Periosteal graft improves healing of bone defects when used together with platelet-rich plasma? Experimental study in rabbits zygomatic arch

Facial fractures with bony defects are common problems faced in the clinic (1,2). The importance of cell-based methods is increase progressively, despite the fact that restoration with bone grafts and flaps is still the most valid method for the treatment of bone defects (1,2). Periosteum cells include multipotent mesenchymal and osteoprogenitor cells. They contribute significantly to bone healing by differentiating into mesenchymal stem cells, osteogenic progenitor cells and osteoblasts with their high proliferation and differentiation rates (3). While tissue engineering has progressed in recent years, periosteum cells have become the focus of interest by being easy to acquire, due to their osteogenic activity and improved response to growth factors (TGF beta-1, PRP, BMP-2) (3). Platelet-rich plasma (PRP) is a material acquired with the centrifugation of blood in two different frequencies. It includes growth factors such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), 'transforming' growth factor α and β (TGF α and β), epidermal growth factor (EGF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF). The platelet amount included in the PRP is 3-5-folds of the amount of platelets in normal plasma. A high number of mediators are secreted from α-granules of platelets with addition of thrombin. Almost all of these mediators play a role in bone healing. It has frequently been used clinically in the last ten years, especially for bone healing, because it is autologous tissue. In the clinical studies conducted, it has been proven to accelerate wound and bone healing, reduce swelling, pain, infection and scarring, and facilitate bleeding control (4,5,6,7,8,9,10). It has also been demonstrated that PRP, the autologus source of growth factors, has a positive effect on bone cycle and the biological behavior of the periosteum cells with bone grafts and periostial cells (3). We have investigated histologically and radiologically whether or not new bone formation of defected membranous bone is enhanced with periostial graft and platelet-rich plasma combination, which is known to have positive effects on bone healing, compared with the control groups, and evaluated the presence of statistical significance. viii We used 12 New Zealand rabbits. The right and left zygomatic arches were exposed through lateral incisions. We created standard 5 mm bone defects on the bilateral zygomatic arches (24 zygomatic bones) of rabbits. We then divided the subjects into 3 groups: Group 1 (Experiment Group): Right zygomatic arches of 12 rabbits in this group, the periosteum graft+PRP application group, were exposed with lateral incisions and 5mm bone defects were formed at the right zygomatic arch midline. 4x1,5cm cranial periosteum grafts were extracted from the craniums of the subjects. Then, 12 cc of blood samples were obtained from the subjects. 8,5cc of the blood were processed with special kits to obtain PRP and the remaining 3,5cc were used to obtain autologous thrombin. PRP and thrombin were mixed in sterile petri dish and activated PRP was obtained. Jelly-activated platelet-rich plasma was placed into the periosteum graft. The periosteum graft was sutured with 6-0 polyglecapron in the form of a tube and sutured to the periosteum at the ends of the zygomatic bones (n:12 zygoma). The periosteum graft was covering the zygomatic bone like a case. It was then closed with 4-0 silk sutures. The blood sample that had been obtained in order to be processed for PRP, was used only for the rabbit from which the sample was obtained. Group 2 (1st control group: 6 zygomatic bones): The left zygomatic arches of 6 rabbits in this group, and the periosteum graft of only the application group, were exposed with lateral incisions, and 5 mm bone defects were created at the midline of the left zygomatic arch, and 4x1,5 cm cranial periosteum grafts were extracted from the craniums of the subjects that had been exposed through midline cranial incisions, and the periosteum graft was sutured with 6-0 polyglecapron in the shape of a tube and fixed around the zygomatic arch defects. PRP was not used in this group (n:6 zygoma). It was then closed with 4-0 silk sutures. Group 3 (2nd control group: 6 zygomatic bones): The left zygomatic arches of 6 rabbits in this group, the silicon drain-only application group, were exposed with lateral incisions and 5mm bone defects were created at the midline of the left zygomatic arch. A silicone tube was placed so as to cover the defect in order to obtain osteoconductive effect (n:6 zygoma). The skin was then closed with 4-0 silk suture. ix The postoperative follow-up period was at the 8th and the 16th weeks. In week 8, three-dimensional computed tomography (3D-CT) was performed on 3 rabbits with imaging of the zygomatic bones for 3 in the experimental group, 2 in the periosteum graft group, and 1 in the silicone tube. The bilateral zygomatic bones of 1 rabbit were removed for histological examination. The remaining 11 rabbits underwent 3D-CT under