Cytology And Animal Experimental Study Of Piezoelectric Properties Bone Substitute Material HA-BaTiO₃

Biological engineering research with human bone properties has been a popular research in the field of orthopedics. However, the related research of biological engineering bone has been focused on the vast majority of the material itself and human bone component simulation, but not the "piezoelectric characteristics" research. Studies have demonstrated that human bone biological phenomena and piezoelectric properties,electrical stimulation can promote bone healing. Therefore, the development of a not only in the structure is similar to human bone, but also has the piezoelectric properties,corresponding to the bone substitute materials simulation has become a new research direction in the field of bone repair and reconstruction.The previous studies have confirmed:（1） The material composition of barium titanate 9:1 hydroxyapatite（BaTiO₃-HA） has better compressive strength, no cell toxicity, and better biocompatibility.（2） The material composition of BaTiO₃-HA 9:1 material has higher compressive strength, good biological activity and biocompatibility than other materials.（3） The charge generated by the BaTiO₃-HA composite in the 9:1 composite afterpolarization has a promoting effect on the osteoblast..This study on the basis of the above study, to further clarify the following questions:（1） The promoting effect of BaTiO₃-HA composites on osteoblasts was caused by the composition of BaTiO₃, or by the surface charge.（2） How much piezoelectric coefficients of BaTiO₃-HA composites can play the best role in promoting bone formation?ObjectiveBy in vitro cytology and in vivo, clear BaTiO₃-HA composite on the mechanism of the role of bone cells screened stimulate bone cell adhesion and growth of best material charge, to explore the for repairing bone defect of effect and influencing factors, for research and development with the piezoelectric properties of bone repair and reconstruction materials to provide experimental basis.Methods Study on the mechanism of action of BaTiO₃-HA composites on osteoblastsOn the basis of previous studies, the quality ratio of BaTiO₃-HA composite and pure BaTiO₃ materials were compared with that of 9:1. Through the electric field of two kinds of materials were polarized, with different materials and electrical properties of three kinds of materials: group A（uncharged BaTiO₃-HA）, B（charged） BaTiO₃-HA, C group（with a charge of pure BaTiO₃）. The above three groups of materials were co cultured,then bone cell surface: MTT method（MTT） co culture test after inoculation 1, 4, 7 days of materials on the proliferation of osteoblasts; using alkaline phosphatase（ALP） staining and ALP co culture quantitative test after inoculation for 1 4, 7 days, materials on osteoblast differentiation; using scanning electron microscopy（SEM） and confocal microscopy（CLSM） cell adhesion and morphology of inoculated cells after 4 days of material surface; the bone cell apoptosis into flow cytometry with Tunel staining on the surface of the material were detected 4 days after inoculation the osteogenic related gene expression; 4 days later by Real time- PCR for the detection of material surface inoculation, including Alp, Collagen, Runx2, Rhoa 1, Gusb and Rpl19; the Western-Blotwas used for detection of osteoblast related genes protein Expression of OPG, Collagen,RANKL, RUNX2, and Osteocalcin（OCN） were detected.In vitro screening of the best electrical quantity of BaTiO₃-HA compositesIn different polarization（0.5kv/mm, 1kv/mm, 1.5kv/mm） BaTiO₃-HA（9:1） composites,BaTiO₃-HA composites with different amount of charge, and will be divided into: group A（with low power group, the remnant polarization of Pr=54.37 c/N）, B group（in charge group, Pr=89.54 c/N, C） group（high power group, Pr=153.50 c/N）, D group（pure HA group with no charge）, respectively: MTT method（MTT） co culture test inoculated cells after 1, 4, 7 days of materials on the proliferation of osteoblasts; using alkaline phosphatase（ALP） co cultured with ALP staining and quantitative test after inoculation for 1, 4, 7 days of osteogenic differentiation effect; with scanning electron microscopy（SEM） and confocal microscopy（CLSM） cell adhesion and morphology of inoculated cells after 4 days of material surface; using flow cytometry Cell apoptosis was detected after 4 days of inoculation with Tunel staining on the surface of the material.Experimental study on the effect of BaTiO₃-HA composite material and motor stimulation on the repair of radius defect in rabbitsAccording to the results of experiment two, the hollow cylindrical BaTiO₃-HA（9:1）composites（highly 12 mm, diameter 5mm） were prepared. 