可注射微尺度支架促进软骨下骨再生对软骨修复的作用机制研究

Knee osteoarthritis (KOA), with the characteristics of highly prevalence, unknown mechanism, and less treatments. The subchondral bone lesion have great relationship with KOA, which was seldom mentioned in other researches. The subchondral bone lesion animal model and the hydroxyapatite-chitosan-gelatin microscaffolds (HaCGMs) were proposed in our previous study. It was shown that HaCGMs to have osteo-inductivity in vitro; and to lead subchondral bone regeneration and protect upper cartilage in vivo. However, the mechanism was needed to be explored. We have found that chondrocytes co-cultured with HaCGMs/MSCs in transwell system were showed great proliferation index and appearance comparing with other groups. The MSCs were seeded on the HaCGMs to assemble as micro-tissure. After scanned by secretome proteomics, more than 3000 proteins were found differently secreted, including highly secreted Thrombospondin-1 (THBS1). THBS1 was shown to upregulate TGF-β of chondrocytes by PPI network prediction and results from other studies. Therefore, we hypothesized that THBS1 could upregulate TGF-β expression of chondrocytes, thus maintain the proliferation and phenotype of chondrocytes. In our study, we will knockdown and rescueTHBS1 expression of MSCs and TGF-β expression of chondrocytes by using siRNA and lentivirus, then using co-culture, Co-IP, and other methods to make sure the relationship between THBS1 and TGF-β. To figure out other OA-related genes expression between different groups, then to test the possible signal pathway. Finally, the bone-cut immunohistochemistry from knee arthroplasty will be used to testify out result. The molecule mechanism after THBS1 upregulate TGF-β is used to illustrate the role of HaCGMs in treating subchondral bone lesions and upper cartilage, which will shed light on another treatment of KOA.

膝骨关节炎（KOA）患病率高、危害重。针对KOA特征之一的软骨下骨损伤，我们研制了动物模型和可注射微尺度支架（HaCGMs），证实其可促进软骨下骨再生，对关节软骨有保护作用，但机制不清。THBS1可促进软骨细胞TGF-β过表达，体外实验证明HaCGMs与MSCs形成微组织块促进THBS1分泌，经Transwell内孔作用于软骨细胞。据此我们提出：HaCGMs介导软骨下骨MSCs高分泌THBS1，上调软骨细胞TGF-β过表达，进而促进软骨细胞增殖和形态维持。本课题拟在细胞水平利用共培养、siRNA、慢病毒过表达、免疫荧光、免疫共沉淀等技术，探索THBS1对软骨细胞增殖与形态起作用的分子机制；证明THBS1/TGF-β二者相互作用及对OA相关基因表达的影响；最后通过选取KOA置换截骨块以及正常的软骨下骨与软骨进行临床组织学验证。阐明HaCGMs软骨修复作用的机制，为KOA预防及治疗提供新思路。

背景：膝骨关节炎（Knee Ostroarthritis, KOA）是一类好发于50岁以上老年人的退行性疾病，对于国家及个人造成严重的疾病负担。如何能做到早期干预，延缓KOA进程，一直是KOA研究的热点。.目的：本课题目的：验证预载MSC的3D可注射微载体具体通过何种机制发挥作用的？ .方法：(1)制作gelatin微尺度载体，并进行材料学表征，同时将MSC种植到微尺度载体上，作为一种“药械”在特制的转瓶中进行3D培养；(2)建立一种大鼠膝骨关节炎模型（Anterior cruciate ligament, ACL切除术），随后通过关节腔注射微载体/MSC以及单独注射MSC进行治疗。术后不同时段利用多种方法进行评估；(3)建立Transwell共培养OA软骨细胞模型，后将细胞上清进行细胞因子高通量筛选检测。并将Transwell内的软骨细胞与MSC进行测序分析，从转录组学水平，探讨外源性MSC作用于原位软骨细胞的具体机制。.结果：(1)gelatin微尺度载体满足如下特性：可注射性、良好的膨胀率，多孔性、具备一定的机械性、良好的生物相容性以及诱导细胞浸润的能力；(2)脐带来源的干细胞具备常见干细胞的特性，即具有成骨、成脂、成软骨三向分化的能力。脐带MSC种植于微尺度载体，后在特制的转瓶中进行3D培养，MSC可在微尺度支架上存活并保持活性达21天以上。RT-PCR显示组织工程化干细胞在不同时间的培养下，MSC均维持了干性基因的表达，同时抑制了MSC的衰老进展；(3)将组织工程化干细胞、单纯MSC、透明质酸分别注入膝骨关节炎的动物模型中。术后12周X光、显微CT、MRI、H&E染色、番红－固绿染色显示：组织工程化干细胞组较单纯MSC组、透明质酸组以及单纯手术组，软骨保护作用明显，表现为：可抑制骨赘产生，维持关节间隙；(4) 共培养细胞上清进行细胞因子高通量筛选显示：3D培养组诸如炎症因子IL-6R等低表达，而GM-CSF、IL-1β、IL-1α等高表达，提示组织工程化干细胞通过提高MSC的抑制炎症反应能力起作用。而共培养细胞RNA测序结果提示：组织工程化干细胞与软骨细胞可以相互作用，促MSC成软骨分化诱导，同时促进软骨细胞表型维持。.结论：构成组织工程化干细胞的这一载体，可以减少细胞使用量以及减少注射次数来发挥作用，未来可获得更多的应用可能。