Preparation Of Cell Composite Thermosensitive Hydrogel And Study On Its Biological Properties

Objective:This study was aimed to prepare a cell composite thermosensitive injectable bioactive hydrogel material and to test its biological properties,initial exploring its possibility for cartilage tissue regeneration.Methods:1.BMSCs and CHs were isolated and cultured in vitro by whole bone marrow adherence method and enzymatic digestion method respectively,and the two cells were identified respectively.Then,the two cells were further co-cultured to obtain CHs-BMSCs;2.Four thermosensitive injectable biologically active hydrogels were prepared by0.4% agarose,10% gelatin,and 10 mg/ml hyaluronic acid with 0,1.0%,2.0%,and 3.0%(w/v)of cartilage extracellular matrix(ECM),respectively;3.The ultrastructure of the four groups of hydrogels was observed by scanning electron microscopy and transmission electron microscopy.The equilibrium swelling rate,biomechanical strength and in vitro degradation rate of the four groups of hydrogels were tested and comparative study on its characterization in vitro.4.In vitro experiments were performed to observe the growth of CHs-BMSCs in thermosensitive agarose/gelatin/hyaluronic acid/ECM hydrogels;5.The composite thermosensitive hydrogel loaded with CHs-BMSCs was constructed,preliminary evaluation of cartilage regeneration ability of the hydrogel composite system in rats by in vivo experiments.Results:(1)We successfully obtained high-purity P3 rat BMSCs and well-characterized P1 rat CHs.After two weeks of co-culture,we successfully obtained high-quality induced differentiation of cartilage-like cells.(2)We have successfully prepared four groups of hydrogels: agarose /gelatin /hyaluronic acid / ECM hydrogel group(S1: ECM 0,w/v),agarose /gelatin / hyaluronic acid / ECM hydrogel group(S2: ECM 1.0%,w/v),agarose /gelatin / hyaluronic acid /ECM hydrogel group(S3: ECM 2.0%,w/v)and agarose /gelatin / hyaluronic acid / ECM hydrogel Glue group(S4: ECM 3.0%,w/v);(3)In vitro characterization results showed that the S3 group hydrogel had a good three-dimensional grid structure and suitable pore size,suitable for cell proliferation and migration;ECM particles were evenly distributed and the addition of 2.0% w/v ECM particles significantly improves the water-swelling ability,mechanical strength and in vitro degradation ability of the hydrogel;(4)The S3 group hydrogel composite CHs-BMSCs were co-cultured for 1,3 and 7days in vitro,and the cell growth was good.The average cell survival rate was all above90%,indicating that the cytocompatibility of the hydrogel was good.(5)Gross anatomy and histology of the site of administration of the body showed that the sub-spheroidal flaxen mass of the S1 group and the S3 group could be formed locally after subcutaneous administration.The mass structure of the hydrogel of the S3 group was more complete,the surface was more smooth and the texture was more flexible.Under the microscope,the neatly arranged fibers with cell clusters and colloidal matrix agglomerates are visible.There were a large number of new cartilage-like cells and a few inflammatory cells in the cell populations.Conclusion:The above studies showed that the ECM-modified thermosensitive agarose /gelatin/hyaluronic acid hydrogel material is a promising medical biomaterial for cartilageregeneration.