Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

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;

Corresponding author：

GUO Yang, Email: drguoyang@njucm.edu.cn

Keywords：

Cartilage tissue engineering; cartilage repair; silk fibroin protein; clematis total saponins; microgravity; cell culture; rabbit

DOI：

10.7507/1002-1892.202107061

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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.

Citation： TU Pengcheng, MA Yong, PAN Yalan, WANG Zhifang, SUN Jie, CHEN Kai, YANG Guanglu, WANG Lining, LIU Mengmin, GUO Yang. Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(3): 343-351. doi: 10.7507/1002-1892.202107061 Copy

1.	Kwon H, Brown WE, Lee CA, et al. Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nat Rev Rheumatol, 2019, 15(9): 550-570.
2.	Rong Y, Zhang J, Jiang D, et al. Hypoxic pretreatment of small extracellular vesicles mediates cartilage repair in osteoarthritis by delivering miR-216a-5p. Acta Biomater, 2021, 122: 325-342.
3.	Chen W, Xu Y, Li H, et al. Tanshinone ⅡA delivery silk fibroin scaffolds significantly enhance articular cartilage defect repairing via promoting cartilage regeneration. ACS Appl Mater Interfaces, 2020, 12(19): 21470-21480.
4.	Castro-Viñuelas R, Sanjurjo-Rodríguez C, Piñeiro-Ramil M, et al. Induced pluripotent stem cells for cartilage repair: current status and future perspectives. Eur Cell Mater, 2018, 36: 96-109.
5.	Hong H, Seo YB, Kim DY, et al. Digital light processing 3D printed silk fibroin hydrogel for cartilage tissue engineering. Biomaterials, 2020, 232: 119679. doi: 10.1016/j.biomaterials.2019.119679.
6.	邓凯烽, 朱英, 廖子龙, 等. 基于复杂网络技术分析中药外用治疗膝骨性关节炎的组方用药规律. 时珍国医国药, 2021, 32(1): 73-76.
7.	涂鹏程, 郭杨, 马勇, 等. 威灵仙提取物可促进体外牵张应力环境下软骨细胞表型的维持. 中国组织工程研究, 2020, 24(8): 1182-1187.
8.	Pan YL, Ma Y, Guo Y, et al. Effects of clematis chinensis osbeck mediated by low-intensity pulsed ultrasound on transforming growth factor-β/Smad signaling in rabbit articular chondrocytes. J Med Ultrason (2001), 2019, 46(2): 177-186.
9.	Khademolqorani S, Tavanai H, Chronakis IS, et al. The determinant role of fabrication technique in final characteristics of scaffolds for tissue engineering applications: A focus on silk fibroin-based scaffolds. Mater Sci Eng C Mater Biol Appl, 2021, 122: 111867. doi: 10.1016/j.msec.2021.111867.
10.	Gavrilova NA, Borzenok SA, Revishchin AV, et al. The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process. Int J Biol Macromol, 2021, 185: 264-276.
11.	Drachuk I, Harbaugh S, Chávez JL, et al. Improving the activity of DNA-encoded sensing elements through confinement in silk microcapsules. ACS Appl Mater Interfaces, 2020, 12(43): 48329-48339.
12.	Li Q, Xu S, Feng Q, et al. 3D printed silk-gelatin hydrogel scaffold with different porous structure and cell seeding strategy for cartilage regeneration. Bioact Mater, 2021, 6(10): 3396-3410.
13.	Sun W, Gregory DA, Tomeh MA, et al. Silk fibroin as a functional Biomaterial for tissue engineering. Int J Mol Sci, 2021, 22(3): 1499. doi: 10.3390/ijms22031499.
14.	涂鹏程, 郭杨, 马勇, 等. 模拟微重力培养环境下载威灵仙丝素蛋白微球对软骨细胞表型分化的影响. 中华中医药杂志, 2021, 36(4): 2038-2043.
