The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 89, Issue 1

Scientific Articles | January 01, 2007

Platelet-Rich Plasma Inhibits Demineralized Bone Matrix-Induced Bone Formation in Nude Mice

Don M. Ranly, DDS, PhD¹; Christoph H. Lohmann, MD, MS²; Domenico Andreacchio, MD³; Barbara D. Boyan, PhD¹; Zvi Schwartz, DMD, PhD¹

¹ Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 315 Ferst Drive N.W., Atlanta, GA 30332-0363. E-mail address for B.D. Boyan: barbara.boyan@bme.gatech.edu
² Department of Orthopaedics, University of Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany
³ Department of Orthopaedics, Fritz-Konig-Stift Bad Harzburg, Ilsenburger Straße 95, 38667 Bad Harzburg, Germany

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Disclosure: In support of their research for or preparation of this manuscript, one or more of the authors received grants or outside funding from DePuy Germany, the National Science Foundation, the Price Gilbert, Jr. Foundation, the Georgia Research Alliance, and the Georgia Tech/Emory Center for the Engineering of Living Tissues. In addition, one or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (DePuy Germany). Also, commercial entities (Elsbeth-Bonhoff Foundation and Plus Orthopaedics) paid or directed, or agreed to pay or direct, benefits to a research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Investigation performed at the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia

The Journal of Bone and Joint Surgery, Incorporated

J Bone Joint Surg Am, 2007 Jan 01;89(1):139-147. doi: 10.2106/JBJS.F.00388

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Abstract

Background: It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix.

Methods: Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 ± 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix.

Results: Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-ß, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma.

Conclusions: Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.

Clinical Relevance: Platelet-rich plasma may not be an appropriate adjunct to demineralized bone matrix in some clinical applications.

Figures in this Article

Augmentation of bone defects with bone graft substitutes to promote healing is a common practice in orthopaedics, dentistry, and maxillofacial surgery. These substitutes include autogenous bone harvested from other osseous sites¹; xenografts, often derived from treated bovine bone²; alloplasts³, usually consisting of polymeric or inorganic matrices used singly or in combination; and allografts⁴, such as nondemineralized freeze-dried bone and demineralized freeze-dried bone allograft, also called demineralized bone matrix. In recent years, autologous platelet-rich plasma has been added to bone substitutes with the expectation that the blood derivative enriched with growth factors would enhance bone-healing⁵.

The impetus for the use of platelet-rich plasma is based on the accumulating knowledge that platelets contain many substances that are released on activation and degranulation and that have been associated with bone formation. In addition to clotting factors, various cytokines including platelet-derived growth factor (PDGF)⁶, transforming growth factors beta-1 and beta-2 (TGF-ß1 and ß2)⁷, insulin-like growth factors I and II (IGF-I and II)⁸, basic fibroblast growth factor (FGF-2)⁹, epidermal growth factor (EGF)¹⁰, vascular endothelial growth factor (VEGF) and VEGF-C¹¹, and bone morpho-genetic protein (BMP)¹² are potentially released into a wound site. Platelet-rich plasma has been used in the treatment of macular holes¹³ and in the topical treatment of dermal ulceration¹⁴. Because of its apparent effectiveness in treating certain types of wounds, its possible use as an aid for the treatment of fractures and bone loss, and as an adjunct to bone graft substitutes, has incurred the interest of maxillofacial⁵, periodontal¹⁵, and orthopaedic surgeons¹⁶.

Although clinical enhancement of bone-healing by platelet-rich plasma has been reported in the orofacial literature⁵, many of the studies were either not controlled or had too many variables to clearly differentiate the role of the autogenous gel. In addition, some studies have suggested that platelet-rich plasma does not enhance bone-healing in oral and maxillofacial applications^17-20. The orthopaedic literature is similarly confusing with respect to the effectiveness of platelet-rich plasma. It has been shown to improve fracture-healing in diabetic rats²¹ and to improve bone formation in necrotic femoral heads in rabbits²². In addition, when platelet-rich plasma was incorporated into a biodegradable gelatin hydrogel, it increased bone formation in a rabbit ulnar bone-defect model²³. In contrast, platelet-rich plasma had no effect on peri-implant bone formation when it was used with allograft in dog femoral condyles²⁴, nor was it effective when it was used with beta-tricalcium phosphate in an anterior lumbar interbody fusion model in pigs²⁵. In a recent study²⁶, we demonstrated that PDGF, a major constituent of platelet releasate, inhibited the osteoinductivity of demineralized bone matrix after implantation in ectopic muscle sites of immunocompromised mice. In a minor part of that study, platelet-rich plasma from one individual demonstrated similar inhibitory properties.

