Platelets and Plasma Proteins Are Both Required
to Stimulate Collagen Gene Expression
by Anterior Cruciate Ligament Cells
in Three-Dimensional Culture
Mingyu Cheng, M.D., Ph.D., Hao Wang, Ph.D., Ryu Yoshida, B.S., and Martha Meaney Murray, M.D.
Collagen–platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate liga-
ment (ACL) healing at early time points in large animal models. However, little is known about the cellular
mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that
the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently
significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this
hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two
(PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and
enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated
collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elon-
gation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma
proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP,
did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful
in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis.
stabilizer of knee motion. The ACL is also the most com-
monly injured knee ligament and is susceptible to ruptures
or tears that can cause pain and discomfort, joint instability,
and eventually degenerative joint disease. The ACL fails to
heal after suture repair, and for this reason, ACL injuries are
commonly treated with ACL reconstruction, where the ACL
is removed and replaced with a graft of tendon. Although
this procedure is excellent at restoring gross knee stability, it
does not restore normal knee kinematics,1–4and the majority
of patients go on to have early premature osteoarthritis (as
many as 78% of patients at 14 years after surgery).5,6
Therefore, there is a great interest in developing new tech-
niques of ACL treatment.
Recently, enhancing healing of ligaments using bioactive
substances has received increasing interest. For example,
growth factors have been shown to influence chemotaxis,
differentiation, proliferation, and synthetic activity of ACL
cells and may potentiate the healing of ligaments.7–10Platelet-
rich plasma (PRP) is a fraction of plasma that can be pro-
he anterior cruciate ligament (ACL) is one of the
four major ligaments of knee and serves as the primary
duced by centrifugal separation of whole blood. It has been
found to be a useful delivery system for growth factors im-
portant in application of tissue engineering. The growth fac-
tors released from platelets (PLs), such as PL-derived growth
factor (PDGF-AA, AB, and BB), transforming growth factor
(TGF)-b1 and b2, PL-derived angiogenesis factor, insulin
growth factor-1 (IGF-1), and PL factor-4, have been noted to
play a pivotal role in initiating and sustaining wound healing
and tissue repair mechanisms.11Additionally, PRP contains
plasma proteins such as fibrin, fibronectin, vitronectin, and
thrombospondin, which are known to act as cell adhesion
molecules important for osteoblast, fibroblast, and epithelial
cell migration and viability.12Therefore, it is suggested that
the bioactive substances included in PRP may activate several
of the cell types involved in ACL healing.
Our previous studies have shown that collagen–PRP
composites can stimulate ACL healing at early time points in
large animal models.8–10However, little is known about the
cellular mechanisms by which the PRP components of these
composites may induce healing. To begin to address this, an
PRP and its two components, PLs and PL-poor plasma (PPP),
on ACL cell behavior. We hypothesized that the addition of
Department of Orthopaedic Surgery, Children’s Hospital of Boston, Harvard Medical School, Boston, Massachusetts.
TISSUE ENGINEERING: Part A
Volume 16, Number 5, 2010
ª Mary Ann Liebert, Inc.
PRP, PLs, or PPP would significantly alter ACL cell prolifer-
ation and viability and would also induce collagen expres-
sion. To test this hypothesis, ACL cells were cultured in a
three-dimensional (3D) scaffold with or without PRP, PPP,
or PLs for 14 days. A collagen type I hydrogel was selected as
scaffold because it has the ability to activate PLs to release
growth factors and also because collagen type I is a major
component of ligament tissue.13,14ACL cell proliferation,
viability, and morphology in the scaffold were investigated.
Collagen expression was also assessed using quantitative
real time-polymerase chain reaction (RT-PCR) and immuno-
Materials and Methods
Soluble type I collagen was used as the basis of the pro-
visional scaffold models used in this study. Four types of
provisional scaffolds were studied: (1) collagen hydrogel
(COL), (2) COL–PPP composite, (3) COL–PL composite, and
(4) COL–PRP composite. All cell culture reagents were ob-
tained from Invitrogen (Carlsbad, CA) or Sigma (St. Louis,
MO), unless otherwise specified.
