Gradient-based strategy for osteochondral regeneration

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，提供精确的时空控制的支架设计，创建信号（生长因子）和机械刚度梯度的任何所需的配置文件。虽然信号梯度对胚胎发育、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，生物材料中的刚度梯度几乎尚未被探索，我们独特的方法引入了一种全新的技术来适应骨骼和软骨的对比机械需求。脐带基质干细胞（UCMSCs）也是肌肉骨骼组织工程的新技术，它具有巨大的潜力，与其他干细胞来源相比具有许多关键优势。因此，本提案的总体目标是采用 结合这些创新方法，设计用于治疗兔膝关节缺损的无缝骨软骨结构。无缝设计的重要性在于能够创建单个、整合的骨软骨组织，而不是离散的骨和软骨区域。主要假设是， 新的梯度驱动的支架设计将导致机械上可行的骨软骨结构，其将模拟天然组织从骨到带状组织化软骨的无缝过渡。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，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，其将包含从软骨区域到骨区域增加的机械刚度梯度，并且还释放精确控制的成软骨和成骨梯度。 分化干细胞的因素。这些梯度是通过改变“成骨”和“软骨”微球的相对数量沿着支架长度，其不同的材料组成和封装的信号。这些微球的材料组成和生长因子载量将在设计驱动的第一个目标中确定。基于梯度的支架将与干细胞一起接种在 接下来的两个目标是将UCMSC与长期存在的金标准骨髓间充质干细胞（BMSC）进行比较，以验证UCMSC在体外和体内都优于BMSC的假设。该项目的成功完成将提供由FDA批准的材料组成的基于梯度的支架，并结合一种现成的、无争议的、免疫相容的人 细胞来源此外，这项技术将在未来对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。

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.