The role of Cx43 and stem cell derived exosomes for the regeneration of cartilage lesioned due to osteoarthritic processes

在骨性关节炎过程中发生的关节软骨损伤是导致马跛行的主要原因之一。骨性关节炎的病因和发病机制仍不清楚，到目前为止，还没有可靠的治疗方案来治疗骨性关节炎，包括软骨下骨。然而，骨髓间充质干细胞(MSC)的治疗应用在人类和兽医中变得越来越重要，因为根据目前获得的信息，MSCs不一定需要原位分化为软骨细胞来替代骨关节炎诱导的软骨缺损。此外，通常认为MSC的治疗作用是基于MSC释放到细胞外空间的外切体。正如以前的研究表明的那样，通过超滤可以从MSC细胞培养上清液中获得外切体。CD9、CD63、CD83等表面标志物的表达可作为其特征。众所周知，作为外体载体的microRNA可能在体内调节干细胞内细胞增殖和细胞分化之间的平衡。此外，根据目前的认识，外周小体和软骨细胞的连接蛋白43在维持健康关节软骨微环境中的组织内稳态方面发挥了重要作用。因此，预期项目的目的是研究从马培养脂肪组织来源的MSC的培养上清液中提取的外切体在使用受损的马软骨细胞建立的体外骨关节炎模型中的保护作用。本研究的主要目的是验证工作假说，即不同的Cx43表达模式和不同的MSC培养条件及其外切体在OA模型中会产生不同的治疗效果。此外，我们将研究在OA过程中诱导的Cx43上调是否可能被外体货物抵消。外切体的治疗效力应通过所包含的治疗物质的特性进行分析，如miRNA、蛋白质、细胞因子和生长因子。该项目的详细数据有能力为兽医和人类医学中有用的基于干细胞但无细胞的OA疗法建立基本原则。

Damaged joint cartilage which occurs in the course of ostoarthritis is one of the main causes for lameness in horses. Etiology and pathogenesis of ostoarthritis are still not clarified and up to now reliable therapeutic options for the treatment of osteoarthritis, which also includes the subchondral bone, are not available. However, the therapeutic use of mesenchymal stem cells (MSC) becomes increasingly important in human and veterinary medicine as according to currently available information MSCs do not necessarily need to differentiate in situ into chondrocytes to replace OA induced cartilage defects. Moreover, it is generally assumed that therapeutic effects of MSC are based on exosomes which are released into the extracellular space by MSC. Exosomes can be harvested from MSC cell culture supernatants by ultrafiltration as has been shown in previous studies. They can be characterized by the expression of surface markers such as CD9, CD63 and CD83. It is well known that micro RNAs as exosome cargo may modulate the equilibrium between cell proliferation and cell differentiation within the stem cell niche in vivo. Additionally, according to the present knowledge, connexin 43 of exosomes and chondrocytes play an instrumental role in the maintenance of tissue homeostasis within the microenvironment of healthy joint cartilage. Hence, the aim of the anticipated project is to investigate the protective effects of exosomes, which are harvested from the supernatant of equine cultivated adipose tissue derived MSC in an in vitro osteoarthritis model using lesioned equine chondrocytes. The main focus of this investigation will be to substantiate the working hypothesis, namely that different expression patterns of Cx43 and various cultivation conditions of MSC including their exosomes will induce different therapeutic effects in the OA model. Furthermore, we will examine whether upregulation of Cx43 induced in the course of OA might be counteracted by the exosome cargo. The therapeutic potency of exosomes shall be analyzed by the characterization of included therapeutic substances such as miRNA, proteins, cytokines and growth factors. Elaborated data of this project have the potency to establish basic principles for a useful stem cell-based but cell-free OA-therapy in veterinary and human medicine.