Preparation of COL
Acid-soluble, type I collagen slurry was made by sterile
harvesting of bovine knee capsular tissue, which was solu-
bilized in an acidic solution as previously described.8Col-
lagen content within the slurry was adjusted to 8mg=mL and
neutralized with 0.1M HEPES (Cellgro; Mediatech, Herndon,
VA), 5? phosphate-buffered saline (PBS; HyClone, Logan,
UT), and 7.5% sodium bicarbonate (Cambrex BioScience
Walkersville, Walkersville, MD).
Preparation of blood fractions
Six-hundred milliliters of whole blood was drawn from
the femoral vein of pigs into tubes containing sodium citrate.
Additional blood was collected in a bag with 10% acid-citrate
dextrose at Children’s Hospital Boston (Boston, MA).
blood was transferred into 15mL centrifuge tubes (10mL per
tube), which were then centrifuged for 6min at 150 g (GH 3.8
rotor, Beckman GS-6 Centrifuge; Beckman, Fullerton, CA).
The supernatant was aspirated and collected as PRP in a
50mL tube. The PL concentration in PRP was 801?106=mL,
whereas in systemic blood it was 277?106=mL, and thus the
process resulted in an enrichment of 2.9?.
To make the PRP fraction, 200mL of whole
blood was used to make PRP as described earlier. The col-
lected PRP was then transferred into 15mL tubes and centri-
fuged at 1000 g for 10min. The top layer was removed and
placed into a new 15mL tube (the old tube with the pellet
after the first centrifugation was saved to make the PL
fraction) and centrifuged again for 10min at 1000 g. The top
layer was removed as PPP and placed in a new 50mL tube.
The PL concentration in the PPP was 19?106=mL.
To make the PPP fraction, 200mL of whole
contained in the 15mL centrifuge tubes saved after the first
centrifugation in the PPP procedure were resuspended in 1?
To make the PL-only fraction, the pellets
PBS (EMD Chemical, Gibbstown, NJ). The PL concentration
PBS was used.
To make the saline fraction, 10mL of 1?
Preparation of ACL cells
Pig ACL explants were obtained from the knee using a
sterile technique. After ligament harvest, explants were cul-
tured in completed medium (Dulbecco’s modified Eagle’s
medium [DMEM]) containing 4.5g=L glucose, 10% fetal
bovine serum, and 1% antibiotic=antimycotic [AB=AM]),
which was changed two times per week. When primary
outgrowth cells were 80% confluent, they were trypsinized
and frozen. All cells used for this experiment were of fifth
passage, and cell viability ranged from 86% to 94% according
to trypan blue exclusion.
Construct preparation and cultivation
Cells were resuspended with PBS or blood fractions (PRP,
PPP, and PL). Eight milliliters of each cell suspension was
mixed with 13mL COL. The final cell density in the mix-
ture was 5?105cells=mL, and the final collagen content in the
mixture was 3mg=mL. For each group, the hydrogel–cell
a polyester mesh at each end to anchor the gels. Each construct
was placed in culture, warmed in a humidified 5% CO2=378C
incubator for 1h to achieve gelation, and then cultured with
completed DMEM. Medium was changed every 3 days during
the culture period. The constructs were assessed on day 14.
Digital pictures of the cultures (n¼6 for each group) were
taken on days 0, 1, 4, 7, and 14, and construct area was mea-
sured using Image J software (NIH, Bethesda, MD). Con-
traction was measured for each construct as the percent
decrease in area at days 1, 4, 7, and 14, with respect to the
time zero value. Collagen gel without cells (cell-free COL)
served as a control in this experiment.
DNA content of the constructs was determined for n¼5
constructs per group by using Quant-iT? PicoGreen dsDNA
Assay Kit at day 14, with type 1 highly polymerized calf
thymus DNA as a standard.
Cell metabolic activity
bromide (MTT) assay was performed on n¼5 constructs per
group to obtain the index of cell metabolic activity. In brief,
constructs were rinsed with PBS and incubated in a solution of
Constructs were then incubated in a solution of 0.1N HCl in
isopropyl alcohol for an additional 4h, and the optical density
of the resulting supernatant was measured at 570nm using a
microplate reader (Molecular Devices, Sunnyvale, CA).
