Demineralized bone matrix (DBM) is used as an adjuvant in orthopaedic surgery because it contains a variety of osteoinductive proteins1. These osteoinductive bone morphogenetic proteins, specifically BMP-2, BMP-4, and BMP-7, initiate stem cell differentiation, which leads to new bone formation2. Platelet-rich plasma has been used as an autologous source of chemoattractant and mitogenic growth factors that are able to enhance the biologic activity of demineralized bone matrix3. Growth factors released from platelets include platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-ß), platelet-derived epidermal growth factor (PDEGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived angiogenesis factor (PDAF), and insulin-like growth factor-I (IGF-I)4-7. The growth factors contained in platelet-rich plasma are believed to enhance important steps of the bone-healing cascade such as stem cell recruitment, angiogenesis, extracellular matrix production, and remodeling8.
The use of platelet-rich plasma to enhance bone regeneration and soft-tissue maturation has increased in many surgical fields over the last decade. However, controversy exists regarding its added benefit. While some authors have reported a stimulatory effect in association with platelet-rich plasma9-11, others did not observe any improvement12,13 or even found inhibitory effects14,15.
Platelet-rich plasma is typically implanted in a gel form enhanced with the addition of a clinical dose of thrombin (1000 U/mL in 10% CaCl2)16. The solution that is used to rehydrate the thrombin contains calcium in order to neutralize the anticoagulant effects associated with the chelating reaction. Thrombin not only catalyzes the conversion of plasma fibrinogen into fibrin to create a platelet-rich plasma gel that has improved handling properties as compared with liquid platelet-rich plasma but also triggers platelet aggregation and degranulation17. Thus, thrombin causes the platelets to release their growth factor content when applied to the wound site. While the typical application of platelet-rich plasma includes activation with thrombin, clinical benefits of platelet-rich plasma have been documented without the use of thrombin activation18.
The purpose of the present study was to determine whether platelet-rich plasma could augment the osteoinductivity of demineralized bone matrix and whether this effect was dependent on thrombin activation. We hypothesized that the addition of thrombin to platelet-rich plasma not only changes the physical property of platelet-rich plasma but may also alter the osteoinductive activity of demineralized bone matrix.
Platelet-Rich Plasma Preparation
Fifty-four milliliters of blood from a healthy, thirty-five-year-old male donor was collected into each of two 60-mL syringes that had been prefilled with 6 mL of anticoagulant citrate dextrose A (ACD-A). Platelet-rich plasma was prepared with the GPS II system (Biomet Biologics, Warsaw, Indiana). After centrifugation at 3200 revolutions per minute for fifteen minutes, three basic components (red blood cells, platelet-rich plasma, and platelet-poor plasma) were separated. This platelet-rich plasma preparation system produces 6 mL of platelet-rich plasma with an eightfold increase in platelet concentration over baseline and 30 mL of platelet-poor plasma5,19. Freshly prepared platelet-rich plasma was used immediately for in vivo implantation.
Aliquots of platelet-rich plasma, platelet-poor plasma, and whole blood were also saved for in vitro studies. In the vial with 2 mL of platelet-rich plasma, 200 µL of thrombin (Jones Pharma, Bristol, Tennessee; bovine origin, 1000 U/mL in 10% CaCl2) was added, followed by vortexing. After incubation for twenty minutes at room temperature, the clotted platelet-rich plasma was centrifuged at 8000 revolutions per minute for ten minutes. This process was repeated with platelet-poor plasma and whole blood. The resulting supernatants were removed to new vials and were used for in vitro studies within two hours (for cell culture) or were stored at -80°C (for ELISA [enzyme-linked immunosorbent assay] tests).
Platelet-Rich Plasma Growth Factor Profiles
The levels of TGF-ß1 (Human TGF-ß1 Quantikine ELISA Kit, DB100B; R&D Systems, Minneapolis, Minnesota), VEGF (Human VEGF Quantikine ELISA Kit, DVE00; R&D Systems), and PDGF-BB (Human PDGF-BB Quantikine ELISA Kit, DBB00; R&D Systems) in the platelet-rich plasma, platelet-poor plasma, and whole blood supernatants were measured with use of commercially available ELISA kits. The platelet-rich plasma and whole blood samples were activated with thrombin as described above. The TGF-ß1 samples were additionally activated with 1N HCl in accordance with the manufacturer's instructions for the ELISA. The optical densities of the microplate wells were measured with a microplate reader (SpectraMax Plus384; Molecular Devices, Sunnyvale, California) at 405 nm. The data were analyzed with SoftMax Pro 4.6 (Molecular Devices). Sample concentrations from duplicate measurements were obtained by interpolating from the standard curve.
