defects
treated with autologous PRP in a poly-lactic-co-glycolic
acid (PLGA) carrier resulted in better cartilage repair
than when treated with PLGA alone. The authors also
observed increased glycosaminoglycan (GAG) and type
II collagen in the regenerative tissue compared with
the PLGA-treated or the untreated group. Conversely,
Kon et al.22 found that PRP that was added to a
collagen-hydroxyapatite scaffold in a sheep osteochondral defect led to poorly organized subchondral
bone and a highly amorphous cartilage repair tissue,
whereas osteochondral lesions treated with the scaffold
alone presented a newly formed hyaline-like cartilage
repair tissue and well-structured subchondral bone.
We have previously developed a method to form
biphasic tissue-engineered constructs composed of
cartilage tissue integrated into the top surface of a porous
calcium polyphosphate (CPP) substrate.23 After implantation of the biphasic construct into a defect, the CPP
and cartilage tissue formed in vitro integrates with the
respective host bone and cartilage.24 Despite the
advantages this design allows, the ECM composition and
mechanical properties of the cartilage are inferior to
those of native tissue.23 However, we have also shown
that the properties of these tissues can be improved
under certain culture conditions.25-27 The purpose of
this investigation was to study the effects of PRP on the

histologic, biochemical, and biomechanical properties of

tissue-engineered cartilage. Given the high concentration of growth factors found in PRP, we hypothesized
that adding PRP to tissue-engineered biphasic constructs
would lead to improved cartilage tissue formation.

Methods
PRP and Platelet-Poor Plasma Preparation
Bovine blood was collected from the jugular vein of
a mature cow in 6 60-mL syringes with citrate dextrose
solution as anticoagulant (1.3 mL/10 mL of blood).
Whole blood was centrifuged at 1,000 rpm for 10 minutes
using a Sorvall RT7 centrifuge (Mandel Scientic, Guelph,
ONT, Canada), leading to separation of plasma and
platelets from red blood cells. Plasma was then
collected and further centrifuged at 3,000 rpm for 5
minutes, producing a concentrate of platelets or PRP.
The supernatant of platelet-poor plasma (PPP) was
collected as well. The separate PRP and PPP fractions
were frozen at 30 C until use. Platelet counts of PRP
and PPP were performed using a hemocytometer and
trypan blue staining.
Tissue-Engineered Cartilage and Culture Conditions
Tissue-engineered biphasic constructs were created
as previously described.23 Briey, chondrocytes were
isolated by enzymatic digestion of articular cartilage
harvested from 6- to 9-month-old bovine metacarpalphalangeal joints. The isolated cells were seeded on
the top surface of CPP cylinders (4 mm diameter 
2 mm height) at a density of 160,000 cells/mm2 in
Hams F12 medium (Wisent Bio Products, Montreal,
Quebec, Canada). The medium was supplemented with
fetal bovine serum (FBS), PRP, or PPP at a starting
concentration of 5%. On day 5, the concentration of
FBS, PRP, or PPP was increased to 20%, and the media
were supplemented with ascorbic acid (100 mg/mL).
The cultures were maintained under standard tissue
culture conditions with media changes every 2 to 3 days
for 2 weeks.
Mechanical Testing
The compressive mechanical properties of the tissueengineered cartilage cultured in media supplemented
with 20% FBS, PPP, or PRP (n 8 for each condition)
were measured using a Mach-1 mechanical tester
(Biosyntech, Laval, Quebec, Canada). Compressive
force was applied in 15 intervals through a 0.65-mm
cylindrically shaped indenter, inducing a 1% to 2%
strain of the relaxed cartilage layer for each deformation. When the compressive load was stopped, an
equilibrium force level was reached. Using LabView
Data Acquisition software (National Instruments, Austin, TX), an equilibrium force-displacement curve was
obtained to estimate compressive stress.

