Functional Construction Of Borosilicate Bioactive Glass (BSG) And Its Mechanism Of Bone Defect Repair

In order to overcome the defect of poor bone induction ability of existing bone cement,a new type of bone cement was prepared,which could moderately promote the polarization of macrophages from M1 to M2,enhance the vascularization of bone defect area and induce new bone formation.Through the functional construction of borosilicate bioactive glass,bone cement composed of borosilicate bioactive glass containing strontium and copper as solid phase and chitosan as liquid phase was prepared.The physical and chemical properties met the application requirements of injectable bone cement,and performed the functions of regulating immunity,angiogenesis and osteogenesis for bone defect repair.This dissertation is divided into three parts.The first part（Chapter 2）describes the preparation and characterization of strontium/copper borosilicate bioactive glass（Sr/Cu-BSG）bone cementObjective:The solid phase and setting solution of Sr/Cu-BSG bone cement was prepared,and the core physical and chemical properties of this injectable bone cement was analyzed by studying the structural characteristics of Sr/Cu-BSG powder and the setting mechanism,bioactivity,degradability,setting time and temperature,injectability and mechanical strength.Methods:Scanning electron microscopy（SEM）,laser particle size analyzer,Inductively Coupled Plasma Optical Emission Spectrometer（ICP-OES）,Fourier transform infrared spectroscopy（FT-IR）and Raman spectrometer were used to detect components,the micro-morphology and structural characteristics of Sr/Cu-BSG.Bone cement was prepared by mixing Sr/Cu-BSG with chitosan curing solution.Syringe,universal tensile testing machine,Gilmore instrument,Probe type thermometer and other instruments were used to test the injectability,initial setting time,compressive strength and setting temperature of bone cement.The p H microelectrode and FT-IR were used to study the setting mechanism of bone cement.XRD,FT-IR and SEM were used to analyze the surface morphology of bone cement samples and the transformation of hydroxyapatite.Acid-base changes of Sr/Cu-BSG bone cement immersed in PBS solution were detected using a p H meter.Results:The particle size of Sr/Cu-BSG was uniform,the actual detection of Sr/Cu-BSG components was almost identical to the design components,and there was no significant difference in the chemical structure of each Sr/Cu-BSG component.The injectability,initial setting time and compressive strength of Sr/Cu-BSG bone cement met the application requirements.The setting mechanism of Sr/Cu-BSG bone cement is as follows:after mixing the solid phase of the cement and the setting solution,the setting solution p H increases to 6.79-7.27,which causes the liquid phase sol-gel transformation of chitosan,the bone cement changes from fluid to solid,and the bone cement is initially solidified.Ca²⁺precipitated from Sr/Cu-BSG bone cement reacts with phosphate in bone cement curing solution to form hydroxyapatite,and chemical chelation is formed between borosilicate bioactive glass with high chemical reaction activity and chitosan to further solidify Sr/Cu-BSG bone cement.The experimental results of SEM,X-ray diffraction and FT-IR showed that Sr/Cu-BSG bone cement can be transformed into hydroxyapatite when immersed in PBS solution.Sr/Cu-BSG bone cement was immersed in PBS solution and degraded to produce an alkaline microenvironment,with a p H value of 9.00-9.26.Conclusion:Sr/Cu-BSG bone cement has self-setting properties and bioactivity,and its physical and chemical properties meet the application requirements of injectable bone cement.The second part（Chapter 3）investigated the mechanism of Sr/Cu-BSG bone cement in repairing bone defects in vitroObjective:Mouse macrophages（RAW264.7）,human umbilical vein endothelial cells and human bone marrow mesenchymal stem cells were cultured in Sr/Cu-BSG bone cement extract medium,and the mechanism of bone defect repair was analyzed in vitro.Methods:In vitro,RAW264.7 cells were cultured in bone cement extract medium for three days,and the effects of bone cement extract on cell polarization and regulation of inflammation-related genes were analyzed by flow cytometry and q RT-PCR.Human umbilical vein endothelial cells were cultured in bone cement extract medium for five days,and the regulation of angiogenesis genes and angiogenesis ability of cells were analyzed by q RT-PCR and in vitro angiogenesis test.Human bone marrow mesenchymal stem cells were cultured in bone cement extract medium for 1,3 and 5 days,and the effects of bone cement extract on the proliferation,activity and morphology of cells were analyzed by CCK-8,live-dead staining and cytoskeleton staining.Human bone marrow mesenchymal stem cells were cultured in bone cement extract medium for seven days,and the effect of bone cement extract on osteogenic differentiation of cells was examined by ALP staining and ALP activity test.Human bone marrow mesenchymal stem cells were cultured in bone cement extract medium for 7 days,14 days and 21 days,and the regulation of bone cement extract on osteogenic genes was analyzed by q RT-PCR.Results:RAW264.7 macrophages were cultured with Sr/Cu-BSG bone cement extract for three days.Sr/Cu-BSG bone cement extract regulated the polarization of RAW264.7 