TGF-ß was selected as a representative growth factor in platelet-rich plasma, and its activity was tested before and after thrombin activation. TGF-ß exists in both a latent and an active form. The latent form has a C-terminal pro-region that must be cleaved in order for the protein to interact with a TGF-ß receptor20. TGF-ß activity in the platelet-rich plasma with and without thrombin activation was assayed with use of mink lung cells transfected with plasminogen activator inhibitor-1 (PAI-1) promoter-luciferase (Luc) reporter21. The cells were provided as a generous gift by Dr. D.B. Rifkin (New York University Medical Center)22. Briefly, PAI-1-Luc-transformed mink lung cells were plated in a forty-eight-well culture plate at a density of 90,000 cells/well in 10% fetal bovine serum (FBS; HyClone, Logan, Utah)/DMEM (Dulbecco modified Eagle medium; Mediatech, Manassas, Virginia) containing 200 µg/mL geneticin (G418; Clontech Laboratories, Mountain View, California) and were allowed to attach for 5.0 hours at 37°C. After washing twice with DMEM, 10µL of 1:2 serial-diluted platelet-rich plasma was added in a 0.5% FBS/DMEM test medium. After seventeen hours, cells were rinsed twice with ice-cold PBS (phosphate-buffered saline solution) before testing for luciferase activity with use of a luciferase assay kit (Promega, Madison, Wisconsin). Only active TGF-ß in platelet-rich plasma contributes to the PAI-1-linked luciferase activity.
Effect of Platelet-Rich Plasma on Osteoblast-Like Cell and Stem Cell Number
Saos-2 cells derived from a human osteosarcoma (HTB-85; ATCC, Manassas, Virginia) and rat bone marrow-derived stromal cells, prepared according to the method described by Hall et al.23, were used for the cell number study. The cell assay was based on the ability of mitochondrial dehydrogenases to oxidize thiazolyl blue (MTT) (Promega). In each ninety-six-well plate, 15,000 cells/well were plated in 10% FBS/DMEM for a twenty-four-hour adhesion period. Right after the growth medium was exchanged to serum-free medium, 10 µL of PBS-serial-diluted platelet-rich plasma or platelet-poor plasma, prepared as described above with and without thrombin activation, was added to each well. Following incubation for forty-five hours, 20 µL of MTT solution (5 mg/mL) was added to the cells. After an additional three hours of incubation, the medium was aspirated and the cells were washed twice with phosphate-buffered saline solution. Cells were lysed with 120 µL of dimethyl sulfoxide (DMSO) for five minutes. The plates were read on a microplate reader (Molecular Devices) with use of test and reference wavelengths of 570 and 620 nm, respectively. The absorbance value is proportional to the number of cells in the dish.
Demineralized Bone Matrix Implant Preparation and Implantation
Three lots of human demineralized bone matrix were acquired from an American Association of Tissue Banks-accredited tissue bank. The biologic activity of each lot was determined in vitro by means of C2C12 rat myoblast cell line (CRL-1772; ATCC) production of alkaline phosphatase (ALP) and in vivo following intramuscular implantation in an athymic rat24. The activity of the demineralized bone matrix was characterized with a bioactive index (BI) calculated from the in vitro assay with use of a known active lot of demineralized bone matrix for the positive control and no demineralized bone matrix for the negative control ([ALPtest-ALPnegative]/[ALPpositive-ALPnegative]). On the basis of the BI value, demineralized bone matrix activity was classified as low (<0.20), moderate (0.20 to 0.79), or high (=0.80) (Table I). This BI value is based on an in vitro experiment and is only a predictor of in vivo activity.
Twenty-seven male athymic rats weighing 150 to 175 g (Harlan Laboratories, Indianapolis, Indiana) were used in the study, which was approved by the University of Southern California Institutional Animal Care and Use Committee. Six pouches were created in the abdominal muscles, three on each side, by means of blunt dissection. Three implantation types were studied: (1) demineralized bone matrix with unactivated platelet-rich plasma, (2) demineralized bone matrix with thrombin-activated platelet-rich plasma, and (3) demineralized bone matrix alone. In each case, 50 mg of demineralized bone matrix was implanted in each site. Types 1 and 2 included 200 µL of platelet-rich plasma. Type 2 also received 20 µL of thrombin (1000 U/mL) reconstituted in 10% CaCl2. Each animal received two implants of each type. The implantation sites with regard to placement in the rat were randomized. Explants were retrieved after fourteen, twenty-eight, and fifty-six days. One-half of each explant underwent histological staining, and the second half was assayed for alkaline phosphatase activity with use of previously described methods24. The histological scoring system and in vivo alkaline phosphatase determination have been found to correlate with the osteoinductive activity of demineralized bone matrix following twenty-eight days of implantation24.
