3D打印钛合金表面多孔涂层促进骨整合的实验研究

发布时间：2018-06-26 07:41

本文选题：3D打印 + 钛合金　；参考：《第二军医大学》2017年博士论文

【摘要】：第一部分3D打印钛合金表面多孔涂层及其机械性能测试目的:探讨利用3D打印技术制作具有多孔涂层钛合金假体的可行性及其机械性能的检测。方法:使用计算机辅助设计建模,导入3D打印机后打印钛合金假体部件;扫描电镜对其进行表征,测量孔径孔隙率;使用电子万能试验机测试打印假体部件的抗压、抗剪切性能。结果:利用3D打印技术制造出4组具有不同孔径、孔隙率多孔涂层的钛合金假体部件,经扫描电镜表征分别为:A组218.7μm,61.2%;B组262μm,68.1%;C组558μm,79.2%;D组753μm,89.0%。4组钛合金多孔涂层的压缩强度和剪切强度分别为A组78.02MPa,44.28 MPa;B组20.62MPa,23.17MPa;C组8.32MPa,12.64MPa;D组4.26MPa,8.53MPa。结论:3D打印技术可精确构建钛合金表面多孔涂层,具有良好的机械性能;随着孔径的逐渐增大,钛合金多孔涂层抗压、抗剪切性能急剧下降,其中孔径262μm,孔隙率68.1%是相对理想内植物微孔参数,可为将来假体设计提供参考。第二部分3D打印钛合金表面多孔涂层对MG-63细胞生物学行为影响目的:研究3D打印钛合金多孔涂层的生物相容性及不同微孔参数对MG-63细胞的影响。方法:以各组钛合金多孔涂层为支架培养MG-63细胞24h、48h、72h后,扫描电镜观察细胞形态;DAPI染色后荧光显微镜下细胞计数;CCK8检测各组细胞成活率;用ALP试剂盒和免疫印迹技术检测各组细胞碱性磷酸酶、骨钙蛋白的表达情况。结果:各时间点均为A组和B组细胞伸展形态较好,伸出更多的伪足。培养24h细胞在A组和B组材料上粘附明显多于C组和D组,差异有统计学意义,培养48h后,B组多于其他3组,差异有统计学意义,培养72h时4组细胞计数无统计学差异。培养24h细胞成活率A组低于空白组(无多孔涂层)、B组、C组、D组,差异均有统计学差异,B组与空白组无统计学差异,低于C组、D组,差异均有统计学差异,C组与空白组无统计学差异,但低于D组,差异有统计学差异,D组高于空白组,差异有统计学差异;培养48h细胞成活率A组低于空白组、B组、C组、D组,差异均有统计学差异,B组与C组无统计学差异,但高于A组,低于空白组,D组,差异均有统计学差异,C组与空白组无统计学差异,低于D组,差异均有统计学差异,D组高于空白组,差异有统计学差异;培养72h细胞成活率A组低于空白组、B组、C组、D组,差异均有统计学差异,B组低于空白组、C组、D组,差异均有统计学差异,C组低于空白组、D组,差异均有统计学差异,D组高于空白组,差异有统计学差异。各时间点各组间细胞碱性磷酸酶和骨钙蛋白表达均无统计学差异。结论:3D打印钛合金多孔涂层可以明显影响MG-63细胞的生物学行为,相对较小的微孔(218.7μm,61.2%;262μm,68.1%)有利于细胞粘附和分化,较大的微孔(753μm,89.0%)有利于细胞增殖;较小孔径的三维多孔涂层培养细胞收集可能存在误差,较低的细胞增值率是否界定为材料毒性反应或抑制增殖还需进一步研究验证。第三部分3D打印钛金属多孔涂层促进骨整合及其机制研究目的:研究3D打印不同微孔参数的钛合金多孔涂层对骨整合及成骨基因表达的影响。方法:3D打印机打印出3组具有不同微孔参数多孔涂层的钛合金假体,分别为(A组262μm,68.1%;B组558μm,79.2%;C组753μm,89.0%)。选择27只新西兰大白兔,随机分为3组,将3种规格假体分别植入各组兔子双侧股骨髓腔,术后4周、8周、12周每组各处死3只兔子,进行X线、Micro-CT扫描并测量骨体积分数和组织矿物密度,制作硬组织切片进行Goldner's染色和荧光显微镜观察,行拔出实验测量最大拔出力,q PCR检测Runx2和Osterix的表达。结果:X线和Micro-CT扫描均显示4周时各组均无明显骨质沉积,随着时间增加,新骨形成逐渐增多,12周时最多,且A组多于B组、C组。荧光显微镜观察术后4周各组均荧光较弱,条带较窄,随时间增加荧光强度增强,条带变宽,黄色与绿色间隙增大,12周时最优,且A组优于B组、C组。Goldner's染色示术后4周各组均以细胞核为主,随时间增加,类骨质逐渐增多,12周时达到顶峰,且A组优于B组、C组。术后4周、8周时,3组间骨体积分数和组织矿物密度差异均无统计学意义;术后12周时A组骨体积分数和组织矿物密度大于B组、C组,差异均有统计学意义,B组和C组差异无统计学意义。术后4周、8周时,A组Runx2和Osterix表达高于B组、C组,差异有统计学意义,B组和C组间无统计学差异;术后12周时,C组Runx2表达低于A组、B组,差异有统计学意义,A组和B组间无统计学差异,Osterix表达3组间无统计学差异。术后4周时,3组间最大拔出力差异均无统计学意义;术后8周时,A组所需最大拔出力均大于B组、C组,差异均有统计学意义,B组和C组差异无统计学意义;术后12周时,任意两组最大拔出力比较,差异均有统计学意义,依次为A组B组C组。结论:3D打印钛合金多孔涂层可以明显促进骨整合;随着时间的延长,骨质形成逐渐增多,沉积速率逐渐加快;孔径262μm,孔隙率68.1%的多孔涂层最有利于促进骨整合;微孔参数的变化对生物力学的影响大于对成骨效应的影响。
[Abstract]:The first part of 3D printing titanium alloy surface porous coating and its mechanical properties test purpose: To explore the feasibility and mechanical properties of porous coated titanium alloy prosthesis by using 3D printing technology. Method: using computer aided design modeling, introducing 3D printer to print titanium alloy prosthesis components; scanning electron microscope Results: 4 groups of titanium alloy prosthesis components with different pore sizes and porous porous coatings were produced by 3D printing technology. The scanning electron microscope was characterized by A group 218.7 m, 61.2%; B group 262 mu m, 68.1%; C group 558 m, 79.2%; D group 753. The compressive strength and shear strength of the porous titanium alloy coating in group 89.0%.4 are A group 78.02MPa, 44.28 MPa, B group 20.62MPa, 23.17MPa, C group 8.32MPa, 12.64MPa, D group conclusion: it can accurately construct porous coating on the surface of titanium alloy, with good mechanical properties; with the increase of pore size, titanium alloy porous coating Layer compression and shear resistance dramatically decrease, the pore diameter is 262 mu m, and the porosity 68.1% is the relative ideal inner plant micropore parameters, which can provide