The use of platelet-rich plasma requires special equipment, a phlebotomy procedure on the patient, and time for preparation, and there is a risk of side effects from the use of bovine thrombin to activate the platelets. Thus, in some instances, it may not be clinically feasible. For these reasons, clinicians should be assured that platelet-rich plasma is a worthwhile adjunct to bone-grafting. Platelet-rich plasma is donor-specific, with consequent differences in the number of platelets and the growth factor content^27,28, and donor variability may be a critical factor in determining its effectiveness experimentally. For this reason, we chose to expand our prior study by increasing the number of subjects used to evaluate the effects of platelet-rich plasma on bone formation induced by demineralized bone matrix. Moreover, donor differences in the osteoinductivity of demineralized bone matrix can also have an impact on the apparent effectiveness of platelet-rich plasma preparations. Thus, the present study was based on the hypothesis that platelet-rich plasma has a negative effect on demineralized bone matrix-induced bone formation and that this effect varies with the donor of the platelet-rich plasma and with the osteoinductivity of the demineralized bone matrix with which it is used. Accordingly, in this report, human demineralized bone matrix from two different batches of commercially available allograft was treated with platelet-rich plasma prepared from six young men and was evaluated in terms of whether osteoinductivity was decreased or increased in immunocompromised mice.

Materials and Methods

Materials and Methods | Results | Discussion | Appendix | References

Preparation and Characterization of Platelet-Rich Plasma

Sixty-five milliliters of venous blood was drawn from each of six healthy men ranging in age from twenty-two to thirty-seven years, with a mean age [and standard error of the mean] of 29.2 ± 2.4 years, after approval was granted from the institutional review board at the Georgia Institute of Technology. Platelet-rich plasma was prepared from each donor with use of the SYMPHONY Platelet Concentrate System (DePuy Spine, Raynham, Massachusetts) according to the manufacturer's instructions. Two fractions were obtained from this apparatus: platelet-rich plasma and platelet-poor plasma. The platelet and growth factor contents in each of these fractions and in the whole blood from the six donors were evaluated by means of platelet counts (Coulter Cell Counter, Multisizer II; Beckman Coulter, Miami, Florida) and kit immunoassays for TGF-ß1 (Quantikine DB100; R&D Systems, Minneapolis, Minnesota), PDGF-AA (Quantikine DAA00), PDGF-AB (Quantikine DHD00B), and PDGF-BB (Quantikine DBB00).

Samples were analyzed before and after platelet activation. After aliquots of platelet-rich plasma, platelet-poor plasma, and whole blood were removed, 200 µL of each of the fractions was treated with 20 µL of sterile bovine thrombin prepared by mixing 25 µL of the enzyme (1000 U/mL) in 10 mL of CaCl₂ under sterile conditions. The addition of thrombin to whole blood elicited clotting. In order to obtain a liquid sample from the whole blood, mild centrifugation (300 × g) was used to precipitate the clot. After activation of the samples, levels of active and latent TGF-ß1 as well as levels of the three isoforms of PDGF were determined.

Formulation of Human Demineralized Bone Matrix

Human demineralized bone matrix particles, prepared for clinical use and ranging in size from 200 to 500 µm, were obtained as a gift from LifeNet (Virginia Beach, Virginia). In a previous study²⁹, we evaluated the osteoinductivity of twenty-seven batches of demineralized bone matrix from this tissue bank in the nude-mouse muscle implantation assay, and from this assortment we selected two batches with moderate osteoinduction activity. These two batches were designated as demineralized bone matrix-1 and demineralized bone matrix-2.

