Mineralized-ecm Cell Spheroid(MECS) As A Bone Graft For Guiding Bone Regeneration

Background:Bone defect caused by tumor,inflammation,trauma,has been a focus in tissue engineering,the treatments including autograft?allograft and scaffolds transplantation all have limitations in a degree.Fabrication of new bone implants is essential for bone regeneration.Osteoblast and mineralized collagen fibril in self-secreted ECM are the basement of bone structure.ECM secreted by osteoblasts has played a crucial role for bone regeneration in tissue engineering.Conventional 2D cell culture system loses a lot of chemical and biology cues for cells to differentiate and proliferate.Scaffold in tissue engineering always fail to mimic the microenvironment of cells and the natural process of bone formation.So it is urgent to create a new implants to mimic bone formation and keep real cell 3D microenvironment for bone regeneration.Objectives:This study aimed to fabricate self-assembly scaffold-free mineralized-ECM cell spheroid to evaluate their characterizations and regeneration potential in vivo and in vitro to provide a new approach for bone regeneration.Methods:We constructed mineralized-ECM cell spheroid of Rat Bone Marrow Mesenchymal Stem Cells?rBMMSCs?with Ca²⁺?PO₄^3-in the culture medium through a micro-well based agarose plate in vitro.SEM,LSM,and qT-PCR were used to observe morphology?viability?mechanical properties and osteogencity of Cell Spheroid?CS?and mineralized-ECM cell spheroid?MECS?.MECS?CS were transferred to subcutaneous of nude mice,and blank,?-TCP,CS and MECS were implanted into a 5mm diameter bone defect in rat bilateral cavarial bone for bone regeneration and repair in vivo.Results:The results indicated that the diameter of MECS is larger than CS,and nano-HA presents in ECM of MECS.Mineralized-ECM microenvironment supported rBMMSCs survival,and upregulated the oeteogenesis-related gene expression,H&E staining showed that Mineralized-ECM cell spheroids achieved a higher amount of new bone-like tissue formation than other groups.Altogether,our results highlight the significance of developing a mineralized microenvironment that supports cell-cell interactions,provides a stiff framework,regulates cells fate,which can in turn contribute toward an optimal atmosphere for successful bone regeneration strategies.Conclusion:1.A novel approach to fabricate mineralized-ECM cell spheroids in vitro;2.ECM with nano-HA improves the stiffness and strenghth of spheroids;3.Mineralized-ECM upregulates the ability of osteogenesis and angiogenesis of spheroids in vitro and in vivo,which provide a new approach for bone regeneration in tissue engineering.