【作者】 李俊辉；

【导师】 徐林；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 背景：目前脊柱外科临床上颈椎病的发病率呈上升趋势,且年轻患者所占比重逐渐增大,对于保守治疗效果不理想的患者需要进行手术治疗,传统的颈椎前路减压植骨融合术存在邻近关节退变加速、取骨处疼痛等并发症,而人工颈椎间盘置换术克服了上述不足,且保留了颈椎的生理活动度,近十年来得到了脊柱外科医师和研究者的认可并得到了较快的发展。我国目前脊柱外科临床上使用的人工颈椎间盘全部被欧美产品所垄断,欧美产品价格昂贵,存在一些不足。在这种形势下,清华大学深圳研究院联合我院及多家院校进行合作,致力于研发符合中国人颈椎生理特征的新型人工颈椎间盘,其上下终板材料为钛合金,钛合金具有良好的生物相容性,但钛合金存在生物活性差、与骨结合强度低、在生理环境下易造成金属离子释放等问题。目的：设计研发出符合中国人生理特征和尺寸的新型人工颈椎间盘,并对间盘上下终板进行不同涂层喷涂改性,观察3种不同涂层的体外细胞相容性和血液相容性,评价新型人工颈椎间盘3种不同涂层的体外生物相容性,为后期的实验研究及临床应用提供实验依据。方法：1、对新型人工颈椎间盘3种不同涂层进行场发射扫描电镜观察及X射线能谱仪检测；2、从Wistar大鼠股骨、胫骨骨髓分离BMSCs,贴壁培养法对其进行传代和纯化,按照1：3比例进行传代,以后4～5天传代1次,对第3代大鼠BMSCs进行鉴定,用符合鉴定标准的大鼠BMSCs进行国产人工颈椎间盘间盘不同涂层体外细胞相容性实验；3、将大鼠BMSCs与3组不同涂层样品共同培养,观察大鼠BMSCs在不同样品表面的黏附分化能力,检测ALP分泌活性及样品的细胞毒性,以评价国产人工颈椎间盘不同涂层体外细胞相容性；4、从大鼠腹主动脉采集3组新鲜血液：第1组、第2组新鲜血与清洁干燥的样品表面接触,分别加入一定量的蒸馏水、生理盐水孵育一定时间,然后收集溶液,利用分光光度计检测溶液吸光度值来评价样品表面动态凝血时间及溶血率；第3组新鲜血离心后收集富含血小板血浆,样品在富含血小板血浆中浸泡一定时间后洗净未黏附的血小板,经固定、脱水、置换及喷金处理后在扫描电镜下观察血小板黏附情况。以上测得的数据用以x±s表示,采用SPSS18.0软件对实验数据进行统计和分析,P<0.05为差异有统计学意义。结果：1、扫描电镜显示3组样品呈蜂窝状多孔表面,但HA、Ti+HA涂层的空隙更加均匀；X射线能谱仪分析显示HA、Ti+HA涂层主要由Ti、Ca、P、Si、O、C元素组成,而Ti涂层主要由Ti元素组成。2、大鼠BMSCs经全骨髓分离后,利用贴壁培养法培养,细胞生长状态良好,原代培养8天后铺满瓶底80%以上,传至第3代时细胞多呈梭形生长,细胞形态均一、生长旺盛,流式细胞仪对第3代大鼠BMSCs进行鉴定,结果显示第3代BMSCs表面抗体CD44、CD106阳性表达率分别为95.0%和81.1%,而造血类细胞表面标志CD45呈阴性,表达率为5.0%,符合作为实验细胞的基本标准。3、细胞体外相容性检测结果：3.1大鼠BMSCs与3组样品分别共同培养24、48h后,扫描电镜下可见材料与细胞复合培养24h后表面即有部分细胞黏附,Ti涂层表面的BMSCs呈圆形,而HA和Ti+HA涂层表面的细胞呈梭形或多角形；48h时可见HA和Ti+HA涂层表面的细胞开始铺展,尤其是在Ti+HA涂层表面,细胞呈多角形,并伸出细长的伪足伸入到材料的微孔内,与材料表面紧密粘附。3.2BMSCs与3组样品分别复合培养2-7d后,细胞分泌的ALP量呈上升趋势,3个观察时间点之间比较有统计学意义(P<0.05)；3种材料对BMSCs分泌ALP均有促进作用,诱导剂组、Ti涂层组组间比较无统计学意义(P>0.05); HA、Ti+HA涂层组组间比较无统计学意义(P>0.05); HA、Ti+HA涂层组与诱导剂组、Ti组两两比较,有统计学意义((P<0.05)。3.3BMSCs与3组样品分别复合培养2-7d后,50%、100%浸提液组吸光度值在各时间段与对照组两两比较,Ti涂层组与对照组比较,在第2d、第4d及第7d时P>0.05,无统计学差异；材料表面细胞相对增殖率在80～96%之间,材料毒性级别为1级；HA涂层组与对照组比较,在第2d时P>0.05,无统计学差异；第4d、7d时P<0.05,有统计学差异；材料表面细胞相对增殖率在89～114%之间,材料毒性级别为0～1级；Ti+HA涂层组与对照组比较,在第2d时P>0.05,无统计学差异；第4d、7d时P<0.05,有统计学差异；材料表面细胞相对增殖率在91～117%之间,材料毒性级别为0～1级。4、血液体外相容性检测结果：4.1动态凝血时间检测,由各个时间点检测的吸光度值结果绘制的折线图可见,HA、Ti+HA涂层组抗凝血效果优于Ti涂层组,在前30min时间段内优势比较明显,30min后时间段内差异稍缩小,但HA、Ti+HA涂层组抗凝血效果仍优于Ti涂层组。4.2溶血率检测,由各样品吸光度值结果绘制的柱状图可见,HA、Ti+HA涂层组溶血率小于Ti涂层组,且溶血率<1%,符合医学标准要求的植入物溶血率<5%的要求。4.3血小板黏附率检测,大鼠抗凝血离心后收集富含血小板血浆,与3组样品共同孵育1h后,经过处理后,扫描电镜下可见Ti涂层表面有一定量的血小板,且有伪足伸出；而HA、Ti+HA涂层组表面血小板数量很少,呈圆盘状,看不到明显的伪足伸出；HA、Ti+HA涂层有较好的血液相容性。结论：新型人工颈椎间盘终板表面喷涂的HA、Ti+HA涂层具有蜂窝状多孔表面,空隙均匀；大鼠BMSCs是良好的骨组织工程细胞,具有取材方便、易于扩增等优点；HA、Ti+HA涂层较Ti涂层具有良好的体外细胞相容性和血液相容性,本实验为假体涂层的初步筛选和改进提供了部分实验资料；后期还要制备其它涂层并进行筛选,以达到获得符合临床应用的最佳涂层的目的。更多还原

【Abstract】 Background:Recently,the incidence rate of cervical spondylosis is increasing year by year in the spine surgery,and the proportion of young patients are increasing.The conservative treatment is not ideal treatment for patients who require surgical treatment.The complications that the traditional anterior cervical decompression and fusion therapy bring are close joint accelerated degeneration and the donor site pain, which can be overcome by the artificial cervical disc replacement surgery and retain the physiological activity of the cervical spine. In the past decade, it has been accepted by the spine surgeons and researchers and got a rapid development. China’s current uses of cervical artificial disc in spinal surgery are all monopolized by European and American products, which are more expensive and there are some other shotage. In this situation, Shenzhen Research Institute of Tsinghua University associated a number of institutions for cooperation, and committed to invent the new artificial cervical disc in accordance with physiological characteristics of the Chinese people. The material of upper and lower endplate of the disc is titanium, which has good biocompatibility, but also weakness of poor biological activity, low