壳聚糖修饰钛骨种植体表面对雪旺细胞的影响

发布时间：2018-07-23 09:01

【摘要】：目的:在新型种植体“钛骨”表面固定壳聚糖涂层,并通过体外细胞实验检测此涂层对雪旺细胞早期粘附、增殖、分泌的影响。方法:通过交联剂将壳聚糖固定在“钛骨”表面,以制成“钛骨”表面的壳聚糖涂层。将实验分为3组:以单纯细胞组作为空白对照组(Blank),以普通钛骨组(Tixos)作为阴性对照组,以壳聚糖涂层组(Tixos-CS)作为实验组。通过扫描电镜(SEM)观察实验组与阴性对照组材料的表面结构;利用悬滴法检测两组材料的接触角以确定其亲水性;通过拉力测试仪检测壳聚糖涂层与钛骨表面的结合强度;通过体外浸泡实验组材料,每日称重,以测量其降解所需的时间。将材料与雪旺细胞共培养,在12小时时通过DAPI染色的方法定性检测细胞在材料表面粘附的量;在24小时时通过扫描电镜观察粘附与材料表面的细胞的形态;于第1天、3天和5天通过CCK-8检测材料对雪旺细胞增殖的影响;细胞与材料共培养后每日进行细胞计数,绘制细胞生长曲线,直观的观察细胞生长状态;在1天、2天和3天通过ELISA检测雪旺细胞分泌NGF和GDNF的情况,并进行统计学分析。结果1.通过扫描电镜观察可见壳聚糖颗粒大小约为2-5μm左右,呈长方形或多边形,颗粒之间排列紧密,紧紧附着于钛骨表面;2.壳聚糖涂层组(Tixos-CS)材料表面接触角小于90°,表现为亲水性;3.通过拉力测试检测,壳聚糖涂层能够稳定的粘附在钛骨的表面;4.降解实验反映了涂层能够匀速缓慢的降解,大概在第8天完成降解;5.人雪旺细胞与两组材料共培养12小时DAPI染色结果显示壳聚糖涂层组(Tixos-CS)细胞粘附数量多;6.共培养24小时后通过扫描电镜观察细胞形态可见壳聚糖涂层组(Tixos-CS)材料表面的细胞伸出的伪足更多更长,细胞伸展更充分;7.CCK-8检测结果,在第1天、3天和5天壳聚糖涂层组(Tixos-CS)吸光度值均高于钛骨组(Tixos);8.绘制生长曲线可见钛骨组(Tixos)细胞传代后1-2天为生长缓滞期,3-6天为快速生长期,6天以后进入平顶期;壳聚糖涂层组(Tixos-CS)第1天为生长缓滞期,2-5天为快速生长期,5天以后进入平顶期;9.流式细胞仪分析细胞周期,在第1天、3天,壳聚糖涂层组(Tixos-CS)中处于S+G2/M期的雪旺细胞百分比较钛骨组(Tixos)增加;10.ELISA检测结果显示,第1天、2天和3天,壳聚糖涂层组(Tixos-CS)分泌的NGF和GDNF均高于钛骨组(Tixos)。结论:1.在钛骨表面固定的壳聚糖涂层具有一定的空间立体结构,涂层具有一定的机械强度,具有一定的稳定性,可缓慢降解。2.壳聚糖涂层可以促进雪旺细胞的粘附和增殖,并且能够促进雪旺细胞分泌NGF和GDNF
[Abstract]:Aim: to fix the chitosan coating on the surface of titanium implant, and to investigate the effect of chitosan coating on the early adhesion, proliferation and secretion of Schwann cells by cell experiments in vitro. Methods: chitosan was immobilized on the surface of titanium bone by crosslinking agent to make chitosan coating on the surface of titanium bone. The experiment was divided into three groups: pure cell group as blank control group, ordinary titanium bone group as negative control group and chitosan coated group as experimental group. The surface structure of the experimental group and the negative control group was observed by scanning electron microscope (SEM), the contact angle of the two groups was determined by hanging drop method, the binding strength of the chitosan coating and the titanium bone surface was measured by tensile force tester. The materials of experimental group were soaked in vitro and weighed daily to measure the time required for degradation. After co-culture with Schwann cells, the quantity of cell adhesion on the surface of the material was detected qualitatively by DAPI staining at 12 hours, and the morphology of the cells on the surface of the material was observed by scanning electron microscope at 24 hours. The effect of CCK-8 on the proliferation of Schwann cells was detected on the 1st day and 5th day, the cell count was carried out daily after co-culture with the material, the cell growth curve was drawn, and the growth state of the cells was observed intuitively. The secretion of NGF and GDNF in Schwann cells was detected by Elisa on day 1, day 2 and day 3, and analyzed statistically. Result 1. Scanning electron microscopy showed that the size of chitosan particles was about 2-5 渭 m, which was rectangular or polygon, closely arranged between particles, and tightly attached to the surface of titanium bone. The surface contact angle of chitosan coated group (Tixos-CS) is less than 90 掳, which is hydrophilic. The results of tensile test showed that the chitosan coating could adhere to the surface of titanium bone steadily. The degradation experiment showed that the coating could degrade at a uniform rate and slowly, and the degradation was completed on the 8th day. Human Schwann cells co-cultured with the two groups for 12 hours DAPI staining results showed that chitosan coated group (Tixos-CS) cell adhesion number of more than 6. After 24 hours of co-culture, the morphology of cells on the surface of chitosan coated group (Tixos-CS) was observed by scanning electron microscope. The absorbance values of the chitosan coated group (Tixos-CS) were higher than that of the titanium bone group (Tixos) on the 1st and 5th day. The growth curve showed that the growth retardation of Tixos cells was from 3 to 6 days after passage, and that of the chitosan coated group (Tixos-CS) from the first day to the first day was the slow growth period (2-5 days), the second day was the rapid growth period (5 days), and the latter was 9 days after the flattened stage. The growth curve showed that the growth retardation period of Tixos group was 3 to 6 days, and that of the chitosan coated group (Tixos-CS) was 2 to 5 days after rapid growth. The percentage of Schwann cells in the S G 2 / M phase in the chitosan coated group (Tixos-CS) was significantly higher than that in the titanium bone group (Tixos). The results of Elisa showed that the percentage of Schwann cells in the chitosan coated group (Tixos-CS) was significantly higher than that in the Tixos group. Both NGF and GDNF secreted by chitosan coated group (Tixos-CS) were higher than those of titanium bone group (Tixos). Conclusion 1. The chitosan coating fixed on the surface of titanium bone has a certain spatial stereoscopic structure, the coating has a certain mechanical strength, a certain stability, and can be degraded slowly. Chitosan coating can promote the adhesion and proliferation of Schwann cells, and promote the secretion of NGF and GDNF by Schwann cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.6

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