Study On Construction Of Hydrogel Phosphorylated Microenvironment And Its Regulation On Osteogenic Differentiation

Bone tissue is a complex phosphorus-containing material system that is precisely assembled by organic matrix and inorganic minerals through biomineralization.How to build a bone extracellular matrix microenvironment through bionics to study the regulation of materials on cell behavior and function is the key and difficult issue in current bone tissue engineering.Phosphorylated polymers,which can simulate the organic matrix microenvironment in the early stage of bone tissue mineralization,provide ideas for solving this problem.Studies have shown that phosphorylated polymers have various biological functions such as promoting protein adsorption,biomineralization,cell adhesion,and osteogenic differentiation.However,the internal law and molecular mechanism of the phosphorylated microenvironment in regulating the osteogenic differentiation have not yet been ascertained.In this study,starting from the bioadaptability of the phosphorylated microenvironment,chemical grafting methods were used to construct mono-phosphorylated and bis-phosphorylated hydrogel systems to systematically study the cellular biological response characteristics of the phosphorylated microenvironment,especially the regulation effect of its osteogenic differentiation.Furthermore,through physical recrystallization technology,a calcium bisphosphate coating was prepared to construct a dual regulation system of bis-phosphorylation microenvironment and the release of osteoinductive factors.The potential molecular mechanism of the coating in regulating the differentiation of stem cells into osteoblasts was explored.It is expected to provide a scientific basis for the design of new bone regeneration and repair materials with biological adaptability.(1)The construction of mono-phosphorylated hydrogel and the study of regulating cell osteogenic differentiationIn this study,the "zero grafting" technology was used to construct a mono-phosphorylated modified chitosan material system.In vitro experiments were conducted to study the effects of mono-phosphorylated modification on the physical and chemical properties of chitosan and the biological properties of osteoblasts,and to explore the regulation of mono-phosphorylation microenvironment on cell osteogenic differentiation and its internal mechanism.The introduction of mono-phosphate groups can effectively improve the protein adsorption and mineralization properties of chitosan,up-regulate the expression of osteogenic-related marker genes and proteins,and promote the osteogenic differentiation of osteoblasts.(2)Study on the construction of bis-phosphorylated hydrogel and its regulation of osteogenic differentiationIn order to enhance the effect of phosphorylated hydrogels on cell osteogenic differentiation,the osteoinductive factor-alendronate was used as the source of bisphosphate groups to construct a bis-phosphorylated modified gelatin hydrogel system.By seeding osteoblasts on the surface of the hydrogel or encapsulating osteoblasts inside of the hydrogel,the effects of the bis-phosphorylation microenvironment on cell viability and osteogenic differentiation were studied from two-dimensional and three-dimensional levels.The introduction of bisphosphonate groups can effectively enhance the mineralization performance of hydrogels,increase the calcium nodule deposition and the expression of osteogenic-related genes and proteins of osteoblasts in two-dimensional environment,increase the secretion of osteogenicrelated proteins of osteoblasts in three-dimensional environment,and promote osteogenic differentiation of osteoblasts.(3)Study on the construction of calcium alendronate coating on the surface of composite hydrogel and its regulation of osteogenic differentiation of stem cellsAlendronate mediates the osteogenic differentiation of stem cells mainly through endocytosis,while chemically grafted alendronate needs to be released with the degradation of the matrix material,which has the problem of insufficient early release.In this study,an calcium alendronate coating(ALC)was prepared on the surface of the composite hydrogel by physical recrystallization,and a dual regulation system of the bis-phosphorylation microenvironment and the release of osteoinductive factors was constructed.ALC promoted the adhesion and spread of stem cells on the surface of the material,significantly increased the alkaline phosphatase activity and the expression of osteogenic-related genes and proteins,and regulated the differentiation of stem cells into osteoblasts.Molecular mechanism studies have found that ALC participates in regulating the osteogenic differentiation of stem cells by activating the integrin-mediated FAK signaling pathway and ERK signaling pathway.All in all,we designed mono-phosphorylated and bis-phosphorylated hydrogel material systems,and systematically explored the control effects of phosphorylated microenvironment on the activity,adhesion,and osteogenic differentiation of osteoblasts and stem cells,and preliminary reveals the cellular biological response characteristics and the signal pathways related to osteogenic differentiation of the phosphorylated microenvironment,providing theoretical guidance for the construction of new bio-adaptable bone regeneration and repair materials.