Abstract
Background: Platelet-derived growth factor (PDGF) has been proposed
as a therapeutic agent to promote bone-healing. The purpose of this study was
to examine the effect of PDGF on the ability of human demineralized bone
matrix to induce bone formation in a nude-mouse muscle-implantation model. We
also examined whether platelet-rich plasma, which contains PDGF, also
modulates osteoinduction in this model.
Methods: Human demineralized bone matrix, previously shown to be
osteoinductive in the calf muscles of nude mice, was mixed with PDGF-BB (0,
0.1, 1, and 10 µg/10 mg of demineralized bone matrix) and was implanted
bilaterally in the calf muscles of immunocompromised (nu/nu) mice (six mice in
each group). Heat-inactivated demineralized bone matrix was used as a control.
Tissue was harvested at fourteen, twenty-eight, and fifty-six days after
implantation. Platelet-rich plasma was prepared from the blood of a healthy
donor with use of the Harvest PRP preparation device, activated with thrombin,
and mixed with active and inactive demineralized bone matrix. Fifty-six days
post-implantation, tissues were harvested. Osteoinduction was assessed with
use of a qualitative scoring system and with quantitative
histomorphometry.
Results: Cartilage was present at fourteen days in all tissues that
had received an implant, but the amount decreased as the PDGF concentration
increased. PDGF reduced bone formation at twenty-eight days in a
dose-dependent manner. This inhibitory effect was resolved by fifty-six days,
except in tissues in which demineralized bone matrix and 10 µg of PDGF had
been implanted. In sites treated with 10 µg of PDGF, the area of new bone
was decreased and the area of bone marrow was reduced at twenty-eight and
fifty-six days. PDGF also appeared to retard resorption of demineralized bone
matrix in a dose-dependent manner. Platelet-rich plasma reduced osteoinduction
by human demineralized bone matrix that had high osteoinductive activity and
had no effect on osteoinduction by demineralized bone matrix with low
activity.
Conclusions: PDGF inhibits, in a dose-dependent manner,
intramuscular osteoinduction and chondrogenesis by demineralized bone matrix
in immunocompromised mice. Platelet-rich plasma also reduces the
osteoinductivity of active demineralized bone matrix.
Clinical Relevance: Osteoinduction in the nude mouse may not reflect
growth-factor effects in bone. However, these data indicate that PDGF and
platelet-rich plasma should not be used with demineralized bone matrix if the
purpose is to increase osteoinduction, although these substances may increase
other aspects of bone-healing. Additional studies are needed to determine the
clinical relevance of these observations.
Demineralized bone matrix has been used as an adjunct in orthopaedic
and dental surgery for many
years1, with the
premise that its inherent osteoinductivity enhances bone-healing. In the years
following the initial finding by
Urist2 that
demineralized bone can induce bone formation when implanted in ectopic sites,
bone morphogenetic protein (BMP) and other biologically active molecules have
been identified as contributors to this
process3. Addition
of exogenous BMP-2 to inactive commercial preparations of human demineralized
bone matrix has restored
osteoinductivity4.
More recently, BMPs have been isolated from human demineralized bone
matrix5, and their
concentration has been correlated with the ability of the demineralized bone
matrix preparation to induce bone formation in a nude-mouse
muscle-implantation
assay6. These
studies and others like them have suggested that the variability in the
osteoinductivity typical of commercial preparations of human demineralized
bone matrix is due to the loss of BMP as a result of donor age and health
and/or the methods of preparation and
sterilization7.
Growth factors other than demineralized bone matrix could also be involved
in the osteoinductive response. It is possible that exposed proteins in the
matrix of the allograft or xenograft interact with factors generated within
the wound site created by the surgery or implantation procedure. It has been
known for years that platelets accumulate at the site of an extravasation and,
when activated by thrombin, secrete alpha
granules8. These
granules release growth factors thought to initiate and promote wound-healing.