Because preparations of demineralized bone matrix, with or without platelet-rich plasma, are not sufficiently cohesive to be implanted into a muscle pouch without dispersion, particles were placed into number-5 gelatin capsules (10 mg of demineralized bone matrix per capsule). The gelatin capsules were not presterilized, and the demineralized bone matrix was not weighed and packaged under clean-room conditions. Therefore, all of the implants were sterilized with ultraviolet light overnight prior to use. Previous studies have demonstrated that implants sterilized by this means do not elicit a microbial infection. Importantly, the osteoinductivity of both batches used in this study was previously determined with the same protocol; consequently, any negative impact of the sterilization was already accounted for.

Immediately prior to implantation, 25 µL of platelet-rich plasma was activated with 2.5 µL of thrombin/calcium chloride and dispensed into a capsule containing the demineralized bone matrix. The demineralized bone matrix and platelet-rich plasma were then mixed with the dispensing pipette tip, and the capsule was implanted. When platelet-rich plasma was implanted without demineralized bone matrix, the same volume of the preparation was used. The platelet-rich plasma employed for these experiments was prepared immediately prior to use.

Implantation Protocol

Inbred male nu/nu mice (nude mice) (Harlan, Indianapolis, Indiana) were used in this study because the compromised immune system in these animals permits an evaluation of the response to xenograft implants with minimal donor-host interactions. Eighty mice were divided into twenty groups of four mice each (see Appendix). Each batch of demineralized bone matrix was evaluated by itself and with each of the six different preparations of platelet-rich plasma. Each preparation of platelet-rich plasma was also implanted without demineralized bone matrix as a control.

Following administration of inhalation anesthesia with isoflurane, the implantation sites were disinfected with povidone iodine. One small skin incision was made over the gastrocnemius muscle in each hindlimb, and a pouch was prepared by blunt dissection. One gelatin capsule was inserted into each pouch, and the incision was closed with clips. This protocol was approved by the Institutional Animal Care and Use Committee at the Georgia Institute of Technology. Each mouse received two identical implants, thereby reducing the possibility of the cross reactivity that might occur with dissimilar formulations. As each group comprised four mice and two implants were used in each animal, the sample size was eight for each treatment group.

Histological Evaluation

Fifty-six days after implantation, the mice were killed by asphyxiation with carbon dioxide. The whole calf muscles were removed to ensure complete recovery of the implant site. Following fixation in 10% neutral buffered formalin, the tissues were decalcified in 5% formic acid and embedded in paraffin. Three consecutive cross-sectional cuts (3 to 4 µm in thickness) were made at each of three different levels of the calf muscle to ensure visualization of residual demineralized bone matrix and any new bone formation. Sections were stained with hematoxylin and eosin, and all were evaluated at 10× magnification for the presence or absence of demineralized bone matrix particles, ossicle formation, and new bone. One section from each implant was selected for further analysis. If more than one section revealed any of these features, the section that had the largest area of demineralized bone matrix or the most induced new bone was chosen for measurement. Because the section with the greatest amount of new bone was evaluated, the results were biased toward success for each formulation.

Each section was evaluated with use of a system for qualitative scoring of osteoinduction²⁶. With this system, sections with no evidence of demineralized bone matrix or new tissue were valued as 0 and not included in any averages. When demineralized bone matrix was observed but no new bone or cartilage was present, the value was 1. When an ossicle was observed, the value was 2. When two or more ossicles were present, the value was 3. In sections with very osteoinductive implants, with 70% of the slide at 10× magnification covered with an ossicle, a value of 4 was ascribed. Two independent examiners evaluated each section with the qualitative scoring system. A third examiner resolved differences in scoring when the two examiners did not agree; this was the case for 11.4% of the sections examined.

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Fig. 1: Qualitative scores for bone induction by demineralized bone matrix-1 (DBM-1) and 2 (DBM-2) with and without platelet-rich plasma (PRP). The values for the implants with platelet-rich plasma were calculated by first separately determining the average value for each of the six donors (eight implants per donor) and then by determining the mean and standard error of the mean of the values for the six donors. This was also the case for platelet-rich plasma alone. ^*p < 0.05 for platelet-rich plasma plus demineralized bone matrix-2 compared with all other samples.