Histological and immunohistological analyses
To identify the collagen deposited in the hydrogel, im-
munohistochemical staining for collagen was performed on
1480CHENG ET AL.
n¼4 constructs per group. In brief, constructs were rinsed in
PBS and cryosectioned at 16 (m. The sections were incubated
for 1h at 378C with monoclonal type I collagen (ab6308;
Abcam, Cambridge, MA) or type III collagen antibody
(MAB3392; Millipore, Billerica, MA), diluted 1:150 in PBS
containing 0.5% Tween 20 and 1.5% horse serum. Subse-
quently, sections were incubated for 30min at room tem-
perature with a secondary antibody (horse anti-mouse
IgG, Standard Elite ABC kit; Vector, Burlingame, CA),
diluted 1:200, and then incubated with an avidin–biotin
complex agent for 30min at room temperature and with 3,30-
diaminobenzidine (D0426; Sigma) for 15min at room tem-
perature. The sections were counterstained with Harris
hematoxylin and coverslipped.
For histological analysis, constructs (n¼4 per group)
cultured for 14 days were rinsed in PBS, fixed for 24h in 10%
neutral-buffered formalin, and embedded in paraffin. Serial
longitudinal sections (6 mm) of the constructs were cut, and
sections at 150mm interval from outer surface to the center
were selected for staining. Cell morphology and distribu-
tions were evaluated with hematoxylin and eosin (H&E)
staining. Collagenous extracellular matrix (ECM) was eval-
uated with Masson’s trichrome staining.
Immunofluorescent staining for the measurement of apo-
ptotic cells and nuclear aspect ratio was performed on n¼4
constructs per group. The sections were stained with termi-
nal deoxynucleotidyl tranferase-mediated dUTP nick-end
labeling (TUNEL) using a commercial available kit (Roche,
Indianapolis, IN), according to the manufacturer’s instruc-
tions. Subsequently, the sections were treated with 40,6-
diamidino-2-phenylindole dihydrochloride (DAPI; Molecular
Probes, Carlsbad, CA) to quantify the total number of nuclei.
The apoptotic (TUNEL positive) and total (DAPI positive) cells
were manually counted for each group, and the fraction of
apoptotic cells were expressed as a percentage of the total cells.
Average nuclear aspect ratio was measured to characterize
ACL cell morphology.15The major axis of cell nucleus iden-
tified by DAPI fluorescence was divided by the minor axis to
Table 1. Real-Time Polymerase Chain Reaction Primer Sequences
GeneForward primer sequencesReverse primer sequences
constructs. (A) Apoptotic (TUNEL-positive) cells expressed
as a fraction of total (40,6-diamidino-2-phenylindole dihy-
drochloride-stained) cells. (B) Cell metabolic activity, assessed
by the MTT assay. Data are the mean?standard deviation
of five to six measurements. *Significantly different from
the COL group (p<0.05). COL, collagen hydrogel; MTT, 3-
TUNEL, terminal deoxynucleotidyl
dUTP nick-end labeling.
Cell apoptosis and metabolic activity in 14-day
evaluated as the percent decrease of a particular construct’s
area at days 1, 4, 7, and 14, with respect to that construct’s
area at day 0. Data are the mean?standard error of six
Construct size as a function of time in culture,
EFFECTS OF PLATELET-RICH PLASMA ON 3D CULTURED ACL CELLS 1481
get nuclear aspect ratio. Three regions were selected from each
test specimen and 150–600 cells were measured in each group.
Transmission electron microscopy
Constructs cultured for 14 days in the PRP and COL
groups were fixed with 10% buffered neutral formalin for
24h, postfixed in 2% osmium tetroxide for 1h, dehydrated
in ethanol, and then embedded in BEEM?vials (BEEM Inc.,
Bronx, NY) with fresh LR white resin (Ted Pella Inc., Redding,
CA). Sections were cut at 60nm, stained with uranyl citrate
and lead citrate, and viewed under a JEM 2011 transmission
electron microscopy (TEM) instruments (Jeol, Tokyo, Japan).
Total RNA was extracted from constructs using an
RNeasy mini kit (Qiagen, Valencia, CA). Briefly, constructs
tudinal section). (A–D) Low-magnification images from the (A) COL, (B) PL, (C) PPP, and (D) PRP groups. Scale bars:
100mm. (E–H) High-magnification images from the (E) COL, (F) PL, (G) PPP, and (H) PRP groups. Scale bars: 50mm. PL,
COL–platelet composite; PPP, COL–platelet-poor plasma composite; PRP, COL–platelet-rich plasma composite. Color images
available online at www.liebertonline.com=ten.