Among these are platelet-derived growth factor (PDGF), transforming growth
factor beta (TGF-ß), fibroblast growth factor (FGF), and epithelial
growth factor
(EGF)9. These and
other factors may be responsible for a variety of healing enhancements,
including neovascularization and collagen
synthesis8.
PDGF exists as three dimers: PDGF-AA, PDGF-BB, and PDGF-AB, which are
encoded by two genes, PDGF-A and
PDGF-B10. The three
isoforms stimulate mitogenesis and migration of osteoblast-like cells in
culture11. PDGF
also inhibits formation of bone matrix in fetal calvarial
cultures12 and
downregulates markers for differentiation such as alkaline
phosphatase13. PDGF
affects chondrocytes in the endochondral lineage as well, stimulating
proliferation but not
differentiation14.
These observations indicate that in vitro PDGF enhances cell replication but
not differentiation, suggesting that, in vivo, PDGF may enhance bone formation
by increasing the number of osteochondral progenitor cells.
In vivo evidence suggests that PDGF is involved in bone formation and
healing. Multiple cell types express mRNAs for PDGF during fracture-healing,
exhibiting a spatial and temporal localization distinct from cells expressing
BMP-2 or TGF-ß1
mRNAs15. Exogenous
application of PDGF has been shown to systemically stimulate the rodent
skeleton and, when delivered in a collagen carrier, to enhance bone-healing in
osteotomy models of osseous
repair16. Several
studies have shown that PDGF can, at least in combination with other agents,
enhance bone regeneration following periodontal and dental implant
surgery17-20.
When combined with demineralized bone matrix, PDGF increases osteoblast
proliferation in
vitro21 and
augments periodontal regeneration following treatment of Class-II furcation
defects in
humans22.
In order to better understand the role that PDGF plays in new bone
formation, we undertook a study of how it might amplify or modulate the
process. We chose demineralized bone matrix, a proven osteoinductive material
when implanted in ectopic sites of host animals, as the initiator of bone
formation and sought to determine how PDGF modulates its effects. Toward this
end, we combined the demineralized bone matrix and growth factor as an implant
to be used in the muscle tissue of immunocompromised mice, a standard practice
in the evaluation of demineralized bone matrix and
carriers7.
Because of our findings regarding the combination of pure recombinant human
PDGF and demineralized bone matrix, we also examined PDGF as it occurs
naturally with other growth factors by using a common clinical preparation,
platelet-rich plasma. The possibility that PDGF and the other growth factors
promote wound-healing has been the impetus for the addition of platelet-rich
plasma releasates to surgical wound sites. While most of the wounds that have
been shown to heal better in the presence of platelet factors have not been
surgical, the presumed role of platelet growth factors in general
wound-healing has prompted the use of platelet-rich plasma as an adjunct in
periodontal, orofacial, and orthopaedic surgical
procedures23-37.
Characteristics of Human Demineralized Bone Matrix
Particles of human demineralized bone matrix, ranging in diameter
from 200 to 500 µm, were donated, in the form that is available for
clinical use, by LifeNet (Virginia Beach, Virginia). Previously, we used the
nude-mouse muscle-implantation assay to study the bone-induction ability of
twenty-seven batches of demineralized bone matrix from this tissue bank, and
from this assortment we selected batches with high and low osteoinduction
activity7. In the
PDGF part of this study, demineralized bone matrix was inactivated by heating
it overnight at 100°C for use as a control.
Preparation of Implants
In order to facilitate the implantation of the test material, particles of
demineralized bone matrix were placed into number-5 gelatin capsules (10
mg/capsule). Because the gelatin capsules were not presterilized and the
demineralized bone matrix was not weighed and packed under clean-room
conditions, all implants were sterilized under ultraviolet light overnight
prior to use. The same sterilization protocol had been used for the previous
determination of the osteoinductivity of the batches in this
study7.
Addition of PDGF
Human recombinant PDGF-BB was provided to us as frozen, sterile saline
solutions (gift of BioMimetic Pharmaceuticals, Franklin, Tennessee) in one of
three concentrations: 4.0, 40.0, or 400 µg/mL. Immediately prior to
implantation, the solutions were thawed and 25-µL aliquots containing 0.1,
1.0, or 10 µg of PDGF were pipetted directly onto the demineralized bone
matrix in each capsule and were mixed with the sterile tip to wet the
particles.