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Fig. 2: Effect of platelet-rich plasma (PRP) on bone induction by demineralized bone matrix-1 (DBM-1) and 2 (DBM-2) as determined by ossicle size and new bone area. The values for the implants with platelet-rich plasma were calculated by first separately determining the average value for each of the six donors (eight implants per donor) and then by determining the mean and standard error of the mean of the values for the six donors. This was also the case for platelet-rich plasma alone. ^*p = 0.05 for platelet-rich plasma plus demineralized bone matrix-2 compared with demineralized bone matrix-1; #p < 0.05 for platelet-rich plasma plus demineralized bone matrix-2 compared with demineralized bone matrix-2.

Histomorphometric analysis with use of a computerized analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, Maryland) was performed on the same histologic sections used for qualitative scoring. 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. The measurements included the ossicle area (as determined by the marrow space), the new bone area (distinct from demineralized bone matrix and limb bones), and the total area of residual demineralized bone matrix particles.

Statistical Analysis

The results of the qualitative and morphometric analyses were calculated as the means and standard error of the mean for each variable. The values for all groups represented the findings from two limbs of each of four animals, providing a sample size of eight. Previous studies demonstrated the validity of treating each implant (at each of two sites per animal), rather than each animal, as a unit²⁹. 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 to be significant. Power calculations showed that the power ranged between 0.54 and 0.89, depending on the parameter being measured.

Results

Materials and Methods | Results | Discussion | Appendix | References

Evaluation of Platelet-Rich Plasma

The SmartPReP system (Harvest Technologies, Plymouth, Massachusetts) used in this study was effective as a platelet concentrator. The platelet count in platelet-rich plasma was amplified approximately fourfold when compared with that in platelet-poor plasma and whole blood (see Appendix). Platelet-rich plasma was also enriched in growth factors, including both active and latent TGF-ß1. The active TGF-ß1 content of whole blood was low, but release of granules following activation of platelets by thrombin more than doubled the amount of active TGF-ß1 in the platelet-rich plasma preparation (see Appendix). The amount of latent TGF-ß1 in whole blood was approximately 150 times greater than the amount of active TGF-ß1, and it doubled on the release of granules (see Appendix). Similarly, the three dimeric forms of PDGF were concentrated in platelet-rich plasma prepared with the SmartPReP apparatus. Comparison of the platelet-rich plasma with the platelet-poor plasma revealed approximately a sixfold increase in the amount of of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold difference in the amount of PDGF-AB (see Appendix). All of these differences were significant (p < 0.05). Degranulation by thrombin significantly increased the amount of each of these PDGFs in the platelet-rich plasma preparation.

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Fig. 3: Effect of platelet-rich plasma (PRP) on the resorption of demineralized bone matrix-1 (DBM-1) and 2 (DBM-2) as determined by residual demineralized bone matrix area. The values for the implants with platelet-rich plasma were calculated by first separately determining the average value for each of the six donors (eight implants per donor) and then by determining the mean and standard error of the mean of the values for the six donors. This was also the case for platelet-rich plasma alone. ^*p < 0.05 for platelet-rich plasma plus demineralized bone matrix-2 compared with demineralized bone matrix-2. No demineralized bone matrix was present in sites implanted with platelet-rich plasma alone.

Histological Evaluation

Validation of the Nude-Mouse Muscle Assay

The two batches of demineralized bone matrix used in this study were both moderately osteoinductive (Fig. 1), with qualitative osteoinductivity scores that were comparable with those reported previously²⁹. Moreover, the qualitative and quantitative results (Fig. 2) were congruent. These observations indicate that the assay is valid and that the effects of platelet-rich plasma (enhancement or inhibition) can be interpreted with confidence. No evidence of pathological change was observed in any of the sections. Newly formed bone had a typical histological appearance and could be differentiated from the implanted bone matrix. The surrounding muscle tissues were normal, and there was no evidence of fibrosis.

