异体滑膜及青少年软骨颗粒移...”缺损及移植细胞的转归研究_闫文强.pdf

中国运动医学杂志2022年12月第41卷第12期 Chin J Sports Med，Dec.2022，Vol.41，No.12异体滑膜及青少年软骨颗粒移植修复兔半月板“白区”缺损及移植细胞的转归研究闫文强吴桐樊逸菲麦合木提麦麦提敏赵逢源李豫皖曹宸喜胡晓青程锦敖英芳北京大学第三医院运动医学科，北京大学运动医学研究所，运动医学关节伤病北京市重点实验室（北京100191）摘要目的：探讨异体滑膜和青少年软骨颗粒移植在半月板“白区”缺损修复上的协同作用；研究移植细胞的转归（存活和分化）；研究滑膜间充质干细胞和软骨细胞间的相互作用。方法：选取2只未成年雄性兔作为滑膜及青少年软骨颗粒供体。选取 24只成年雌性兔（共48个膝关节）制备半月板缺损模型，分为以下几组：空白组，半月板缺损无任何处理；单纯纤维蛋白胶组，在半月板缺损处填充单纯纤维蛋白胶；纤维蛋白胶+滑膜移植组，纤维蛋白胶包裹异体滑膜碎片填充到半月板缺损处；纤维蛋白胶+滑膜+异体青少年软骨颗粒移植组，纤维蛋白胶包裹异体滑膜和异体青少年软骨颗粒填充到半月板缺损处，每组样本量为12个膝关节。分别在术后8周和16周取材，通过组织学和免疫组织化学染色评估半月板缺损修复程度，针对雄性个体特有的Y染色体性别决定区基因（SRY）进行核酸原位杂交，并结合免疫荧光共定位评估移植细胞的存活和分化。其次，建立体外兔滑膜间充质干细胞和软骨细胞共培养系统，检测半月板纤维软骨细胞相关基因转录水平，并探索细胞间相互作用的可能机制。结果：在术后8、16周，相较于空白组、单纯纤维蛋白胶组及纤维蛋白胶+滑膜移植组，纤维蛋白胶+滑膜+异体青少年软骨颗粒移植组的缺损表现出良好的结构完整性、表面光滑度和边缘整合度，修复组织中可见较多的半月板样纤维软骨细胞，并且，细胞周围的基质在甲苯胺蓝和II型胶原免疫组织化学染色中表现为强阳性。在术后16周，纤维蛋白胶+滑膜移植组及纤维蛋白胶+滑膜+异体青少年软骨颗粒移植组的SRY核酸原位杂交信号表现为强阳性，表明移植细胞在受体中至少可存活16周。其次，核酸原位杂交和SOX9免疫荧光共定位显示表面移植滑膜组织中的细胞表现为SOX9阳性，表明移植滑膜组织中的细胞可分化为半月板纤维软骨细胞。滑膜间充质干细胞和软骨细胞共培养后半月板基质相关基因的转录水平与半月板纤维软骨细胞相似，并且，滑膜间充质干细胞和软骨细胞之间可通过细胞外囊泡相互作用。结论：异体滑膜和青少年软骨颗粒移植可协同促进半月板“白区”缺损修复；移植细胞在受体中至少可存活16周；移植滑膜组织中的细胞在半月板缺损处可分化为半月板纤维软骨细胞。关键词半月板损伤；半月板修复；滑膜；异体青少年软骨颗粒The Fate of Donor Cells in Synovium and Particulated Juvenile Allograft Cartilage for the Repair ofMeniscal“white zone”DefectsYan Wenqiang，Wu Tong，Fan Yifei，Maihemuti Maimaitimin，Zhao Fengyuan，Li Yuwan，Cao Chenxi，HuXiaoqing，Cheng Jin，Ao YingfangDepartment of Sports Medicine，Peking University Third Hospital，Institute of Sports Medicine of PekingUniversity，Beijing Key Laboratory of Sports Injuries，Beijing 100191，ChinaCorresponding Author:Ao Yingfang，Email:Abstract Objective To explore the synergistic effect of synovium and particulated juvenile allograftcartilage（PJAC）on repairing meniscal defects in the avascular zone，track the survival and differentia收稿日期：2022.04.05基金项目：国家自然科学基金（82172420，81972101，81802161，81871770）第1作者：闫文强，Email：；通信作者：敖英芳，Email： 941DOI:10.16038/j.1000-6710.2022.12.012中国运动医学杂志2022年12月第41卷第12期 Chin J Sports Med，Dec.2022，Vol.41，No.12tion of donor cells and interactions between synovial cells and chondrocytes.Methods In this study，2immature male rabbits were raised as synovium and particulated cartilage donors.Meanwhile，24 adultfemale rabbits were selected and induced cylindrical full-thickness defects measuring 2.0 mm in theavascular portion of medial menisci in both knees.They were then randomly divided into a blankgroup，a fibrin gel group，a fibrin gel+synovium transplant group as well as a fibrin gel+synoviumand PJAC transplant