Synthesis Of Carboxymethyl Chitosan-strontium Complex And Its Therapeutic Effects On Relieving Osteoarthritis

Objectives:1.Preparation method and properties of carboxymethyl chitosan-strontium complex;2.To study the biological effect of carboxymethyl chitosan-strontium complex on chondrocytes;3.To investigate the overall efficacy of carboxymethyl chitosan-strontium complex in animals.Methods:1.Carboxymethyl chitosan strontium was synthesized by chelating carboxymethyl chitosan with strontium ion;2.SEM/EDS was used to analyze the micromorphology and element composition of carboxymethyl chitosan-strontium particles.FTIR/1HNMR was used to separate the functional groups that could be bound by strontium ions.TGA/DSC was used to detect the thermal stability of carboxymethyl chitosan strontium and the endothermic and exothermic properties of the material.XRD was used to detect the chemical synthesis properties of carboxymethyl chitosan-strontium.AFM was used to detect the surface morphology and particle size of carboxymethyl chitosan-strontium.3.Cell proliferation assay(MTT)and cell viability staining were used to detect the biological toxicity of carboxymethyl chitosan-strontium on osteoarthritis chondrocytes.Flow cytometry was used to qualitatively and quantitatively detect the effect of carboxymethyl chitosan-strontium on osteoarthritis chondrocytes against programmed death.RT-qPCR was used to determine the regulation of carboxymethyl chitosan-strontium on OA chondrocytes by detecting the expression level of inflammation-related genes.Immunofluorescence staining of type 2 collagen fiber was used to detect the effect of carboxymethyl chitosan-strontium on secretion of extracellular matrix of OA chondrocytes.4.SPF rat anterior cruciate ligament is broken and the medial meniscus free bone arthritis of rats model was constructed,articular cavity injection of rats with Micro syringe gait analysis experiments on animals,after four weeks after will kill rats,rats of knee joint,the related follow-up experiments:the knee joint cartilage general pictures,Micro indentation mechanical test instrument,Micro-CT analysis.Results:1.We use the mature principle and method of strontium ion chelating carboxymethyl chitosan to prepare carboxymethyl chitosan-strontium,strontium ion successfully chelated carboxymethyl chitosan,and uniformly distributed on carboxymethyl chitosan;According to Fourier transform infrared spectroscopy(FTIR),strontium ions are probably bound to the hydroxyl,amino and carboxyl groups of carboxymethyl chitosan by coordination bonds.2.Strontium carboxymethyl chitosan has no toxic effect on chondrocytes,and can promote the proliferation and differentiation of chondrocytes,and can inhibit the apoptosis of chondrocytes to a certain extent.RT-qPCR results confirmed that carboxymethyl chitosan strontium can significantly inhibit the expression of MMP9,IL-1β,IL-6 and other pro-inflammatory genes.In addition,it may inhibit the apoptosis of chondrocytes by down-regulating p53 gene and up-regulating Bcl-2 gene.The results of flow cytometry showed that carboxymethyl chitosan strontium could significantly inhibit the early apoptosis of OA chondrocytes.Immunofluorescence staining showed that carboxymethyl chitosan strontium could also promote the secretion of type 2 collagen by chondrocytes.3.Through animal experiments,we further verified the efficacy of carboxymethyl chitosan strontium:carboxymethyl chitosan-strontium can significantly increase the thickness of knee cartilage in OA rat model,and at the same time,it can significantly inhibit the erosion of inflammation to subchondral bone in the early stage of arthritis.Conclusion:We use the mature principle and method of strontium ion chelating carboxymethyl chitosan to prepare carboxymethyl chitosan-strontium,and through the related cell experiments further confirmed the biological function of carboxymethyl chitosan strontium anti-osteoarthritis,to provide a certain experimental basis for the clinical treatment of osteoarthritis in the future.