Enhancing Bone Regeneration By Combining Mesenchymal Stem Cell Sheets With β-tricalcium Phosphate/Type Ⅰ Collagen Fiber(β-TCP/COL-Ⅰ)Scaffold

Introduction:The combination of bone marrow-derived mesenchymal stem cells(BMSCs)and biological scaffold have been demonstrated as a promising strategy for bone regeneration.In this study,the distinctly porous β-TCP/COL-Ⅰ composites 3D scaffold combined with BMCSs sheet were applied to evaluate the bone regeneration capacity.Method:To prepare an osteogenic matrix cell sheet,BMSCs were plated at a high density and cultured with osteogenic medium.After cultured for 6-8 days,the cell sheet was harvested by a mechanical method.The obtained BMSCs sheet was then wrapped around the β-TCP/COL-Ⅰ scaffold(8 mm diameter and 2 mm thickness).To evaluated the osteogenic property among sheet,sheet+scaffold,BMSCs+scaffold,the activity of alkaline phosphatase(ALP),osteocalcin(OCN),osteopontin(OPN)protein and mRNA expression levels was meassured in vitro.After Sheet,sheet+scaffold,BMSCs+scaffold and scaffold itself implanted subcutaneously on the back of nude mice for 4weeks,the morphology of the specimens were assessed using Mansson staining and micro-CT system.Result:In vitro evaluation results showed that β-TCP/COL-Ⅰ scaffold combined with BMSCs sheet significantly upregulated the protein expression of ALP,osteocalcin(OCN)and osteopontin(OPN),compared with BMSCs sheet and β-TCP/COL-Ⅰscaffold combined with scattered BMSCs.The expression level of OCN and OPN gene markers also demonstrated the similar trend.Moreover,the histological and micro-computed tomography(micro-CT)analysis further demonstrated the β-TCP/COL-Ⅰ scaffold combined with BMSCs sheet was the only group that showed new bone formation after 4 weeks implantation in vivo.Conclude:These results emphasized the critical requirement of the synergistic effects of β-TCP/COL-Ⅰ scaffold and BMSCs sheet,and thus provided a promising candidate and novel design strategy for bone tissue engineering.