EFFECTS OF PRP ON TISSUE-ENGINEERED CARTILAGE

Macroscopic and Histologic Analysis

After 2 weeks of culture, constructs were photographed and cartilage tissue heights determined using
Adobe Photoshop CS5 (Adobe Systems, San Jose, CA).
The cartilage layer was removed from the top surface of
the CPP (n 8 for each culture condition), xed in
buffered 10% neutral formalin (EMD Chemicals,
Gibbstown, NJ), and embedded in parafn. Fivemicronethick sections were then cut and stained with
H&E and analyzed histologically by light microscopy.
Representative samples were immunostained for
collagen types I and II. Briey, parafn-embedded tissue
sections were deparafnized with xylene and rehydrated in a graded series of ethanol. Rehydrated sections
were digested sequentially with 2.5 mg/mL pepsin
(Sigma-Aldrich, Oakville, ONT, Canada) for 10 minutes
at room temperature (pH, 2.0), 2.5 mg/mL trypsin
(Sigma-Aldrich) for 30 minutes at room temperature
(pH, 7.3), and lastly 25 mg/mL hyaluronidase (SigmaAldrich) for 30 minutes at 37 C (pH, 7.3). After blocking
with 20% (v/v) goat serum and 0.1% (v/v) Triton X100, the sections were incubated with rabbit polyclonal
anticollagen type I antibody (1:100; Meridian Life
Science, Cincinnati, OH) and mouse monoclonal anticollagen type II (clone 6B3, 1:100; LabVision/Thermo
Fisher Scientic, Kalamazoo, MI) overnight at 4 C. The
sections were then incubated with goat antirabbit
IgG (conjugated with Alexa Fluor 488) and goat
antimouse IgG (conjugated with Alexa Fluor 594) secondary antibodies (Invitrogen/Life Technologies, Grand
Island, NY), and counterstained with 4,6-diamidino2-phenylindole. Images of stained sections were visualized and captured with a Leica Microsystems (type
090-135.002) microscope (Leica Microsystems, Buffalo
Grove, IL) and a Hamamatsu digital CCD camera
(Hamamatsu Photonics, Hamamatsu City, Japan).

1687

Table 1. Compressive Mechanical Properties and Tissue

Thickness of the Tissue-Engineered Cartilage After 2 Weeks of
Culture in 20% PRP, 20% PPP, or 20% FBS
Compressive
Mechanical Properties
Tissue
(Youngs Modulus, kPa) Thickness (mm)
20% PRP-supplemented
38.1  3.6
1.6  0.2
media
20% PPP-supplemented
15.6  1.5
0.8  0.2
media
20% FBS-supplemented
20.4  3.5
0.7  0.008
media
FBS, fetal bovine serum; PPP, platelet-poor plasma; PRP, plateletrich plasma.

nm). The standard curve was generated using bovine

tracheal chondroitin sulfate (Sigma-Aldrich).
Collagen content was calculated from the hydroxyproline content. Aliquots of the papain digest were
hydrolyzed in 6 M HCl at 110 C for 18 hours and
hydroxyproline content of the hydrolysate determined
using the chloramine-T/Ehrlichs reagent assay and
spectrophotometry (wavelength, 561 nm). L-hydroxyproline (Sigma-Aldrich) was used to generate the standard curve.
Statistical Analysis
The experiment for each condition (FBS, PRP, and
PPP) was repeated 3 times and the data combined. The
data are expressed as the mean  standard deviation.
The statistical analysis was performed using a rankbased test, the Wilcoxon rank sum test. Multiple pairwise Wilcoxon tests (2-sided) were performed and
adjusted for multiple testing. Signicance was assigned
to P less than .05.

