复合骨碎补总黄酮的丝素蛋白/壳聚糖支架在兔关节软骨损伤中的应用研究

doi:10.13418/j.issn.1001-165x.2022.6.12

摘要/Abstract

摘要： 目的以丝素蛋白/壳聚糖支架为载体将骨碎补总黄酮应用于兔软骨损伤局部，观察修复效果，为临床提供实验数据。方法制备丝素蛋白/壳聚糖支架、骨碎补总黄酮缓释微球与负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架，扫描电子显微镜下观察支架形貌，同时检测该支架的体外缓释能力。24只新西兰大白兔随机分3组，利用电钻在股骨滑车部位构建直径3.5 mm、深1.5 mm的软骨损伤模型，空白组软骨缺损处不植入任何材料，对照组植入单纯的丝素蛋白/壳聚糖支架，实验组植入负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架，术后12周、24周行标本大体与组织学观察，RT-PCR检测修复组织Sox-9、II型胶原与聚集蛋白聚糖mRNA的表达量，Western blot检测软骨缺损部位II型胶原蛋白表达，分析软骨修复效果。结果丝素蛋白/壳聚糖支架具有良好的三维孔隙结构，孔洞之间相互联通；制备的载药微球表面较光滑，为较规则的圆球形；载药微球均匀分散于丝素蛋白/壳聚糖支架基质中。丝素蛋白/壳聚糖支架可在体外持续稳定地释放骨碎补总黄酮，实验组软骨损伤修复效果优于对照组，对应的ICRS评分与Wakitani组织学评分高于对照组（P<0.05）。实验组的软骨基因Sox-9、Ⅱ型胶原与聚集蛋白聚糖mRNA的表达量高于对照组（P<0.05），Ⅱ型胶原蛋白的表达高于对照组（P<0.05）。结论负载骨碎补总黄酮的丝素蛋白/壳聚糖支架植入软骨损伤部位可持续缓慢地释放骨碎补总黄酮，为种子细胞的生长与分化提供适合的微环境，有效促进软骨组织再生。

关键词: 丝素蛋白/壳聚糖支架； , , 关节软骨组织； , , 损伤修复

Abstract: Objective To observe the repair effect of cartilage injury, Drynaria total flavonoids was applied to the cartilage injury site by using silk fibroin/chitosan scaffold as the carrier, and provide experimental data for clinical application. Methods Silk fibroin / chitosan scaffolds, Drynaria total flavonoids sustained-release microspheres and silk fibroin / chitosan scaffolds loaded with Drynaria total flavonoids sustained-release microspheres were prepared. The microstructure of scaffolds was observed under scanning electron microscope, and the in vitro sustained-release ability of silk fibroin / chitosan scaffolds loaded with Drynaria total flavonoids sustained-release microspheres was detected. Twenty-four New Zealand white rabbits were randomly divided into three groups. A cartilage injury model with a diameter of 3.5 mm and a depth of 1.5 mm was established in the trochlear region of femur by electric drill. In the blank group, no material was implanted in the cartilage defect. In the control group, a simple Silk Fibroin / chitosan scaffold was implanted. In the experimental group, a silk fibroin / chitosan scaffold loaded with osteoclast total flavonoids sustained-release microspheres was implanted. At 12 and 24 weeks after operation, gross and histological observations were performed. The mRNA expressions of Sox-9, type II collagen and aggrecan were detected by RT-PCR, and the expression of type II collagen was detected by Western blot. Results Silk fibroin / chitosan scaffolds had good three-dimensional pore structure, and the pores connected with each other. The surface of the microspheres was smooth. The drug loaded microspheres were evenly dispersed in the silk fibroin / chitosan scaffold matrix. Silk fibroin / chitosan scaffolds could release Drynaria flavonoids in vitro. The results of gross and histological observation showed that the cartilage injury repair effect of the experimental group was better than that of the control group, and the corresponding ICRs score and Wakitani histological score were higher than those of the control group (P<0.05). The expressions of Sox-9, collagen II and collagen aggregation in the experimental group were higher than those in the control group (P<0.05), and the expression of collagen II was higher than that in the control group (P<0.05). Conclusions The silk fibroin / chitosan scaffold loaded with total flavonoids of osteoclast can release the total flavonoids of osteoclast slowly and continuously after being implanted into the damaged part of cartilage, which provides suitable microenvironment for the growth and differentiation of seed cells and effectively promotes the regeneration of cartilage tissue.