Bone Induction Score

The qualitative osteoinductivity scores are presented in Figure 1. The values for the implants in which platelet-rich plasma was used represent the averages from the six different preparations of platelet-rich plasma (i.e., the plasma from the six donors). Both batches of demineralized bone matrix implanted without platelet-rich plasma exhibited moderate osteoinduction. None of the platelet-rich plasma samples implanted without demineralized bone matrix elicited any kind of osteoinductive activity. The average score for demineralized bone matrix-1 implanted with platelet-rich plasma was very similar to the value for the untreated demineralized bone matrix-1. The average score for demineralized bone matrix-2 implanted with platelet-rich plasma was lower than that for the untreated demineralized bone matrix-2. These qualitative scores suggest that platelet-rich plasma does not enhance the osteoinductivity of demineralized bone matrix in nude mice and that the effectiveness of platelet-rich plasma may depend on the properties of the demineralized bone matrix with which it is used.

Histomorphometric Analysis

The measurements of ossicle and new bone formation are shown in Figure 2. Again, the values for the groups in which platelet-rich plasma was used represent the averages of the values for the six preparations of platelet-rich plasma. Both control samples of demineralized bone matrix generated new bone in the form of ossicles. Demineralized bone matrix-2 induced the formation of smaller ossicles (p < 0.05), although the amount of new bone within the ossicle was comparable with that found in the ossicles induced by demineralized bone matrix-1. The addition of platelet-rich plasma reduced the size of the ossicles induced by both demineralized bone matrix batches, but the effect of the platelet-rich plasma was significant only when it was used with demineralized bone matrix-2 (p < 0.05). Moreover, platelet-rich plasma reduced the amount of new bone within the ossicles induced by demineralized bone matrix-2 (p < 0.05). These data reinforce the qualitative finding that platelet-rich plasma does not enhance the osteoinductivity of demineralized bone matrix in the nude mouse model and suggest that it may reduce osteoinductivity in a donor-dependent manner.

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Fig. 4: Treatment/control ratios for demineralized bone matrix-1 and platelet-rich plasma (DBM-1+PRP) compared with demineralized bone matrix-1 as well as for demineralized bone matrix-2 and platelet-rich plasma (DBM-2+PRP) compared with demineralized bone matrix-2. The effects of platelet-rich plasma on the qualitative osteoinductivity score, ossicle area, new bone area, and area of residual demineralized bone matrix are shown. The values for the implants with platelet-rich plasma were calculated by first separately determining the average value for each of the six donors (eight implants per donor) and then by determining the mean and standard error of the mean of the values for the six donors. This was also the case for platelet-rich plasma alone. ^*p < 0.05 for the addition of platelet-rich plasma compared with a ratio of 1.

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Fig. 5: Effect of individual platelet-rich plasma preparations on the qualitative osteoinductivity score (Panel A), ossicle area (Panel B), and new bone area (Panel C) associated with demineralized bone matrix-1 (DBM-1) and 2 (DBM-2). Each donor platelet-rich plasma was tested in eight implants for each batch of demineralized bone matrix, and the values are given as the mean and standard error of the mean. ^*p < 0.05 for the addition of platelet-rich plasma compared with a ratio of 1.

The areas of residual demineralized bone matrix and platelet-rich plasma-treated demineralized bone matrix remaining after fifty-six days are shown in Figure 3. The values are means of the mean results for each donor. Platelet-rich plasma delayed the resorption of demineralized bone matrix-2 but did not significantly alter the amount of demineralized bone matrix-1 present in the harvested tissues. When the values from the qualitative and quantitative assessments are plotted as treatment/control ratios (Fig. 4), the impact of platelet-rich plasma on the osteoinductivity of demineralized bone matrix can be fully appreciated. Platelet-rich plasma had a more pronounced inhibition effect on demineralized bone matrix-2, but it affected both bone preparations.

Individual Effects of Platelet-Rich Plasma

Figure 5 presents the scores for each of the six blood donors in a treatment/control ratio format. In panel A, the majority of the qualitative scores had a ratio of <1 when demineralized bone matrix-2 with platelet-rich plasma was used. In contrast, when demineralized bone matrix-1 with platelet-rich plasma was used, none of the platelet-rich plasma preparations showed a significant effect, either positive or negative. In panel B, ossicle formation induced by demineralized bone matrix-1 and platelet-rich plasma is shown to be comparable with that induced by demineralized bone matrix-1 alone, but platelet-rich plasma from donors 2, 4, 5, and 6 significantly decreased the ossicle area induced by demineralized bone matrix-2. Panel C shows that none of the platelet-rich plasma preparations were capable of significantly enhancing new bone formation when implanted with demineralized bone matrix. None of the platelet-rich plasma preparations significantly reduced the bone formed in response to demineralized bone matrix-1, but platelet-rich plasma from donors 2, 4, 5, and 6 significantly reduced the new bone formed in response to demineralized bone matrix-2. Although the two demineralized bone matrix samples differed slightly with regard to their osteoinductivity, and individual responses to the six platelet preparations were exhibited, collectively the findings indicate that platelet-rich plasma does not amplify the osteoinductivity of demineralized bone matrix in muscle sites of nude mice.

