Synthesis Of Magnesium Hyaluronate And Its Application In Osteoarthritis Treatment

Objectives:1.To synthesize magnesium hyaluronate and characterize its physicochemical properties.2.To investigate the biological effect of magnesium hyaluronate on human chondrocytes and.mouse MC3T3-E1 cells.3.To explore the biofunction of magnesium hyaluronate in the mouse osteoarthritis model.Methods:1.Cation exchange resin was used to replace sodium ions with hydrogen ions,and then magnesium ions were combined with the hyaluronic acid.2.X-ray Diffraction(XRD)was applied to detect the presence of uncombined metal ions in magnesium hyaluronate.Fourier Transform Infrared Spectrometer(FTIR)was used to explore the new functional groups generated with the binding of magnesium ions.Scanning Electron Microscope(SEM)and Energy Dispersive Spectroscopy(EDS)were taken to evaluate the surface morphology and the element composition of magnesium hyaluronate.Thermogravimetry Analysis(TGA)was performed to assess the thermostability of magnesium hyaluronate.3.Cell proliferation assay was performed to investigate the proliferative effect of magnesium hyaluronate on human chondrocytes and mouse MC3T3-E1 cells.Quantitative real-time PCR(RT-qPCR)was performed to detect the regulatory effect on chondrocytes and MC3T3-E1 cells at the genetic transcription level.Immunofluorescence was conducted to evaluate the regulatory effect on chondrocytes at the protein expression level.Alkaline phosphatase(ALP)staining was performed to investigate the effect of magnesium hyaluronate on inducing osteogenic differentiation of MC3T3-E1 cells.4.OA animal model was established using C57 female mice(8 weeks)through anterior cruciate ligament transection(ACLT).Intra-articular injection of magnesium hyaluronate was performed once a week after operation.Gait analysis was performed at week 4 and 8.The mice were euthanized by CO2 inhalation at 8 weeks post-operation,and the joint tissue on the affected side were obtained for micro-CT analysis and histologic analysis.Results:1.Magnesium hyaluronate was successfully synthesized and characterized.Sodium ions were totally replaced with magnesium.No new functional groups were observed in magnesium hyaluronate,indicating that the combination mode of magnesium ion was similar to the sodium ion.TGA results revealed that magnesium hyaluronate had better thermostability.2.Magnesium hyaluronate promoted chondrocytes and MC3T3-E1 cell proliferation at the optimal concentration.RT-qPCR results revealed that magnesium hyaluronate effectively increased the expression level of anti-apoptosis gene BCL-2,anti-inflammation gene IL-10 and type Ⅱ collagen related gene Col2A1,and inhibited expression of apoptosis gene BAX,inflammation gene IL-6 and MMP9 of chondrocytes.Besides,expression of osteogenic genes ALP,Col-I and Runx2 was found to be up-regulated by magnesium hyaluronate in MC3T3-E1 cells.Immunofluorescence results showed that magnesium hyaluronate protected the cellular skeleton and promoted type Ⅱ collagen expression in chondrocytes.ALP staining indicated that magnesium hyaluronate showed better effect on promoting osteogenic differentiation of MC3T3-E1 cells.3.Animal experiments revealed that magnesium hyaluronate could significantly prevent OA progression,inhibit degeneration of articular cartilage,and delay the degradation of subchondral bone at the same time.Conclusion:We firstly synthesized and characterized a novel metal ion-polysaccharide compound,magnesium hyaluronate.In vitro and in vivo experiments indicated that magnesium hyaluronate had satisfying biofunction in anti-osteoarthritis,and it might provide a potential strategy for clinical OA treatment.