3D打印仿生钛骨表面复合涂层对成骨细胞相容性及抑菌性能的研究
发布时间：2018-08-09 14:16
【摘要】：目的:应用3D打印技术制作具有三维孔隙相互通连结构的仿生钛骨,分别复合壳聚糖-甘油磷酸温敏水凝胶涂层和壳聚糖纳米粒两种生物活性涂层,观察仿生钛骨及不同涂层表面对成骨细胞黏附、增殖和分化的影响以及对牙龈卟啉单胞菌的抑菌效果。方法:3D打印制作孔隙大小为300um的仿生钛骨试件;将壳聚糖溶解在乙酸溶液中与甘油磷酸溶液按8:2混合形成壳聚糖-甘油磷酸温敏水凝胶(CS/α,β-GP),与三聚磷酸钠(TPP)溶液按10:1混合形成壳聚糖纳米粒(CSn)。实验分组:Ti:无涂层的仿生钛骨组;Ti+CS/GP:复合CS/α,β-GP涂层的仿生钛骨组;Ti+CSn:复合CSn涂层的仿生钛骨组;Contr:空白孔板组,作为阳性对照。⑴.SE M观察各组3D打印仿生钛骨的结构形态;⑵.体外培养MC3T3-E1细胞,并接种于不同涂层仿生钛骨上,荧光技术观察细胞的黏附情况,CCK-8法检测细胞的增殖,ALP试剂盒检测细胞的分化状况;⑶.体外培养牙龈卟啉单胞菌,细菌培养计数法检测其在不同生物活性涂层仿生钛骨上的黏附情况。所得数据均采用SPSS18.0统计软件处理。结果:⑴.3D打印仿生钛骨表面为尺寸一致的网格状结构,孔隙分布均匀,相互贯通;内部多孔隙结构能够促进血液快速附着,增强血块稳定,缩短种植愈合时间。扫描电镜显示:3D打印仿生钛骨呈均匀分布的网格状结构,网格间交汇相通;CS/α,β-GP及CSn在仿生钛骨上均匀分布。⑵.荧光显微镜显示:MC3T3-E1在各组试件表面及孔隙内黏附生长;Contr组胞体突触伸展呈多角型,可见细长的突触,Ti+CSn组与Ti+CS/GP组细胞黏附在钛骨表面,胞体伸展呈类圆形,突触伸展呈多角型,但触角伸展不明显,Ti组胞体呈类圆形,无明显的多角形态;MC3T3-E1接种后分别培养1、3、5、7天,CCK-8检测显示:ContrTi+CSnTi+CS/GPTi,各组间存在明显统计学差异;ALP检测:Ti+CSn组与Ti+CS/GP组的ALP分泌均高于Ti组,存在明显的统计学差异;Contr组的ALP分泌均低于Ti+CSn组、Ti+CS/GP组,具有显著的统计学差异。⑶.细菌黏附实验结果显示:Ti+CS/GP组、Ti+CSn组表面细菌黏附数均明显少于Ti组(P0.05),且Ti+CSn组Ti+CS/GP组(P0.05)。结论:(1)3D打印仿生钛骨有利于血液快速的进入仿生钛骨内,促进血液在表面的附着,增强血块稳定性,促进骨血管化,缩短骨结合时间;(2)3D打印仿生钛骨结构具有良好的细胞相容性,复合壳聚糖纳米粒涂层后更有利于成骨细胞早期的黏附生长、增殖及成骨分化;(3)壳聚糖涂层可以有效抑制牙龈卟啉单胞菌的黏附;壳聚糖纳米粒抑菌效果优于壳聚糖-甘油磷酸温敏水凝胶。
[Abstract]:Objective: to fabricate bionic titanium bone with three-dimensional porous interconnecting structure by using 3D printing technology, and composite chitosan glycerophosphoric acid thermo-sensitive hydrogel coating and chitosan nanoparticles bioactive coating, respectively. The effects of biomimetic titanium bone and different coatings on the adhesion proliferation and differentiation of osteoblasts and the bacteriostatic effect on porphyromonas gingivalis were observed. Methods the biomimetic titanium bone specimens with pore size of 300um were made by using 30% 3D printing. Chitosan was dissolved in acetic acid solution and mixed with glycerophosphoric acid solution at 8:2 to form CS/ 伪, 尾 -GP, and then mixed with sodium tripolyphosphate (TPP) solution to form chitosan nanoparticles (CSn). At 10:1 The experiment was divided into two groups: TICs / GP: CS/ 伪, 尾 -GP coated bionic titanium bone group: CSn coating bionic titanium bone group: blank hole plate group, as positive control. 1. SE M was used to observe the structure and morphology of 3D printed biomimetic titanium bone in each group. MC3T3-E1 cells were cultured in vitro and inoculated on different coating biomimetic titanium bone. The cell adhesion was observed by fluorescence technique and the proliferation of MC3T3-E1 cells was detected by CCK-8 method. Porphyromonas gingivalis was cultured in vitro. The adhesion of Porphyromonas gingivalis on biomimetic titanium bone with different bioactive coatings was detected by bacterial culture counting method. All the data were processed by SPSS18.0 software. Results: 1. The surface of the biomimetic titanium bone was of uniform size, the pore distribution was uniform, and the porous structure could promote the rapid blood adhesion, enhance the stability of blood clot and shorten the time of implant healing. Scanning electron microscope (SEM) showed that: 3D printed bionic titanium bone showed a uniform distribution of mesh-like structure, and the intermeshes of CSA / 伪, 尾 -GP and CSn were evenly distributed on the biomimetic titanium bone. Fluorescence microscope showed that the cell body synaptic extension of the Contr group was polygonal on the surface and pore of the specimens. The elongated cells of Ti CSn group and Ti CS/GP group adhered to the surface of titanium bone, and the cell body stretched round. The synaptic extension was polygonal, but the cell bodies in the Ti group were round, but the antennal extension was not obvious. After inoculation with MC3T3-E1 without obvious polygonal morphology, CCK-8 was detected in 10% ContrTi CSnTi CS-GPTi. there was significant statistical difference in ALP secretion between the two groups. The ALP secretion in the two groups was higher than that in the Ti CS/GP group, and the ratio of ALP secretion between the two groups was higher than that in the Ti group. The ALP secretion in Contr group was significantly lower than that in Ti CSn group and Ti CS/GP group, and there was significant statistical difference between Contr group and Ti CS/GP group. The results of bacterial adhesion test showed that the number of bacteria adhesion in Ti CSn group was significantly lower than that in Ti group (P0.05), and that in Ti CSn group was significantly lower than that in Ti CS/GP group (P0.05). Conclusion: (1) 3D printing of the biomimetic titanium bone is beneficial to the rapid entry of blood into the biomimetic titanium bone, promoting the blood adhesion on the surface, enhancing the stability of the blood clot, and promoting the vascularization of the bone. (2) 3D printing biomimetic titanium bone structure has good cell compatibility, the composite chitosan nanoparticles coating is more conducive to the early adhesion growth of osteoblasts. Proliferation and osteogenic differentiation; (3) chitosan coating could effectively inhibit the adhesion of Porphyromonas gingivalis; chitosan nanoparticles had better bacteriostatic effect than chitosan glycerophosphoric acid thermo-sensitive hydrogels.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1