Results
Analysis of ECM Accumulation
Analysis of ECM accumulation was conducted on 8
samples. Cartilage tissue was removed from the surface
of the CPP after 2 weeks of culture and lyophilized
overnight; dry weights were then recorded. The tissue was then digested with papain (Sigma-Aldrich; 40
mg/mL in 20 mM ammonium acetate, 1 mM ethylenediaminetetraacetic acid, and 2 mM dithiothreitol)
for 48 hours at 65 C as previously described.23,24
DNA content was determined from aliquots of the
papain digest using the Hoechst dye 33258 assay (Polysciences, Warrington, PA) and uorometry (excitation, 365 nm; emission, 458 nm). Calf thymus DNA was
used to generate the standard curve.
Proteoglycan content was calculated by quantifying
the amount of sulfated GAGs through the use of the
dimethylmethylene blue dye binding assay (Polysciences) and spectrophotometry (wavelength, 525

Platelet Count
PRP had greater platelet numbers than did the PPP
fraction. The concentration of platelets in PRP was
1.22  106/mL, whereas in PPP it was 0.03  106/mL.
Mechanical Properties
The samples cultured with 20% PRP exhibited the
highest Youngs modulus, with an equilibrium modulus
equal to 38.1  3.6 kPa. Constructs cultured with 20%
PPP and 20% FBS had inferior equilibrium moduli
(15.6  1.5 kPa; P .0002 and 20.4  3.5 kPa;
P .007, respectively) (Table 1).
Macroscopic Evaluation and Histologic Examination
Gross observation of the 3 constructs revealed that
samples cultured with 20% PRP formed signicantly
thicker cartilage tissue (1.6  0.2 mm) than did samples
cultured with 20% FBS (0.7  0.008 mm; P .002)

1688

M. PETRERA ET AL.

Fig 1. Gross appearance of tissue-engineered cartilage on

calcium polyphosphate (CPP) after 2 weeks of culture in 20%
fetal bovine serum (FBS), 20% platelet-rich plasma (PRP), or
20% platelet-poor plasma (PPP). Layer of tissue can be seen
on the biomaterial (CPP).

and 20% PPP (0.8  0.2 mm; P .03) (Table 1, Fig 1).
Examination under light microscopy showed a cartilage
tissue with viable round or oval cells within lacunae in
all culture conditions. An increase in ECM in constructs
cultured with 20% PRP was observed despite no noticeable differences in cellularity among the 3 different
culture conditions. Moreover, samples cultured in 20%
PRP showed a more homogeneous cell distribution than
did 20% FBS and 20% PPP samples (Fig 2). Although
immunohistochemical staining showed positivity for
both type I and II type collagen, type II collagen appeared
to be prevalent in all culture conditions. Within the 3
groups, the samples cultured in 20% PRP showed
a higher content of type II collagen compared with the
20% FBS and 20% PPP cultures based on positivity for
staining (Fig 3).
Biochemical Testing
The GAG content was increased in tissue-engineered
constructs supplemented with 20% PRP (176.1 
18.8 mg, GAG/mg dry weight) compared with those
supplemented with 20% FBS (112  10.6 mg; P .01)
or 20% PPP (131.5  14.8 mg; P .11) (Fig 4A).
Hydroxyproline content was similar whether the media
was supplemented with 20% PRP (8.7  0.9 mg/mg
dry weight), 20% FBS (7.6  0.9 mg/mg; P .37;

condence interval [CI]  0.62), or 20% PPP (6.4  1 mg/

mg; P .28, CI  0.69) (Fig 4B).
There was no signicant difference in DNA content
whether the tissue was formed in the presence of 20%
PRP (11.9  3.5 mg/mg dry weight), 20% FBS (9.3 
2.5 mg/mg; P .99; CI  1.73), or 20% PPP (7.2 
1.3 mg/mg dry weight; P .78; CI  0.9) (Fig 4C).