Cell distribution and morphology in 14-day constructs, as evaluated with hematoxylin and eosin staining (longi-
1482CHENG ET AL.
that had been cultured for 14 days were rinsed in PBS, cut
into small pieces, lysed with supplied buffer (Qiagen), and
transferred to RNeasy spin columns. RNA concentration and
purity were determined at 260 and 280nm, respectively. The
RNA samples were reverse transcribed into cDNA using
RETROscript Kit (Ambion, Austin, TX), following the sup-
plier’s instructions. Real-time PCR was performed in ABI
PRISM 7900 Sequence Detection System (Applied Biosystems,
Foster City, CA) using SYBRGreen PCR Master Mix Kit
(Applied Biosystems). Targeted genes were types I and III
procollagens (COL1A1 and COL3A1), and GAPDH was se-
lected as a reference gene. The primer sequences of selected
genes for real-time PCR are listed in Table 1. The transcript
level of target genes normalized to GAPDH was calculated
using the 2?DCtformula.
Data were calculated as means?standard deviation and
were considered significant.
Cell viability and metabolism
The amount of DNA in the constructs was similar in all
the groups (4.42?1.34, 4.69?1.14, 5.46?0.65, and 5.95?
0.89 (g=construct in the COL, PL, PPP, and PRP groups, re-
spectively; p>0.6). The apoptotic rate in the COL group was
more than double that in the PL, PPP, and PRP groups, with
the percentage of apoptotic (TUNEL positive) cells in the
COL group at 10.8%?2.5%, whereas the PL, PPP, and PRP
groups had 5.2%?0.9%, 4.1%?1.7%, and 3.2%?0.5%, re-
spectively (Fig. 1A) (p<0.01). There was no significant dif-
ference in apoptosis among the PL, PPP, and PRP groups
(p>0.3). The cellular metabolic activity of the PL, PPP, and
PRP groups were all significantly higher than the COL group
(p<0.05), and there was no significant difference among
these three groups (p>0.5) (Fig. 1B).
The cell-free COL gel contracted 14.1%?2.6% over 14
days of culture (Fig. 2). The contraction rate of the ACL cell-
seeded COL gels were significantly higher than that in the
cell-free COL group after 7 days culture (18.9%?2.7%,
p<0.05) and also after 14 days (39.7%?5.7%, p<0.01). The
contraction rate in the PL group was significantly higher
than that in the COL group at all time points (p<0.05). The
contraction rates in the PPP and PRP groups after 14 days
were 17.1%?9.8% and 25.8%?3.9%, respectively, which
were significantly lower than that in the cell-seeded COL
Histological and TEM analysis
Cell distribution and morphology were evaluated by H&E
staining and the representative images are shown in Figure
3. ACL cells were distributed throughout the entire volume
of 14-day constructs from all the groups (Fig. 3A–D). ACL
cell morphology was very similar in each group. In the PRP
and PPP groups, most of the cells appeared elongated and
contained centrally positioned elongated nuclei, suggestive
of early differentiation of these cells (Fig. 3G, H). Most of the
cells were oriented along the longitudinal axis of the con-
structs. In contrast, most of the cells in the COL and PL
groups appeared round (Fig. 3E, F). ACL cell morphology
was further evaluated by the measurement of average nu-
clear aspect ratio (Fig. 4). The PRP group had the highest
average nuclear aspect ratio among all the groups (2.74?
1.13; p<0.001). The average nuclear ratio in the PPP group
(2.51?0.85) was significantly higher than that in the COL
and PL groups (1.47?0.27 and 1.58?0.31; p<0.001), and
the ratio was similar in the COL and PL groups (p>0.2).
