课题背景：关节软骨缺损的修复是南京军区骨科研究所全军“十一五”资助项目，目前进行了系列研究，主要围绕支架材料复合不同细胞修复软骨缺损。取得了一定成就，但材料对机体的负面效应限制其在临床的应用，在此情况下，本实验进行了无支架条件下构建组织工程软骨研究。

应用要点: 实验初步在琼脂糖表面构建了组织工程软骨，是无支架条件下构建组织工程软骨的新方法。应用该方法构建的组织工程软骨与三维支架构建组织工程软骨相比，有其自身的优势和研究价值。

相关链接：目前常用的细胞-材料复合物构建组织工程软骨还存在以下问题：①接种细胞时的温度、pH及剪切应力等环境影响。②复合物生长阶段，细胞的形态和表型改变。③凝胶类材料对机械刺激的应力遮蔽效应。④细胞材料复合物在体内的降解产物安全性等。

摘要
背景：实验表明，软骨细胞在体外培养时，经常发生表型的丢失。但形成软骨细胞团块时可以维持软骨细胞表型，由此人们探讨无支架结构培养组织工程软骨的技术。
目的：拟建立软骨细胞聚集培养体系，观察在琼脂糖表面培养软骨细胞，无支架条件下构建组织工程软骨的特点。
设计、时间及地点：单一样本观察，于2006-10/2007-04在解放军南京军区总医院骨科研究所完成。
材料：SPF级新西兰白兔4只，2周龄，雌雄不限。
方法：分离、提取兔关节软骨原代软骨细胞，将低熔点琼脂糖铺于24孔培养板表面，按每孔2.5×105接种提取的兔原代软骨细胞，在培养箱内培养，定期换液。
主要观察指标：取10 d标本行组织学、Ⅱ型胶原免疫组织化学检测。
结果：10 d时，软骨细胞自聚集形成有一定形状、大小的类软骨组织，苏木精-伊红染色可见软骨陷窝结构，甲苯胺蓝染色见紫红色异染，说明有蛋白聚糖的分泌。钙染色未见钙化，Ⅱ型胶原免疫组织化学染色有棕黄色颗粒，说明有Ⅱ型胶原分泌。
结论：在琼脂糖表面构建组织工程软骨的培养体系，琼脂糖膜阻止软骨细胞的黏附和伸展，从而保持软骨细胞的圆形形态；软骨细胞在琼脂糖表面直接接触，加强了细胞之间的信息交流，有利于维持细胞的表型，维持软骨细胞分泌细胞外基质的特征。
关键词：无支架；组织工程软骨；琼脂糖；免疫组织化学；组织构建

王金良，赵建宁，郭亭，岳鹏举，何劼，何志伟.在琼脂糖表面无支架条件下构建组织工程软骨[J].中国组织工程研究与临床康复，2008，12(19):3625-3628 [www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3625(ps).pdf]

Constructing tissue engineering cartilage on the agarose surface without scaffolds

Abstract
BACKGROUND: Studies demonstrate that, the phenotype loss frequently occurs during chondrocytes are cultured in vitro. However, chondrocyte aggregation can maintain the phenotype of chondrocytes, thus exploring the techniques of culturing tissue engineering cartilages without scaffold.
OBJECTIVE: To establish the chondrocyte aggregation culture system, to observe the culture of chondrocytes on the agarose surface, and to explore a new method to construct tissue engineering cartilage without scaffold.
DESIGN, TIME AND SETTING: A single sample observation was carried out in the Orthopaedics Institute, Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA between October 2006 and April 2007.
MATERIALS: Four New Zealand rabbits were adopted, SPF grade, two months old, irrespective of genders.
METHODS: Primary chondrocytes were isolated from rabbit articular cartilages, and then the low melting-point agarose spread the surface of 24-well culture plate, and 2.5×105 chondrocytes were introduced to each well. Chondrocytes were cultivated in incubator, and medium was changed regularly.
MAIN OUTCOME MEASURES: The examples were detected with histology and collagen type Ⅱ immunohistochemistry at day 10.
RESULTS: Chondrocytes formed cartilage-like tissues with proper shape and size at day 10. Hematoxylin-eosin staining appeared cartilage lacuna, and toluidine blue staining showed metachromatic prunosus reaction, which indicated the secretion of proteoglycans. Calcium staining presented no calcification, and collagen type Ⅱ immunohistochemistry was stained as yellow, which indicated the secretion of collagen type Ⅱ.
CONCLUSION: During the construction of tissue engineering cartilage on the agarose surface without scaffold, the agarose membrane may prevent the attachment and extension of chondrocytes, thus sustaining the round shape of chondrocytes; the direct contact of chondrocytes on the agarose surface leads to enhance the communication between cells, and benefit for the phenotype stabilization; the chondrocytes also keep their secretion function of extracellular matrix.

Wang JL, Zhao JN, Guo T, Yue PJ, He J, He ZW.Constructing tissue engineering cartilage on the agarose surface without scaffolds.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(19):3625-3628(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3625(ps).pdf]