Gradient Hydrogels to Promote MSC Differentiation for Osteochondral Defect Repair

骨软骨（OC）缺损是指关节软骨损伤或退化的局部区域， （软骨下）骨导致局灶性和退行性病变，如果不治疗，会导致不可逆的 以及导致骨关节炎（OA）的进行性关节恶化。OC缺陷具有挑战性， 损伤发生在关节软骨和软骨下骨中，或者更具体地在OC界面处， 涉及不同形态和分子组成的组织。临床治疗，如骨髓 刺激、清创、自体软骨细胞植入、OC自体移植和同种异体移植 可以改善临床症状，但不能治疗潜在的病理学。可植入组织工程 递送间充质干细胞（MSC）的支架对于基于细胞的OC缺陷修复具有巨大潜力， 它们可以从多种成体组织中分离，容易在培养物中扩增，并且已经显示， 经历成骨和成软骨分化。嵌入有扩散生长梯度的支架 因子、粘附配体和基质硬度已经显示促进MSC分化为 然而，这些研究中的大多数仅限于 基于单一因子的梯度刺激OC分化。我们以前开发了一种新的 聚合方法，其允许产生具有可调和连续的生物相容性的合成水凝胶支架。 交联密度和/或弹性模量梯度、蛋白水解介导的降解和固定化细胞 粘附肽（RGD）。此外，我们已经表明，细胞响应这些梯度，通过定向 并在三维培养中引导入侵和芽的形成。我们假设， 蛋白水解降解、弹性模量和整合素结合肽配体可用于调节MSC 分化成成骨和软骨谱系。这一假设将通过以下方式加以解决： 具体目标：目标1。为了确定蛋白水解降解的时空梯度的影响，固定化 RGD浓度和基质硬度对MSC分化的影响。目标2.为了评估是否时空 呈递的整合素特异性肽配体差异性地促进MSC分化为成骨细胞或成纤维细胞。 软骨形成谱系。这些研究将提供第一个证据，说明如何时空调节多个 生理相关梯度的类型在3D中调节MSC整合素表达和谱系定型 在没有诱导因子的情况下培养。这些发现将有助于植入式 组织工程支架可用作MSC递送载体，以促进软骨形成和成骨， 分化为软骨和骨的再生。

Osteochondral (OC) defects are localized areas of injury or degeneration of articular cartilage and underlying (subchondral) bone resulting in focal and degenerative lesions which if left untreated contribute to irreversible and progressive joint deterioration leading to osteoarthritis (OA). OC defects are challenging to treat as the damage occurs in both articular cartilage and subchondral bone, or more specifically at the OC interface, involving tissues of distinct morphologic and molecular composition. Clinical treatments such as bone marrow stimulation, debridement, autologous chondrocyte implantation, and OC autograft and allograft transplantation may improve clinical symptoms, but do not treat the underlying pathology. Implantable tissue engineered scaffolds that deliver mesenchymal stem cells (MSCs) hold great potential for cell-based OC defect repair as they can be isolated from a variety of adult tissues, are readily expanded in culture, and have been shown to undergo osteogenic and chondrogenic differentiation. Scaffolds embedded with gradients of diffusible growth factors, adhesion ligands, and matrix stiffness have been shown to promote MSC differentiation into chondrogenic and/or osteogenic lineage in 3D culture, however, most of these studies have been limited to stimulating OC differentiation based on gradients of a single factor. We have previously developed novel polymerization approaches that allow for the creation of synthetic hydrogel scaffolds with tunable and continuous gradients of crosslink density and/or elastic modulus, proteolytically mediated degradation, and immobilized cell adhesive peptides (RGD). Furthermore, we have shown that cells respond to these gradients through directed and guided invasion and sprout formation in 3D culture. We hypothesize that spatiotemporal gradients of proteolytic degradation, elastic modulus and integrin-binding peptide ligands can be used to regulate MSC differentiation into osteogenic and chondrogenic lineage. This hypothesis will be addressed by the following specific Aims: Aim 1. To define the effect of spatiotemporal gradients of proteolytic degradation, immobilized RGD concentration, and matrix stiffness on MSC differentiation. Aim 2. To evaluate whether spatiotemporally presented integrin-specific peptide ligands differentially promote MSC differentiation into osteogenic or chondrogenic lineage. These studies will provide the first evidence of how spatiotemporal regulation of multiple types of physiologically relevant gradients modulate MSC integrin expression and lineage commitment in 3D culture in the absence of induction factors. These findings will contribute towards the development of implantable tissue engineered scaffolds that can be used as MSC delivery vehicles to facilitate chondrogenic and osteogenic differentiation for the regeneration of cartilage and bone.