Collagenous ECM was evaluated with Masson’s trichrome
staining. Hydrogels in the PRP group showed a wave fiber-
like structure of collagenous ECM aligned with the longitu-
dinal axis of the constructs (Fig. 5D), as is typical for ligament
tissue (Fig. 5E). Hydrogels in the PPP group also showed a
similar wave fiber-like structure but with less density and
alignment when compared with the PRP group (Fig. 5C). In
contrast, the aligned wave-like structure was not seen in the
COL and PL groups, which exhibited only a homogenous
structure (Fig. 5A, B). Ultrastructure of the constructs from the
PRP and COL groups was evaluated by TEM. Classic collagen
fibrils were observed in both groups (Fig. 6). Collagen fibrils
were prevalent in the PRP group (Fig. 6B, D), and most of
them were aligned with the cells and packed into collagen
fibers. In contrast, the collagen fibrils in the COL group were
sporadic and less oriented (Fig. 6A, C).
mRNA transcript expressions of types I and III collagen
were evaluated by real-time PCR after 14 days in culture
(Fig. 7). The analysis indicated that hydrogels in the PRP
group had the highest transcript level of both types I and III
collagen among all the groups. The transcript level of type I
collagen in the PRP hydrogels was 9.4 times higher than that
in the COL group (p<0.01). The transcript level of type III
collagen in the PRP group was 11.2 times higher than that in
the COL group (p<0.01). The PL and PPP groups showed
slightly increased transcript levels of both types I and III
constructs. *Significantly higher than the COL and PL groups
(p<0.001).#Significantly higher than the PPP group (p<
0.001). ACL, anterior cruciate ligament.
Average nuclear aspect ratio of ACL cell in 14-day
EFFECTS OF PLATELET-RICH PLASMA ON 3D CULTURED ACL CELLS1483
collagen when compared with the COL group, but there was
no significant difference among these three groups.
Deposition of types I and III collagen in the scaffold was
evaluated by immunohistochemical staining (Fig. 8). For each
both cases showed similar staining intensity to the constructs
from the COL group. Hydrogels in the PRP group showed a
more intense staining for collagen types I and III when com-
pared with the other groups (Fig. 8). The PL and PPP groups
had similar staining intensity for collagen types I and III, and
both of them were more intense than the COL group.
PRP is thought to facilitate successful wound healing and
is likely to be useful in stimulating healing of tissues that
have an impaired ability to heal, like the ACL.8–10Under-
standing the influence of PRP and its components, PL and
PPP, on fundamental ACL cell behaviors is important for
optimizing the use of these stimulatory cells in translational
applications of these materials.
Many previous studies have reported that PLs can stim-
ulate the proliferation of various cells, for example, osteo-
blast, gingival fibroblast, endothelial cells, and stromal stem
cells.16–20Most of these studies were carried out in two-
dimensional culture systems. In this study, ACL cells were
section). (A) COL group, (B) PL group, (C) PPP group, (D) PRP group, and (E) intact pig ACL tissue. Scale bars: 100mm. Color
images available online at www.liebertonline.com=ten.
Collagenous extracellular matrix in 14-day constructs, as evaluated with Masson’s trichrome staining (longitudinal
1484 CHENG ET AL.
cultured in 3D collagen type I hydrogel, and no significant
difference of cell proliferation was observed among all the
four groups after 14 days culture. According to previous
studies, the stimulating effect of PL on cell proliferation is in
a dose-dependent manner.21Arpornmaeklong et al. reported
that when cells were cultured in a 3D collagenous composite,
only highly concentrated PRP (250?106PL in a carrier at
5mm diameter?3mm thick, about 4400?106=mL) showed a
stimulation of cell proliferation, whereas lower concentra-
tions of PLs (62.5?106=carrier and 16?106=carrier) did not
result in increased levels of cell proliferation. Therefore, the
relatively low PL concentration (300?106=mL) used in this
study might be the reason for the lack of significant cellular
proliferative response in our PL hydrogels.