group，each of 12 rabbits.The blank group was left untreated，while the fibringel group，fibrin gel+synovium transplant group as well as fibrin gel+synovium and PJAC transplantgroup were filled into the meniscal defects with fibrin gel，fibrin gel encapsulating synovium fragments，as well as fibrin gel encapsulating synovium fragments and PJAC respectively.Rabbits were sacrificed 8 and 16 weeks postoperatively，and the histological and immunohistological methods were applied to assess the degree of meniscal repair.The in situ hybridization（ISH）for male-specific sex-determining region Y-linked（SRY）gene and immunofluorescence for Sry-type HMG-box 9（SOX9）wereconducted to evaluate the survival and differentiation of donor cells.Moreover，in vitro cell coculturesystemofsynovium derived mesenchymal stem cells（SMSCs）and chondrocytes was built，and their interaction was analyzed.Results Eight and 16 weeks after the operation，better structural integrity，superficial smoothness and marginal integration were observed in the fibrin gel+synovium and PJAC transplantgroup，compared with the other 3 groups，with more meniscus-like fibrochondrocytes in the repairedtissue，and the matrix around such cell clusters being strongly positive after the toluidine blue andtype 2 collagen immunohistochemistry staining.The SRY signals of ISH were positive in both the fibrin gel+synovium transplant and fibrin gel+synovium and PJAC transplant groups 16 weeks postoperatively，which indicated that donor cells survived for at least 16 weeks in the recipient.Moreover，thetransplanted synovial cells were found differentiating into meniscal fibrochondrocytes as they demonstrated the positive SOX9 expression.The transcriptional levels of meniscal matrix related genes after thecoculture of SMSCs and chondrocytes were similar to meniscal fibrochondrocytes.Meanwhile，SMSCsand chondrocytes could interact through extracellular vesicles.Conclusion The synovium and PJACtransplant facilitates meniscal repair synergistically.The donor cells survive for at least 16 weeks inthe recipient and synovial cells differentiate into fibrochondrocytes in the meniscal defect.Key wordsmeniscal injuries;meniscal repair;synovium;particulated juvenile allograft cartilage半月板是股骨髁和胫骨平台之间的纤维软骨组织，在膝关节的承重、载荷传递、润滑和减震中起关键作用1。半月板损伤是最常见的膝关节相关损伤之一，可发生于各个年龄人群，包括儿童、年轻人、老年人以及普通人群和运动员2。对于发生在半月板内侧无血管区域（“白区”）的损伤，由于其愈合能力有限，半月板切除术仍是目前主要的治疗手段3。半月板切除会导致继发性软骨退变，甚至发展为骨关节炎（osteoarthritis，OA）4。然而，目前对半月板损伤的治疗理念是促进半月板愈合，保持结构完整性，防止继发性软骨退化和改善关节功能。因此，亟需开发新的治疗方法以促进半月板愈合。Kim等5采用兔内侧半月板“白