Discussion

Materials and Methods | Results | Discussion | Appendix | References

The philosophy behind the use of platelet-rich plasma as an adjunct to bone surgery is based on reasonable assumptions. Platelets are a normal component of clotting and wound-healing and have been shown to promote certain types of ocular or dermal healing. Growth factors present in platelet-rich plasma have been shown to increase osteoblastic activity in vitro in a number of studies³⁰. However, these growth factors have pleiotropic effects and may also inhibit osteoblastic differentiation depending on the concentration and the state of maturation of the cells within the osteoblast lineage. For example, TGF-ß1 increases alkaline phosphatase activity in cultures of osteoblast-like cells^31,32, but it also inhibits terminal differentiation of these cells³³. The effects of the growth factors may also be time-dependent. Ogino et al.³⁴ showed that platelet-rich plasma stimulates proliferation of osteoblast-like cells at early time-points and this effect can be suppressed by neutralizing antibodies to PDGF, TGF-ß1, and IGF-I, indicating that they are involved. However, at later time-points, antibodies to IGF-I no longer block the stimulatory effects of the preparation. Effects also vary with the target cell. IGF-I has been shown to upregulate osteoblastic markers in human mesenchymal stem cells³⁵, to have no effect on osteogenic differentiation of human bone-marrow stromal cells³⁶, and to inhibit osteoblastic differentiation of bovine calcifying vascular cells³⁷. Factors in platelet-rich plasma may also affect the activity of each other³⁸. Thus, the variability in the human platelet-rich-plasma growth-factor content observed in our study as well as in other laboratories³⁹ is a confounding factor in predicting clinical effectiveness.

Our results showed that, if platelet-rich plasma does enhance bone-healing in some applications, the effect is not due to an osteoinductive property of the platelet-rich plasma itself. Platelet-rich plasma alone did not induce osteogenesis when implanted in nude-mouse muscle under the same conditions used to demonstrate osteoinductivity of protein extracts that contain bone morphogenetic proteins^40,41. In addition, platelet-rich plasma did not enhance the osteoinductivity of demineralized bone matrix in the nude-mouse muscle-implantation assay. It is unlikely that the failure to detect enhancement was due to a lack of sensitivity of the assay. We previously showed that concentrated extracts of deproteinized bovine bone, which contain TGF-ß1 and BMP-2, can increase osteoinductivity⁴², as can porcine enamel matrix proteins, which consist primarily of amelogenins⁴³. These former studies also showed that the muscle environment is adequate to support osteoinduction if appropriate signals are present.

The results of the present study indicate that platelet-rich plasma exerts an inhibitory effect on the osteoinduction process, confirming our previous observation regarding the effects of platelet-rich plasma from a single donor²⁶. In the present study, we demonstrated that the magnitude of the inhibitory effect varied from one platelet-rich-plasma donor to another. Although a major reason for using autologous platelet-rich plasma is that the patient is not exposed to exogenous infectious or immunogenic agents, specific differences in the platelet content and growth factor composition of platelet-rich plasma^27,28,39 could affect the ability of an osteogenic material like demineralized bone matrix to induce bone formation. Our results support this hypothesis.

The failure of the platelet-rich plasma to augment demineralized bone matrix activity cannot be ascribed to poor preparation of the platelet-rich plasma. Blood was deliberately drawn from young healthy men, and platelet counts and growth factor analysis proved that the methodology used to concentrate the platelets was successful. The findings of comparisons of commercial systems with regard to their ability to concentrate platelets and growth factors illustrate the variability of the process^44,45. Our results also showed donor variation in the composition of the platelet-rich plasma, but the differences were not significant. In all cases, TGF-ß1 and PDGF isoforms were substantially increased during platelet-rich-plasma preparation and activation.

