Repairing The Rat Critical-sized Calvarial Defects Using Pre-vascularized Tissue-engineered Periosteum And β-TCP

Purpose:This study aims to explore the feasibility of repair the rat Critical-sized calvarial defects using the pre-vascularized tissue-engineered periosteum and P-TCP based on cell sheet engineering technology..Methods:(1) Established fibrous vascular layer of the biomimetic periosteum:isolated and cultured the rat bone marrow mesenchymal stem cells using whole bone marrow adherence method, MTT assay was performed to observe the cell growth circle; rBMSCs were cultured in M-199 medium containinglO μg/L Rat-VEGF and 2μg/L Rat-BFGF; after 14 days, flow cytometric was used to check the adhesion molecule CD31 of ECs, cell morphology was observed under the microscope. Then, The rBMSC were cultured continuously for 14 days to obtain an undifferentiated rBMSC sheet.after that, aprevascularized cell sheet in vitro was fabricated by seeding the ECs onto the rBMSC sheet. After 7 days, CD31 immunohistochemical staining was used to evaluate vascularized networks formation. Established cambium layer of biomimetic periosteum:The rBMSCs were cultured in osteogenic medium for 21 days to obtain the osteogenic differentiated sheet. Alkaline phosphatase staining(ALP) and Alizarin red staining were performed to identify osteogenic characteristics. This mineralized rBMSC sheet was first wrapped onto a cylindrical P-TCP scaffold followed by wrapping the prevascularized sheet, thus generating a biomimetic periosteum on the P-TCP scaffold.scanning electron microscopy(SEM) was used to observe the construction of biomimetic periosteum/β-TCP cultured in vitro for 3 days.(2) The biomimetic periosteum compound with β-TCP to repair the critical skull defect of rat (CSD):Then the compound structure was implanted into the 8mm rat skull defects, autogenous periosteum/β-TCP,P-TCP and the blank group as the control group; After implantation at the time point of 2,4,8 weeks, the rats were sacrificed, Gross observation, histological staining, immunohistochemistry staining and X-ray testing were performed to evaluate the functional blood vesseels formed and the new bone deposition.Results:(1) The morphology of primary rBMSC were triangle, spindle and the growth patten showed clone-like growth. After passage, the rBMSC proliferated rapidly and became long-spindle-shaped. MTT test growth curve showed "S" type. The rBMSC cultured in the endothelial induction medium appeared short, round shape gradully, and showed cobblestone morphology.conversion rate of ECs was 35.1%. When the rBMSC were cultured for 14 days at a high density, an intact sheet was formed, which could be lifted up by a sharpened point forceps. After seeding the ECs onto the undifferentiation cell sheet, the cells were realignment. CD31 staining showed that ECs migrated, gatheredand lumen formation. ALP staining and Alizarin red staining for the osteogenic cell sheets werer positive after 21 days. The biomimetic periosteum wraped β-TCP 3 days later, the SEM showed that cells grew into the scaffold. (2)After transplantation 2 weeks, immunohistochemistry staining for CD31 showed the functional blood vessels existed in biomimetic periosteum/β-TCP group, autogenous periosteum/β-TCP group and β-TCP group. The number of blood vessels in biomimetic periosteum/β-TCP group, autogenous periosteum/β-TCP group were higher than β-TCP group and blank group obviously(P<0.05), After implantation 8 weeks, some part of the materials were absorbed in biomimetic periosteum/β-TCP group and autogenous periosteum/β-TCP group, while the scaffold group showed a slight absorption. Van Geison staining showed that the formation of new bone in biomimetic periosteum /β-TCP group and autogeneic periosteum/β-TCP group (P>0.05), and amount of the new bone formation was significantly more than that of in β-TCP scaffold group and the blank group(P<0.05).Conclusions:(1) The method of whole bone marrow adherent cultre to get rBMSC is relatively simple and feasible; rBMSC can be induced to form ECs in the endothelial induction medium. Furthermore, seeding ECs onto the rBMSC cell sheets can form vascularized networks in vitro. rBMSC continuously cultured in osteogenic differentiation medium can form osteogenic cell sheet. (2) The biomimetic periosteum -covered β-TCP graft is a promising approach for repairing bone defect.