Experimental Study On KGN Loaded Hydrogel For Promoting Cartilage Regeneration

Objective:A new composite hydrogel was constructed based on hydrazide modified gelatin（Gelatin-ADH）and sulfonated aldehyde modified hyaluronic acid（HA-CHO-SO₃）.At the same time,transforming growth factor beta 1（TGF-β1）and small molecule drug Kartogenin（KGN）were used to synergistically promote cartilage tissue regeneration and repair.Methods:Gelatin,hyaluronic acid andβ-cyclodextrin were modified into Gelatin-ADH,HA-CHO-SO₃ andβ–CD-CHO,respectively.The modified materials were characterized by FTIR.Rabbit Bone Marrow Stem Cells（r BMSCs）was extracted from rabbit bone marrow mesenchymal stem cells by whole bone marrow method.The blank control group（Hydrogel group）,the separate application group（Hydrogel+KGN group,Hydrogel+TGFβgroup）and the combined application group（Hydrogel+KGN+TGFβgroup）were prepared respectively for four different types of hydrogels,and the cartilage formation effect of different groups was observed.The biocompatibility of four groups of hydrogels was detected by living dead cell staining and cytoskeletal staining.In order to evaluate the feasibility of inducing r BMSCs to differentiate into cartilage in vitro,four groups of hydrogels were cultured in vitro for 4 weeks,and the soft bone related proteins（SOX-9,ACAN and COL Ⅱ）were detected by type Ⅱ collagen immunofluorescence staining and tissue protein quantification.In order to evaluate whether hydrogels can regenerate cartilage tissue with specific shape and proper mechanical strength in vivo,we loaded r BMSCs four groups of hydrogels to form cylindrical and tubular morphology and implanted them into nude mice.After 6weeks,the samples were taken for detection.HE staining,Safranin-O staining and type Ⅱ collagen immunohistochemical staining were used to evaluate cartilage of regenerated cartilage.The expression of extracellular matrix and phenotype were measured by Western blot.Finally,the combined group was selected to regenerate the tubular trachea tissue after loading r BMSCs,and repair the cartilage defect model of rabbit tracheal defect model.After 12 weeks,the materials were taken and the results were evaluated by gross and tissue staining.Results:（1）Bothβ-cyclodextrin and hyaluronic acid were successfully modified.The general results showed that the modified gelatin and hyaluronic acid could form stable hydrogels.（2）Staining of living dead cells and cytoskeletal staining showed that hydrogels had good biocompatibility in the process of gelatinization and cell proliferation.In combination group,r BMSCs showed the most homogeneous cell distribution,morphology and proliferation.（3）After 4 weeks of culture in vitro,the expression of type Ⅱ collagen was the highest in the combined application group.Protein quantitative analysis showed that the expression of cartilage characteristic genes and proteins（SOX-9,ACAN and COL Ⅱ）was the highest in the combined application group.（4）When cultured in nude mice,the hydrogel composite r BMSCs successfully regenerated cartilage like tissue,but the combined application group showed a more white cartilage like texture than the single application group and the blank control group.HE staining,Safranin-O staining and collagen Ⅱ staining showed the same trend.The combined group had more typical cartilage lacuna structure and more abundant cartilage.The results of Western blot showed that cartilage marker type Ⅱ collagen protein appeared in both groups,and the expression of SOX-9 protein and ACAN protein was higher than that in the blank control group.（5）r BMSCs loaded hydrogel successfully regenerated tubular tracheal cartilage tissue in nude mice.Histology showed that it had typical cartilage lacuna structure and cartilage specific extracellular matrix deposition.（6）After tracheal cartilage defect modeling and implantation of r BMSCs-hydrogel complex,12 weeks later,the trachea tissue healed well with the surrounding normal tissue and maintained tubular shape.Histological results showed that there were new cartilage formation and lacuna structure in the repair trachea,and the rest was filled with fibrous tissue.Conclusion:（1）Through the modified gelatin and hyaluronic acid,we successfully prepared a new type of hydrogel with stable structure,good biocompatibility and easy molding.（2）The hydrogel mixed with TGF-β1 and KGN has cartilage induction function,which can induce cartilage like tissue in vitro and in vivo,and can be used as a carrier of r BMSCs for repairing cartilage defects.（3）The combination of TGF-β1 and KGN can promote r BMSCs to differentiate into chondrocytes and regenerate mature cartilage lacunae.（4）Hydrogels combined with growth factor TGF-β1 and small molecule KGN can be used to repair tracheal defects in rabbits.