Effect Of Microcapsule Matrix Stiffness On Osteogenic Differentiation Of Mesenchymal Stem Cells And Its Mechanobiological Mechanism

Mesenchymal stem cells(MSCs)can be expanded and induced in vitro or in vivo,and can differentiate into osteoblasts,adipocytes,myotubes,nerve cells,and other tissue cells.They are the most common seed cells in bone tissue engineering,and have great application prospects in the field of bone repair.However,after directly injecting MSCs into damaged tissue or implanting MSCs onto two dimensional scaffold materials,the survival rate of transplanted cells is severely affected due to hypoxia,poor material transplant ability,and lack of site specificity for host immune response transplantation,making cell based therapy often ineffective.Cell microencapsulation is a bioengineering technology,a strategy for implanting allogeneic and xenogeneic cells into damaged tissues and organs.It can provide environmental protection for cell proliferation and the production of therapeutic agents.Cells are isolated from the host immune response through a semi permeable membrane,allowing the diffusion of oxygen and nutrients,but not the diffusion of host immune cells.In addition,it creates a specific microenvironment that can support the formation of three-dimensional structures,cell to cell interactions,or directed cell differentiation into corresponding cell types.Therefore,this thesis avoids the above problems by constructing three-dimensional microcapsules to wrap stem cells.At the same time,it has been found that appropriate matrix stiffness can regulate the osteogenic differentiation of mesenchymal stem cells without the need for additional inducing factors.Therefore,this topic aims to explore the effects of matrix stiffness of microcapsules on the encapsulation efficiency,survival,and osteogenic differentiation of mesenchymal stem cells by constructing three-dimensional microcapsules with different matrix stiffness.In this paper,based on the excellent physical strength of carbon nanofibers,carbon nanofibers(CNF)and sodium alginate(SA)were used as substrates to synthesize microcapsules with similar three-dimensional spatial structures and different stiffness characteristics,and their physical and chemical properties were characterized.The selected CNF/SA microcapsules with different stiffness characteristics were further used for the in vitro and in vivo culture of MSCs to investigate their effects on the adhesion,survival,and osteogenic differentiation of MSCs.Finally,the in vivo histocompatibility of microcapsules with different matrix stiffness was investigated by subcutaneous implantation in rats.The results showed that three groups of cell microcapsules with different stiffness were prepared by adding carbon nanofibers,and the stiffness of the three groups of microcapsules was verified to be 27.04 k Pa,35.90 k Pa,and 52.40 k Pa,respectively,using a texture analyzer compression test.The morphology and size of the three groups of microcapsules were uniform and regular,and they had good water absorption and in vitro degradability.The results of Live/Read staining showed that the microcapsules in each group had good cell compatibility,and the high stiffness matrix microcapsules promoted the adhesion and growth of MSCs.The detection of alkaline phosphatase(ALP)activity and immunohistochemical staining of osteopontin(OPN)and osteocalcin(OC)showed that high stiffness microcapsules were most beneficial for promoting the osteogenic differentiation of MSCs when cultured in vitro for 7 days.Subcutaneous implantation experiments showed that the three groups of cell microcapsules had different cell infiltration abilities,and the higher the stiffness,the higher the infiltration ability.Masson staining showed that the stent in the low stiffness group significantly promoted the formation and deposition of collagen fibers after one month of subcutaneous implantation.Immunohistochemical tests showed that the stent in the high stiffness group had the strongest ability to promote the positive expression of OPN and OC after one month of subcutaneous implantation.In summary,the stiffness of the three groups of cell microcapsules prepared in this paper has significant differences after testing by texture analyzer,indicating that the superior mechanical properties of CNF alone can enhance the physical properties of SA microcapsules;After further water absorption,biodegradability,environmental SEM observation,and Fourier transform infrared spectroscopy scanning,it was confirmed that there were no significant differences among the three groups of microcapsules in the above aspects;The experimental results of Live-dead staining,ALP staining,OPN and OCN detection in vitro indicate that high stiffness microcapsules can promote the osteogenic differentiation of MSCs,expression levels of YAP and integrin-β.This indicates that stiffness can activate YAP transcription;In vitro subcutaneous implantation experiments,H-E and Masson staining showed that the three groups of microcapsules had good histocompatibility.The detection of alizarin red S,OPN,and OCN further showed that high stiffness was most beneficial for promoting osteogenic differentiation in MSCs.The above results indicate that the cell microcapsules prepared in this paper have good biocompatibility and histocompatibility,which provides a new platform for the study of matrix mechanics in bone tissue repair.