Mo Xiangtao, Deng Li, Li Xiuqun, Xie Huiqi, Luo Jingcong, Guo Shangchun, Yang Zhiming
Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu Sichuan 610041, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Aug;23(8):974-9.
To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects.
SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articular cartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3 weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multiplication method were diluted by sodium alginate to (5-7) x 10(7) cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n = 20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was established in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation.
Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the interface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in the implant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly and active cell metabolism and proliferation were evident 9 months after operation. While in the control group, no repair of the defect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9 months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P < 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P < 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P < 0.05).
Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in line with physiological repair process of articular cartilage.
探讨以海藻酸钠水凝胶与小肠黏膜下层（SIS）复合物为支架材料、软骨细胞为种子细胞构建的组织工程软骨对全层关节软骨缺损的修复作用。
采用定制机器及去污剂-酶处理法制备SIS。取8只2-3周龄新西兰白兔肱骨头及股骨髁负重面的全层关节软骨进行体外软骨细胞培养。将经传统传代培养法培养至第4代的兔软骨细胞用海藻酸钠稀释至（5-7）×10⁷个细胞/mL，然后接种于SIS上制备软骨细胞-海藻酸钠水凝胶-SIS复合物。将40只6月龄、体重3.0-3.5 kg的清洁级新西兰白兔按不同手术方法随机分为两组（每组20只），为每只兔建立单侧膝关节（右侧或左侧）全层软骨缺损模型。实验组将复合物逐层植入缺损处构建组织工程软骨，并在表面覆盖SIS膜以完全填充缺损；对照组用海藻酸钠水凝胶填充软骨缺损，覆盖SIS膜后缝合，不接种软骨细胞。观察术后兔的一般情况。两组兔分别于术后1、3、5、7、9个月处死，进行大体及组织学观察。
因麻醉死亡、伤口感染及腹泻死亡等原因排除8只兔。每组纳入实验16只兔，每组分别随机选取3、3、3、3、4只兔于术后1、3、5、7、9个月处死。大体观察及组织学Masson三色染色：实验组，植入物表面的SIS与宿主组织融合，术后1个月二者界面消失；术后3个月部分植入物软骨化，植入物与宿主组织界面融合；术后5个月植入物变为纤维软骨；术后7个月植入物内软骨纤维排列接近宿主组织；术后9个月植入物内软骨纤维排列紊乱，细胞代谢及增殖活跃。而对照组术后9个月缺损未见修复。术后9个月内对照组缺损无明显修复。组织形态计量学评价显示，实验组缺损处修复组织单位面积染色强度在各时间点均显著高于对照组（P<0.05），实验组术后3、5、7个月软骨化逐渐增加（P<0.05），术后9个月与术后7个月相比有所下降（P<0.05）。
软骨细胞-海藻酸钠水凝胶-SIS复合物联合SIS膜表面缝合构建的组织工程软骨可原位修复软骨缺损，促进软骨组织再生，符合关节软骨的生理修复过程。