strength of combination with bone and easily release of metal ions under physiological conditions.Objects:To design and development a kind of new type artificial cervical disc to meet Chinese people’s physical characteristics and dimensions, we used different modified coatings of disc endplate, and observed the cell compatibility and hemocompatibility of the three kinds of coatings in vitro, and evaluated the biocompatibility of the three kinds of different coatings in vitro of new type artificial cervical disc, and then try to provide experimental evidences for the latter part of the experimental study and clinical application.Methods:1, The three different coatings of new type artificial cervical disc are observed by field emission scanning electron microscopy and detected by X-ray spectrometer;2, Isolating BMSCs from the femur, tibia bone marrow of Wistar rat. BMSCs were passaged and purificated by adherent culture method, and passaged by the proportion of1:3. We can got another generation after four to five days, and the third-generation rat BMSCs were identified. The new type of artificial cervical disc compatibility of different coatings were tested in vitro experiments;3,The rat BMSCs were co-cultured with3different coated samples, observing adhesion and differentiation ability of rat BMSCs on different samples surface, and detecting cytotoxicity and ALP secretion to evaluate the cytocompatibility of different coatings in vitro;4,Collecting three groups of fresh blood from the rat abdominal aortic:the first and second group of fresh blood were touched with the clean, dry surface of different samples, and then added a certain amount of distilled water or physiological saline respectively, and incubated for a certain of period. The solutions were collected to evaluate the dynamic coagulation time and hemolysis of the different samples using a spectrophotometer; the third group of fresh blood was centrifugaled and collected platelet-rich plasma. Different samples were immersed in platelet-rich plasma for certain of period, and then the non-adherent platelets were washed off. After fixation, dehydration, replacement and sprayed gold processions, platelet adhesion proportion was observed by scanning electron microscopy. The measured data were expressed by x±s,using SPSS18.0software for statistical analysis of the experimental data, P<0.05was considered statistically significant.Results:1The result of scanning electron microscopy showed that three kinds of samples have honeycomb porous surface, but there are more uniform gaps on HA and Ti+HA coatings; X-ray spectrometer analyzed that HA, Ti+HA coatings are mainly composed of Ti, Ca, P, Si, O, Celements, and the Ti coating is mainly composed of Ti element.2BMSCs were cultured after separation from rat bone marrow by adherent method, the cells grew well. More than80%of the bottom was covered by BMSCs eight days after primary culture. The third generation appeared fusiform growth, forming a uniform, vigorous growth. The third generation rat BMSCs were identified by flow cytometry and the surface antibody CD44, CD106positive expression rate were95.0%and81.1%respectively. While the hematopoietic cell surface marker CD45negative expression rate was5.0%, in line with the basic criteria as the experimental cells.3Cell compatibility test results in vitro:3.1Rat BMSCs and3different kinds of samples were co-cultured for24,48h respectively. After24h, the result of scanning electron microscope showed that few BMSCs adhesion on bare titanium and the cells were round, while the cells on HA and Ti+HA coating surfaces were fusiform or polygonal; after48h, cells on HA and Ti+HA coating surfaces began to spread, especially polygonal cells on the