macrophages from M1 phenotype to M2 phenotype and significantly increased the relative expression levels of anti-inflammatory cytokines IL-1Ra and TGF-β1,and decreased the relative expression levels of pro-inflammatory cytokines IL-1βand IL-6.Human umbilical vein endothelial cells were cultured with Sr/Cu-BSG bone cement extract for five days.Sr/Cu-BSG bone cement extract increased the relative expression levels of angiogenic genes VEGF,b FGF,Veger1（FLT1）and VEGFR2（KDR）,and significantly enhanced the angiogenic ability of human umbilical vein endothelial cells in vitro.Sr/Cu-BSG bone cement extract had no adverse effect on the proliferation,activity and morphology of human bone marrow mesenchymal stem cells,and significantly promoted the secretion of alkaline phosphatase,after culturing for seven days.Human bone marrow mesenchymal stem cells were cultured with Sr/Cu-BSG bone cement extract for 7,14 and 21 days.Sr/Cu-BSG bone cement extract significantly increased the relative expression levels of osteogenic genes such as Runx-2,OCN and OPN.In the designed cement components,the 5Sr3Cu-BSG bone cement group had the best effect on immunity,angiogenesis and osteogenesis.Conclusion:In vitro studies showed that Sr/Cu-BSG bone cement had good biocompatibility in vitro,modulated the formation of anti-inflammatory immune microenvironment,induced the vascular differentiation of human umbilical vein endothelial cells,and promoted the osteogenic differentiation of bone marrow mesenchymal stem cells.The third part（Chapter 4）examines the mechanism of Sr/Cu-BSG bone cement in repairing bone defects in vivoObjective:The femoral condylar defect model was established in SD rats and New Zealand rabbits.Thereafter,the Sr/Cu-BSG cement and calcium sulfate cement were implanted into the two animal models to analyze the mechanism and effect of bone defect repair in each group.Methods:Two animal models of femoral condylar bone defect were established in vivo.Bone cement was implanted into the femoral condylar bone defect of SD rats.The mechanism of bone defect repair was studied by immunohistochemical staining at three time points of 3 days,7 days and 28 days.Bone cement was implanted into the femoral condylar bone defect of New Zealand rabbits.The repair effect of bone defect in vivo was studied by micro CT,HE and Masson staining sections at three time points of 8 weeks,12 weeks and 16 weeks.At the time point of 16 weeks,blood samples were taken from the vein of the ear margin of the rabbits for routine tests and blood biochemical analysis,and the viscera of the rabbits such as heart,liver,spleen,lung and kidney were taken for HE staining analysis to test the biocompatibility of Sr/Cu-BSG bone cement.Results:In vivo study of femoral condylar bone defect in SD rats showed that after three days of bone defect implantation,Sr/Cu-BSG bone cement played an immunomodulatory role at the bone implant interface,induced macrophages to polarize to M2 type,and significantly improved the anti-inflammatory IL-1Ra and TGF-β1 expression rates and reduced the pro-inflammatory IL-1βand IL-6 expression rates to induce anti-inflammatory immune microenvironment.After seven days of implantation,Sr/Cu-BSG bone cement significantly increased the positive expression rate of angiogenesis indexes such as VEGF,b FGF,VEGFR1（Flt1）and VEGFR2（KDR）at the bone implant interface,and promoted angiogenesis.After 28 days of implantation,Sr/Cu-BSG bone cement significantly increased the positive expression rate of osteogenic markers such as Runx-2,OCN and OPN at the bone implant interface.In the long-term implantation experiment in New Zealand rabbits,the new bone tissue grew into the degradation region of Sr/Cu-BSG bone cement,indicating that the degradation rate of Sr/Cu-BSG bone cement exactly matches the osteogenesis rate.Compared with other bone cements,the bone-implant interface of the5Sr3Cu-BSG bone cement group showed the best bone formation and bone repair effect around the implant,with significantly increased bone mineral density（BMD）,bone volume fraction（BV/TV）,number of trabeculae（Tb.N）,trabecular thickness（Tb.Th）,tissue area（T.Ar）,bone mineralization deposition rate（MAR）and bone implant contact rate（BIC）.After bone cement was implanted into the femoral condyle of New Zealand rabbits for 16 weeks,the blood routine and blood biochemical indexes were within the normal range,and the HE staining sections of heart,liver,spleen,lung and kidney showed no obvious abnormalities,indicating that the strontium/copper borosilicate bioactive glass cement is biocompatible in vivo.Conclusion:The short-term implantation of femoral condyle in SD rats showed that Sr/Cu-BSG glass bone cement modulated the formation of anti-inflammatory immune microenvironment,induced angiogenesis at the bone defect site,and facilitated the formation of new bone tissue.Further long-term in vivo experiments of femoral condylar bone defects in New Zealand rabbits showed that Sr/Cu-BSG bone cement had good biocompatibility,moderate degradation,and the degradation rate completely matched the osteogenesis rate.Compared with other bone cement groups,the 5Sr3Cu-BSG bone cement group showed the best bone repair effect.Taken together,these results showed that Sr/Cu-BSG bone cement has good physical,chemical and biological properties,and is an ideal bone repair material.