Preparation and Characterization of Platelet-Rich Plasma
Thirty milliliters of blood was drawn from a thirty-year-old man who had no
known history of medical disease. The blood sample was processed in the
Harvest PRP preparation device (Harvest Technologies, Plymouth, Massachusetts)
to obtain platelet-rich plasma comparable with that used clinically. This
apparatus separates the blood into three fractions: red blood cells,
platelet-poor plasma, and platelet-rich plasma. The third fraction was
activated with thrombin according to the manufacturer's instructions just
prior to implantation of the demineralized bone matrix.
To ensure that the platelet-rich plasma was prepared with adequate
growth-factor content, TGF-ß1 was measured in the donor blood,
platelet-poor plasma, and platelet-rich plasma as well as in the thrombin
activator. We elected to quantify TGF-ß1 rather than another growth
factor present in platelet-rich plasma for two reasons: TGF-ß1 has been
used by others to evaluate the effectiveness of the platelet-rich-plasma
preparation38, and
we were able to compare the concentrations of latent and active TGF-ß1 as
an indicator of platelet activation. Before and after treatment with thrombin,
the levels of active TGF-ß1 in the samples were measured with use of a
commercially available enzyme-linked immunoassay (ELISA) kit (Promega,
Madison, Wisconsin) specific for human TGF-ß1 according to the
manufacturer's directions. In addition, aliquots of each sample were treated
with acid to convert all latent TGF-ß1 to the active
form39. We
determined that whole blood and platelet-rich plasma without thrombin
activation contained 16.7 and 20.5 ng/mL of latent TGF-ß1, respectively,
but in neither could active TGF-ß1 be detected. Following activation with
thrombin, whole blood contained 16.5 ng/mL of latent TGF-ß1 and 1.2 ng/mL
of the active form, whereas platelet-rich plasma had 35.1 ng/mL of latent
TGF-ß1 and 3.1 ng/mL of the active form. This enriched plasma was
subsequently used for further experimentation.
Implantation Protocols
PDGF
Sixty-eight inbred male nu/nu mice (nude mice) (Harlan, Indianapolis,
Indiana) were used in this study. The compromised immune system of these
animals permits evaluation of the response to xenograft implants with minimal
donor-host interactions. The mice were divided into fifteen groups
(Table I). The same batch of
demineralized bone matrix was used in all groups, with demineralized bone
matrix that had been heat-inactivated at 100°C for twenty-four hours used
as the negative control. Three concentrations of PDGF were evaluated at three
post-implantation time-periods.
After administration of inhalation anesthesia with isoflurane, the
implantation sites were disinfected with povidone iodine. A small skin
incision was made in each hindlimb, and a pouch in the calf muscle was
prepared with blunt dissection. One gelatin capsule was inserted into each
pouch, and the incision was closed with clips. Each mouse received two
identical implants, thereby reducing the possibility of cross-reactivity,
which might occur with dissimilar formulations. Previous studies have shown
that an implant placed in one limb causes systemic effects in the
contralateral limb that are specific to the chemistry of the
implant40. To
ensure that the statistical power of the study was not compromised by this
design, we compared data derived by using each animal as an individual n value
with data derived by using each implant as an individual n value; we found
that significant differences between groups could be identified with either
approach7.
Therefore, to limit the number of animals that needed to be used for the
study, we opted for the bilateral implant study design.
The mice were allowed water and food ad libitum for fourteen, twenty-eight,
or fifty-six days. This protocol was approved by the Institutional Animal Care
and Use Committee at the Georgia Institute of Technology. Six mice were used
for each PDGF treatment group and positive control group, and four mice were
used for each negative control group. Two identical capsules were implanted
into each mouse, with one placed into each hindlimb calf muscle. This resulted
in twelve implants for each PDGF variable and eight negative controls.