Alkaline Phosphatase Evaluation
One-half of each explant was homogenized in extraction buffer (1% Triton X-100). Alkaline phosphatase activity was assayed with 50 µL of this solution with use of 150 µL of 0.3-mM pNPP (Sigma-Aldrich, St. Louis, Missouri) as a substrate25. Absorbance was detected at 410 nm after thirty minutes of incubation. The alkaline phosphatase activity was normalized to the total protein content of the extract (BCA Protein Assay; Pierce, Rockford, Illinois)24.
Histological Evaluation
Explants were fixed in 10% neutral buffered formalin, decalcified in 5% formic acid, embedded in paraffin, sectioned, and stained with either hematoxylin or eosin or safranin-O. Three consecutive cross-sectional cuts were made at each of three different levels of the explant to visualize any induced cartilage and/or bone formation. Each section was evaluated for evidence of chondrogenesis, osteoinduction, and/or inflammation with use of a modified qualitative scoring system as described previously24. Briefly, de novo bone formation was scored on a 5-point scale as 1 (fibrous tissue only), 2 (cartilage only), 3 (new bone and bone marrow involving 10% of the area), 4 (new bone and bone marrow involving 20% of the area), or 5 (new bone and bone marrow involving =40% of the area). All slides were viewed at 100× magnification, and each section was evaluated with the qualitative scoring system by two blinded, independent examiners (B.H. and Z.Y.).
Statistical Analysis
All of the results were expressed as the mean and the standard deviation. Significant differences were determined with use of a one-way analysis of variance followed by a Student-Newman-Keuls test (a = 0.05). Additionally, the 95% confidence interval for the difference in the means was calculated for the histological observations.
Source of Funding
Financial support for this study was provided by Biomet (Warsaw, Indiana) and the Wright Foundation (University of Southern California, Los Angeles, California). The funding was used to support the salary of a research associate who performed cell culture and biochemical analysis. In addition, the funding was utilized for the purchase of materials and supplies.
Growth Factors in Platelet-Rich Plasma
Aliquots of thrombin-activated platelet-rich plasma, unactivated platelet-poor plasma, and thrombin-activated whole blood were tested for growth factor concentrations with ELISA kits. There was a fourfold to sevenfold increase in growth factor concentration in the platelet-rich plasma samples as compared with the baseline whole blood (Table II). These values are consistent with the growth factor content seen in clinically derived platelet-rich plasma with use of the same platelet-rich plasma preparation system5.
Thrombin Activation of Platelet-Rich Plasma
The extent of TGF-ß activation in platelet-rich plasma was measured with a specific and sensitive cell-culture method for luciferase activity with use of mink lung epithelial cells transfected with PAI-1 promoter-luciferase reporter. Only active TGF-ß can contribute to luciferase activity in platelet-rich plasma preparations. Before thrombin activation, TGF-ß in platelet-rich plasma was in a latent form. The thrombin-activated platelet-rich plasma preparations significantly increased luciferase activity. However, when freshly prepared platelet-rich plasma was not activated with thrombin, the TGF-ß activity was as low as that in platelet-poor plasma in the cell culture assay (Fig. 1). This is in contrast to the ELISA preparations, in which TGF-ß in both the thrombin-activated and unactivated platelet-rich plasma samples was converted to the active form by means of 1N hydrochloric acid treatment; thus, differences in activity were not detected. With an increase in storage time, the platelet-rich plasma began to exhibit spontaneous activation and consequently increased activity (data not shown). These results suggest that exogenous addition of thrombin activates TGF-ß from the latent to the active form immediately, as tested in the present study and in others14.
Effect of Platelet-Rich Plasma on Cell Number
When platelet-rich plasma was tested for its biologic effect in an in vitro cell culture system, it demonstrated a dose-dependent increase in the number of marrow stromal cells at forty-eight hours. However, when thrombin-activated platelet-rich plasma was evaluated, no increase in cell number was seen (Fig. 2, A).
When the biologic potential of platelet-rich plasma was assessed on osteosarcoma cells (Saos-2), a dose-dependent increase in cell number was seen at forty-eight hours. When the supernatant of thrombin-activated platelet-rich plasma was added at low doses (0 to 125µL), a positive effect was observed, although the effect was significantly less than that noted in association with platelet-rich plasma without thrombin activation. Furthermore, high doses of thrombin-activated platelet-rich plasma decreased Saos-2 cell number (Fig. 2, B). These results suggest that the growth factors released from the platelet-rich plasma have different dose-dependent effects on different cell types; thrombin present in the activated samples also may have deleterious effects on the viability and/or growth of the cells.