Discussion
Tissue-engineered cartilage implants are a novel
strategy for the treatment of cartilage injuries. This
study shows that a tissue-engineered biphasic implant
cultured in media supplemented with 20% PRP had
signicantly thicker cartilage tissue, with increased
ECM content and improved mechanical properties
compared with implants cultured with 20% FBS or
20% PPP. The increase in matrix predominantly
resulted from increased proteoglycan content.
The results of our study are consistent with other
in vitro studies that showed a stimulatory effect of PRP
on matrix synthesis.28-30 Akeda et al.28 studied the
effects of PRP in porcine chondrocytes cultured in
alginate beads. They showed that supplementation with
PRP stimulates ECM production while maintaining
hyaline phenotype. Spreaco et al.29 studied the effects
of human platelet releasates on cultures of human
chondrocytes. The authors reported a marked increase
in proteoglycan synthesis in chondrocytes grown in 5%
PRP compared with those grown in 5% PPP. Similarly,
Gaissmaier et al.30 reported that seeding chondrocytes
in alginate beads in the presence of human platelet
supernatant led to increased matrix production but
induced a dedifferentiation of chondrocytes toward
a broblast-like phenotype. The stimulatory activity on
matrix synthesis mediated by growth factors released
from activated platelets was not found in previous
studies in which the medium was supplemented with

Fig 2. Histologic appearance of cartilage

tissue after 2 weeks of culture on 20%
fetal bovine serum (FBS), 20% plateletrich plasma (PRP), or 20% plateletpoor plasma (PPP) (*, where CPP
would have been located; H&E).

EFFECTS OF PRP ON TISSUE-ENGINEERED CARTILAGE

1689

Fig 3. Immunohistochemical staining

showing more intense staining for type II
collagen (Col II) (red) in cartilage formed
in the presence of 20% platelet-rich plasma
(PRP) compared with 20% platelet-poor
plasma (PPP) and 20% fetal bovine serum (FBS).

platelet lysate.31,32 This might be related to the different

protocol used in the preparation of platelet lysate,
obtained by a centrifugation and freeze-thaw process,
which may have an inuence on the concentration and
activity of the growth factors released (Choi et al.,32
Kaps et al.31).
The improved mechanical properties are likely related
to the increased GAG content. It is known that the high

Fig 4. (A) Analysis of proteoglycan

accumulation in cartilage cultured in
presence of 20% platelet-rich plasma
(PRP), 20% platelet-poor plasma (PPP),
or 20% fetal bovine serum (FBS) after
2 weeks of culture (*, statistically
signicant difference). (B) Analysis of
collagen accumulation in cartilage
cultured in the presence of 20% PRP,
20% PPP, or 20% FBS. (C) DNA content
of cartilage cultured for 2 weeks in presence of 20% PRP, 20% PPP, or 20% FBS.
(GAG, glycosaminoglycan; wt., weight.)

afnity for water of keratan sulfate and chondroitin

sulfate side-chain groups on GAGs makes articular
cartilage biomechanically efcient in compression. The
increased tissue thickness of the implants cultured in
20% PRP can be related to the signicantly increased
GAG content and associated water afnity. This leads to
greater compressive mechanical properties. The equilibrium modulus (38.1  3.6 kPa) of constructs cultured

