软骨细胞-海藻酸钠水凝胶-SIS复合体修复全层关节软骨缺损的实验研究

EXPERIMENTAL STUDY OF REPAIRING FULL-THICKNESS ARTICULAR CARTILAGE DEFECT WITH CHONDROCYTE-SODIUM ALGINATE HYDROGEL-SIS COMPLEX

目的研究利用海藻酸钠水凝胶和SIS复合作为支架材料,接种软骨细胞构建组织工程软骨修复兔全层关节软骨缺损的效果。方法采用机械法和去垢剂-酶消化法制备SIS。取8只2～3周龄新西兰白兔,肱骨头和股骨髁负重面全层关节软骨,体外培养软骨细胞。将常规扩增培养的第4代软骨细胞用海藻酸钠稀释,软骨细胞浓度为(5～7)×107个/mL,均匀涂刷于SIS表面,制备软骨细胞-海藻酸钠水凝胶-SIS复合体。取6月龄清洁级新西兰白兔40只,体重3.0～3.5kg,根据处理方法不同随机分为实验组和对照组(n=20),建立单侧膝关节4mm×4mm全层软骨缺损模型(左右不限)。实验组将复合体通过逐层叠加法置于缺损处,构建组织工程软骨,表面缝合覆盖一层SIS薄膜将缺损完全填充;对照组以单纯海藻酸钠水凝胶填充软骨缺损、覆盖SIS薄膜缝合。术后观察动物一般情况,于术后1、3、5、7、9个月,各组分别处死动物,行大体和组织学观察。结果由于麻醉死亡、切口感染和腹泻死亡,8只动物被排除实验,两组分别余16只动物进行取材观察。术后1、3、5、7、9个月,每组分别随机处死3、3、3、3、4只动物。大体观察和组织学Masson染色结果显示:实验组术后1个月,植入体表面的SIS与宿主软骨组织融合,边界消失;3个月,植入体部分软骨化,界面融合;5个月,植入体演变为纤维软骨;7个月植入体内软骨组织中纤维排列接近宿主软骨;9个月植入体内软骨纤维排列紊乱,细胞代谢增生活跃。对照组观察9个月内缺损无明显修复。组织学染色结果经图像量化分析显示,实验组各时间点缺损内修复组织单位面积染色强度均较对照组显著升高(P<0.05);术后3、5、7个月,实验组软骨化程度逐渐增大(P<0.05),9个月较7个月有所降低(P<0.05)。结论利用软骨细胞-海藻酸钠水凝胶-SIS复合体表层缝合SIS薄膜术构建的组织工程软骨原位修复兔软骨缺损,能促进软骨组织再生,符合关节软骨的生理性修复过程。

Objective 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. Methods 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） × 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. Results 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 inferface 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）. Conclusion 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 1 ine with physiological repair process of articular cartilage.