Study On The Role And Mechanism Of Lithium Borate-containing Bioactive Glass Composites For Repairing Bone Defects

Objective:In this study,we investigated the effect of Li-BG/PMMA on repairing bone defects and its mechanism by synthesizing poly(methyl methacrylate)composites containing lithium borate bioactive glass to provide relevant experimental data and scientific basis for the development of new artificial composites.(I)In vivo study of Li-BG/PMMA to promote the repair of early bone defects in rabbit femoral condylesMethods:Li-BG/PMMA composite scaffolds were implanted into cancellous bone defects in the distal femoral condyles of rabbits,and the ability of the composite materials to repair bone defects and the safety of the materials were assessed by imaging,histology and hematobiochemistry.Twenty-four healthy adult New Zealand rabbits were surgically implanted with 0Li-BG/PMMA,1Li-BG/PMMA,3Li-BG/PMMA,and5Li-BG/PMMA composite scaffolds after anesthesia to establish an animal model of cancellous bone defects in the femur.The animals were evaluated at 4 weeks,8 weeks and 12 weeks after surgery,and the growth of bone tissue was determined by gross observation,micro-CT scan was performed to analyze the growth of new bone and the degradation of the material,the blood biochemical indexes were measured by enzyme-linked immunosorbent assay(Elisa),the levels of calcium,potassium,phosphorus,chloride,magnesium,sodium and iron were measured by automatic biochemical analyzer,and the safety of the material on liver and kidney tissues was evaluated by HE staining.Results:The material implantation position healed well in all groups after surgery,and the composite material showed no signs of loosening and displacement,and the bone defects were repaired to different degrees;micro-CT results showed that the 5Li-BG/PMMA group had more new bone at the edges and voids of the material,and the material degraded faster;the differences in blood biochemical indexes between all material groups before and after surgery with time were not statistically significant(P>0.05);the kidney and Histological HE staining of the liver showed no adverse effects of scaffold implantation in rabbits.Conclusion:Li-BG/PMMA can effectively promote the formation of new bone in local cancellous bone defects in rabbit femoral condyles and accelerate the repair and reconstruction of bone defects with good biocompatibility and no adverse reactions in vivo by implantation.(Ⅱ)Experimental study of Li-BG/PMMA to promote the proliferation,differentiation and polarization of human bone marrow mesenchymal stem cells(h BMSCs)and macrophages in vitroMethods:MTT assay was used to detect the proliferation ability of h BMSCs and macrophage cells.Alkaline phosphatase(ALP)assay kit was used to detect the ALP activity of h BMSCs.The osteogenic differentiation ability and extracellular matrix mineralization ability of h BMSCs were assessed by ALP staining method and alizarin red staining method.The M1 and M2 phenotypes of macrophages were detected by flow cytometry and cell polarization was observed;the levels of inflammatory factors in macrophages were detected by real-time fluorescent quantitative PCR(RT-q PCR).Results:MTT assay results showed that on day 1,0Li-BG/PMMA,1Li-BG/PMMA,3Li-BG/PMMA and5Li-BG/PMMA significantly promoted the proliferation of h BMSCs in a concentration-dependent manner.On days 3,5 and 7,5Li-BG/PMMA promoted the proliferation of h BMSCs most significantly compared with the negative control group,and the difference was statistically significant(P<0.05).The MTT assay results of macrophages showed that 0Li-BG/PMMA,1Li-BG/PMMA,3Li-BG/PMMA,and5Li-BG/PMMA significantly promoted macrophage proliferation at 48 h and 72 h,with the 5Li-BG/PMMA group being the most significant and the difference was statistically significant(P<0.01).The results of alkaline phosphatase quantification showed that alkaline phosphatase activity was significantly higher in the 3Li-BG/PMMA and 5Li-BG/PMMA groups(P<0.05 and P<0.01,respectively),and the color degree of alkaline phosphatase staining was also significantly higher than that of the control group and the0Li-BG/PMMA and 1Li-BG/PMMA groups;alizarin red staining experiments also showed that the5Li-BG/ PMMA group showed the deepest red color.The results of cell polarization indicated that the5Li-BG/PMMA group could promote the conversion of macrophages from M1 pro-inflammatory phenotype to M2 anti-inflammatory phenotype.RT-q PCR results showed that the 5Li-BG/PMMA group could upregulate the levels of anti-inflammatory factors(CD206,transforming growth factor-β,arginase)while downregulating the levels of pro-inflammatory factors(inducible nitric oxide synthase,tumor necrosis factor-α,interleukin-1β).Conclusion:Li-BG/PMMA extract promotes proliferation and differentiation of h BMSCs with good osteoinductivity and biocompatibility,promotes M1/M2 transformation of macrophages,improves the proliferation of macrophages,and decreases the expression of pro-inflammatory genes while increasing the expression of anti-inflammatory genes.