Successful clinical use of platelet-rich plasma may be site and application-specific. While the present study showed that platelet-rich plasma does not enhance osteoinduction at an ectopic site, it may be an effective additive at bone sites where the supply of progenitor cells is adequate. Our observation that demineralized bone matrix itself may play a role is an important one. Human demineralized bone matrix preparations may vary not only in their content of BMPs⁴⁰, but also in their physical properties²⁹. Both of the batches used in the present study were from the same bone bank, limiting differences in osteoinductivity due to processing or sterilization. Both batches had comparable qualitative osteoinduction scores based on the numbers of ossicles formed, and these scores were comparable with scores obtained for the same batches in other groups of mice. Histomorphometric analysis of the ossicles showed that demineralized bone matrix-1 induced more new bone than did demineralized bone matrix-2, suggesting that there may have been quantitative differences in the factors contributing to osteogenic properties. The interaction of platelet-rich plasma with demineralized bone matrix, either in terms of physically coating the demineralized bone matrix particles or in terms of the combination of growth factors that ultimately modulate host progenitor cells, is not yet well understood.

Our findings suggest that concentration of PDGF, one of the principal factors in platelet-rich plasma, may have contributed to the reduced osteoinductivity. We showed that PDGF-BB causes a dose-dependent inhibition of demineralized bone matrix-induced ossicle formation in nude-mouse muscle²⁶. The platelet concentration method that we used resulted in a sixfold increase in PDGF-BB content and increased the amounts of the AA and AB dimeric forms of the growth factor as well. PDGF increases mesenchymal cell proliferation, but it does not induce progression. In the absence of an adequate concentration of appropriate progression factors, high levels of PDGF may actually reduce endochondral differentiation^46,47. Active TGF-ß1 was also increased during platelet concentration and was further increased by thrombin activation, but the levels of the active form of the growth factor were relatively low in both instances. In contrast, high levels of latent TGF-ß1 were achieved during platelet concentration and further increases were seen following platelet activation. TGF-ß1 stimulates proliferation of mesenchymal cells and increases early differentiation events in committed chondrocytes⁴⁸ and osteoblasts⁴⁹ in a concentration-dependent manner. However, it is not known if the latent TGF-ß1 was activated following implantation of the platelet-rich plasma in the mouse muscle or what the final local levels of the growth factor were.

Our study raises the issue of the clinical utility of platelet-rich plasma in applications where demineralized bone matrix-induced bone formation is a critical requirement. Previous investigators showed that platelet-rich plasma was ineffective when used with calcium phosphate granules for spine fusion applications²⁵. Similarly, concentrates of autologous growth factors consisting of platelet-rich plasma and whole blood cells reduced the effectiveness of demineralized bone matrix in lumbar spine fusions⁵⁰. Our results indicate that this was not necessarily due to the lack of an osteoinductive stimulus; platelet-rich plasma had an overall negative effect even in the presence of demineralized bone matrix with a proven osteoinduction ability. The results also indicate that selection of demineralized bone matrix is an important variable. Similarly, criteria other than platelet counts should be established for predicting whether donor platelet-rich plasma will be a useful additive in some applications.

Appendix

Materials and Methods | Results | Discussion | Appendix | References

A table showing the study design and figures presenting the specimen platelet counts and the amounts of active TGF-ß1 and of the three isomeric forms of PDGF are available with the electronic versions of this article, on our web site at (go to the article citation and click on "Supplementary Material") and on our quarterly CD-ROM (call our subscription department, at 781-449-9780, to order the CD-ROM). ?

References

Materials and Methods | Results | Discussion | Appendix | References

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Topics

blood platelets ; bone matrix ; growth factor ; mice, nude ; osteogenesis ; platelet-derived growth factor ; tissue donors ; platelet rich plasma

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Don M. Ranly, Christoph H. Lohmann, Domenico Andreacchio, Barbara D. Boyan, Zvi Schwartz; Platelet-Rich Plasma Inhibits Demineralized Bone Matrix-Induced Bone Formation in Nude Mice. The Journal of Bone & Joint Surgery. 2007 Jan;89(1):139-147.

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