15.	Qu J, Wang L, Niu L, et al. Porous silk fibroin microspheres sustainably releasing bioactive basic fibroblast growth factor. Materials (Basel), 2018, 11(8): 1280. doi: 10.3390/ma11081280.
16.	李维嘉, 王志强, 许泽群, 等. 分光光度法测定灵芝孢子油中总三萜的含量. 食品研究与开发, 2019, 40(17): 165-170.
17.	Suderman MT, Temeyer KB, Schlechte KG, et al. Three-dimensional culture of rhipicephalus (Boophilus) microplus BmⅧ-SCC cells on multiple synthetic scaffold systems and in rotating bioreactors. Insects, 2021, 12(8): 747. doi: 10.3390/insects12080747.
18.	Ao Y, Li Z, You Q, et al. The yse of particulated juvenile allograft cartilage for the repair of porcine articular cartilage defects. Am J Sports Med, 2019, 47(10): 2308-2315.
19.	Campos Y, Almirall A, Fuentes G, et al. Tissue engineering: an alternative to repair cartilage. Tissue Eng Part B Rev, 2019, 25(4): 357-373.
20.	Pan T, Cheng TF, Jia YR, et al. Anti-rheumatoid arthritis effects of traditional Chinese herb couple in adjuvant-induced arthritis in rats. J Ethnopharmacol, 2017, 205: 1-7.
21.	Xiong Y, Ma Y, Kodithuwakku ND, et al. Protective effects of clematichinenoside AR against inflammation and cytotoxicity induced by human tumor necrosis factor-α. Int Immunopharmacol, 2019, 75: 105563. doi: 10.1016/j.intimp.2019.04.010.
22.	Lin TF, Wang L, Zhang Y, et al. Uses, chemical compositions, pharmacological activities and toxicology of Clematidis Radix et Rhizome-a Review. J Ethnopharmacol, 2021, 270: 113831.doi: 10.1016/j.jep.2021.113831.
23.	潘娅岚, 马勇, 涂鹏程, 等. 低频超声促透威灵仙对早期兔膝骨关节炎的干预作用及机制研究. 中国中西医结合杂志, 2020, 40(4): 470-475.
24.	Qian KY, Song Y, Yan X, et al. Injectable ferrimagnetic silk fibroin hydrogel for magnetic hyperthermia ablation of deep tumor. Biomaterials, 2020, 259: 120299. doi: 10.1016/j.biomaterials.2020.120299.
25.	Zhao Y, Zhu ZS, Guan J, et al. Processing, mechanical properties and bio-applications of silk fibroin-based high-strength hydrogels. Acta Biomater, 2021, 125: 57-71.
26.	Zhang W, Chen L, Chen J, et al. Silk fibroin biomaterial shows safe and effective wound healing in animal models and a randomized controlled clinical trial. Adv Healthc Mater, 2017, 6(10). doi: 10.1002/adhm.201700121.
27.	Crivelli B, Bari E, Perteghella S, et al. Silk fibroin nanoparticles for celecoxib and curcumin delivery: ROS-scavenging and anti-inflammatory activities in an in vitro model of osteoarthritis. Eur J Pharm Biopharm, 2019, 137: 37-45.
28.	Zhang X, Zhou J, Xu Y. Optimized parameters for the preparation of silk fibroin drug-loaded microspheres based on the response surface method and a genetic algorithm-backpropagation neural network model. J Biomed Mater Res B Appl Biomater, 2021, 109(1): 6-18.
29.	Wuest SL, Caliò M, Wernas T, et al. Influence of mechanical unloading on articular chondrocyte dedifferentiation. Int J Mol Sci, 2018, 19(5): 1289. doi: 10.3390/ijms19051289.
30.	Onitsuka K, Murata K, Kokubun T, et al. Effects of controlling abnormal joint movement on expression of MMP13 and TIMP-1 in osteoarthritis. Cartilage, 2020, 11(1): 98-107.
31.	Maenohara Y, Chijimatsu R, Tachibana N, et al. Lubricin contributes to homeostasis of articular cartilage by modulating differentiation of superficial zone cells. J Bone Miner Res, 2021, 36(4): 792-802.

