Zhou Libin, Ding Ruiying, Li Baowei, Han Haolun, Wang Hongnan, Wang Gang, Xu Bingxin, Zhai Suoqiang, Wu Wei
Department of Otolaryngology-Head and Neck Surgery, 306 Hospital of PLA Beijing 100101, P.R China.
Institute of Otolaryngology, Chinese PLA General Hospital Beijing 100853, P.R China.
Int J Clin Exp Pathol. 2015 Jan 1;8(1):73-80. eCollection 2015.
The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.
支架材料的缺陷阻碍了软骨组织工程的临床应用。最近开发的细胞片技术被用于构建无支架材料的组织，因此被认为有可能克服与支架缺陷相关的问题。本研究构建了单层和双层软骨细胞片，并使用细胞刮匙而非温度响应培养皿收获细胞片。通过体外和体内研究进一步探讨了培养的软骨细胞片的特性以及使用软骨细胞片进行软骨工程的可行性。在体外培养期间，在细胞片中检测到初级细胞外基质（ECM）形成和II型胶原蛋白表达。植入裸鼠8周后，收获成熟的软骨盘。源自单层和双层软骨细胞片的构建体中，新形成软骨的形态相似。软骨细胞位于均匀分布的卵圆形陷窝内。在新形成的软骨中，在均匀分布的软骨细胞周围观察到强大的ECM形成和强烈的II型胶原蛋白表达。生化分析表明，新形成软骨的DNA含量高于天然人肋软骨；而ECM主要成分糖胺聚糖和羟脯氨酸的含量与天然人肋软骨相似。总之，使用简单且低成本技术构建的软骨细胞片与在温度响应培养皿中培养和收获的细胞片基本相同，可用于软骨组织工程。
