pvaι-ca软骨组织工程支架对atdc-5细胞生物学行为和组织相容性的影响

PVA/-CA 软骨组织工程支架对 ATDC-5细胞生物学行为和组织相容性的影响 车鹏程 车轩 李硕峰 张亚彬 熊艳杰 崔曼 崔菁 姚芳莲 孙红 华北理工大学基础医学院河北省慢性病重点实验室唐山市慢性病临床基础研究重点实验室 湖南师范大学医学院医学检验学系 天津大学化工学院高分子科学与工程系 华北理工大学附属医院病理科 摘 要： 目的:探讨 PVA/-CA 软骨组织工程支架对 ATDC-5 细胞生物学行为的影响, 评价其用于构建组织工程软骨的可行性。方法:采用物理共混技术和反复冷冻-解冻方法, 将聚乙烯醇 (PVA) 和卡拉胶按照一定比例制作成复合支架材料PVA/-CA 并测定其孔径和孔隙率。将 ATDC-5 细胞接种于支架材料, 观察细胞的黏附生长情况;免疫组织化学法和免疫荧光法检测 ATDC-5 细胞中型胶原的表达情况;甲苯胺蓝检测 ATDC-5 细胞形态表现;扫描电镜 (SEM) 下观察细胞生长及细胞外基质 (ECM) 分泌情况。将 ATDC-5 细胞分为阴性对照组 (加入空白培养液) 和实验组 (加入含材料的培养液) , MTT 法检测支架材料上 2 组 ATDC-5 细胞的增殖率。将支架材料植入 SD 大鼠皮下, 评价该支架材料的组织相容性和血管化能力。结果:PVA/-CA 软骨组织工程支架平均孔隙率为 (86.88±3.88) %, 平均孔径为 2040m。HE 染色, ATDC-5 细胞在支架材料上贴附生长良好, 呈多角形, 形态饱满;免疫组织化学和免疫荧光染色, ATDC-5 细胞分泌型胶原蛋白;甲苯胺蓝染色, ATDC-5 细胞在支架材料上保持软骨细胞的特性, 随着培养时间的延长, 支架材料上阳性细胞数量明显增加;ATDC-5 细胞与支架材料共培养 7d 时, SEM 下可见少量细胞呈多角形;培养 14d 时细胞数量增多, 形态饱满、相互融合, 伸入材料间形成锚状结构, 牢固地黏附于材料表面;培养 2128d 时材料上附着的细胞分泌大量的 ECM 包裹材料;ATDC-5 细胞在材料上于 714d 增殖较快, 21 28d 增殖较慢, 实验组增殖率与对照组比较差异无统计学意义 (P>0.05) 。皮下包埋早期有轻微的炎症反应, 随着时间延长而消退;后期有血管化发生, 材料降解吸收比较缓慢。结论:PVA/-CA 支架材料有望成为的软骨组织工程支架材料。关键词： 软骨组织工程; 聚乙烯醇; 卡拉胶; 生物相容性; 作者简介：车鹏程 (1970-) , 男, 河北省唐山市人, 医学博士, 主要从事组织工程和再生医学方面的研究。作者简介：孙红, 教授, 主任医师, 硕士研究生导师 (Tel:0315-8805157, E-mail:xsun1hotmail.com) 收稿日期：2017-01-04基金：国家自然科学基金资助课题 (81101448) Effect of cartilage tissue engineering scaffolds PVA/-CA on biological behavior and biocompatibility of ATDC-5 cellsCHE Pengcheng CHE Xuan LI Shuofeng ZHANG Yabin XIONG Yanjie CUI Man CUI Jing YAO Fanglian SUN Hong School of Basic Medical Sciences, North China University of Science and Technology, Key Laboratory for Chronic Diseases of Hebei Province, Key Laboratory for Preclinical and Basic Research on Chronic Diseases of Tangshan City; Department of Medical Laboratory Technology, School of Medical Sciences, Hunan Normal University; Department of Polymer Science and Engineering, School of Chemical Engineering and Technology, Tianjin University; Department of Pathology, Affiliated Hospital, North China University of Science and Technology; Abstract： Objective:To investigate the effect of cartilage tissue engineering scaffold PVA/-CA on the biological behavior of the ATDC-5 cells, and to evaluate its feasibility on constructing tissue engineering cartilage.Methods:The polyvinyl alcohol (PVA) and carrageenan were used to make the composite scaffold material PVA/-CA according to a certain proportion by physical blending technology and repeated freezing thawing method, and the porosity and pore size of PVA/-CA were detected.The ATDC-5 cells were seeded into the composite scaffold and its growth was observed;the expressions of collagen type in the ATDC-5 cells were tested by immunohistochemical staining and immunofluorescence staining;the morphology of the ATDC-5 cells was confirmed by Toluidine blue staining.The growth and secretion of extracellular matrix of the ATDC-5 cells were observed under scanning electron microscope (SEM) ;the proliferative rates of ATDC-5 cells in composite scaffold materials in negative control group (added with DMEM culture media) and experimental group (added with DMEM contain scaffold) were determined by MTT assay.The composite scaffolds were implanted subcutaneously in the SD rats.The histocompatibility and vascularization in vivo of the composite scaffolds were evaluated.Results:The average porosity of cartilage tissue engineering scaffold PVA/-CA was (86.88±3.88) %, and the average pore size was 20-40m.The HE staining results showed that the ATDC-5 cells grew well with the polygon and plumpness morphology.All the samples were stained positive for collagen type by immunohistochemistry and immunofluorescence staining, which verified the normal phenotype of chondrocytes on the scaffolds.All the sample were stained positive for toluidine blue staining, which verified ECM deposition of the ATDC-5 cells on the scaffolds.The number of the positive cells was significantly increased with the prolongation of time.After cultured for 7 d, few of the ATDC-5 cells presented polygonal;after cultured for 14 d, the ATDC-5 cells distributed more densely, and contacted with each other on the scaffold;after cultured for 21-28 d, the ATDC-5 cells filled the interconnected pores of the scaffolds, synthesizing a significant amount of neo-formed ECM.The proliferation of ATDC-5 cells in PVA/-CA grew fast during 7-14 d, and it became slow during 21-28 d;the difference was not statistically significant compared with control group (P>0.05) .The subcutaneous implantation results showed the inflammatory reactions were slight at the early stage and eviated gradually, there was an increasing angiogenesis at the late stage, and the degradation and absorption of the meterial were slight.Conclusion:PVA/-CA composite material will be an ideal material for the cartilage tissue engineering.Keyword： cartilage tissue engineering; polyvinyl alcohol; carrageenan; biocompatibility; Received： 2017-01-04成熟的关节软骨缺少血供, 软骨细胞在无血运环境中不能迁徙, 关节软骨损伤后缺乏自愈能力, 目前临床上的治疗方法均有其局限性1。组织工程是一门新兴的生物技术, 为关节软骨修复提供了新的思路2。在各种关节软骨