40 adult male New Zealand white rabbits were selected to establish bilateral middle forearm bone defect model（12mm）. Implant materials is charged and motion stimuli, randomly divided for four groups: group A（with charge + motion stimulation group） according to the of and group B（without charge + motion stimulation group）, C group charged, non motor stimulation group), group D（uncharged + non motion stimuli group）. 2 weeks after the operation, 1times a day, 30 minutes of exercise every day. Respectively after operation in 6 weeks, 12 weeks, drawn by von Gieson staining and tetracycline and calcein pigment fluorescence double staining observed between the different groups of new bone ingrowth and bone mineral apposition rate.Results1.The MTT results showed that cell proliferation was polarization group OD value was significantly higher than the other two groups at first days and fourth days（P < 0.05）, but in seventh days had no significant difference; the detection of ALP activity is similar with the results of MTT, on the first day and the fourth day group was significantly higher than the activity of cell polarization in other groups（P < 0.05）, but no difference between the groups on the seventh day of cell activity; the scanning electron microscope: group A（uncharged BaTiO₃-HA） cells compared to the B group（charged BaTiO₃-HA） and group C（with a charge of pure BaTiO₃） form better and more full and a large number of pseudopodia; phalloidin labeled Luo Danming staining and quantitative analysis of cell size and cell density of the cytoskeleton, results show that the difference between the A group and B group significantly（P < 0.05）, and A group than in B and C two groups have a better cell surface spread Product with the number of cells;（4） flow cytometry instrument apoptosis experiment and TUNEL staining results showed that in group a compared to group B and group C significantly reduced apoptosis（P < 0.05）; real time PCR results showed, into the bone related genes, ALP, Col1a1 and Runx2 expression in group A was significantly higher than that of group B and group C（P < 0.05）, and Rhoa,Gusb and expression of the gene of Rpl19 no statistically significant difference in. The Wstern-Blot detection showed on the expression of OPG, Collagen1 and rankle and Runx2 protein in group A was significantly better than the rest of the group B and the statistical difference（P < 0.05）.2.MTT results showed that: on day 1 and day 4, a charged group（Pr=89.54 mu c/N） and high consumption group（mu the Pr=153.50 c/N） cell proliferation quantity OD value was significantly higher than that of low charged group（Pr=54.37 mu c/N）（P < 0.05）, but in between day 7, high consumption group no statistically difference; II ALP activity test results with the MTT results coincide, on day 1 and 4 days of charged group and highly charged group cell activity was significantly higher than that of low charged group（P <0.05）, but in the 7th day of activity of cells in each group no significant difference. The detection results of scanning electron microscopy showed that: in charge group and cellmorphology with high consumption group is superior to the low voltage coefficient of material group, in charge group and high consumption group with no significant differences between the groups, the best HA cell morphology; phalloidin labeled Luo Danming staining and quantitative analysis of cell size and cell density were the results showed that the cytoskeleton, in charge group and high consumption group is higher than that with low voltage coefficient of material group（P < 0.05）, but in charge group and high consumption group with pure HA and there is no significant difference; the flow cytometry results showed that cell apoptosis in charge group and high band compared to the low power group material charge group material group significantly decreased cell apoptosis（P < 0.05）.3.Von Gieson staining results showed that 12 weeks. Group A（polarization and motion stimulus group） of new bone ingrowth was also was significantly higher than that in group B(polarization and non motor stimulation group（P < 0.05）, a group and B group new bone ingrowth was higher than group C（non polarization and motion stimulus group） and group D(non polarized and non motor stimulation group（P < 0.05）. Fluorescent double labeling method was used to measure bone mineral apposition rate results in 6 weeks, 12 weeks in group A was significantly higher than that in group B（P < 0.05）. In group B was significantly higher than that of D group and there was significant difference（P < 0.05）. C group D group and no significant difference（P > 0.05）.Conclusion BaTiO₃-HA composite into bone cells promote function is mainly derived from material surface to carry negative charge and charge must be adequate amounts above mu the Pr=89.54 c/N can play a better promote the function of bone cell activity. The piezoelectric effect of BaTiO₃-HA composite in the motor stimulation can promote the new bone formation and accelerate the healing of bone.