1690

M. PETRERA ET AL.

in PRP was signicantly higher than in constructs

cultured in FBS or PPP.
Unlike other studies,28,29,31,32 we did not observe
a marked increase in cellularity in cartilage tissue
formed in the presence of PRP. This is contrary to what
was described in the study by Akeda et al.,28 who reported a signicant increase in DNA in porcine chondrocytes cultured in 10% PRP. It is possible that this
difference may arise as a result of the high initial
cellularity of the tissue-engineered implant used in this
study. Importantly, the total content of DNA did not
decrease, suggesting that PRP has no inhibitory effect
on cell proliferation.
Although immunohistochemical staining showed
a prevalence of type II over type I collagen, compatible
with hyaline tissue, we cannot conclude that the
hyaline phenotype was unaffected by the 2-week
culture period, because a quantication of type I and
type II collagen content was not possible with the
method used. Spreaco et al.29 and Akeda et al.28 reported ndings consistent with hyaline phenotype in
their studies. Conversely, the studies by Gaissmaier
et al.30 and Kaps et al.31 showed a dedifferentiation
of chondrocytes toward a broblast-like phenotype, a
feature that was not seen in our study.
To our knowledge, this is the rst experiment that
analyzes the effects of PRP on the mechanical properties
of a tissue-engineered cartilage implant that is suitable
for transplantation. The availability of tissue-engineered
implants with hyaline phenotype and improved mechanical properties are essential for use in the treatment of
cartilage injuries.
Clinically, the use of PRP as a supplement to current
cartilage restoration techniques presents advantages
over the use of recombinant or animal origin growth
factors related to cost and availability. Autologous
growth factors avoid the potential risk of disease
transmission and reduce the cost related to the use of
recombinant growth factors. It can also be collected at
the time of cartilage harvest. Moreover, the use of PRP
may represent a potential advantage over mechanical
stimulation in terms of avoiding the inhibition of
proliferation or decline of cell viability (caused by the
application of mechanical stimuli) that has been shown
in some in vitro studies.33,34 In addition, the use of
mechanical loading regimens requires customized
equipment to deliver the appropriate uniform forces.
PRP supplementation does not require any specialized equipment other than a source of blood and a
centrifuge.
Disadvantages to the use of PRP might be related to
the absence of established protocols for PRP preparations. Currently, different ways of PRP preparation
exist. This variability in the preparation process includes
the use of exogenous or endogenous platelet activation
(with thrombin or CaCl2), centrifugation speed, volume

of blood drawn, and use of single versus double

spin. Moreover, the use of thrombin as activating agent
might inuence cell metabolism, inhibiting protein
synthesis.35 In our experiment, we did not use an
activating agent to avoid any potential inuence.
Limitations
DeLong et al.36 described a system to classify PRP
preparations (PAW classication) based on the absolute
number of platelets, the manner in which the activation
of platelets occurs, and the presence or absence of white
cells. In our experiment, conducted before this classication was published, the leukocyte counts were not
obtained; therefore, we cannot apply the PAW system
to classify the kind of PRP preparation we used. This
may represent a limitation, making our experiment
difcult to replicate and results difcult to compare. A
second limitation might be found in the method of
histologic analysis. Although immunostained images
showed a prevalence of type II collagen, an objective
quantication of the amount of type II collagen amount
could not be done. In this respect, pepsin extraction of
collagen and Western blotting would have been a more
appropriate method.
Other limitations in our study include the small
number of samples studied (n 8), the fact that it was
a nonblinded analysis, and the short culture period.
However, regarding the culture period, current literature supports our methods. Others have used a 2-week
culture period for successful in vitro implantation and
integration of tissue-engineered constructs.24 Moreover, the stimulatory effect of PRP on articular chondrocyte proliferation and ECM production has been
shown in other studies after a shorter (2 to 7 days)
culture period.28-30 Only Kaps et al.31 used a longer
culture period (30 days), and these authors did not
obtain the same favorable results. We believe that the
use of PRP allows for a shorter culture period, therefore
allowing faster manufacture of the implant. This would
make the implant more commercially feasible.

Conclusions
The presence of PRP in the culture media enhances
the in vitro formation of cartilage with increased GAG
content and greater compressive mechanical properties
while maintaining characteristics of the hyaline
phenotype.

References
1. Schlegel W, Nurnberger S, Hombauer M, Albrecht C,
Vecsei V, Marlovits S. Scaffold-dependent differentiation
of human articular chondrocytes. Int J Mol Med 2008;22:
691-699.
2. Ahmed N, Gan L, Nagy A, Zheng J, Wang C, Kandel RA.
Cartilage tissue formation using redifferentiated passaged
chondrocytes in vitro. Tissue Eng Part A 2009;15:665-673.

