| Skull defect is a common complication of craniocerebral trauma and surgery,which affects the physical and mental health of patients.At present,methods and materials for repairing clinically applied skull defects include:autologous bone,allogeneic bone,xenogeneic bone,non-metallic materials,metallic materials,and the like.Among them,autologous bone and allogeneic bone are still considered to be the most ideal bone graft materials,which are believed to be the"gold standard"for bone transplantation.However,these methods are not ideal due to their limited sources,secondary damage to the donor,immune rejection,high costs of manufacture,and high difficulty in shaping.In recent years,the rapid development of regenerative medicine and tissue engineering has provided a new direction for tissue repair.Tissue engineering materials are mainly divided into three categories:natural materials,synthetic materials,and bioactive ceramic materials,in which natural materials are used more widely in tissue engineering.Gelatin is one kind of natural material.It has a wide range of sources,low price,strong physical and chemical properties,biocompatibility,biodegradability,cytocompatibility and injectability.It has become a research hotspot in recent years.Object:One of the main challenges of bone tissue engineering is to explore the ideal cell delivery vehicle.In this study,covalently cross-linked gelatin microspheres(ccG-MSs)were explored as bone tissue engineering scaffolds,moreover mouse osteoblast MC3T3-E1was used as model cell.After co-culture,to explore the feasibility of covalently cross-linked gelatin microsphere scaffolds used as cells delivering vector.Methods:The gelatin microspheres were prepared by water-in-oil single emulsion method,then the selected gelatin microspheres are cross-linked by EDC.The photocrosslinkable precursor of micro-cavity gel scaffold which called methacrylated gelatin(Gel-MA)was prepared by chemical crosslinking method.The osteoblast/covalently gelatin microsphere scaffold complex group and the osteoblast/micro-cavity gel scaffold complex group were constructed.And then the proliferation and osteogenic induction culture were performed.The structure of gelatin microspheres was observed under inverted phase contrast microscope and the particle size distribution of gelatin microspheres was measured.The cross-linking effect of methacrylated gelatin was detected by nuclear magnetic resonance spectroscopy(~1H MR).Cell viability was detected by fluorescent staining with live/dead cells assay kit.H&E staining was carried out to detect the growth of osteoblasts in gel scaffolds.Alizarin red-S staining,type I collagen(Col1),alkaline phosphatase(ALP),osteocalcin(Ocn)immunofluorescence staining as well as quantitative detection of ALP and calcium were performed to detect the osteogenic ability of osteoblasts in the scaffold.Results:The prepared gelatin microspheres have a round appearance,good dispersibility,and concentrated distribution of particle size.The chemical properties of covalently crosslinked gelatin microspheres by EDC are relatively stable.The methacrylated gelatin is well crosslinked under irradiation of ultraviolet light.MC3T3-E1 can adhere to gelatin microsphere scaffolds and stretch well,meanwhile,the cells on the microspheres proliferate actively.Compared with the corresponding MCG group,MC3T3-E1 cells of ccG-MSs construct in vivo showed higher cell viability and proliferative activity than MC3T3-E1 cells encapsulated in MCG construct.The ccG-MSs construct provides a better cellular microenvironment for MC3T3-E1 proliferation and osteogenic differentiation.In addition,the ccG-MSs group produced more calcium nodules than the MCG group.Moreover,the expression of type I collagen(Col1),alkaline phosphatase(ALP),and osteocalcin(Ocn)was also stronger than that of the MCG group.Conclusion:The covalently cross-linked gelatin microsphere scaffold has good cytocompatibility and ability to induce osteogenic differentiation.It can be used as a cell delivery carrier in bone tissue engineering scaffolds.Moreover,it is expected to become a new type of bone tissue engineering scaffold as well as a skull defect repair material which lays the foundation for the development of bone tissue engineering. |
PDF Full Text Request