Platelet-Rich Plasma
Sixteen inbred nu/nu mice (nude mice) (Harlan) were used in this study. The
mice were divided into four groups, of four mice each, according to whether
they were treated with high-activity demineralized bone matrix, low-activity
demineralized bone matrix, high-activity demineralized bone matrix and
platelet-rich plasma, or low-activity demineralized bone matrix and
platelet-rich plasma. Two identical capsules were implanted into each mouse,
with one placed into each hind-limb calf muscle. This resulted in eight
implants for each variable. The mice were maintained for fifty-six days in the
vivarium, where they were provided food and water ad libitum. This protocol
was approved by the Institutional Animal Care and Use Committee at the
University of Texas Health Science Center at San Antonio prior to commencement
of the study.
Histologic Evaluation
At fourteen, twenty-eight, and fifty-six days post-implantation in the PDGF
study and at fifty-six days in the platelet-rich-plasma study, animals were
killed by asphyxiation with carbon dioxide. The whole calf muscle, including
the limb bones, was excised to ensure complete recovery of the implant site.
Following harvest, the tissues were fixed in 10% neutral buffered formalin,
decalcified in 5% formic acid, embedded in paraffin, sectioned, and stained
with hematoxylin and eosin. Three consecutive cross-sectional cuts (3 to 4
µm each) were made at each of three different levels of the calf muscle to
ensure visualization of the implant site.
All of the sections were evaluated, at 10× magnification, for the
presence or absence of demineralized bone matrix particles, new bone, and new
cartilage. Particles of demineralized bone matrix were present in all
implanted sites at the time of harvest. One section of each implant was
selected for further analysis. Because the selected section was the one with
the greatest amount of new bone and/or cartilage, the results were biased
toward success for each formulation.
The osteoinduction in each section was graded with use of a qualitative
score and was measured with quantitative histomorphometry. The qualitative
score indicated whether osteoinduction had occurred as well as the number of
ossicles that had formed. The quantitative score provided an assessment of the
amount of new bone but did not indicate if that bone was present in one large
ossicle or if it represented smaller amounts of bone in multiple ossicles. The
qualitative score was 1 when demineralized bone matrix, but no new bone, was
present; 2 when an ossicle was observed; 3 when two or more ossicles were
present; and 4 when at least 70% of the slide (at 10× magnification) was
covered by an ossicle (a very osteoinductive implant). There are no
"ossicle equivalents" for cartilage, so the qualitative score for
cartilage was determined by the number of cartilage sites in the histologic
section. The score was 1 when demineralized bone matrix, but no new cartilage,
was present; 2 when one cartilage site was observed; 3 when two or more sites
were present; and 4 when at least 70% of the slide (at 10×
magnification) was covered with cartilage. Each section was evaluated with the
qualitative scoring system by two independent examiners.
Histomorphometric analyses were performed on the same histologic sections
with use of a computerized analysis system (Image-Pro Plus; Media Cybernetics,
Silver Springs, Maryland). Areas of the sections to be measured were captured
at the appropriate magnification by a video camera. Calibration was performed
according to the instructions accompanying the software. Measurements were
made of the ossicle area (marrow space and associated new bone), marrow area,
new-bone area (distinct from demineralized bone matrix and limb bones),
cartilage area, and total area of residual particles of demineralized bone
matrix.
Statistical Analysis
The results of the qualitative and histomorphometric analyses were
calculated as the mean and standard error of the mean for each variable. The
values for all groups represented findings from either four or six animals,
providing a sample size of eight or twelve. Previous studies have shown the
validity of treating each implant, rather than each animal, as a unit (two
sites per animal)7.
Significant differences between groups were determined with analysis of
variance and the use of the Bonferroni modification of the Student t test. For
both statistical tests, p values of =0.05 were considered significant.
Power calculations showed that the power ranged between 0.54 and 0.89,
depending on the parameter being measured.