In addition, the culture environment is known to signifi-
cantly affect cell viability and metabolic activity. In general,
in vitro statically cultured constructs rely on diffusion to
supply oxygen and nutrients. Within the body, most cells are
found no more than 100–200 mm from the nearest capillary,
with this spacing providing sufficient diffusion of oxygen.22
In vitro, likewise, sufficient oxygenation of cells by diffusion is
limited to a distance of 100–200 mm.23,24It has been well rec-
ognized that oxygen concentration gradient exists in statically
cultured tissue constructs that are larger than few hundred
micrometers in thickness.25,26Previous studies also showed
that hypoxia existedinsizable constructsseeded with cells.27,28
In this work, the constructs 2–5mm in thickness were stati-
cally cultured for 14 days in vitro. Although oxygen concen-
tration was not measured, we believe that hypoxia existed in
the statically cultured constructs. Many previous studies have
shown that hypoxia is a sufficient trigger for cell apoptosis
in vitro and in vivo,29,30and future studies to correlate the
degree of hypoxia and the apoptotic mechanism in ACL cells
are needed. In this work, cell apoptosis was found in all the
from the (A, C) COL and (B, D) PRP groups. Longitudinal section: (A) COL group and (B) PRP group; Cross section: (C) COL
group and (D) PRP group. Scale bars: 500nm. ACL cells and collagen fibrils are indicated by asterisks and arrows,
Microstructure in 14-day constructs, as evaluated with transmission electron microscopy. Representative images
EFFECTS OF PLATELET-RICH PLASMA ON 3D CULTURED ACL CELLS1485
groups, and we speculate that hypoxia might be one of the
major reasons for cell apoptosis. PLs are a biological reservoir
of various growth factors, and high concentrations of some
growth factors, such as PDGF, IGF-I, and TGF, are found in
PLs. Our previous studies showed that the release of these
growthfactors fromPLcanbe triggeredbycontactwithtypeI
collagen.13Some of the released growth factors, such asPDGF
and IGF, can suppress cell apoptosis and enhance cell via-
bility.31,32Some plasma proteins, such as fibronectin, were
this study, as we expected, PL, PPP, and PRP all significantly
reduced apoptosis in the day-14 constructs.
Fibroblasts are also known to induce collagen type I hy-
drogel contraction, which was originally reported by Bell
et al.34In a 3D collagen gel system, the ability of fibroblasts
to contract the gels is dependent on a variety of factors in-
cluding fibroblast strain, cell density, collagen concentration,
and the presence of soluble factors.35,36The contraction rate
of the COL group in this study is much lower than that in
previous reports,37which may be due to the higher collagen
concentration of the gel used here than that of previous re-
ports (3mg=mL here vs. 1.95mg=mL previously). In addition,
PLs are known to potentiate fibroblast-mediated contraction
of collagen gels.38Our results are consistent with these re-
ports, in that the contraction rate of the PL group was higher
than the COL group at all time points evaluated. This is also
consistent with our previous results.39In contrast, the PPP
hydrogels showed an inhibitory effect on collagen gel con-
traction. The contraction rate of hydrogels in the PPP and
PRP groups was significantly lower than that in the COL and
PL groups. One of the possible reasons for this inhibition of
contraction in the PRP and PPP groups is the high concen-
tration of fibrinogen in collagen gel, which contained pig
plasma (PRP and PPP groups). Fibrinogen is a soluble pro-
tein in pig plasma at concentrations of 2–3mg=mL.40The
final concentration of fibrinogen in the PRP and PPP groups
was 0.75–1.15mg=mL, whereas no plasma fibrinogen was
contained in the PL and COL groups. Previous studies
have reported that fibrinogen decreases the contraction of
fibroblast-populated collagen gels in a dose-dependent man-
ner, and the contraction of collagen gel was partially in-
hibited by fibrinogen at 0.5mg=mL and was completely
blocked at 3mg=mL.41However, the mechanism remains
unknown, and the principal factor behind the observed dif-
ferences requires further study.
ACL cell morphology was characterized by H&E staining
and average nuclear aspect ratio (Figs. 3 and 4). Most ACL
cells in the collagen-only and PL-only hydrogels cultured for
14 days exhibited a rounded-cell phenotype. The rounded-
cell phenotype might be due to the method of obtaining cells
where freshly digested cells were mixed with collagen gel
and then cultured in the gel for 14 days. During digestion,
cells undergo extensive morphological and biochemical chan-
ges including the loss of normal morphology and reduced
marker protein synthesis.42Compared with other scaffolds,
this gel inoculation method enables rapid and spatially uni-
form seed at high cell density but may restrict cell elongation.
In the PL group, many growth factors, such as PDGF, IGF-I,
and TGF-b, are released by PLs; however, the growth factors
released by the PLs in these constructs did not appear to in-
fluence cell morphology, and there was no significant differ-
ence in average aspect ratio between the COL and PL groups.
