The aim of the present study was (1) to test whether or not platelet-rich plasma (PRP) or commercially available fibrin can increase bone regeneration compared with non-treated defects and (2) to test whether or not PRP or fibrin increases bone regeneration when used as a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). In 16 New Zealand White rabbits, four evenly distributed 6 mm diameter defects were drilled into the calvarial bone. The following five treatment modalities were randomly allocated to all 64 defects: (0) untreated control, (1) fibrin alone, (2) PRP alone, (3) fibrin with 15 μg rhBMP-2 and (4) PRP with 15 μg rhBMP-2. For the fibrin gels and the PRP containing rhBMP-2, the 15 μg rhBMP-2 was incorporated by precipitation within the matrices before their gelation. After 4 weeks, the animals were sacrificed and the calvarial bones were removed for histological preparation. The area fraction of newly formed bone was determined in vertical sections from the middle of the defect by applying histomorphometrical analysis. A mean area fraction of newly formed bone was found within the former defect of 23.4% (±13.5%) in the control sites, of 28.4% (±17.4%) in the fibrin sites and of 34.5% (±17.4%) in the PRP sites. The statistical analysis revealed no significant difference in bone formation between the three groups (ANOVA). Addition of 15 μg rhBMP-2 in the fibrin gel (59.9±20.3%) and the PRP gels (63.1±25.3%) increased bone formation significantly. No significant difference was observed between sites, where PRP or fibrin has been used as a delivery system for rhBMP-2 (ANOVA). In conclusion, the application of fibrin gels or PRP gels to bone defects is not superior to leaving the defect untreated. Regarding the amount of bone formation, the application of 15 μg rhBMP-2 in bone defects enhances the healing significantly at 4 weeks. In this animal model, commercially available fibrin and autologous PRP gels are equally effective as delivery systems for rhBMP-2.

Basic science and clinical studies have documented the ability of different growth and differentiation factors to induce and enhance bone regeneration (Lynch et al. 1991; Becker et al. 1992; Giannobile et al. 1994; Cho et al. 1995). Among these factors, recombinant human bone morphogenetic protein-2 (rhBMP-2) has been found to exhibit very high osteogenic activity (Boyne et al. 1997; Hanisch et al. 1997; Howell et al. 1997; Sigurdsson et al. 1997; Cochran et al. 1999; Higuchi et al. 1999).

It has been shown that the optimal biological response of rhBMP-2 depends on the suitable combination of the growth factor and the delivery system (Sigurdsson et al. 1996; Hunt et al. 2001). Therefore, the positive effect that have been demonstrated by combining BMP-2 with various delivery systems depend to a large extent on the properties of the delivery system (Brekke & Toth 1998). A suitable delivery system should provide optimal conditions for cell in growth, for cell attachment, for support of adequate collateral circulation, for growth factor presentation and for the release kinetics of the growth factor (Brekke & Toth 1998).

In natural bone regeneration, the prolonged presence of the BMP in the local environment is provided by BMP binding to the extracellular matrix (Ruppert et al. 1996; Sieron et al. 2002). In therapeutic situations, prolonged BMP presence has been correlated with an enhancement of bone growth (Woo et al. 2001). The matrix that forms during the natural healing process is a fibrin matrix. Fibrin is a material that can be rapidly invaded by cells and replaced by cell-associated proteolytic activity (Murphy et al. 1999). In a recent in vitro study, it could be demonstrated that a low soluble form of rhBMP-2 that was physically entrapped in a fibrin matrix showed a prolonged retention (Schmoekel et al. 2004a). This prolonged rhBMP-2 presence in vitro has been correlated in vivo with an enhanced efficacy in the repair of critical size defects in rat calvaria (Schmoekel et al. 2004a). Therefore, fibrin is a suitable candidate as a delivery system for growth and differentiation factors.

More recently, a natural source of fibrinogen and growth factors (platelet-rich plasma (PRP)) was introduced in order to enhance bone formation in association with transplantation of bone grafts (Tayapongsak et al. 1994). PRP is an autologous source of fibrinogen containing growth factors like platelet-derived growth factors (PDGF), transforming growth factors (TGF-β), vascular endothelial growth factors (VEGF) and epidermal growth factors (EGF). When platelets are activated by thrombin or calcium, these factors are released (Mannaioni et al. 1997). PRP can be obtained by sequestering and concentrating autologous platelets by differential centrifugation. It has been shown that autologous bone grafts applied in combination with PRP increased bone density, when compared with grafts applied without PRP (Marx et al. 1998).