PDGF Study
Histologic Observations
Validation of the nude-mouse muscle-implantation assay: The
demineralized bone matrix used in the study was osteoinductive, with a
qualitative score identical to that derived by previous assessments of the
same batch. Heat-inactivation resulted in a loss of osteoinduction. Moreover,
the qualitative and quantitative results were congruent. These observations
indicate that the assay was valid and, therefore, that the effects of PDGF on
new cartilage and bone formation in the nude-mouse muscle-implantation assay
can be interpreted with a high degree of certainty. There was no evidence of
pathologic change in the sections. The cartilage and bone had a normal
histologic appearance. Surrounding tissues were normal, and there was no
evidence of fibrosis.
Bone-Induction Score
The qualitative scores for bone induction are graphed in
Figure 1. Demineralized bone
matrix induced bone formation by day 28, and the amount of induced bone
increased by day 56. This finding is in agreement with that in an earlier
study demonstrating that eight weeks allows more time for the full development
of ossicles after use of commercial preparations of human demineralized bone
matrix7. When PDGF
was added to the implants, there was a small dose-dependent decrease in the
bone-induction score as the amount of factor was increased. This was evident
at both day 28 and day 56.
Histomorphometry
The quantitative changes in ossicle formation brought about by PDGF are
depicted in Figure 2. PDGF
reduced the quantity of new bone in a dose and time-dependent manner. At a
dose of 0.1 µg per implant, PDGF did not significantly affect the ossicle
area. However, at higher concentrations, there was a decrease in ossicle area.
At a concentration of 1 µg per implant, the reduction was significant at
twenty-eight days only. However, at a concentration of 10 µg per implant,
the ossicle area was reduced by >80% at twenty-eight days and by 75% at
fifty-six days.
The marrow space, used to determine the ossicle area in
Figure 2, is the area
encompassed by a combination of demineralized bone matrix and new bone. At
twenty-eight days, there was a significant dose-dependent decrease in the area
of bone marrow in the implants treated with 1 or 10 µg of PDGF
(Fig. 3). This effect was
evident at fifty-six days only in the implant treated with 10 µg of the
growth factor. The area of new bone was also affected, but to a lesser extent
than the marrow space (Fig. 4).
The amount of new bone was significantly reduced only in tissues in which 10
µg of PDGF had been implanted.
As bone formation in an ectopic site follows the classic endochondral
pathway, we measured cartilage as well as bone in order to better monitor
changes due to PDGF. The effect of PDGF on cartilage induction is graphed in
Figure 5. As expected,
cartilage appears early in the induction process (by day 14) and essentially
becomes nonexistent as the conversion to bone occurs in the following weeks.
According to our qualitative scoring, the two lower doses of PDGF did not
modify cartilage response, but the higher dose significantly altered the
process. The cartilage induction score was reduced, and the cartilage
persisted through twenty-eight days. The area of cartilage was affected by
PDGF in a dose and time-dependent manner
(Fig. 6). At fourteen days, as
the concentration of PDGF increased, the area of new cartilage decreased. At
twenty-eight and fifty-six days, there was little cartilage remaining,
regardless of the dose of PDGF.
The effect of PDGF on resorption of demineralized bone matrix was evaluated
indirectly by measuring the area of residual particles
(Fig. 7). The area of residual
particles in tissues in which heat-inactivated demineralized bone matrix had
been implanted was comparable with that in the positive control specimens.
Addition of PDGF reduced resorption of demineralized bone matrix at fourteen
and twenty-eight days. However, by fifty-six days, these effects were no
longer evident.
Platelet-Rich-Plasma Study
TGF-ß1 Content
In this study, we determined that the total TGF-ß1 content was 18.7
ng/mL in the blood of the donor, 10.4 ng/mL in the platelet-poor plasma, and
38.2 ng/mL in the platelet-rich plasma. No active TGF-ß1 could be
detected in the blood, platelet-poor plasma, or platelet-rich plasma prior to
treatment with thrombin. After coagulation, however, 5% of the total
TGF-ß1 in the donor's blood was activated; 2% was activated in the
platelet-poor plasma, but 71% was activated in the platelet-rich plasma.
