Compressible bone grafts supported by platelet rich plasma

This study was financially supported by Hacettepe University Scientific Research Projects Coordination Unit with a graduate project entitled "Compressible Bone Grafts Supported By Platelet Rich Plasma" (FYL-2018-16974). The aim of this thesis is to develop compressible 3-dimensional (3D) nanofibrous bone grafts supported by platelet rich plasma (PRP) for use in treatment of irregular bone damage. Firstly, hydroxyapatite (HAp) nanoparticles were produced from synthetic body fluid (SBF) and added to poly (L-lactic acid) (PLLA) to increase the osteoinductive effect. Alloplastic graft materials were fabricated by using three-dimensional (3B) electrospinning method and modification processes were carried out in different concentrations of NaOH solutions at different times. The water contact angle of PLLA-HAp group was measured as 128.2 ± 3.1°, while the water contact angle was 73.8 ± 12.3° after the modification performed with 0.1 M NaOH solution for 6 hours. As a result, it was observed that the hydrophilicity increased with the modification period. In addition, it was determined with scanning electron microscope (SEM) images that the surface roughness increased and the materials protecting the fiber morphology were produced successfully. According to Hacettepe University Noninvasive Clinical Research Ethics Committee approval of the report dated 27.09.2017 and numbered GO17/671-23, blood of volunteers was taken to isolate PRP with centrifugation (1,500 g, 8 min). PRP was loaded into the grafts by pipetting and activated with 10% CaCl2. The efficiency of the activation was evaluated with flow cytometry data which give the percentages of CD62 (P-selectin) and CD41/61 (glycoprotein IIb/IIIa) proteins. After activation, it was seen that the percentages of proteins increased approximately 4-fold. In vitro cell culture studies were carried out with using mouse preosteoblast cells (MC3T3-E1). MTT (3-[4,5-Dimethylazol-2-yl]-Diphenyltetrazolium Bromide) analysis for cell viability, confocal microscopy for cell morphology, Alkaline Phosphatase (ALP) activity and Real Time Polymerase Chain Reaction (RT-PCR) analyzes were used to examine for osteogenic differentiation of cells cultured on graft materials. At the end of the cell culture studies, it was observed that graft materials supported by PRP increased cell proliferation and osteogenic differentiation significantly. According to Hacettepe University Animal Experiments Local Ethics Committee approval of the report dated 26.02.2019 and numbered 2019/02-15, in vivo animal experiments were performed with using of 24 Wistar Albino rats. Defects of 5 mm diameter were created in both parietal calvarium bones independently. Bone tissues were obtained at the end of 8th week and 20th week for early and late ossification respectively, also the ossification rate was compared with computed tomography (micro-CT) images. The highest bone formation was observed in the gold standard autograft, but it was found that the graft materials supported by PRP could be used as an alternative to autograft in bone regeneration. In conclusion, it was determined that developed PRP+PLLA-HAp nanofiber composite bone grafts supported the attachment and differentiation of preosteoblasts. In animal experiments, it was shown that biomaterials were easily shaped for defect geometry because of their compressible form. Thus, the grafts supported by PRP can be used as an alternative to autograft by accelerating bone regeneration.