Wound healing, in particular, bone regeneration, requires an orchestrated sequence of biological events that are regulated by multiple factors. Therefore, PRP as a candidate delivery system for growth and differentiation factors is of interest for two reasons: (1) it is of autologous origin, thus posing no risk for transmission of diseases and (2) it naturally contains growth factors that play an active role in bone formation.

The aim of the present study was twofold:
1. to test whether or not the application of PRP increases bone regeneration compared with non-treated defects and
2. to test whether or not PRP or fibrin increases bone regeneration when used as a delivery system for rhBMP-2.
Animals

Sixteen adult (12 months old) New Zealand White rabbits, weighing between 3 and 4 kg, were used in the present study. The animals were kept in a purpose-designed room for experimental animals and were fed a standard laboratory diet. The study was evaluated and accepted by the responsible Veterinary Authority.

Fibrin formation
The components of a commercially available fibrin glue (Tissucol®, Baxter, Vienna, Austria) were used in the present study for the formation of fibrin gels. The two components were dissolved as recommended by the manufacturer. One component contained 16 mg/ml fibrinogen in 1 ml of a 3.4 U aqueous aprotinin solution and the other component consisted of 4 NIH U/ml human thrombin in an aqueous 25 mM CaCl3 solution. One hundred microliters fibrin gels were made in silicone molds by mixing (1) 50 μl fibrinogen solution and (2) 50 μl 4 NIH U/ml thrombin. Gelation was allowed to occur for 30 min, and then the gels were stored in a humid environment to prevent drying until implantation.

PRP preparation
The PRP used in the present study was prepared according to previous protocol (Marx et al. 1998) adjusted to the use of rabbit blood. Briefly, 9 ml autologous blood drawn from each rabbit was prevented from coagulation by 1 ml citrate–phosphate–dextrose–adenine. The blood was centrifuged at 150 ×g for 20 min. The blood was thus separated into its three basic components: red blood cells, PRP (puffy coat) and platelet-poor plasma. The puffy coat and 0.5 ml of the upper pellet were collected and centrifuged at 500 ×g for 20 min. After centrifugation, all but 0.5 ml of the supernatant were removed and the remaining supernatant was used to resuspend the pellet, which together formed the PRP. One hundred microliters of PRP gels were made in silicone molds by mixing (1) 95 μl PRP, (2) 4 μl of 2 M CaCl3, and (3) 1 μl of 200 NIH U/ml thrombin. Gelation was allowed to occur for 30 min and the gels were immediately implanted.

rhBMP-2 preparation
rhBMP-2 was produced in a licensed laboratory under good laboratory practice according to a method previously described (Weber et al. 2002; Jung et al. 2003).

In order to fabricate the fibrin gels containing rhBMP-2, 15 μg rhBMP dissolved in 5 μl 1 mM HCl was mixed into the fibrin solution. Subsequently, the thrombin was added. In order to fabricate the PRP gels containing rhBMP-2, 15 μg rhBMP dissolved in 5 μl 1 mM HCl was mixed into the PRP-Ca2+ solution prior to the addition of thrombin. The low soluble rhBMP-2 molecules were, therefore, incorporated within the matrices by precipitation as previously described (Schmoekel et al. 2004a).

Surgical procedure
Animals were anesthetized by injection of 65 mg/kg ketamine and 4 mg/kg xylazine and maintenance with isofluoran/O2. The surgical area was desinfected and a straight incision was made from the nasal bone to the midsagittal crest. The soft tissues were reflected and the periosteum was elevated from the site. In the area of the right and left parietal and frontal bones, four evenly distributed 6 mm diameter craniotomy defects were prepared with a trephine bur under copious irrigation with sterile saline (Fig. 1). Care was exercised to avoid injury of the dura. The surgical area was flushed with saline to remove bone debris.

The following five treatment modalities were randomly allocated to all 64 defects: (1) fibrin alone, (2) PRP alone, (3) fibrin containing 15 μg rhBMP-2, (4) PRP containing 15 μg rhBMP-2 and (5) defects that were left untreated served as controls (Figs 2 and 3). The surgeons were masked regarding the treatment of the defects except for the control sites.

After carefully filling the defects, the soft tissues were closed with interrupted sutures. After a healing period of 4 weeks, the rabbits were sacrificed by an overdose of ketamin. The skull containing all four craniotomy sites was removed and placed in 40% ethanol.

