Study On Seed Cells(ASCs&BMSCs) And HHK Hydrogel Scaffolds For Bone Tissue Engineering

Background:How to repair large area bone defects is a difficult problem in clinic,and the traditional methods have their own shortcomings.At present,the use of bone tissue engineering technology for repair,whether in seed cells or in the scaffold materials are not perfect.Bone marrow stromal cells are the main source of seed cells in bone tissue engineering.However,the process of harvesting them is suffering.Adipose tissue derived stromal cells are easy to obtain and large in quantity,which can meet the need of the source of autologous seed cells.But compared with bone marrow stromal cells,their osteogenic ability is relatively weak.Therefore,how to foster strengths and circumvent weaknesses means that not only makes use of the advantages of adipose derived stromal cells but also further enhance the osteogenic ability become an urgent problem.Traditional scaffold materials can not simulate the 3D microenvironment properly in vivo.Therefore,the research on the bio-derived scaffold materials is in the ascendant.However,how to obtain and prepare a large number of autologous or allogeneic biological source scaffold material is still quite limited.The scaffold of human hair keratin is a new strategy because of its extensive sources,convenient materials,good biocompatibility,economic and environmental protection,small immune response and so on.In this study,both from seed cells and three-dimensional hydrogel scaffolds are discussed on better utilization of fat and hair tissue in the human body which had been considered as waste tissue for bone tissue engineering resources to achieve bone regeneration and repairObjects:1)To study the feasibility of bone marrow stromal cells(BMSCs)and its secretion of extracellular matrix(BM-ECM)to promote the osteogenic ability of adipose tissue derived stromal cells(ASCs);2)To investigate the safety and efficacy of human hair keratin(HHK)three-dimensional hydrogel scaffolds as scaffold material for bone tissue engineering in vitro and in vivo;3)To investigate the effect of microenvironment on cell-based bone tissue engineering;Methods:1)ASCs and BMSCs were isolated from the same rat.To compare their ability of proliferation and osteogenic differentiation after amplification2)ASCs and BMSCs from the same rat were seeded in 6-well plates for direct co-culture and Transwell dishes for indirect co-culture.Then osteogenic related histochemistry stainings were processed and related genes were detected by real-time quantitative PCR after osteogenesis induced for 14 days3)To prepare the BM-ECM and to direct co-culture with the ASCs in 6-well plates Alizarin staining were processed after osteogenesis induced for 14 days4)Brown alginate,porcine derived collagen and human hair derived keratin were prepared to form 3D hydrogel and their relevant characterization were detected in vitro;5)Rat BMSCs were encapsulated in three hydrogel materials for three-dimensional co-culture and induction.The live and dead of the cells(AM/PI)and bone differentiation ability(ALP activity)were detected6)Three kinds of hydrogel encapsulated rat BMSCs which were osteogenesis induced for 14 days in vitro were implanted subcutaneously in nude mice or at critical sized calvarial defects of SD rat respectively.They were sacrificed after 1 month and microCT detection and relevant histological(HE,Masson and Sirius Red)staining were proceeded to evaluate the ability of bone formation among them.Results:1)BMSCs relative to ASCs has better osteogenic ability,and ASCs relative to BMSCs has stronger ability of proliferation and colony formation ability;2)The proliferation ability of ASCs and BMSCs co-culture group was increased after cultured for a long period.3)ASCs and BMSCs were co-cultured with two-dimensional plates,appropriate amount addition of BMSCs can greatly improve the ability of bone fomationg for ASCs,and it will achieve the best osteogenic effect when the ratio of ASCs:BMSCs is 4:3;4)The Alizarin Red staining result of ASCs and BM-ECM co-cultured is better than simply ASCs,but simply BM-ECM is even better;5)Including keratin hydrogel,three kinds of 3D biological derived hydrogel may mimic the micro environment in vivo.All of them have the ability to carrier BMSCs in a 3D culture system to guide their proliferation and differentiation in vitro;6)The safety and eff-icacy of human hair keratin(HHK)three-dimensional hydrogel as a scaffold material for bone tissue engineering is certified since there is no inflammatory or immune responses,and they can promote the regeneration of bone tissue in vivo.Conclusion:1)It is available to improve the capability of osteogenic differentiation of ASCs by co-culture ASCs and BMSCs,and ASCs:BMSCs=4:3 in the highest effectiveness;2)Human hair derived keratin hydrogel can be used alone to encapulate BMSCs for bone tissue engineering,but its osteogenesis ability is relatively weaker than calcium alginate hydrogel;3)The microenvironment for cell-based bone tissue engineering is critical and it can be simulated and controlled by physical,chemical and biological cues.