Key words: Silk fibroin / chitosan scaffold; , , Articular cartilage tissue; , , Damage repair

中图分类号:

R322.7+1

于洋, 宋永才, 杨立峰. 复合骨碎补总黄酮的丝素蛋白/壳聚糖支架在兔关节软骨损伤中的应用研究[J]. 中国临床解剖学杂志, 2022, 40(6): 696-703.

Yu Yang, Song Yongcai, Yang Lifeng. Application of silk fibroin / chitosan scaffold combined with total flavonoids of Drynaria fortunei on repair of articular cartilage injury[J]. Chinese Journal of Clinical Anatomy, 2022, 40(6): 696-703.

参考文献 26

[1]	Matsuoka M, Onodera T, Homan K, et al. Depletion of gangliosides enhances articular cartilage repair in mice[J]. Sci Rep, 2017, 7: 43729. DOI: 10.1038/srep43729.
[2]	Simon TM, Jackson DW. Articular cartilage: injury pathways and treatment options[J]. Sports Med Arthrosc Rev, 2018, 26(1): 31-39. DOI: 10.1097/JSA.0000000000000182.
[3]	Hong H, Seo YB, Kim DY, et al. Digital light processing 3D printed silk fibroin hydrogel for cartilage tissue engineering[J]. Biomaterials, 2020, 232: 119679. DOI: 10.1016/j.biomaterials.2019.119679.
[4]	Chen W, Xu Y, Li H, et al. Tanshinone IIA delivery silk fibroin scaffolds significantly enhance articular cartilage defect repairing via promoting cartilage regeneration[J]. ACS Appl Mater Interfaces, 2020, 12(19): 21470-21480. DOI: 10.1021/acsami.0c03822.
[5]	Qi Y, Zhang W, Li G, et al. An oriented-collagen scaffold including Wnt5a promotes osteochondral regeneration and cartilage interface integration in a rabbit model[J]. FASEB J, 2020, 34(8): 11115-11132. DOI: 10.1096/fj.202000280R.
[6]	Shi W, Sun M, Hu X, Ren B, et al. Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo[J]. Adv Mater, 2017, 29(29): 1-7. DOI: 10.1002/adma.201701089.
[7]	Oryan A, Sahvieh S. Effectiveness of chitosan scaffold in skin, bone and cartilage healing[J]. Int J Biol Macromol, 2017, 104(Pt A): 1003-1011. DOI: 10.1016/j.ijbiomac.2017.06.124.
[8]	Shamekhi MA, Mirzadeh H, Mahdavi H, et al. Graphene oxide containing chitosan scaffolds for cartilage tissue engineering[J]. Int J Biol Macromol, 2019,127:396-405. DOI: 10.1016/j.ijbiomac. 2019. 01.020.
[9]	李晋玉, 赵学千, 孙旗, 等. 骨碎补总黄酮的实验及临床研究概况[J]. 中国骨质疏松杂志,2018, 24(10): 1357-1364. DOI: 10.3969/j.issn. 1006-7108.2018.10.019.
[10]	韩亚力, 罗奕, 曾佳学. 骨碎补总黄酮基于Notch信号通路改善骨质疏松的作用及机制研究[J]. 中国免疫学杂志, 2018, 34(2): 261-266. DOI: 10.3969/j.issn.1000-484X.2018.02.021.
[11]	Bhardwaj N, Kundu SC. Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends[J]. Biomaterials, 2012, 33(10): 2848-2857. DOI: 10.1016/j.biomaterials.2011.12.028.
[12]	郭英, 李佩芳, 舒晓春, 等. 骨碎补总黄酮对骨髓间充质干细胞成骨分化过程中Wnt/β-catenin信号通路的影响[J]. 中华医学杂志, 2012, 92(32): 2288-2291. DOI: 10.3760/cma.j.issn.0376-2491.2012.32.017.