Histologic Observations
High-activity demineralized bone matrix consistently induced formation of
one or more ossicles of new bone (Fig. 8,
A) in at least one section of the implant. Typical of
this assay system, only a small portion of the demineralized bone matrix
particles in any cross section was associated with an ossicle. The ossicles
and the unincorporated demineralized bone matrix were surrounded by normal
connective tissue. Low-activity demineralized-bone-matrix implants were also
surrounded by connective tissue, but virtually no osteoinduction was observed.
While the addition of platelet-rich plasma to the low-activity demineralized
bone matrix incited no side effects such as inflammation, the platelet-rich
plasma did not increase osteoinductivity either. On the other hand, the
addition of platelet-rich plasma to high-activity demineralized bone matrix
appeared to delay ossicle formation and to encourage the persistence of
cartilage (Fig. 8, B and
C).
Bone-Induction Scores
The mean bone-induction score was 1.27 ± 0.22 for the implants
treated with low-activity demineralized bone matrix, whereas it was 2.67
± 0.19 for those treated with high-activity demineralized bone matrix
(Fig. 9, A). The
addition of platelet-rich plasma to low-activity demineralized bone matrix had
no effect on the bone-induction score. In contrast, the bone-induction score
of the implants treated with platelet-rich plasma and high-activity
demineralized bone matrix decreased to 2.0 ± 0.45. While this score
remained higher than that for the low-activity demineralized bone matrix, it
was lower than the value for the high-activity demineralized bone matrix
alone. In both cases, the changes were not significant.
Histomorphometry
The trends indicated by the bone-induction scores were confirmed by
histomorphometry. The area of new bone formation induced by high-activity
demineralized bone matrix was more than 3.5 times greater than that induced by
low-activity demineralized bone matrix
(Fig. 9, B). Addition
of platelet-rich plasma to low-activity demineralized bone matrix had no
effect. The difference in the amounts of demineralized bone matrix, as
measured by area, was striking after fifty-six days in situ. It appeared that
low-activity demineralized bone matrix was resorbed more slowly than the
highly active material and that platelet-rich plasma significantly increased
the resorption of both (Fig. 9,
C). In both cases, the decrease in the area of
demineralized bone matrix that had been treated with platelet-rich plasma was
significant.
Following Urist's elucidation of the osteoinductivity of
demineralized bone
matrix2, this graft
material has been widely used in orofacial and orthopaedic surgical procedures
in which additional bone formation is deemed
critical41.
Presumably, the osteoinductivity derives from the presence of bone
morphogenetic proteins (BMPs) and their interaction with other factors at the
implant site, including TGF-ß and PDGF, among
others42. Proteins
present in demineralized bone matrix can be heat-inactivated, thereby
destroying the osteoinductivity of the
matrix43. More
importantly, demineralized bone matrix is produced in various forms by many
bone banks, in many cases without any substantiation of
activity42.
Because the osteoinductivity of demineralized bone matrix is so
variable42, the
possibility of replacing or adding growth factors to make it more uniformly
osteoinductive is
attractive16,22.
Our findings demonstrate that PDGF affects new bone formation induced by
demineralized bone matrix, at least in the nude-mouse muscle-implantation
model used in this study. PDGF caused a dose and time-dependent suppression of
ossicle formation, particularly when high concentrations were used. Addition
of PDGF to the demineralized bone matrix reduced the amount of new bone
formation. This effect was seen twenty-eight days post-transplantation. The
highest dose (10 µg) virtually obviated bone induction. In contrast, on the
fifty-sixth day, bone formation in tissues in which demineralized bone matrix
mixed with 0.1 or 1 µg of PDGF had been implanted were at or near the
control level. In the implants treated with 0.1 µg of PDGF, there was a
slight increase in new bone formation, but it was not significant.
These data suggest that lower doses of PDGF delay the osteoinduction
process but do not lessen the amount of new bone formation. Only the highest
level of the mitogen seems to inhibit the entire cascade. It could be argued
that the effects of PDGF are nonspecific and the delay reflects the time
needed for the factor to be removed from the implant site before the
demineralized bone matrix can become optimally inductive. However, the almost
complete suppression of events at the higher dose suggests that a mitotic or
metabolic component is involved.
