Preparation Of Dual-Regulatory Composite Hydrogel And Study On Osteochondral Repair

Articular cartilage disease is a common disease.Due to the low bioactivity and metabolic capacity of cartilage,the regenerative repair capacity is quite limited,and current methods cannot provide a complete solution strategy.Due to its network structure and high water content,hydrogels are very similar to cartilage.Their excellent biocompatibility,combined with growth factors,makes them widely applied in tissue engineering.However,the use of growth factor will cause some problems,including pain,swelling or ectopic bone formation after use,which makes it difficult to control the balance between drug efficacy and adverse reactions,and also makes its clinical conversion difficult.Traditional hydrogels lack the function in stimulating cell proliferation and differentiation,making the repair effect not very ideal.Considering the insufficient mechanical properties of scaffolds,the lack of effective stimulation of pure scaffolds,the unsafe use of growth factors,and the difficulty of clinical conversion,we designed a therapeutic ions–based method to regulate the mechanical properties of scaffolds on the one hand,and improve the biological properties of scaffolds on the other hand,to actively stimulate cells,so as to achieve the purpose of enhancing tissue regeneration and repair.In this study,we introduced Mg²⁺ions into the polyglutamic acid/carboxymethyl chitosan/bacterial cellulose（PGA/CMCS/BC）composite hydrogel to achieve the purpose of regulating the mechanical properties of the hydrogel.On this basis,in order to better simulate the osteochondral structure,this article designed a double-layer bionic scaffold,which introduced Mg²⁺ions into the cartilage layer and Cu²⁺ions into the bone layer.The repair effect of scaffolds would be finally tested through the in vivo experiments for the repair of osteochondral defects.The content and results of this study are as follows:1.Firstly,the PGA/CMCS/BC composite hydrogel was prepared through the amidation reaction catalyzed by NHS/EDC.The results of Fourier transform infrared analysis showed that PGA and CMCS were successfully cross-linked under the action of the catalyst,and BC was also successfully introduced into PGA/CMCS hydrogels.The compression test results showed that the compressive strength of the material was affected by the CMCS content and the BC content with the rigid chains synergistically.The DN-8 group showed the best compression performance in the orthogonal test,and the final swelling test also showed that the DN-8 group had the lowest swelling rate.Therefore,we selected the DN-8 group as the base material group for late experiments.2.Then,this article regulated the PGA/CMCS/BC composite hydrogel by using Mg²⁺ions.The compressive modulus of the PGA/CMCS/BC composite hydrogels was significantly improved by the introduction of Mg²⁺ions into the PGA/CMCS/BC composite hydrogels,and the swelling ratios also decreased.When the Mg SO₄content reached 3%,the compressive modulus was 0.405 MPa,which reached the modulus of natural cartilage.Finally,we introduced Mg²⁺ions into the cartilage layer and Cu²⁺ions into the bone layer,and successfully prepared a double-layer biomimetic scaffold.The microscopic morphology showed that the hydrogel bone layer was a porous structure with a pore size range of 200 to250μm,while the cartilage layer was relatively dense.In addition,the energy spectrum showed that Ca²⁺,Mg²⁺and Cu²⁺were uniformly distributed in the material.The results of ion release experiments showed that effective ion release could provide beneficial elements for cell differentiation and proliferation,which could more efficiently stimulate cells and regulate their differentiation.3.This article also characterized the biocompatibility,hemocompatibility,in vitro degradation,and antibacterial properties of the materials.The results,from the co-culture of cells and PGA/CMCS/BC composite hydrogel regulated by Mg²⁺ions,showed that the proliferation and growth of cells were normal while the hemolysis experiment showed that the hemolysis rate was only 2.85%.The results of the antibacterial experiment showed that the antibacterial rate of the material against both bacteria reached more than 95%.4.This article established a rabbit osteochondral defect model and implanted the material into the defect site to verify the promoting effect of the composite hydrogel material on the repair of osteochondral tissue.The macroscopic observation results at three time points after operation showed that,compared with the blank group,for the PGA/CMCS/BC composite hydrogel group regulated by Mg²⁺ions,the boundary between the defect site and the host could not be found,and that the fusion between the new tissue and the host was good,and that their color was basically the same.Histological staining also showed the same result,the defect had been completely repaired,and was basically consistent with the surrounding normal cartilage and subchondral bone structure.