1. Kwon H, Brown WE, Lee CA, et al. Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nat Rev Rheumatol, 2019, 15(9): 550-570.
2. Rong Y, Zhang J, Jiang D, et al. Hypoxic pretreatment of small extracellular vesicles mediates cartilage repair in osteoarthritis by delivering miR-216a-5p. Acta Biomater, 2021, 122: 325-342.
3. Chen W, Xu Y, Li H, et al. Tanshinone ⅡA delivery silk fibroin scaffolds significantly enhance articular cartilage defect repairing via promoting cartilage regeneration. ACS Appl Mater Interfaces, 2020, 12(19): 21470-21480.
4. Castro-Viñuelas R, Sanjurjo-Rodríguez C, Piñeiro-Ramil M, et al. Induced pluripotent stem cells for cartilage repair: current status and future perspectives. Eur Cell Mater, 2018, 36: 96-109.
5. Hong H, Seo YB, Kim DY, et al. Digital light processing 3D printed silk fibroin hydrogel for cartilage tissue engineering. Biomaterials, 2020, 232: 119679. doi: 10.1016/j.biomaterials.2019.119679.
6. 邓凯烽, 朱英, 廖子龙, 等. 基于复杂网络技术分析中药外用治疗膝骨性关节炎的组方用药规律. 时珍国医国药, 2021, 32(1): 73-76.
7. 涂鹏程, 郭杨, 马勇, 等. 威灵仙提取物可促进体外牵张应力环境下软骨细胞表型的维持. 中国组织工程研究, 2020, 24(8): 1182-1187.
8. Pan YL, Ma Y, Guo Y, et al. Effects of clematis chinensis osbeck mediated by low-intensity pulsed ultrasound on transforming growth factor-β/Smad signaling in rabbit articular chondrocytes. J Med Ultrason (2001), 2019, 46(2): 177-186.
9. Khademolqorani S, Tavanai H, Chronakis IS, et al. The determinant role of fabrication technique in final characteristics of scaffolds for tissue engineering applications: A focus on silk fibroin-based scaffolds. Mater Sci Eng C Mater Biol Appl, 2021, 122: 111867. doi: 10.1016/j.msec.2021.111867.
10. Gavrilova NA, Borzenok SA, Revishchin AV, et al. The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process. Int J Biol Macromol, 2021, 185: 264-276.
11. Drachuk I, Harbaugh S, Chávez JL, et al. Improving the activity of DNA-encoded sensing elements through confinement in silk microcapsules. ACS Appl Mater Interfaces, 2020, 12(43): 48329-48339.
12. Li Q, Xu S, Feng Q, et al. 3D printed silk-gelatin hydrogel scaffold with different porous structure and cell seeding strategy for cartilage regeneration. Bioact Mater, 2021, 6(10): 3396-3410.
13. Sun W, Gregory DA, Tomeh MA, et al. Silk fibroin as a functional Biomaterial for tissue engineering. Int J Mol Sci, 2021, 22(3): 1499. doi: 10.3390/ijms22031499.
14. 涂鹏程, 郭杨, 马勇, 等. 模拟微重力培养环境下载威灵仙丝素蛋白微球对软骨细胞表型分化的影响. 中华中医药杂志, 2021, 36(4): 2038-2043.
15. Qu J, Wang L, Niu L, et al. Porous silk fibroin microspheres sustainably releasing bioactive basic fibroblast growth factor. Materials (Basel), 2018, 11(8): 1280. doi: 10.3390/ma11081280.
16. 李维嘉, 王志强, 许泽群, 等. 分光光度法测定灵芝孢子油中总三萜的含量. 食品研究与开发, 2019, 40(17): 165-170.
17. Suderman MT, Temeyer KB, Schlechte KG, et al. Three-dimensional culture of rhipicephalus (Boophilus) microplus BmⅧ-SCC cells on multiple synthetic scaffold systems and in rotating bioreactors. Insects, 2021, 12(8): 747. doi: 10.3390/insects12080747.
18. Ao Y, Li Z, You Q, et al. The yse of particulated juvenile allograft cartilage for the repair of porcine articular cartilage defects. Am J Sports Med, 2019, 47(10): 2308-2315.
19. Campos Y, Almirall A, Fuentes G, et al. Tissue engineering: an alternative to repair cartilage. Tissue Eng Part B Rev, 2019, 25(4): 357-373.
20. Pan T, Cheng TF, Jia YR, et al. Anti-rheumatoid arthritis effects of traditional Chinese herb couple in adjuvant-induced arthritis in rats. J Ethnopharmacol, 2017, 205: 1-7.
21. Xiong Y, Ma Y, Kodithuwakku ND, et al. Protective effects of clematichinenoside AR against inflammation and cytotoxicity induced by human tumor necrosis factor-α. Int Immunopharmacol, 2019, 75: 105563. doi: 10.1016/j.intimp.2019.04.010.
22. Lin TF, Wang L, Zhang Y, et al. Uses, chemical compositions, pharmacological activities and toxicology of Clematidis Radix et Rhizome-a Review. J Ethnopharmacol, 2021, 270: 113831.doi: 10.1016/j.jep.2021.113831.
23. 潘娅岚, 马勇, 涂鹏程, 等. 低频超声促透威灵仙对早期兔膝骨关节炎的干预作用及机制研究. 中国中西医结合杂志, 2020, 40(4): 470-475.
24. Qian KY, Song Y, Yan X, et al. Injectable ferrimagnetic silk fibroin hydrogel for magnetic hyperthermia ablation of deep tumor. Biomaterials, 2020, 259: 120299. doi: 10.1016/j.biomaterials.2020.120299.
25. Zhao Y, Zhu ZS, Guan J, et al. Processing, mechanical properties and bio-applications of silk fibroin-based high-strength hydrogels. Acta Biomater, 2021, 125: 57-71.
26. Zhang W, Chen L, Chen J, et al. Silk fibroin biomaterial shows safe and effective wound healing in animal models and a randomized controlled clinical trial. Adv Healthc Mater, 2017, 6(10). doi: 10.1002/adhm.201700121.
27. Crivelli B, Bari E, Perteghella S, et al. Silk fibroin nanoparticles for celecoxib and curcumin delivery: ROS-scavenging and anti-inflammatory activities in an in vitro model of osteoarthritis. Eur J Pharm Biopharm, 2019, 137: 37-45.
28. Zhang X, Zhou J, Xu Y. Optimized parameters for the preparation of silk fibroin drug-loaded microspheres based on the response surface method and a genetic algorithm-backpropagation neural network model. J Biomed Mater Res B Appl Biomater, 2021, 109(1): 6-18.
29. Wuest SL, Caliò M, Wernas T, et al. Influence of mechanical unloading on articular chondrocyte dedifferentiation. Int J Mol Sci, 2018, 19(5): 1289. doi: 10.3390/ijms19051289.
30. Onitsuka K, Murata K, Kokubun T, et al. Effects of controlling abnormal joint movement on expression of MMP13 and TIMP-1 in osteoarthritis. Cartilage, 2020, 11(1): 98-107.
31. Maenohara Y, Chijimatsu R, Tachibana N, et al. Lubricin contributes to homeostasis of articular cartilage by modulating differentiation of superficial zone cells. J Bone Miner Res, 2021, 36(4): 792-802.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair

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