Ti+HA coating surface, and the outstretched slender pseudopodia extended into the material within the pores, and tight adhesion on surface of the material.3.2Rat BMSCs and3different kinds of samples were co-cultured for2-7d. The amount of ALP secretion was upward trend and there was statistically significant between the three time points in the same group(P<0.05);3kinds of materials have the characteristic of promoting ALP secretion, and there was no statistically significant between the inducer group and Ti coating group(P>0.05); it was the same between HA and Ti+HA coating groups(P>0.05); HA,Ti+HA coating groups pair-wise compared with inducer, Ti coating groups, and there was statistically significant(P<0.05).3.3Rat BMSCs and3different kinds of samples were co-cultured for2-7d.50%,100%soak extract group absorbance values at each time point pair-wise compared with the control group, and there was no significant difference between Ti coating group and the control group in2d and7d, P>0.05; there was significant difference between the100%soak extract group and the control group in4d. P<0.05; the relatively cell proliferation rate on the material surface was80-96%, and material toxicity was level1; HA coating group compared with control group, and there was no significant difference in the first2d, P>0.05; in the4d and7d, there was significant difference between them, P<0.05; the relatively cell proliferation rate on the material surface was89-106%, and material toxicity was level0-1; Ti+HA coating group compared with the control group, and there was no significant difference in the first2d, P>0.05; in the4d and7d, there was significant difference between them, P<0.05; the relatively cell proliferation rate on the material surface was91-109%, and material toxicity was level0-1.4Hemocompatibility test results in vitro:4.1Dynamic coagulation time test, from the line chart drawn from the absorbance values at each time point, we can see that HA, Ti+HA coating groups anticoagulant effect are better than Ti coating group. In the first period of30min, the advantages are more obvious. After30min, the differences between them is slightly narrowing, but the HA, Ti+HA coating groups anticoagulant effects are still better than the Ti coating group.4.2Hemolysis rate detection, from the histogram drawn from the absorbance values at each time point, we can see that the hemolysis rate of HA, Ti+HA coating groups are less than Ti coating group, and the hemolysis rates of them are<1%, in line with the requirement of medical implant hemolysis rate<5%.4.3Platelet adhesion rate detection, platelet-rich plasma was collected from rat anti-coagulation blood after centrifugation.3kinds of samples were co-incubated with platelet-rich plasma for1h. After treatment, the result of scanning electron microscope showed that Ti coating surface had a certain amount of platelets, and the pseudopodia of the platelet outstreched; while on the HA, Ti+HA coating surface the platelet amount was seldom, disc-shaped, and the pseudopodia of the platelet cannot be seen nearly; HA, Ti+HA coatings have good hemocompatibility.Conclusion:The new type artificial cervical disc endplate surface are coated with HA, Ti+HA, which have honeycomb porous surface, and the gaps are uniform; Rat BMSCs are good bone tissue engineering cells, with the advantages of low-cost, easily obtained, and easy amplification, etc; the HA,Ti+HA coatings have good cell compatibility and hemocompatibility in vitro than the Ti coating, this experiment provided some data for the prosthesis coating initial selection and improvement; in the later period, other coatings will also be prepared and selected in order to getting optimum coating for clinical application.更多还原