Interestingly, most cells in the PPP and PRP groups appeared
elongated, and theaverage nuclear aspect ratio inthesegroups
were much higher than that in the COL and PL groups. Also
most cells in the PRP and PPP groups were oriented along the
longitudinal axis of the constructs. One possible reason for
the cell morphology change in the PPP and PRP groups is
the presence of cell adhesion proteins in plasma. For example,
fibronectin is one ofthe major adhesive proteins in plasma that
can mediate cell attachment and spreading on various sub-
strates. Collagen molecules have specific binding sites for this
mediumcontaining10% bovine fetalserum, 3D type I collagen
scaffolds can absorb high amounts of fibronectin, and cell ad-
hesion to the collagen scaffold is partially blocked by specific
antifibronectinreceptorantibodies.44Inthis work, thePRP and
PL groups did not. Based on previous studies, the presence of
adhesive proteins in the plasma is one possible reason for ACL
cell attachment and spreading in the PRP and PPP groups.
structs, as measured with real-time polymerase chain reac-
tion. (A) Procollagen I gene and (B) procollagen III gene.
Data are the means?standard deviation of three measure-
ments. *Significantly higher than the COL group (p<0.05).
Transcript level of collagen gene in 14-day con-
1486 CHENG ET AL.
(B) COL, (C) PL, (D) PPP, and (E) PRP groups. (F–J) Images of collagen III staining from the (F) cell-free COL, (G) COL, (H)
PL, (I) PPP, and (J) PRP groups. Scale bars: 100mm. Color images available online at www.liebertonline.com=ten.
Immunohistochemical analysis of 14-day constructs. (A–E) Images of collagen I staining from the (A) cell-free COL,
EFFECTS OF PLATELET-RICH PLASMA ON 3D CULTURED ACL CELLS1487
Intrinsic ACL cells are believed to be an important cell in
tissue remodeling during stimulated repair as well as during
the incorporation of a tendon graft after ACL reconstruction.
In the PRP group, the ECM had a wavy appearance, which
was aligned with the longitudinal axis of the constructs. This
newly deposited ECM was populated by ACL cells, which
had cytoplasmic extensions that were also aligned with the
longitudinal axis. The PPP group had some similar regions,
which occupied less area of the constructs. This structure was
not observed in the COL and PL groups. As types I and III
are the major matrix components of the natural ligament, we
hypothesized that PRP has a stronger stimulating effect on
collagen synthesis of ACL cells than either PPP or PL. Real
time-polymerase chain reaction and immunohistochemical
analyses confirmed this hypothesis. Cell culture environment
is an important factor that can affect cell functions, including
protein synthesis. Many studies reported that some growth
factors, such as PDGF and TGF-b, have the ability to pro-
mote the synthesis of collagen types I and III by fibro-
blasts.45,46In the PL group, PDGF and TGF were released
from the PLs, and its collagen synthesis was increased when
compared with the COL group, with the transcript level of
type III collagen 180% higher than that in the COL group
(p>0.05). This increase was even more notable in the group
combining PLs and plasma proteins (the PRP group): a sig-
nificant increase in collagen synthesis was observed in the
PRP group; specifically, the transcript level of type I collagen
was 9.4 times and that of type III collagen was 11.2 times
higher than the COL groups, and also much higher than both
PL and PPP groups (p<0.05). Immunohistochemical stain-
ing also revealed the increased deposition of collagen types I
and III in the scaffold when compared with the other groups.
As the PRP group contains PLs and plasma proteins that
allowed the cells to obtain a more elongated shape, we
suggest that both the cytokines and the ECM composition
are able to influence collagen synthesis by ACL cells, and
therefore, for ACL cells.
In conclusion, this study demonstrated that PRP and its
two components, PLs and plasma proteins, can enhance the
viability and metabolic activity of ACL cells in 3D culture.
However, only the combination in PRP has significant ben-
eficial effect on the collagen synthesis of ACL cells.
This study was supported by a grant from the National
Institutes of Health (NIH RO1 AR052772 to M.M.M.).
Dr. Murray is a paid consultant, founder, and stockholder
in Connective Orthopaedics. No other competing financial
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Address correspondence to:
Martha Meaney Murray, M.D.
Department of Orthopaedic Surgery
Children’s Hospital of Boston
Harvard Medical School
300 Longwood Ave.
Boston, MA 02115
Received: March 23, 2009
Accepted: December 3, 2009
Online Publication Date: January 11, 2010
EFFECTS OF PLATELET-RICH PLASMA ON 3D CULTURED ACL CELLS1489
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