Bioinspired Mechanically Stiff Hydrogels for Osteochondral Tissue Regeneration

关节软骨和底层软骨下骨的损伤最终会导致骨关节炎，这是一种衰弱的疾病 无法治愈的疾病。成功的治疗需要促进组织再生，支持综合修复， 并保护周围的组织不会进一步退化。该项目的总体目标是开发 一种具有机械能力的、基于干细胞的再生方法，用于治疗骨软骨(OC)缺陷。在.期间 在最初的资金阶段，我们的团队开发了一种模拟OC的水凝胶，其设计可以分离负载- 承载(即，结构)来自软细胞仿生成分的成分。这使我们能够创建一个 功能分级、僵硬的结构，具有与软骨匹配的机械硬度，同时创造柔软的细胞壁龛 支持间充质干细胞(MSC)分化。从关键的体外里程碑开始，这次更新 目的是在体内翻译模拟OC的水凝胶。我们将检验一种假设，即模拟OC水凝胶 在体内诱导外源性MSCs快速和靶向分化，使其能够直接参与OC- 组织再生，同时保护和支持与周围组织的整合。一个 我们设计的新特点是在结构支撑内模仿水泥线，类似于本土 骨水泥线不会影响细胞在软骨-骨界面上的迁移，但会对营养物质产生渗透作用。 运输。这将保护软骨层中的MSCs，使其能够快速分化并对 再生。我们将在三个具体目标上检验这一总体假设。在Aim1中，我们将确定 在模拟OC的水凝胶中，不同地控制MSC命运的机械转导通路将 允许我们建立对实现强大的MSC的物理化学线索的机械性理解 在有负载的动态环境中实现差异化。在目标2中，我们将确定MSC在体内的命运 示踪分离的差异标记MSCs模拟OC水凝胶植入大鼠OC缺损区模型 来自DsRed+和GFP+大鼠。这一目标将确定MSC的命运及其对OC组织的直接和间接贡献 再生。在目标3中，我们将创建一个经过表面退化的结构支撑，以保持其 机械性能。我们将评估这一完全可降解和机械的有效性 使用三种增加复杂性的模型来模拟OC水凝胶：OC外植体缺陷模型 监测作为支撑结构的邻近缺损区的关节软骨的健康状况和与之的结合情况 降解；用于纵向研究的大鼠OC缺陷模型，以监测体内结构的降解 伴随着组织再生和综合修复；以及在临床前动物(猪)模型中进行测试。 在本项目结束时，我们希望(1)加深我们对 骨髓间充质干细胞的力学转导途径及其在体内的去向；(2)建立了机械能力强的 和可降解的OC模拟水凝胶，实现OC组织再生和综合修复，同时 保持关节健康。

Lesions to articular cartilage and underlying subchondral bone eventually lead to osteoarthritis, a debilitating disease with no cure. A successful therapy will need to promote tissue regeneration, support integrative repair, and protect the surrounding tissue from further degeneration. The overarching goal for this project is to develop a mechanically competent, stem cell-based regenerative approach to treat osteochondral (OC) defects. During the initial funding period, our team developed an OC-mimetic hydrogel with a design that decoupled the load- bearing (i.e., structural) component from the soft cellular biomimetic component. This allowed us to create a functionally graded, stiff structure with cartilage-matched mechanical stiffness, while creating soft cellular niches that supported mesenchymal stem cell (MSC) differentiation. Building from key in vitro milestones, this renewal aims to translate the OC-mimetic hydrogel in vivo. We will test the hypothesis that the OC-mimetic hydrogel induces rapid and targeted differentiation of exogeneous MSCs in vivo, enabling their direct participation in OC- tissue regeneration while simultaneously protecting and supporting integration with the surrounding tissue. A new feature of our design is a cement line-mimetic within the structural support that similar to the native cement line will be impervious to cell migration across the cartilage-bone interface, but pervious to nutrient transport. This will protect the MSCs in the cartilage layer, enabling their rapid differentiation and contribution to regeneration. We will test the overarching hypothesis in three specific aims. In Aim1, we will identify mechanotransduction pathways that differentially control MSC fate in the OC-mimetic hydrogel, which will allow us to establish a mechanistic understanding of the physiochemical cues that achieve robust MSC differentiation in a dynamic environment with loading. In Aim 2, we will determine MSC fate in vivo within the OC-mimetic hydrogel after implantation in a rat OC defect model by tracking differentially labeled MSCs isolated from DsRed+ and GFP+ rats. This aim will confirm MSC fate and their direct and indirect contribution to OC-tissue regeneration. In Aim 3, we will create a structural support that undergoes surface degradation to maintain its mechanical properties. We will evaluate the effectiveness of this fully degradable and mechanically competent OC-mimetic hydrogel using three models of increasing complexity: an OC explant defect model to monitor the health of and integration with articular cartilage adjacent to the defect as the support structure degrades; a rat OC defect model for longitudinal studies to monitor in vivo degradation of the structure concomitant with tissue regeneration and integrative repair; and, testing in a pre-clinical animal (swine) model. At the conclusion of this project, we expect to have (1) advanced our fundamental understanding of the mechanotransduction pathways in MSCs and their fate in vivo and (2) established a mechanically competent and degradable OC-mimetic hydrogel that achieves OC-tissue regeneration and integrative repair, while maintaining joint health.