Experimental Study On Osteogenesis And Subchronic Toxicity Of Silicon-based Bone Repair Materials

Bone defect is a common clinical disease with characteristics of large base,wide range and diversified condition.Therefore,there are many requirements for the performance of bone repair materials.It is well known that silicon is an important trace element for the bone regeneration and repair,which can up-regulate the expression of osteogenesis-related genes,activate the osteogenesis related signaling pathways and promote the osteogenic differentiation of bone marrow mesenchymal stem cells,which has good biological activity and osteogenic induction.There has been great progress in the development of silicate-based bioactive ceramics in the past decade.Base on the characteristics of bone repair materials,the research is carried out with silicate based cement,which has moderate hydration speed and batter bioactivity.The mechanical property ?bioactivity of materials and the possible subchronic toxicity of akermanite was systematically studied to provide reference and date support for the clinical application of silicate based materials.Part ?:Evaluation of Silicate Absorbable Bone Cement in vivoObjective: The biocompatibility,osteogenesis and degeneration characteristics of calcium phosphate-tricalcium silicate cement(CPC-C3S) were evaluated in vivo,aiming to provide animal evaluation reference in vivo for the potential cilinical application of such materials.Methods: Bone defects of 5 mm×10 mm were made at both femoral condyles of 24 rabbits,and CPC-C3 S or CPC were implanted into bone defects of rabbit randomly.We observed the degradation of material in the body and new bone formation through X-ray and HE staining in 4,8,12 weeks after operation.Results: Immediate X-ray examination after operation showed that the filling condition was good and four weeks later,the boundary between the defect site and the material was clear.Both group of materials were non-degradable while the white granules could be seen in the defect site of control group.Eight weeks after operation,the material in CPC-C3 S was degraded and new bone formed while the boundary between the material and bone defect was not clear.As to control group,the implanted material was wrapped by fibrous scar tissue,and the boundary of the bone defect was still clear.Twelve weeks after operation,the degradation rate of CPC-C3 S group was 45.4% while the CPC group was 32.1%.Conclusion: The biocompatibility of CPC-C3 S composite is similar to CPC bone cement while CPC-C3 S composite has better mechanical properties,surface osteogenic activity and degradation performance.Through further optimization,CPC-C3 S composite is a new kind of bioactive bone repair material with good application prospects.Part ?:Preparation and in vivo evaluation of a silicate-based composite bone cementObjective: Bioglass 45S5 was added to silica-based bone cement as bioactive filler in order to further improve the mechanical strength of silica-based bioactive materials.The setting time,mechanical strength and the ability of materials to induce hydroxyapatite deposition were evaluated.The biocompatibility,biodegradability and osseointegration were evaluated in vivo experiments,providing more reference data for the clinical application of these materials.Methods: The Ca₃SiO₅ was prepared by sol-gel method.Ca₃SiO₅ was mixed with CaSO₄ and 45S5 at the weight ratio of 35%,35% and 30%,then the mixture was milled for 24 h obtaining CSC cement,and the mixture of Ca₃SiO₅ and CaSO₄ was used as control group.Mixed material was added into the mould to prepare sample and the inject ability of mixed material slurry were evaluated.The self-setting time was measured according to ISO9597-1989 E standard.Compressive strength of the sample was measured after curing and the ability of hydroxyapatite deposition induced by the material was tested in simulated body fluid(SBF).The silicate based cement samples were prepared with a liquid to powder ratio of 0.35 ml/g(L/P=0.35).BG and CPC were used as control group.Cylindrical bone defects of 5 mm×10 mm were drilled at both femoral condyles,then CSC sample was implanted to one side and CPC or BG sample was implanted to the other side as control.Rabbits were executed respectively at 3,6 and 12 months after operation to observe the appearance of the operation area.Then took samples for histological analysis.Results: The initial and final setting time of CSC bone cement were 13 and 26 min respectively,comparing 18 and 36 min respectively of Ca₃Si O₅/CaSO₄ bone cement in control group.The injectability of CSC slurry deteriorated with time delay,and 70% slurry can be injected within 5 minutes while only 43% slurry can be injected in 10 minutes without solid-liquid separation.The compressive strength of CSC samples increased rapidly in the early stage,higher than 10 MPa at 1 h and 30 MPa after 24 h.The characteristic absorption peaks of PO₄^3- ions appeared at 1044 cm^-1 in infrared spectra,which indicated that bone-like hydroxyapatite was formed on the surface of CSC materials.Histological analysis showed that the degradation rate of BG group was the fastest,while that of CSC group was the slowest,and no stress fracture occurred during the process.Von-Gieson staining showed that three months after implantation,new bone formed on the surface of CSC material.Bone tissue was contact with the material directly and with the time went on,bone contact area increased continuously.After 12 months,most of the material surface was covered by new bone tissue.Bone contact rates of CSC samples at 3,6 and 12 months were 40%,70% and 80% respectively.Conclusion: The introduction of BG improves the mechanical strength of CSC materials while CSC material has the ability to induce the deposition of hydroxyapatite on its surface.Experiments in vivo proved that CSC material has good bonding ability with surrounding bone tissue and promotes new bone formation.Part ?:The Research about Subchronic Toxicity of Akermanite Bioactive Ceramics Degradation Products in vivoObjective: As to bioactivity silicate based cements,the possible subchronic toxicity of the ionic products of akermanite was systematically studied,aiming to provide important evidence for the clinical transformation of akermanite and other silicon-based bioceramics in terms of biological safety.Methods: Sample extracts were prepared at a ratio of 0.2 g/mL.Animals in the experimental group were injected with the sample extract through intravenous injection once a day,20 mL/kg each time.Animals in control group were injected with 0.9% NaCl solution in the same way,and the state of the animals was observed regularly.The blood cytology and biochemical indexes were tested after the end of dose.After the animals were executed,recording the wet weights of brain,lung,heart,liver,spleen,stomach,kidney and gonad respectively,and the pathological changes of each organ were recorded.Light microscopy was used to observe the histopathological changes of various organs in rats which were stained by HE.Scanning Electron Microscope(SEM) was used to observe the tissue states of liver,kidney and heart and Energy Dispersive X-ray spectroscopy(EDS)was used to analyze the content of elements in tissues.Results: The ion concentration of Ca,Mg and Si in the extract of akermanite were significantly higher than that of corresponding elements in saline solution.During the dose period,all rats survived well and weight gained normal without poisoning.After administration,there were no significant decreases in hemoglobin concentration,erythrocyte count,platelet count,white blood cell count,white blood cell classification and hematocrit between the experimental group and the control group,and there was no significant difference between the two groups.Serological indexes between the experimental group and the control group was no significant difference.The negative changes such as congestion,hemorrhage,edema and necrosis were not observed in brain,lung,heart,liver,spleen,stomach,kidney and reproductive organs bot in experimental group and control group.There was no significant difference in viscera/body ratio between experimental group and control group.The results of HE staining showed that there were no abnormal phenomena in organs of the experimental group and the control group.EDS results showed that the elements were not enriched in liver,kidney and heart organs,and there was no significant difference in the distribution of elements.Conclusion: Akermanite has no negative effect on the cells in blood and the shape as well as function of each organs.Ion products released during material degradation can be efficiently and safely utilized,and metabolized in vivo.Akermanite bioactive ceramics is a kind of bioactive ceramics with high biological safety,which has no subchronic toxicity to the body.