In Vivo Chondrogenic and Osteogenic Effects of Platelet-Rich Plasma
Chondrogenesis and Osteogenesis on Day 14
Ectopic bone formation induced by demineralized bone matrix with the addition of platelet-rich plasma or thrombin-activated platelet-rich plasma was tested in an athymic rat intramuscular model. After fourteen days, no new bone formation could be observed and only cartilage was seen. The addition of platelet-rich plasma to demineralized bone matrix did not show a significant effect on cartilage scores at fourteen days in comparison with demineralized bone matrix alone (Fig. 3, A). However, there was a significant decrease in cartilage formation in the thrombin-activated platelet-rich plasma-demineralized bone matrix group when compared with either the demineralized bone matrix-alone group or the demineralized bone matrix with platelet-rich plasma group (p < 0.05). Cartilage exhibiting positive safranin-O staining of proteoglycans was found within and around the demineralized bone matrix particles (Fig. 3, B). In both the demineralized bone matrix group and the platelet-rich plasma-demineralized bone matrix group, the quantity of cartilage and the morphology of chondrocytes were similar, although the cartilage looked more hypertrophic in the platelet-rich plasma group. In contrast, new cartilage was found only in very small and local areas when thrombin-activated platelet-rich plasma was added. Moreover, more inflammatory cells, including macrophages and monocytes, were found between demineralized bone matrix particles in the thrombin-activated platelet-rich plasma group (data not shown). This trend held for every lot of demineralized bone matrix combined with thrombin-activated platelet-rich plasma.
Platelet-rich plasma increased alkaline phosphatase activity in comparison with corresponding demineralized bone matrix controls (lot 1 and lot 2), whereas thrombin-activated platelet-rich plasma actually decreased alkaline phosphatase activity. The decrease, however, was only significant for demineralized bone matrix lot 1 (Fig. 3, C). These results, combined with the cartilage scores, suggest that the activation of platelet-rich plasma in the presence of demineralized bone matrix either inhibits chondrogenesis or stimulates the removal of calcified cartilage at Day 14. The increased inflammation seen in association with the thrombin-activated platelet-rich plasma may have contributed to the decrease in cartilage seen at Day 14.
Bone Formation Potential of Demineralized Bone Matrix on Days 28 and 56
In the athymic rat, most cartilage is replaced by osteoblasts, osteocytes, and bone marrow by twenty-eight days following the intramuscular implantation of demineralized bone matrix26. In the present study, platelet-rich plasma enhanced demineralized bone matrix bone formation as determined histologically and on the basis of alkaline phosphatase activity (p < 0.05) (Fig. 4). As an example, in the experiments involving demineralized bone matrix lot 1, the 95% confidence interval for the difference in means was 0.296 = 1.875 = 3.454 for the comparison between demineralized bone matrix alone and demineralized bone matrix with platelet-rich plasma and -0.317 = 1.5 = 3.317 for the comparison between demineralized bone matrix alone and demineralized bone matrix with platelet-rich plasma and thrombin. Histologically, demineralized bone matrix alone did exhibit osteoinductivity with islands of bone marrow and new bone forming between the residual demineralized bone matrix particles. In the platelet-rich plasma group, much more bone marrow and new bone was evident. However, thrombin activation of the platelet-rich plasma dramatically decreased bone formation as determined histologically and on the basis of alkaline phosphatase activity (p < 0.05). No bone marrow or new bone was seen, and the residual demineralized bone matrix particles were surrounded by fibrous tissue. Subchronic inflammation, which was rated as medium to severe, was again persistent in the thrombin-activated groups (Fig. 5).
Results at the fifty-six-day harvest period were similar to those at the twenty-eight-day time point. By fifty-six days, the results for the demineralized bone matrix-alone group appeared to be similar to those for the unactivated platelet-rich plasma-demineralized bone matrix group. The unactivated platelet-rich plasma-demineralized bone matrix group showed the highest new bone formation score, whereas the thrombin-activated platelet-rich plasma-demineralized bone matrix group continued to have the lowest score (Fig. 6). As an example, in the experiments involving demineralized bone matrix lot 1, the 95% confidence interval for the difference in the means was -0.787 = 0.75 = 2.287 for the comparison between demineralized bone matrix alone and demineralized bone matrix and platelet-rich plasma and -0.998 = 0.5 = 1.998 for the comparison between demineralized bone matrix alone and demineralized bone matrix with platelet-rich plasma and thrombin.