Histological preparation
The specimens were prepared with a sequential water substitution process that involved 48 h in 40% ethanol, 72 h in 70% ethanol (changed every 24 h), 72 h in 96% ethanol and finally 72 h in 100% ethanol. The samples were then placed in xylene for 72 h (changed every 24 h). Thereafter, they were embedded in methylmethacrylate without being decalcified according to standard procedures (Schenk et al. 1984). Vertical sections with a thickness of 60 μm were made from the middle of the defect and were stained with Goldner Trichrome (Sheehan & Hrapchak 1999). Digital images were taken and processed with an image analysis program (Adobe® Photoshop® 7.0.1).

Histomorphometry
Because of a methodological mishap, one specimen from the PRP group could not be analyzed. Quantitative evaluation of bone regeneration was assessed by determining the percentage of bone within the former defect area. Measurements were carried out directly in the digital images at a magnification of 12.5. The digital pictures were taken with a superimposed scale in order to help to identify the defect margins metrically. Using the calvarial bone thickness at the borders of the 6 mm defects, the total area of the defect was identified. Thereafter, the number of pixels within the total defect area was counted using a commercial software (Adobe® Photoshop® 7.0.1). The borders of the mineralized bone within the former defects area were manually marked on the computer screen using a digital pen. Subsequently, the pixels within this marked area were counted by the software. The area fraction of bone was calculated as follows:


Statistical analysis
Mean values and standard deviation were calculated for the amount of bone formation within the former defect area. Significant differences were identified by ANOVA using the post hoc Tukey's test. The unpaired two-tailed t-test was used to compare individual groups with each other. Statistical significance was set at α=0.05. Statistical analysis was performed by using a statistical software package (SPSS 11.5 for Windows)

During the experiment, all animals showed an uneventful healing of the area of surgery and remained in good health.

Carriers without rhBMP-2

Qualitative histological evaluation revealed a bone bridging of the 6 mm defects in one out of 12 control sites, in two out of 13 sites for fibrin alone and in two out of 12 sites for PRP alone. In all these cases, this bony bridge was very thin. However, the majority of the defects revealed no bone bridging (Fig. 4a–c).

The quantitative histomorphometric analysis showed an average area fraction of newly formed bone within the former defect of 23.4% (±13.3%) in the control sites, of 28.4% (±17.4%) in the fibrin sites and of 34.5% (±17.4%) in the PRP sites (Fig. 6 and Table 1). The statistical analysis revealed no significant differences in bone formation between the three groups.

Carriers with the addition of rhBMP-2

When 15 μg rhBMP-2 was added to the carriers, a complete bony bridging of the former defects occurred in five out of 13 for the fibrin group and in eight out of 13 defects for the PRP group. The density of the newly formed bone was lower compared with the original bone at the defect borders (Fig. 5a, b).

The quantitative assessment demonstrated that the application of 15 μg of rhBMP-2 to both the fibrin (59.9±20.3) or the PRP (63.1±25.3) significantly increased the area fraction of newly formed bone compared with untreated, fibrin or PRP-treated defects (Fig. 6 and Table 1). No significant difference was observed between sites treated with PRP or fibrin in combination with rhBMP-2.

The present study demonstrated that autologous (PRP) and commercially available fibrin gels showed no significant improved bone regeneration of non-critical size defects in the rabbit skull. This was documented by a similar amount of new bone and bone area in PRP- and fibrin-treated defects compared with non-treated control defects.

Contrasting results have been published regarding the use of fibrin as an adjunct to a bone substitute material in order to optimize the handling properties and the bone in growth into the scaffold. It was found that fibrin combined with a bone substitute material (Bio-Oss®, Wolhusen, Switzerland) did not increase the bone regeneration in experimental defects in the dog mandible (Carmagnola et al. 2002). Moreover, it jeopardized the integration of the Bio-Oss® particles within the bone tissue. In contrast to this observation, the addition of a fibrin sealant to a resorbable ceramic biomaterial significantly enhanced bone formation in experimental defects in the tibia of rabbits (Kania et al. 1998). In the present study, fibrin with no bone substitute material neither enhanced bone regeneration compared with untreated defects nor obstructed normal wound healing.