EFFECTS OF PRP ON TISSUE-ENGINEERED CARTILAGE

3. Schulze-Tanzil G, de Souza P, Villegas Castrejon H, et al.
Redifferentiation of dedifferentiated human chondrocytes in high-density cultures. Cell Tissue Res 2002;308:
371-379.
4. Hauselmann HJ, Fernandes RJ, Mok SS, et al. Phenotypic stability of bovine articular chondrocytes after longterm culture in alginate beads. J Cell Sci 1994;107(Pt 1):
17-27.
5. Vunjak-Novakovic G, Martin I, Obradovic B, et al.
Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered
cartilage. J Orthop Res 1999;17:130-138.
6. Sah RL, Trippel SB, Grodzinsky AJ. Differential effects of
serum, insulin-like growth factor-I, and broblast growth
factor-2 on the maintenance of cartilage physical properties during long-term culture. J Orthop Res 1996;14:
44-52.
7. Martin I, Obradovic B, Treppo S, et al. Modulation of the
mechanical properties of tissue engineered cartilage. Biorheology 2000;37:141-147.
8. Dunkelman NS, Zimber MP, Lebaron RG, Pavelec R,
Kwan M, Purchio AF. Cartilage production by rabbit
articular chondrocytes on polyglycolic acid scaffolds in
a closed bioreactor system. Biotechnol Bioeng 1995;46:
299-305.
9. Smith RL, Donlon BS, Gupta MK, et al. Effects of uidinduced shear on articular chondrocyte morphology and
metabolism in vitro. J Orthop Res 1995;13:824-831.
10. Schmidt MB, Chen EH, Lynch SE. A review of the effects
of insulin-like growth factor and platelet derived growth
factor on in vivo cartilage healing and repair. Osteoarthritis
Cartilage 2006;14:403-412.
11. Pecina M, Jelic M, Martinovic S, Haspl M, Vukicevic S.
Articular cartilage repair: The role of bone morphogenetic
proteins. Int Orthop 2002;26:131-136.
12. Ellman MB, An HS, Muddasani P, Im HJ. Biological
impact of the broblast growth factor family on articular
cartilage and intervertebral disc homeostasis. Gene
2008;420:82-89.
13. Goodrich LR, Hidaka C, Robbins PD, Evans CH, Nixon AJ.
Genetic modication of chondrocytes with insulin-like
growth factor-1 enhances cartilage healing in an equine
model. J Bone Joint Surg Br 2007;89:672-685.
14. Middleton J, Manthey A, Tyler J. Insulin-like growth
factor (IGF) receptor, IGF-I, interleukin-1 beta (IL-1
beta), and IL-6 mRNA expression in osteoarthritic and
normal human cartilage. J Histochem Cytochem 1996;44:
133-141.
15. Mishra A, Pavelko T. Treatment of chronic elbow tendinosis with buffered platelet-rich plasma. Am J Sports Med
2006;34:1774-1778.
16. de Vos RJ, Weir A, van Schie HT, et al. Platelet-rich
plasma injection for chronic Achilles tendinopathy: A
randomized controlled trial. JAMA 2010;303:144-149.
17. Randelli P, Arrigoni P, Ragone V, Aliprandi A, Cabitza P.
Platelet rich plasma in arthroscopic rotator cuff repair: A
prospective RCT study, 2-year follow-up. J Shoulder Elbow
Surg 2011;20:518-528.
18. Sampson S, Reed M, Silvers H, Meng M, Mandelbaum B.
Injection of platelet-rich plasma in patients with primary

