Gradient-based strategy for osteochondral regeneration

描述（由申请人提供）：本申请的长期目标是开发一种基于干细胞的骨软骨生物材料，可用于重建骨关节炎（OA）和创伤损伤的关节。为了实现这一目标，我们开发了一种新的梯度支架技术，可以对支架设计进行精确的时空控制，创建任何所需轮廓的信号（生长因子）和机械刚度梯度。尽管信号梯度对胚胎发生、伤口愈合和无数其他生物过程至关重要，但它们在肌肉骨骼组织工程中尚未得到系统的研究。此外，刚度梯度在生物材料中几乎没有被探索过，我们独特的方法引入了一种全新的技术来适应骨和软骨的不同机械需求。脐带基质干细胞（UCMSCs）也是肌肉骨骼组织工程的新成员，与其他干细胞来源相比，它具有许多关键优势，具有巨大的潜力。因此，本建议的总体目标是采用这些创新方法的组合来设计无缝骨软骨结构，用于治疗兔膝关节缺陷。无缝设计的意义在于能够创建一个单一的、集成的骨软骨组织，而不是离散的骨和软骨区域。主要假设是，UCMSCs在一种新型梯度驱动支架设计中，将导致机械上可行的骨软骨结构，模仿天然组织从骨到带状组织软骨的无缝过渡。为了验证这一假设，我们提出了以下具体目标：1)开发和表征含有刚度和生长因子梯度的新型支架，2)在体外设计无缝骨软骨结构，以及3)确定骨软骨结构在兔膝关节缺损模型中的功效。我们的总体策略是开发一种异质支架，它将包含一个机械刚度梯度，从软骨区域到骨区域增加，并释放精确控制的软骨因子和成骨因子梯度，以分化干细胞。这些梯度是通过改变沿支架长度的“成骨”和“软骨”微球的相对数量来实现的，这些微球的材料组成和包封信号不同。这些微球的材料组成和生长因子载荷将在设计驱动的第一个目标中确定。在接下来的两个目标中，基于梯度的支架将植入干细胞，其中UCMSCs将与长期存在的金标准骨髓来源间充质干细胞（BMSCs）进行比较，以验证UCMSCs在体外和体内都优于BMSCs的假设。该项目的成功完成将提供基于梯度的支架，该支架由FDA批准的材料与现成的、无争议的、免疫兼容的人类细胞来源相结合。此外，这项技术将对未来需要梯度或集成界面的其他领域产生重大影响，例如神经再生，韧带/骨界面等。

DESCRIPTION (provided by applicant): The long-term objective of this application is to develop a stem-cell based osteochondral biomaterial that can be used for reconstructing joints damaged by osteoarthritis (OA) and trauma. Toward this objective, we have developed a novel gradient scaffold technology that affords precise spatiotemporal control of the scaffold design, creating both signal (growth factor) and mechanical stiffness gradients of any desired profile. Although signal gradients are vital to embryogenesis, wound healing, and countless other biological processes, they have yet to be systematically investigated in musculoskeletal tissue engineering. Moreover, stiffness gradients remain virtually unexplored in biomaterials, and our unique approach introduces an entirely new technology to accommodate the contrasting mechanical demands of bone and cartilage. Also new to musculoskeletal tissue engineering are umbilical cord matrix stem cells (UCMSCs), which possess tremendous potential with numerous key advantages over other stem cell sources. The overall goal of this proposal is thus to employ a combination of these innovative approaches to engineer seamless osteochondral constructs for the treatment of rabbit knee defects. The significance of the seamless design lies in the ability to create a single, integrated osteochondral tissue instead of discrete bone and cartilage regions. The chief hypothesis is that UCMSCs in a novel gradient-driven scaffold design will lead to a mechanically viable osteochondral construct that will mimic the seamless transition of native tissue from bone to zonally organized cartilage. To test this hypothesis, we propose the following specific aims: 1) to develop and characterize novel scaffolds containing stiffness- and growth factor-gradients, 2) to engineer seamless osteochondral constructs in vitro, and 3) to determine the efficacy of osteochondral constructs in a rabbit knee defect model. Our overall strategy is to develop a heterogeneous scaffold that will contain a mechanical stiffness gradient, increasing from the cartilage region to the bone region, and also release precisely-controlled and opposing gradients of chondrogenic and osteogenic factors to differentiate stem cells. These gradients are accomplished by varying the relative numbers of "osteogenic" and "chondrogenic" microspheres along the scaffold length, which differ in material composition and encapsulated signal. The material composition and growth factor loading for these microspheres will be determined in the design-driven first aim. The gradient-based scaffolds will be seeded with stem cells in the next two aims, where UCMSCs will be compared to the long standing gold standard, bone-marrow derived mesenchymal stem cells (BMSCs), to test the hypothesis that UCMSCs will outperform BMSCs both in vitro and in vivo. Successful completion of this project will deliver gradient-based scaffolds comprised of FDA- approved materials in combination with a readily available, non-controversial, and immune-compatible human cell source. Moreover, this technology will have a high impact on other fields in the future where a gradient or integrated interface is desired, such as nerve regeneration, the ligament/bone interface, and beyond. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the most common form of arthritis, affecting approximately 21 million Americans at an annual cost of over $60 billion. Given the severely impaired quality of life and the limitations of current treatments, there is an urgent need to explore new treatment strategies for the steadily growing number of OA patients. Toward that end, the proposed research will produce a continuous osteochondral tissue to heal defects in joints ravaged by arthritis and trauma.