reference for the future prosthesis design. Second part 3D printing titanium alloy surface porous coating on the biological behavior of MG-63 cells: the study of the biocompatibility and different microcompatibility of the porous coating of the 3D titanium alloy The effect of pore parameters on MG-63 cells. Methods: the cell morphology was observed by scanning electron microscope after MG-63 cells 24h, 48h and 72h were cultured with porous titanium alloy coating as scaffold. After DAPI staining, the cell counts were counted under fluorescence microscope; CCK8 was used to detect the survival rate of each cell, and the cell alkaline phosphatase, bone calcium egg was detected by ALP kit and immunoblotting technique. Results: at all time points, the cells in group A and group B extend well and extend more pseudo foot. The adhesion of 24h cells in A and B groups is more than that of C group and D group. The difference has statistical significance. After developing 48h, the B group is more than the other 3 groups. The difference has the significance of total count, and there is no statistical difference between the 4 groups when developing 72h. The survival rate of 24h cells in A group was lower than that of blank group (no porous coating), group B, C group and D group, and there was no statistical difference between group B and blank group. There was no statistical difference between group B and D group. There was no statistical difference between group C and group D, but there was no statistical difference between the C group and the blank group, but the difference was statistically different from the D group, and the D group was higher than the blank group. There was statistical difference between the group and the group of D. The difference was statistically significant Difference: the survival rate of 48h cells in A group was lower than that of blank group, B group, C group and D group had statistical difference, but there was no statistical difference between group B and C group, but higher than that of group A, lower than that of blank group and D group, there was no statistical difference, there was no statistical difference between the C group and the blank group, and the difference was statistically different from that in the D group. The D group was higher than the blank group, and the difference was statistically significant. Difference: the survival rate of 72h cells in A group was lower than that of blank group, group B, C group and D group, and there were statistical differences in group B, group B, C group and D group, and C group was lower than that of blank group, D group, and D group was higher than that of blank group, the difference was statistically different. The cell alkaline phosphatase and bone in each group of time points were different. There is no statistical difference in the expression of calcium protein. Conclusion: 3D printing titanium alloy porous coating can obviously affect the biological behavior of MG-63 cells. Relatively small micropores (218.7 mu m, 61.2%; 262 mu m, 68.1%) are beneficial to cell adhesion and differentiation, larger micropores (753 mu m, 89%) are beneficial to cell proliferation; small pore size porous coating culture cells are beneficial to cell proliferation. To collect possible errors, whether the lower cell value added rate is defined as material toxicity or inhibition of proliferation needs further research. Third part 3D printing porous titanium porous coating to promote bone integration and its mechanism: the study of bone integration and osteogenesis gene expression of titanium alloy porous coating with different micropore parameters in 3D printing Methods: 3D printers print 3 groups of titanium alloy prosthesis with porous coating with different microporous parameters, which are (group A, 262, m, 68.1%; B 558 mu m, 79.2%; C group 753 m, 89%). Select 27 New Zealand white rabbits randomly into 3 groups. 