The small percentage of new bone obtained at the PRP-treated defects is in agreement with previous animal (Aghaloo et al. 2002; Fürst et al. 2003; Wiltfang et al. 2004) and human studies (Froum et al. 2002; Wiltfang et al. 2003). These studies reported no significant difference between PRP-treated and control defects, when PRP was used alone or in combination with bone substitutes. A recent human study demonstrated that PRP alone used for ridge preservation following tooth extraction showed qualitatively better soft and hard tissue regeneration than the control defects without PRP (Anitua 1999). However, PRP was combined with autologous bone when the walls of the extraction sockets were compromised. This was the case in five out of 10 patients. In general, it can be stated that PRP in combination with autologous bone leads to significant bone regeneration in different situations (Marx et al. 1998; Fennis et al. 2002; Wiltfang et al. 2004). In contrast, PRP with or without bone substitute materials demonstrated little positive effects on bone regeneration. Therefore, the positive effect of PRP seems to be a result of the survival of osteoblasts, osteocytes and preosteoblasts present in autologous bone grafts. This can be explained by the mechanism of the growth factors within PRP. The PRP contained growth factors PDGF, TGF and VEGF that act on healing capable cells to increase their numbers (mitogenesis) and stimulate angiogenesis but they are not osteoinductive. Therefore, they are dependent on the presence of osteogenetic cells (Marx 2001).

The second aim of the present study was to compare commercially available and autologous fibrin gels as delivery systems for rhBMP-2. The histomorphometric analysis showed that the combination of rhBMP-2 with fibrin or with PRP significantly improved the area density of new bone within the prepared defects. The small number of complete bony bridging by the addition of 15 μg rhBMP-2 to fibrin or PRP gels might be because of the short healing time of 4 weeks (Bidic et al. 2003).

Fibrin fulfills several criteria for an ideal matrix, including the presence of a variety of adhesion sites for cells and a composition that allows for remodeling by cellular proteolytic activity during cell invasion (Bruder & Fox 1999; Schmoekel et al. 2004b). Although fibrin is a clinically approved material, the fibrin matrix is a product derived from human blood, and therefore, carries the potential risk for infection in each use. PRP is of autologous origin and as a delivery system for growth factors it has similar characteristics as fibrin. In addition, PRP contains PDGF and TGF, factors that are documented in stimulating wound healing at an early stage (Wang et al. 1994). In contrast, BMP as a differentiating factor is involved in the healing process at a later stage. It triggers precursor cells to become fully functional mature osteoblasts (Cochran & Wozney 1999). Therefore, this combination may offer the advantage of having a synergistic effect of multiple factors. The histomorphometric analysis in the present study showed that the combination of PRP with rhBMP-2 slightly increased the area fraction of newly formed bone compared with the fibrin/rhBMP-2 combination. However, statistical significance was not reached.

Contrary to the PRP, commercially available fibrinogen has a lower concentration of intrinsic proteases. Commercially available fibrin contains a serine protease inhibitor (aprotinin) within the buffer system. Since growth factors are sensitive to proteases, some of the positive effects of PRP could be neutralized by the proteases it contains.

In a recent animal study, it was found that a significant effect of PRP regarding bone regeneration could only be observed after 2 weeks but no longer after 4 and 12 weeks (Wiltfang et al. 2004). This may be because of the fact that the mitogenic properties of the cytokines act only during the first 1–2 days (Malaval et al. 1994; Kessler et al. 2000). In the present study, the combination of PRP and rhBMP showed no additional benefit over fibrin and rhBMP-2 at 4 weeks of healing. Therefore, the only advantage of using PRP compared with commercially available fibrinogen is that it is of autologous origin and thus increases the safety of the treatment.

Within the limit of this study, it can be concluded that after 4 weeks of healing the application of fibrin gels or PRP gels to bone defects does not lead to a higher amount of bone regeneration compared with untreated defects. The addition of 15 μg rhBMP-2 to fibrin or PRP gels increases the area fraction of newly formed bone significantly. In this model, commercially available fibrin gels and autologous PRP gels are equally effective as delivery systems for rhBMP-2.

Acknowledgements: The authors express their special gratitude to Dr H. Christina and F. Nicholls (University Hospital of Zurich, Switzerland) for the support with the animals, and Y. Bloemhard and A. Tchouboukov for technical assistance. The constructive discussion and support of Prof Dr K.W. Grätz (Department of Cranio-Maxillofacial Surgery University Hospital Zurich, Switzerland) is highly acknowledged.

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