One possibility is that PDGF increased the pool of progenitor cells, as it
has been reported to do in soft-tissue
wound-healing44. If
this had been the case, bone formation should have been delayed but ultimately
there should have been a greater amount of new bone. The mouse
muscle-implantation model may have been limiting in this regard, in that the
amount of demineralized bone matrix that we could use may have been
insufficient to provide enough osteoinductive factors to induce chondrogenesis
in an enlarged pool of undifferentiated mesenchymal cells. The muscle
environment may also have lacked the necessary cofactors. It is also possible
that, at the highest concentration of PDGF used in the present study, muscle
mesenchymal cells were induced to enter other mesenchymal lineages. This
hypothesis is supported by the observation that ultimately the same amount of
bone was present in all implant sites, suggesting that the amount of
demineralized bone matrix was the overriding factor. However, when
BMP-24 or enamel
matrix
derivatives45 are
added to demineralized bone matrix in the same model, bone formation is
increased, indicating that muscle tissue can support enhanced osteoinduction
in the presence of appropriate stimuli.
Our findings regarding the modulation of bone induction by PDGF in this
system cannot be used to generalize how this growth factor might influence
bone growth in situ, such as with fracture-healing or bone-remodeling. The
present study was designed to evaluate PDGF as a modulator of demineralized
bone matrix-induced osteoinduction in muscle. It is clear that the effects of
PDGF depend on the model used. In contrast to our observations in the muscle
assay, others have found that PDGF enhanced regeneration of periodontal
tissues in defects treated with demineralized bone
matrix46,47
and Nash et al.16
observed an increase in bone-healing in osteotomy defects treated with PDGF in
a collagen carrier. These findings suggest that other osteogenic factors in a
bone site may work synergistically with exogenous PDGF to increase bone
formation. The format in which PDGF is delivered and the time course of
delivery may also be factors. It is likely that, in our study, PDGF was
released from the implants during the early inflammatory phase of
osteoinduction, whereas, in a bone environment, osteoblasts continue to
produce PDGF as a paracrine factor during osteogenesis. Recently, Kubota et
al.48 observed that
PDGF is also secreted by osteoclasts and acts as an inhibitor of osteogenesis,
which implicates it as a factor in bone-remodeling.
Bone induction in nude mice follows the common pathway of bone formation
through the endochondral
stage49. The effect
of PDGF on bone formation was secondary to its effect on cartilage formation.
Histomorphometric measurements showed that PDGF reduced the amount of
cartilage in a dose-dependent manner, with little cartilage present in the
implants treated with the highest concentration. The relative lack of
cartilage at the higher dose is in keeping with the lack of bone at the same
dose. This finding suggests that the role of PDGF at high concentrations is at
the earliest stages of the osteoinductive process. Interestingly, although
only minimal cartilage was present in the sites treated with high
concentrations of PDGF, that cartilage persisted, whereas it was converted to
bone in the presence of lower concentrations of the mitogen. The reason for
this is not known.
The rate of resorption of demineralized bone matrix and the release of its
growth factors could, theoretically, influence the osteoinductive process. The
data suggest that PDGF slightly inhibited the resorption of demineralized bone
matrix particles, but only at days 14 and 28. By day 56, the area of implant
material left in the sections was essentially the same as that in the controls
at all dose levels. The retardation of particle resorption at days 14 and 28
might explain some of the enhanced new-bone formation seen at the lesser doses
at the same time-periods, but it certainly cannot explain the profound
inhibition seen at day 56. We assume that, in all cases, the demineralized
bone matrix was accepted by and integrated into the host tissue and that any
loss of inductivity was after the release of biologically active
molecules.