3 kinds of prosthesis were implanted in each group of rabbit bilateral femur medullary cavity, 4 weeks, 8 weeks, 12 weeks in every group. 3 rabbits were killed, X-ray, Micro-CT scan, bone volume fraction and tissue mineral density were measured. Hard tissue sections were made by Goldner's staining and fluorescence microscopy. The maximum pulling force was drawn out, and the expression of Runx2 and Osterix was detected by Q PCR. Results: both X-ray and Micro-CT scans showed no obvious bone deposition at 4 weeks. With the increase of time, the formation of new bone gradually increased, at 12 weeks at most, and group A was more than group B, group C. The fluorescence microscope observed that the fluorescence intensity was weaker and the band was narrower in 4 weeks after the operation. The fluorescence intensity increased, the band widened, the yellow and green space increased, the best in the 12 week, and the A group was superior to the B group, and the C group.Goldner's staining showed that all groups after 4 weeks after the.Goldner's staining were all in all groups. With the nucleus based, the osteoid increased gradually with time, reaching the peak at 12 weeks, and the A group was superior to the B group. The difference of bone volume fraction and tissue mineral density between the 3 groups was not statistically significant at the time of 4 weeks and 8 weeks after the operation, and the bone volume fraction and tissue mineral density of group A were greater than that of group B and group C at 12 weeks after operation, and the difference was statistically significant, B group and C were significant. At 4 weeks and 8 weeks after operation, the expression of Runx2 and Osterix in group A was higher than that in group B, in group C, there was no statistical difference between group B and C group. At 12 weeks after operation, the Runx2 expression of group C was lower than that of A group and B group. There was no statistical difference between the A group and the group, and there was no statistical difference between the A group and the group. 4 weeks after the operation, there was no statistical difference between the group of A and the group. The maximum pulling force difference between the 3 groups was not statistically significant. At 8 weeks after the operation, the maximum pulling force needed in group A was greater than that of group B, and the difference was statistically significant in group C, and there was no statistical significance in group B and C group. The difference was statistically significant at 12 weeks after the operation, and the difference was statistically significant in group A B group C. Conclusion: 3D printing titanium alloy. Porous coating can obviously promote bone integration. As time prolongs, the formation of bone is increasing and the deposition rate is gradually accelerated; porous coating with a pore size of 262 mu m and 68.1% porosity is most conducive to promoting bone integration, and the changes in microporous parameters have greater effect on biomechanics than on the effect of osteogenesis.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TG174.4;R318.08

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