Preparation Of Injectable Hydrogels Loaded With Bone Marrow Mesenchymal Stem Cells Based On Enzyme Catalytic Reaction And Their Osteogenic Properties

Objective:To prepare hyaluronic acid（HA）/alginic acid sodium salt（ALG）injectable hydrogel by enzyme catalytic reaction,and to study the micromorphology,swelling,in vitro degradation,rheological and mechanical properties of HA/ALG injectable hydrogel,and then implant rabbit bone marrow mesenchymal stem cells（r BMSCs）into HA/ALG hydrogel.The proliferation,survival and osteogenic differentiation of r BMSCs in hydrogel were investigated and evaluated.Methods:Based on EDC and NHS,Tyramine（TA）modified HA and ALG were synthesized by chemical reaction and characterized by ¹HNMR and UV.Then HA/ALG in-situ injectable hydrogel was prepared by enzyme-catalyzed reaction using HRP as a catalyst and H₂O₂ as an oxidant.FT-IR and TGA were used to characterize the hydrogel.The internal micro-morphology of the hydrogel was observed by SEM,the swelling and in vitro degradability of the hydrogel were detected by gravimetric method,and the rheological and mechanical properties of the hydrogel were tested by rotary rheometer and universal material testing machine.In addition,r BMSCs were extracted by whole bone marrow adherent method and identified by cell surface markers and stem cell differentiation.r BMSCs were encapsulated in HA/ALG hydrogel.The proliferation,survival and adhesion of r BMSCs in the hydrogel were observed by CCK-8,FDA staining and SEM.Then the osteogenic differentiation of r BMSCs-hydrogel complex in vitro was evaluated by alkaline phosphatase（ALP）quantitative analysis,alizarin red staining and calcium nodule concentration quantitative analysis.Results:¹HNMR results showed that the chemical shift of 6.8-7.2ppm belonged to the absorption peak of the benzene ring group in TA,and UV results showed significant TA ultraviolet signal at 275nm,which indicated that HA-TA and ALG-TA were successfully prepared.The results of FT-IR and TGA showed that HA/ALG hydrogels have been successfully prepared.The swelling test results showwd that the hydrogels had a good swelling rate,which expanded rapidly at first,and then gradually reached the expansion equilibrium.The results of in vitro degradation experiments showed that the prepared hydrogels showed good stability and degradability.SEM images showed that the hydrogel showed a good interconnected porous microstructure with a pore size in the range of 30-110μm.The test results of rheological and mechanical properties showed that the storage modulus（G’）,compressive strength and compression modulus of the hydrogels would change with the change in the composition and mass ratio of the precursor materials in the hydrogels.The results of r BMSCs identification showed that the positive rates of CD29 and CD44 were as high as 94.88%and 99.92%,while those of CD45 and CD34were only 4.58%and 0.08%.Red calcified nodules could be observed after alizarin red staining,and red round lipid droplets could be observed after oil red O staining.CCK-8,FDA staining and SEM results showed that the hydrogels had good biocompatibility and could provide a suitable microenvironment for cell survival and proliferation,and the cells in the hydrogel attached well and maintained their good morphology.The results of ALP quantitative detection and alizarin red staining showed that hydrogel could promote the expression of alkaline phosphatase and extracellular matrix mineralization of r BMSCs,especially HA50/ALG50 hydrogel.Conclusion:In this study,injectable hydrogels loaded with r BMSCs were successfully prepared based on the enzyme catalytic reaction mediated by HRP and H₂O₂.The hydrogels have both good swelling and degradability,and their interior shows a connected porous structure,and the rheological and mechanical properties of the hydrogel can be adjusted by changing the mass ratio of the precursor material.In addition,HA/ALG hydrogel has excellent biocompatibility,which can provide a good microenvironment for the growth of r BMSCs and promote osteogenic differentiation.Therefore,as an effective three-dimensional scaffold,this injectable hydrogel has potential application value in the field of bone repair and regeneration.