[Abstract]:Objective: to fabricate bionic titanium bone with three-dimensional porous interconnecting structure by using 3D printing technology, and composite chitosan glycerophosphoric acid thermo-sensitive hydrogel coating and chitosan nanoparticles bioactive coating, respectively. The effects of biomimetic titanium bone and different coatings on the adhesion proliferation and differentiation of osteoblasts and the bacteriostatic effect on porphyromonas gingivalis were observed. Methods the biomimetic titanium bone specimens with pore size of 300um were made by using 30% 3D printing. Chitosan was dissolved in acetic acid solution and mixed with glycerophosphoric acid solution at 8:2 to form CS/ 伪, 尾 -GP, and then mixed with sodium tripolyphosphate (TPP) solution to form chitosan nanoparticles (CSn). At 10:1 The experiment was divided into two groups: TICs / GP: CS/ 伪, 尾 -GP coated bionic titanium bone group: CSn coating bionic titanium bone group: blank hole plate group, as positive control. 1. SE M was used to observe the structure and morphology of 3D printed biomimetic titanium bone in each group. MC3T3-E1 cells were cultured in vitro and inoculated on different coating biomimetic titanium bone. The cell adhesion was observed by fluorescence technique and the proliferation of MC3T3-E1 cells was detected by CCK-8 method. Porphyromonas gingivalis was cultured in vitro. The adhesion of Porphyromonas gingivalis on biomimetic titanium bone with different bioactive coatings was detected by bacterial culture counting method. All the data were processed by SPSS18.0 software. Results: 1. The surface of the biomimetic titanium bone was of uniform size, the pore distribution was uniform, and the porous structure could promote the rapid blood adhesion, enhance the stability of blood clot and shorten the time of implant healing. Scanning electron microscope (SEM) showed that: 3D printed bionic titanium bone showed a uniform distribution of mesh-like structure, and the intermeshes of CSA / 伪, 尾 -GP and CSn were evenly distributed on the biomimetic titanium bone. Fluorescence microscope showed that the cell body synaptic extension of the Contr group was polygonal on the surface and pore of the specimens. The elongated cells of Ti CSn group and Ti CS/GP group adhered to the surface of titanium bone, and the cell body stretched round. The synaptic extension was polygonal, but the cell bodies in the Ti group were round, but the antennal extension was not obvious. After inoculation with MC3T3-E1 without obvious polygonal morphology, CCK-8 was detected in 10% ContrTi CSnTi CS-GPTi. there was significant statistical difference in ALP secretion between the two groups. The ALP secretion in the two groups was higher than that in the Ti CS/GP group, and the ratio of ALP secretion between the two groups was higher than that in the Ti group. The ALP secretion in Contr group was significantly lower than that in Ti CSn group and Ti CS/GP group, and there was significant statistical difference between Contr group and Ti CS/GP group. The results of bacterial adhesion test showed that the number of bacteria adhesion in Ti CSn group was significantly lower than that in Ti group (P0.05), and that in Ti CSn group was significantly lower than that in Ti CS/GP group (P0.05). Conclusion: (1) 3D printing of the biomimetic titanium bone is beneficial to the rapid entry of blood into the biomimetic titanium bone, promoting the blood adhesion on the surface, enhancing the stability of the blood clot, and promoting the vascularization of the bone. (2) 3D printing biomimetic titanium bone structure has good cell compatibility, the composite chitosan nanoparticles coating is more conducive to the early adhesion growth of osteoblasts. Proliferation and osteogenic differentiation; (3) chitosan coating could effectively inhibit the adhesion of Porphyromonas gingivalis; chitosan nanoparticles had better bacteriostatic effect than chitosan glycerophosphoric acid thermo-sensitive hydrogels.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1
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