anesthesia and their zygomatic bones were removed at the end of 16 weeks. Bone defect areas on the zygomatic bones were examined with microtomography (micro-CT) to evaluate new bone formation. The histological data were evaluated as the last step. The histological examination results were classified as "Modified Heiple" and "Bosch" classifications, and the results were compared statistically. The quantities of newly formed bone at the end of 16 weeks were compared among the groups with 3D-CT evaluation. This was determined as 2,5±1,51 mm in Group 1 (periosteum+PRP group (experiment group)), 1,9±1,53 mm in Group 2 (periosteum graft group (1st Control group)), and 3,7±2,02 mm in Group 3 (Silicone tube group (2nd Control group)). No statistically significant difference was determined among the groups with regard to the quantity of newly formed bone (p=0.232). A significant difference was determined among the groups when the mean Bone Volumes (BV)( mm3 ) were compared (P=0.029): No significant difference was determined between Group 1, the periosteum+PRP (experiment group) and Group 2, the periosteum graft group (1st Control group) (P=0.137). A significant difference was determined between Group 1, the periost+PRP group (experiment group), Group 3, and the Silicone tube group (2nd Control group) (P=0.028). No significant difference was determined between Group 2, the periosteum graft group (1st Control group) and Group 3, the Silicone tube group (2nd Control group) (P=0.967).x A significant difference was found among the groups when the mean Bone Mineral Density (BMD)(mm 2 ) obtained from micro-CT results were compared No significant difference was detected between Group 1, the periosteum+PRP (experiment group) and Group 2, the periosteum graft group (1st Control group) (P=0.953). A Significant difference was detected between Group 1, the periosteum +TRP group (experiment group), and Group 3, the Silicone tube group (2nd Control group) (P=0.001). No significant difference was found between Group 2, the periosteum graft group (1st Control group) and Group 3, the Silicone tube group (2nd Control group) (P=0.166). The groups were compared according to the histological scoring system at the end of 16 weeks for the histological evaluation. A statistically significant difference was determined among the groups according to the Modified Heiple Classification (P=0.015). A statistically significant difference was determined among the groups according to the Bosch Bone Regeneration Classification (P=0.015). Grade 2 (%45.5), grade 1 (%54.5), grade 0 (%0) histological healing percentages were higher in Group 1 (the periosteum+PRP (experiment group)) than in the other groups. And it was grade 2 (% 0), grade 1 (%40), grade 0 (%60) in Group 2 (periosteum graft group (1st Control group)), and grade 2 (%0), grade 1 (%33.3), grade 0 (%66.7) in Group 3 (Silicone tube group (2nd Control group)), respectively. Although the positive effects of PRP and periosteum graft combination on bone healing have been determined as a result of this study, despite bone regeneration being observed to be higher in the periost graft+PRP (experiment group), no statistically significant difference was determined when compared to the periosteum graft only. A significant difference between the silicone tube (2nd control group) and the periosteum graft+PRP groups has rendered the consideration that both PRP and periosteum graft contribute to bone healing. We have concluded that periosteum grafts enhance bone healing with osteoconductive, in addition to osteoinductive effects, as histological bone healing was observed to be significantly higher in groups with periosteum graft than the silicone tube group, and at the same time, the periosteum grafts and silicone tubes xi were placed as they could exhibit osteoconductive activity. It is thought that PRP increases the osteoinduction in a positive direction due to further enhancement of bone healing with addition of PRP to the periosteum graft formed as a tube. Similar studies in which PRP is prepared in higher concentrations and applied repeatedly are required to determine the benefits on bone healing and to put forth the statistical significance. We think that cell-based methods may be used, as they are prepared from autologous tissues, easy to obtain and cheap, in spite of the fact that the results were not so statistically significant in our study, compared to the periosteum graft-only group, and further studies in this matter will enlighten the subject further. We think that PRP, one of the cellular-based methods, positively contributes to healing of bony defects. We observed that it provided quick healing, especially in the early period (8th week). Repeated doses were not possible in our experimental model. In other studies, too, a single dose was applied, due to the vital risk of the subjects. We think that 3-4 stepped repeated doses, as known from clinical applications, may provide more favorable results. Any method that may improve bone healing is meaningful for reconstructive surgery. We believe that cell-based methods enhancing bone healing will be important in the future, too.