| The material researchers and the doctors in clinic focused on the research to innovate and apply the ideal biomaterial for the bone defects. The ideal material should include: the good biocompatibility, degradable and absorbable, osteoconduction, osteoinduction, similar to natural bone structure, high cost-performance, easy to operate. Nowadays, bioglass is well known by material scientists, because bioglass has good biocompatibility and osteoconductive, and also can stimulate bone growth, and is widely used in clinic. However, poor mechanical property of bioglass has limited its application into the non-load bearing condition. Therefore, it is necessary to improve the biomechanics properties and maintain the stimulate bone growth ability for the material researchers and the doctors. Our team colleagues cooperate with the Nova Bone Products Limited Liability Company, designed and made macro-pore bone block bioglass(MPBB) by improve the ingredient and fabricating technique. Our research is going to study the mechanical property biocompatibility and the stimulate bone growth ability of MPBB.Influence of macro-pore bone block on osteoblast behavior in vitroObjective:To observe and identify the impact of a type of macro-pore bone block bioactive glass on osteoblast in vitro.Methods:1. Choose the ?-TCP as the control group, and MPBB as the experimental group. Analysis and compare the morphological structure and the main components of MPBB and ?-TCP through energy spectrum analysis under the scanning electron microscopy. 2. Soak the MPBB and ?-TCP in simulated body fluid, Observe the surface of the two materials under the scanning electron microscopy. 3. Cultivation of MC3T3 E1 osteoblast in mice with extract liquid of MPBB and ?-TCP, Giemsa staining was performed to detect the cell numbers under the optical microscope. 4. Put the MC3T3 E1 osteoblast directly grown on MPBB and ?-TCP material, observe the situation of cell adhesion on materials, and also cultivation of MC3T3 E1 osteoblast in mice with extract liquid of MPBB and ?-TCP, Immunofluorescence staining was performed to detect the Cell overall aera to nuclear aera, also the Average fluorescence intensity per cell. 5. Cell proliferation and viability in different groups were detected by MTT assay, Brd U assay respectively. 6. OSX immunofluorescent staining and ALP staining, alizarin red staining, the real-time PCR were performed to assess the OSX positive cell rate, ALP activity rate, mineralization and relative transcript level in gene of MC3TC-E1 cells in different groups.Results:1. The surface of MPBB are more rough and porous than ?-TCP, and the energy spectrum analysis showed that the MPBB possess Si element, and there are more Ca and P element in ?-TCP.2. The result of the experiment simulated body fluids showed that the surface of MPBB appeared hydroxyapatite deposition in 6 hours, hydroxyapatite gradually increase as the soaking time extended, there were no hydroxyapatite deposition on ?-TCP. 3. In Giemsa staining, the cell numbers in 400 times under a microscope were(106.0±6.025)in MPBB group and(40.20±3.639) in ?-TCP group, which was statistically significant(P<0.01). 4. The adhesion state of MC3T3 E1 osteoblasts on MPBB were better than ?-TCP group, it had more pseudopod. In the immunofluorescence of Venculin, the ratio of cell total area to nucleus area and the expression of Venculin were higher in MPBB group than in ?-TCP group. which had statistically significant(P<0.05). 5. In the Brd U and MTT assay, MC3T3-E1 cells showed a higher proliferation rate in bioactive medium group respectively, all of which was statistically significant(P<0.05). 6. In the OSX assay, the MPBB group had more OSX positive cell rate than ?-TCP group(P<0.01), in the ALP staining and alizarin red staining, the expression of ALP and the mineralization rate were higher in MPBB group than in?-TCP group, which were all statistically significant(P<0.01, P<0.05). In MC3T3-E1 cells cultured with the MPBB group, osteogenesis-related genes expression was higher than those cultured with?-TCP solution.Conclusion:The MPBB promotes MC3T3-E1 cell proliferation and osteoblastic differentiation, and have much more Si element, which indicates that MPBB glass can promote cell proliferation and differentiation and has promising bioactivity.Influence of macro-pore bone block on osteogenic activity and mechanical property in vivoObjective:To observe and identify the osteogenic activity, biocompatibility and mechanical property of a type of macro-pore bone block bioactive glass on in rabbit.Methods:Establish the femoral condyle defect model with New Zealand white rabbit. Implant in the defect with macro-pore bone block bioglass, ?-TCP and NOVABONE? respectively. According to the different materials implanted in the defect, three groups were divided as MPBB, ?-TCP group and Nova Bone group. After the surgery, X-ray examination were performed to confirm the location and fixation of the materials and to observe the femoral condyle fracture. The specimens were harvested in 4, 12 and 24 weeks after the surgery respectively. Micro-CT was performed to assess the new bone formation and degradation of the materials. Tetracycline-calcein double labeling was used to detect the mineral apposition rate of new bone. Van Gieson staining was to assess the new bone formation percentage, Biomechanical markers including the compress strength and elasticity modulus was also measured.Results:X-ray examination showed that each femoral defect was filled fully with materials and the materials were all in proper position. As indicated by Micro-CT results, at 12 weeks, the bone regeneration volume fraction of MPBB group, ?-TCP group and Nova Bone group was 16.83±1.01,9.88±1.23,21.35±1.27 respectively, the residual material volume fraction of each group was 50.96±1.37,68.29±3.93,19.37±1.40. at 24 weeks, the bone regeneration volume fraction of each group was 37.48±0.70,25.29±1.45,27.03±1.25 respectively and the difference between MPBB group and ?-TCP group or Nova Bone group was statistically significant(P<0.001). the residual material volume fraction of each group was 34.67±3.52,55.66±2.05,7.52±1.15 respectively and the difference between MPBB group and ?-TCP group or Nova Bone group was statistically significant(P<0.01). The results of tetracycline-calcein double labeling showed that the mineral apposition rate in MPBB group, ?-TCP group and Nova Bone group at 4 weeks was(1.577±0.045,2.064±0.068,1.19±0.09) um/d respectively and the difference between MPBB group and ?-TCP group was statistically significant. As shown by the results of Van Gieson staining, the new bone area percentage of MPBB group, ?-TCP group and Nova Bone group was 5.43±1.25,2.77±0.85,6.51±1.21 at 4 weeks, 8.48±0.84,2.94±0.65,11.42±2.66 at 12 weeks,23.55±1.13,12.92±0.45,19.53±0.91 at 24 weeks. The difference between MPBB group and ?-TCP group or Nova Bone group at 24 weeks was statistically significant(P<0.001). By biomechanical test, the compress strength of specimens in MPBB group and ?-TCP group increased as time prolonged, with no statistically significant between the two groups. The elasticity modulus of specimens in MPBB group and Nova Bone group was stable after surgery, closer to the rabbit bone. While elasticity modulus of the ?-TCP group increased a lot, unsuit to the rabbit bone.Conclusion:MPBB presented good biological activity, biocompatibility and suitable biomechanical properties. This research loaded foundation for the application in weight-bearing sites of this new material.Conclusion:Studies have shown through the above two parts, the MPBB promotes MC3T3-E1 cell adhere and proliferation, and osteoblastic differentiation through gene and protein level. which indicates that MPBB glass can promote cell proliferation and differentiation and has promising bioactivity. At the same time, MPBB possess good biocompatibility and osteoconductive, and also can stimulate bone growth, has certain mechanical strength and elasticity modulus, our study provide theoretical foundation for the bone graft in load bearing bone defects. |
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