Hyaluronic Acid/Chitin Thermo-sensitive Hydrogel Loaded With Mg²⁺ Was Applied In Cartilage Repair

Objective: To prepare an injectable temperature-sensitive natural polymer hydrogel for cartilage repair and cartilage formation.Methods :1)Preparation and characterization of hydrogel: HA/HPCH hydrogels with mass ratios of m（HA）/m（HPCH）=1:6 and m（HA）/m（HPCH）=1:12 were prepared with gel concentrations of 2%（m/v）,and were named HA/ HPCH-1 and HA/ HPCH-2 respectively.The surface morphologies of the gel samples HPCH,HA/HPCH-1 and HA/HPCH-2 were observed by SEM.Mg²⁺ was introduced into the hydrogels of HA/HPCH-1 and HA/HPCH-2,with Mg²⁺ concentration of 5 m M,and were named Mg²⁺/HA/HPCH-1 and Mg²⁺/HA/HPCH-2 respectively.The sol-gel phase transition temperature of several hydrogels in PBS solution was determined by bottle inversion method.2)Cell experiments: the biocompatibility of HPCH,HA/ HPCH-2 and Mg²⁺/HA/ HPCH-2 gels were evaluated by the（cck-8）method.In order to explore the adhesion ability of the material to cells,the material was co-incubated with HUVEC cell suspension.The un-adsorbed cells were washed with PBS,and the density of the remaining cells was observed by live-dead cell staining.BMSC cells were co-cultured with HPCH,HA/HPCH-2 and Mg²⁺/HA/HPCH-2 hydrogels,then,the surface morphology of Mg²⁺/HA/ HPCH-2 gel-BMSC cells was observed by SEM.3)In vivo study: 32 New Zealand white rabbits weighing 3 kg were randomly divided into A,B,C and D groups with 8 rabbits in each group after knee cartilage defect model was made.Group A as injected with saline,group B was injected with HPCH hydrogel,group C was injected with HA/ HPCH-2,and group D w was injected with Mg²⁺/HA/HPCH-2.HE staining and Saffron O-Fast green staining were performed at 12 W and 24 W respectively,and histological score of articular cartilage defect was obtained for each section.Results: SEM showed that the hydrogel material presented a porous,loose scaly structure,which was connected to each other and conducive to cartilage adhesion.The SEM images of each group after three-dimensional culture of hydrogel and BMSC showed that there were many filamentous structures,and the lines were pseudopodia of mesenchymal stem cells,indicating that the cells could adhere to the gel well.The hydrogels in each group were temperature-sensitive.The sol-gel phase transition temperature of the gel was about 22℃,and the sol-gel phase transition temperature of the gel increased with the increase of HA content in the gel.The degradation rate of each group was above 60% after 28 days.The cytotoxicity of the materials in each group was small,and the biosafety was good.A small amount of HA and Mg²⁺ added to HPCH had no significant effect on the adhesion of the material.However,compared with HPCH,HA/HPCH-2 and Mg²⁺/HA/HPCH-2 groups,the adhesion of HA cells was significantly reduced.After gel-cell co-culture for 24 h,the number of cells in each group increased by about 15%,and after culture for 48 h,the number of cells in each group increased by 60%,indicating that 2% scaffold materials formed the gel at 37℃ did not damage BMSC cells,and the cells grew well in the gel network.After 24 W in animal experiments,gross observation,HE staining and Saffron O-Fast green staining showed that the HPCH/HA/Mg²⁺ group had the best effect in repairing cartilage defects,followed by HPCH/HA repair.HPCH also had certain cartilage repair effect,and the histological score of each group was significantly different.Conclusion: Mg²⁺/HA/HPCH composite injectable hydrogel has good temperature sensitivity,degradability,cell adhesion and biocompatibility,which can effectively promote the repair of cartilage defects and has a good application prospect.