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

1691

and secondary knee osteoarthritis: A pilot study. Am J

Phys Med Rehabil 2010;89:961-969.
Kon E, Buda R, Filardo G, et al. Platelet-rich plasma:
Intra-articular knee injections produced favorable results
on degenerative cartilage lesions. Knee Surg Sports Traumatol Arthrosc 2010;18:472-479.
Filardo G, Kon E, Buda R, et al. Platelet-rich plasma intraarticular knee injections for the treatment of degenerative
cartilage lesions and osteoarthritis. Knee Surg Sports Traumatol Arthrosc 2011;19:528-535.
Sun Y, Feng Y, Zhang CQ, Chen SB, Cheng XG. The
regenerative effect of platelet-rich plasma on healing
in large osteochondral defects. Int Orthop 2010;34:
589-597.
Kon E, Filardo G, Delcogliano M, et al. Platelet autologous
growth factors decrease the osteochondral regeneration
capability of a collagen-hydroxyapatite scaffold in a sheep
model. BMC Musculoskeletal Disord 2010;11:220.
Waldman SD, Grynpas MD, Pilliar RM, Kandel RA.
Characterization of cartilagenous tissue formed on
calcium polyphosphate substrates in vitro. J Biomed Mater
Res A 2002;62:323-330.
Theodoropoulos JS, De Croos JN, Park SS, Pilliar R,
Kandel RA. Integration of tissue-engineered cartilage with
host cartilage: An in vitro model. Clin Orthop Relat Res
2011;469:2785-2795.
Gan L, Tse C, Pilliar RM, Kandel RA. Low-power laser
stimulation of tissue engineered cartilage tissue formed on
a porous calcium polyphosphate scaffold. Lasers Surg Med
2007;39:286-293.
Waldman SD, Couto DC, Grynpas MD, Pilliar RM,
Kandel RA. A single application of cyclic loading can
accelerate matrix deposition and enhance the properties
of tissue-engineered cartilage. Osteoarthritis Cartilage
2006;14:323-330.
Waldman SD, Spiteri CG, Grynpas MD, Pilliar RM,
Hong J, Kandel RA. Effect of biomechanical conditioning
on cartilaginous tissue formation in vitro. J Bone Joint Surg
Am 2003;85-A(suppl 2):101-105.
Akeda K, An HS, Okuma M, et al. Platelet-rich plasma
stimulates porcine articular chondrocyte proliferation and
matrix biosynthesis. Osteoarthritis Cartilage 2006;14:
1272-1280.
Spreaco A, Chellini F, Frediani B, et al. Biochemical
investigation of the effects of human platelet releasates on
human articular chondrocytes. J Cell Biochem 2009;108:
1153-1165.
Gaissmaier C, Fritz J, Krackhardt T, Flesch I, Aicher WK,
Ashammakhi N. Effect of human platelet supernatant on
proliferation and matrix synthesis of human articular
chondrocytes in monolayer and three-dimensional alginate cultures. Biomaterials 2005;26:1953-1960.
Kaps C, Loch A, Haisch A, et al. Human platelet supernatant promotes proliferation but not differentiation of
articular chondrocytes. Med Biol Eng Comput 2002;40:
485-490.
Choi YC, Morris GM, Sokoloff L. Effect of platelet lysate
on growth and sulfated glycosaminoglycan synthesis in
articular chondrocyte cultures. Arthritis Rheum 1980;23:
220-224.

1692

M. PETRERA ET AL.

33. Huselstein C, de Isla N, Kolopp-Sarda MN, Kerdjoudj H,

Muller S, Stoltz JF. Inuence of mechanical stress on cell
viability. Biorheology 2006;43:371-375.
34. Waldman SD, Couto DC, Grynpas MD, Pilliar RM,
Kandel RA. Multi-axial mechanical stimulation of tissue
engineered cartilage: Review. Eur Cell Mater 2007;13:
66-73; discussion 73-74.

35. Fujii N, Kaji T, Akai T, Koizumi F. Thrombin reduces large

heparan sulfate proteoglycan molecules in cultured
vascular endothelial cell layers through inhibition of core
protein synthesis. Thromb Res 1997;88:299-307.
36. DeLong JM, Russell RP, Mazzocca AD. Platelet-rich
plasma: The PAW classication system. Arthroscopy
2012;28:998-1009.

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