Our results also suggest that the effects of PDGF on demineralized bone
matrix-induced bone formation are not mitigated by the presence of other
growth factors in platelet-rich plasma. PDGF is a major growth factor found in
platelets, and it is currently made available by concentrating platelets with
an appropriate device. Published studies have indicated that the PDGF content
of platelet-rich plasma ranges from approximately 85 to 254
ng/mL50. Typically,
the platelets are concentrated from freshly drawn blood, by a preparation
device, to produce platelet-rich plasma; their activity is subsequently
released by thrombin or other
agents3. Our results
show that the levels of active TGF-ß1 are increased by this process.
TGF-ß1 has been shown to potentiate the activity of
BMPs51, but it has
also been shown to reduce the effectiveness of
BMP52 and to
increase
fibrosis53.
Platelet-rich plasma has been shown to improve healing when compared with
standard
therapies23-27,
and it has been used as an adjunct in orofacial and orthopaedic surgical
procedures23-31.
However, its actual effectiveness has been obscured by the lack of
well-designed study protocols. One exception is a randomized controlled trial
by Marx et al.29,
who reported that mandibular bone grafts augmented with platelet-rich plasma
matured earlier than unaugmented bone grafts. One group reported that
platelet-rich plasma does not enhance bone-healing, but neither of their
studies were controlled clinical
trials32. Also, the
effects of platelet-rich plasma on bone-healing in animals have not been
consistent. While many studies have demonstrated enhanced bone-healing
following the addition of platelet-rich plasma to grafts or graft
substitutes33-35,
others have shown less favorable
results36,37.
Because platelet-rich plasma has been studied at disparate sites, has often
been combined with other materials, and in some cases has had negative
effects, the exact potential of platelet-rich plasma to improve bone-healing
has been difficult to
distill54.
In this study, we demonstrated that platelet-rich plasma cannot transform
low-activity demineralized bone matrix into a graft material with higher
osteoinductivity, at least at the single time-point that we examined. The
osteoinductivity of even the highly active demineralized bone matrix was
slightly inhibited, with the process of endochondral bone formation seemingly
delayed, as demonstrated by residual cartilage in some sections. These
findings were similar to those associated with the PDGF, suggesting that the
other growth factors found in platelet-rich plasma releasates do not alter the
final outcome due to PDGF alone. However, as we mentioned, only a single
time-point was examined; transient events occurring early in the
osteoinduction process were not assessed and we did not determine the amount
of PDGF in the platelet releasate, so a direct comparison cannot be made.
In contrast to the reduced rate of resorption of demineralized bone matrix
noted in the presence of PDGF, particle resorption was increased in the
presence of platelet-rich plasma. The areas of both low and high-activity
particles were reduced by supplementation with platelet-rich plasma,
suggesting that something in the platelet-rich plasma stimulated osteoclastic
resorption of the implants. Logically, a process that freed BMPs and other
factors would be expected to promote osteogenesis. However, there is a
possibility that a slower removal of the implant matrix provides a continuum
of scaffolding for new bone formation. Although PDGF decreased osteogenesis in
a dose-dependent manner, it did not promote the resorption of demineralized
bone matrix particles. The combined findings suggest that PDGF in the
platelet-rich plasma would not be expected to enhance the osteoinductivity of
this type of allograft, but neither should it affect resorption. Because
platelet-rich plasma contains so many growth factors and their concentration
varies with the method of preparation, more studies of individual components
are necessary to understand the soup that is platelet-rich plasma and in what
kind of clinical situations it will prove beneficial.
We concluded from these findings that PDGF can affect demineralized bone
matrix-induced osteoinduction in muscle and that, at high concentrations, it
can inhibit the process. Any statement regarding whether this observation can
be transferred to other kinds of bone formation would be premature. However,
there does appear to be some consistency of findings in the nude-mouse model.
Our study demonstrated that the inhibitory effects of PDGF are dose-sensitive,
and it may be that nature regulates local concentrations far more optimally
than does an implant study such as this one. The results indicate that PDGF
and platelet-rich plasma are unlikely to improve the osteoinductive ability of
demineralized bone matrix at a non-bone site. Additional studies are needed to
determine the clinical relevance of our observations. ?
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