[13]	尚平, 贺宪, 安耀武, 等. 骨碎补总黄酮对骨关节炎家兔骨髓间充质干细胞软骨定向分化的实验研究[J]. 生物骨科材料与临床研究, 2009, 6(6): 10-13. DOI: 10.3969/j.issn.1672-5972.2009.06.003.
[14]	Guo W, Shi K, Xiang G, et al. Effects of rhizoma drynariae cataplasm on fracture healing in a rat model of osteoporosis[J]. Med Sci Monit, 2019, 25: 3133-3139. DOI: 10.12659/MSM.914568.
[15]	李明, 李君, 付昆. 骨碎补总黄酮对膝骨关节炎模型兔HIF-1α和VEGF表达的影响[J]. 中国药房, 2018, 29(18): 2484-2488. DOI: 10.6039/j.issn.1001-0408.2018.18.09.
[16]	Li DW, Lei X, He FL, et al. Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells[J]. Int J Biol Macromol, 2017, 105(Pt 1): 584-597. DOI: 10.1016/j.ijbiomac.2017.07.080.
[17]	刘亚珍, 邱晓明, 李松凯. 正交设计优化万古霉素/聚乳酸-羟基乙酸共聚物微球的制备及体外药物释放[J]. 中国组织工程研究, 2019, 23(2): 211-217. DOI: 10.3969/j.issn.2095-4344.1509.
[18]	薛鹏, 杜斌, 王礼宁, 等. 可控释淫羊藿苷-β-磷酸三钙复合支架的制备[J]. 中国组织工程研究, 2018, 22(6): 865-870. DOI: 10.3969/j.issn.2095-4344.0060.
[19]	Widhiyanto L, Utomo DN, Perbowo AP, et al. Macroscopic and histologic evaluation of cartilage regeneration treated using xenogenic biodegradable porous sponge cartilage scaffold composite supplemented with allogenic adipose derived mesenchymal stem cells (ASCs) and secretome: an in vivo experimental study[J]. J Biomater Appl, 2020, 35(3): 422-429. DOI: 10.1177/0885328220934938.
[20]	徐立岩, 马剑雄, 王颖, 等. 关节制动对大鼠膝关节软骨缺损修复的影响[J]. 中国组织工程研究, 2016, 20(37): 5496-5503. DOI: 10.3969/j.issn.2095-4344.2016.37.004.
[21]	Borrelli J Jr, Olson SA, Godbout C, et al. Understanding articular cartilage injury and potential treatments[J]. J Orthop Trauma, 2019, 33 Suppl 6: S6-S12. DOI: 10.1097/BOT.0000000000001472.
[22]	Krych AJ, Saris DBF, Stuart MJ, et al. Cartilage injury in the knee: assessment and treatment options[J]. J Am Acad Orthop Surg, 2020, 28(22): 914-922. DOI: 10.5435/JAAOS-D-20-00266.
[23]	柳海峰, 吴冰, 梁达强, 等. 基质诱导自体软骨细胞移植术治疗距骨软骨损伤临床疗效观察[J]. 中国临床解剖学杂志, 2019, 37(1): 91-96. DOI: 10.13418/j.issn.1001-165x.2019.01.019.
[24]	吴紫璇, 郝征, 郭亚萍, 等. 骨碎补总黄酮通过SDF1/CXCR4信号途径促进小鼠骨髓基质细胞ST-2迁移[J]. 天津医药, 2019, 47(10): 1009-1014. DOI: 10.11958/20192305.
[25]	Yang S, Qian Z, Liu D, et al. Integration of C-type natriuretic peptide gene-modified bone marrow mesenchymal stem cells with chitosan/silk fibroin scaffolds as a promising strategy for articular cartilage regeneration[J]. Cell Tissue Bank, 2019, 20(2): 209-220. DOI: 10.1007/s10561-019-09760-z.
[26]	Maity PP, Dutta D, Ganguly S, et al. Isolation and mass spectrometry based hydroxyproline mapping of type II collagen derived from Capra hircus ear cartilage[J]. Commun Biol, 2019, 2: 146. DOI: 10.1038/s42003-019-0394-6.