Engineering Developmental Microenvironments: Cartilage Formation and Maturation

描述（由申请人提供）：关节软骨排列在关节表面，并传递载荷产生的力。由于软骨的自然愈合能力的限制，并且鉴于骨关节炎的发病率增加，存在对用于修复的基于细胞的策略的日益增长的需求。组织工程，特别是那些基于自体间充质干细胞（MSC）的方法，正在发展成为促进软骨再生的临床相关技术。然而，用于植入的工程化结构的力学和ECM组织尚未与天然组织的力学和ECM组织相匹配，并且不足以支持关节负荷。大多数软骨组织工程的努力复制软骨发育的早期阶段，隔离高密度的软骨ECM生产细胞在一个确定的体积。然而，在软骨形成的这些早期快速阶段和使其能够发挥成年功能的逐渐重塑（成熟）之间存在显著差异。随着承重的使用，软骨ECM被完全重塑成成人组织的独特组成和结构。这一转变过程是由多种时间因素（化学、机械和可溶性）驱动的。我们的软骨组织工程方法采用了这种复杂的发育模式（从胚胎到成人）的概念，并采用了基于天然ECM成分的合成水凝胶，该成分提供了初始受体-基质结合、受控力学和定制的降解特性。这些凝胶，再加上生理负荷和相关可溶性因子的临时应用，将重新创建发育微环境，使能和鼓励MSC在3-D培养中产生功能性软骨。在第一个目标中，MSC将被封装在先前作为软骨细胞载体研究的HA水凝胶中，所述HA水凝胶具有一系列可以影响细胞活力、受体结合、扩散和最终新软骨性质的结构，并在一系列条件下培养（即，细胞密度和可溶性因子的时间变化）。在第二个目标中，我们实验室最近开发的新型HA水凝胶通过水解和酶促机制降解，将作为3D网络进行研究，以促进MSC软骨形成和构建成熟。降解的时间变化将通过可降解基团的类型、交联密度以及与没有水解降解基团的HA大分子单体的共聚来改变。在第三个目标中，将对水凝胶结构施加滑动接触的机械加载生物反应器应用于目标1和2中确定的载有MSC的水凝胶，重现软骨的正常生理加载模式。将在短期（基因表达）和长期（基质生产、组织和力学）评价机械预处理参数。这些目的旨在允许测试我们的假设，即控制MSC微环境并包含正常发育期间存在的信号，这些信号对软骨形成和成熟既允许又有指导意义，将导致最终构建体具有类似于天然组织的特性。公共卫生相关性：该项目开发了一种临床相关的软骨修复方法，使用MSC负载水凝胶进行生理机械调节，以模拟从胚胎到成年发生的组织形成和成熟的发育模式。该生物材料系统提供受体-基质相互作用、受控力学和定制的降解谱，以增强包封的MSC的分化以及形成的细胞外基质（ECM）的积累和分布。如果成功的话，这种方法将克服软骨组织工程中的一个主要障碍，提供一种结构上更相关的ECM，具有增强的机械性能，以支持关节中发现的强烈负荷。这种软骨组织工程技术可以帮助治疗数百万因创伤或疾病而导致软骨丧失和退化的患者。

DESCRIPTION (provided by applicant): Articular cartilage lines the surfaces of joints and transmits the forces generated with loading. Due to limitations in cartilage's natural healing capacity, and given the increasing incidence of osteoarthritis, there exists a growing demand for cell-based strategies for repair. Tissue engineering, and particularly those approaches based on autologous mesenchymal stem cells (MSCs), is evolving as a clinically relevant technique to promote cartilage regeneration. Yet, the mechanics and ECM organization of engineered constructs for implantation have yet to match those of the native tissue and are insufficient to support joint loading. Most cartilage tissue engineering efforts replicate early stages of cartilage development, sequestering a high density of cartilaginous ECM producing cells in a defined volume. However, significant differences exist between these early rapid stages of cartilage formation and the gradual remodelling (maturation) that enables its adult function. With load-bearing use, cartilage ECM is wholly remodelled into the unique composition and architecture of the adult tissue. This transformative process is driven by a multitude of temporal factors (chemical, mechanical, and soluble). Our approach to cartilage tissue engineering adopts concepts of this complex developmental paradigm (from embryo to adult) with a synthetic hydrogel based on a natural ECM component, which provides initial receptor-matrix binding, controlled mechanics, and tailored degradation profiles. These gels, coupled with physiologic loading and temporal application of relevant soluble factors will re-create developmental microenvironments that both enable and encourage functional cartilage production by MSCs in 3-D culture. In the first Aim, MSCs will be encapsulated in HA hydrogels previously investigated as chondrocyte carriers with a range of structures that can influence cell viability, receptor-binding, diffusion, and ultimately, neocartilage properties and cultured under a range of conditions (i.e., cellular density and temporal variations in soluble factors). In the second Aim, novel HA hydrogels recently developed in our laboratory that degrade via both hydrolytic and enzymatic mechanisms will be investigated as 3D networks to promote MSC chondrogenesis and construct maturation. Temporal changes in degradation will be altered through the type of degradable group, crosslinking density, and copolymerization with HA macromers without hydrolytically degradable groups. In the third Aim, a mechanical loading bioreactor that applies sliding contact to hydrogel constructs will be applied to MSC-laden hydrogels identified in Aims 1 and 2, recapitulating normal physiologic loading patterns of cartilage. Mechanical preconditioning parameters will be evaluated in the short term (gene expression) and long term (matrix production, organization, and mechanics). These Aims were designed to allow the testing of our hypotheses that control over the MSC microenvironment and inclusion of signals present during normal development that are both permissive and instructive for cartilage formation and maturation will lead to final constructs with properties akin to native tissue. PUBLIC HEALTH RELEVANCE: This project develops a clinically-relevant approach to cartilage repair using MSC-laden hydrogels with temporally controlled structures subjected to physiologic mechanical conditioning to mimic the developmental paradigm of tissue formation and maturation that occurs from the embryo to adulthood. This biomaterial system provides receptor-matrix interactions, controlled mechanics, and tailored degradation profiles to enhance the differentiation of encapsulated MSCs and the accumulation and distribution of formed extracellular matrix (ECM). If successful, this approach would surmount a major hurdle in cartilage tissue engineering to provide a more structurally relevant ECM with enhanced mechanical properties to support the intense loads found in the joint. This cartilage tissue engineering technique could aid in the treatment of millions of patients afflicted with debilitating cartilage loss and degeneration due to trauma or disease.