Preconditioning of mesenchymal stem cells with mechanobiological load and hypoxia for joint regeneration in moderate osteoarthritis

目前的组织工程方法主要用于局部创伤性骨软骨病变的再生，而关节中存在骨关节炎的改变是该方法的禁忌。我们的长期目标是修复退行性骨软骨病变，除此之外，在OA环境中治疗软骨和骨软骨缺陷。为了取得成功，我们需要了解软骨形成、机械应力、氧张力和炎症之间的建设性和破坏性相互作用。我们建议间充质干细胞可以在植入前进行机械生物学负荷和/或缺氧预处理，以实现稳定的软骨表型。即使在骨关节炎环境下，这种预处理也可以使骨软骨缺损再生。生理范围内的机械负荷是维持关节软骨健康状态的重要因素。另一方面，超载被认为会增加患骨关节炎的风险。因此，我们将在不同的机械生物学加载机制下对在体外3d聚集体培养系统中进行软骨形成的MSCs施加静水压力。我们将确定加载和卸载条件下显示最强软骨生成（合成代谢增加和分解代谢减少）差异的加载条件。我们计划在生理条件下实现这一目标，但也可以在OA条件下实现，这将通过在培养基中添加IL-1ß来模拟。低氧培养条件也被证明对软骨细胞有有益的预处理。我们提出，在健康和骨关节炎培养条件下，缺氧会促进分化并抑制肥大标志物。我们将应用缺氧对MSCs进行体外软骨形成，并分析其合成代谢和分解代谢作用。我们将专注于抑制肥大，以实现稳定的软骨表型。我们计划确定参与预处理过程的关键信号通路。特别是，我们将分析PI3K/ akt依赖通路，因为这个级联通过机械转导和缺氧参与软骨形成的调节。最后，我们想在健康和早期骨关节炎的动物模型中检验msc -海绵结构在机械生物学负荷和/或缺氧的前提下修复创伤后骨软骨缺损的效果。

The presence of osteoarthritic changes in a joint is a contraindication for current tissue engineering approaches, which are designed for regeneration of localized traumatic osteochondral lesions. Our long-term goal is the restoration of degenerative osteochondral lesions and beyond that, the treatment of chondral and osteochondral defects in an OA environment. To be successful we need to understand the constructive and destructive interactions between chondrogenesis, mechanical stress, oxygen tension and inflammation. We propose that mesenchymal stem cells can be preconditioned with mechanobiological load and/or hypoxia prior to implantation in order to achieve a stable chondrogenic phenotype. This preconditioning could allow regeneration of osteochondral defects even in an OA environment. Mechanical load in physiological range is an important factor for maintenance of the healthy status of joint cartilage. On the other hand, overloading is believed to increase the risk of osteoarthritis. Therefore we will apply hydrostatic pressure in different mechanobiological loading regimes to MSCs undergoing chondrogenesis in an in vitro 3D-aggregate culture system. We will identify loading conditions that show the strongest chondrogenic (increased anabolism and decreased catabolism) difference between loaded and unloaded conditions. We plan to achieve this under physiological conditions, but also in OA conditions, which will be mimicked by addition of IL-1ß to the culture medium.Hypoxic culture conditions have also been shown to have beneficial preconditioning for chondrogenic cells. We propose that hypoxia will promote differentiation and suppress markers of hypertrophy in both healthy and osteoarthritic culture conditions. We will apply hypoxia to MSCs undergoing in vitro chondrogenesis and analyse for anabolic and catabolic effects. We will focus on the suppression of hypertrophy in order to achieve a stable chondrogenic phenotype. We plan to identify key signaling pathways involved in the preconditioning process. In particular, we will analyse the PI3K/Akt-dependent pathway, because this cascade is involved in the modulation of chondrogenesis through mechanotransduction and also hypoxia. Finally we want to examine the effect of MSC-sponge constructs, preconditionend with mechanobiological load and/or hypoxia, to repair posttraumatic osteochondral defects in an animal model in a healthy and an early osteoarthritic condition.