Objective: To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair.
Methods: CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups ( n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining.
Results: The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes ( P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers ( P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B ( P<0.05), and the level of TIMP-1 in group C was significantly higher ( P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B ( P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B ( P<0.05).
Conclusion: CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
目的: 制备负载威灵仙总皂苷（clematis total saponins，CTS）丝素蛋白微载体，探讨其复合软骨细胞后促进兔膝关节软骨缺损修复的效果。.
方法: 取5%丝素蛋白溶液与10 mg/mL CTS溶液、甘油混匀后，利用高压静电场结合冷冻干燥方法制备负载CTS丝素蛋白微载体，扫描电镜对样本表征并检测CTS累积释放量；同时制备丝素蛋白微载体。取6只4周龄新西兰大白兔膝关节软骨，分离培养软骨细胞并传代。取第3代细胞分别与两种微载体在微重力条件下共培养7 d，期间倒置相差显微镜及扫描电镜观察软骨细胞在微载体上黏附情况，细胞计数试剂盒8（cell counting kit 8，CCK-8）检测细胞增殖活性，并与正常培养细胞比较。取30只3月龄新西兰大白兔制备双侧膝关节软骨缺损模型后随机分为3组（ n=20），A组膝关节软骨缺损不作任何处理，B、C组分别采用丝素蛋白微载体-软骨细胞复合物、负载CTS丝素蛋白微载体-软骨细胞复合物填充关节软骨缺损。术后12周取材，ELISA检测关节液基质金属蛋白酶9（matrix metalloproteinase 9，MMP-9）、MMP-13、MMP组织抑制因子1（tissue inhibitor of MMP 1，TIMP-1）水平；大体观察及组织学观察（HE、甲苯胺蓝染色）评估软骨缺损修复情况；Western blot检测Ⅱ型胶原及蛋白聚糖表达；组织学及诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）免疫组织化学染色观察关节滑膜炎症程度。.
结果: 负载CTS丝素蛋白微载体呈类球形，直径主要在300～500 μm 之间，表面呈多孔结构，孔隙率达35.63%±3.51%；药物缓释检测示微载体中CTS可长期缓慢释放。微重力条件下随培养时间延长，两种微载体表面黏附的软骨细胞逐渐增多，培养24 h时软骨细胞增殖活性均较正常培养细胞提高（ P<0.05），两种微载体间差异无统计学意义（ P>0.05）。动物体内实验观察，与A、B组相比，C组关节液中MMP-9、MMP-13含量均降低（ P<0.05），TIMP-1含量上调（ P<0.05）。与A组相比，B、C组软骨缺损均有组织填充，且C组修复表面更完整，与周围软骨结合较好；组织学观察及Western blot检测显示B、C组国际软骨修复评分准则（ICRS）评分以及Ⅱ型胶原、蛋白聚糖相对表达量均优于A组，C组优于B组，差异均有统计学意义（ P<0.05）。关节滑膜组织学观察显示，与A、B组相比，C组滑膜炎症细胞浸润及小血管增生减少，免疫组织化学染色示C组iNOS表达水平降低（ P<0.05）。.
结论: 负载CTS丝素蛋白微载体具有良好的CTS缓释效果及生物相容性，在微重力条件下能促进共培养的软骨细胞增殖，植入体内后能促进兔膝关节软骨缺损修复。.
Keywords: Cartilage tissue engineering; cartilage repair; cell culture; clematis total saponins; microgravity; rabbit; silk fibroin protein.