TU Pengcheng 1,2 , MA Yong 1,2,3 , PAN Yalan 2,4 , WANG Zhifang 5 , SUN Jie 1,2 , CHEN Kai 2 , YANG Guanglu 1,2 , WANG Lining 2,3 , LIU Mengmin 2,3 , GUO Yang 1,2
  • 1. Department of Orthopedics, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing Jiangsu, 210029, P.R.China;
  • 2. Laboratory of New Techniques of Restoration and Reconstruction of Orthopedics and Traumatology, Nanjing University of Chinese Medicine, Nanjing Jiangsu, 210023, P.R.China;
  • 3. School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing Jiangsu, 210023, P.R.China;
  • 4. Nursing Institute of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing Jiangsu, 210023, P.R.China;
  • 5. Department of Orthopedics, Zhangjiagang Affiliated Hospital of Nanjing University of Chinese Medicine, Suzhou Jiangsu, 215699, P.R.China;
GUO Yang, Email: drguoyang@njucm.edu.cn
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Objective  To design and prepare the silk fibroin microcarrier loaded with total saponins from clematis chinensis osbeck (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 microscopy and the release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from keen 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 microscopy, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=10). 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 cartilage defects were collected for gross observation and histological observation (HE and toluidine blue staining). Western blot was used to detect the expression of collagen type Ⅱ and proteoglycan. 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 can 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, 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) criteria score and the relative expression levels of collagen type Ⅱand proteoglycan in group B and C were significantly better than those in group A, and group C was significantly better than group 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 reactor.