Peptide Discovery for Chondrogenesis

项目摘要 在治疗患有局灶性软骨损伤的患者时，防止进展为 骨关节炎是实现真正的功能性透明软骨。R21项目的长期目标是 创造一种现成的生物材料，可以填充任何形状的软骨损伤，在关节镜下植入， 再生透明样软骨，而不需要昂贵的生长因子或外源细胞。的秘诀 获得软骨诱导生物材料的成功在于肽， 并与生物材料结合以引导内源性骨髓源性间充质细胞的分化 干细胞（BMSCs）。目前缺乏严格的、系统的和可重复的方法来鉴定新的肽 软骨再生在这一空白中，我们介绍了一种新的肽发现策略，以再生医学。 我们的方法采用肽微阵列，这是劳动密集度低，成本低，速度快得多， 传统方法（例如，噬菌体展示）来快速地筛选大量的肽序列。 在我们的初步研究中，我们用肽微阵列方法检测了TGF-β3， 新的候选肽，我们很高兴地报告，这些新发现的肽中有三个导致了 显著上调大鼠BMSCs II型胶原基因表达。我们现在有一个激动人心的机会 将这项技术扩展到其他增长因素。例如，BMP-6和BMP-7已经展示了强大的 和TGF-β驱动的软骨形成的有效放大。因此，本提案的目的是评估 我们最近鉴定的TGF-β3肽以及鉴定的有希望的新肽的软骨诱导性 从BMP-6或BMP-7通过肽微阵列方法，然后评估领先的肽在3D 用于BMSC软骨形成水凝胶。主要假设是软骨诱导肽将优于TGF-β。 β3在软骨形成中的作用，将通过以下具体目的进行测试：1）发现额外的新肽 从肽微阵列序列，2）筛选基于软骨诱导性的高通量肽 细胞球体，和3）评估精制肽在3D软骨组织工程中的功效。 目的1将鉴定来自BMP-6和BMP-7的肽，以与上述TGF-β3激发的细胞协同作用。 我们初步研究的肽。Aim 2利用细胞球体作为高通量工具来筛选 软骨诱导，并验证肽的作用机制是否与其宿主蛋白一致。后 目标2筛选步骤，目标3将在3D水凝胶系统中用快速- 交联戊烯酸酯修饰的透明质酸（PHA）生物材料。该项目的预期成果将是 具有结合的软骨诱导肽的有效组合的PHA水凝胶，用于未来的探索， 体内软骨缺损修复。这个R21项目的成功完成为肽发现提供了新的工具 其他人可以很容易地适应，改变了再生医学研究人员 世界各地都在开发自己的生物活性材料，以引导人体自身的干细胞再生组织。

PROJECT SUMMARY In treating a patient with a focal cartilage injury, the greatest challenge in preventing the progression to osteoarthritis is achieving true functional hyaline cartilage. The long-term goal of this R21 project is therefore to create an off-the-shelf biomaterial that will fill a cartilage injury of any shape, be implanted arthroscopically, and regenerate hyaline-like cartilage without the need for costly growth factors or exogenous cells. The secret to success in achieving a chondroinductive biomaterial resides in peptides, which can be reproducibly synthesized and conjugated to biomaterials to guide the differentiation of endogenous bone marrow-derived mesenchymal stem cells (BMSCs). There is a lack of rigorous, systematic, and reproducible methods to identify new peptides for cartilage regeneration. In this void, we introduce a new peptide discovery strategy to regenerative medicine. Our approach employs peptide microarrays, which are less labor intensive, less costly, and much faster than traditional methods (e.g., phage display) to quickly iterate vast numbers of peptide sequences. In our preliminary studies, we examined TGF-β3 with the peptide microarray approach to identify eight unique new candidate peptides, and we are pleased to report that three of these newly discovered peptides led to remarkable upregulation of collagen II gene expression in rat BMSCs. We now have the exciting opportunity to expand this technology to other growth factors. BMP-6 and BMP-7 for example have demonstrated a powerful and potent amplification of TGF-β-driven chondrogenesis. The objective of this proposal is therefore to evaluate the chondroinductivity of our recently-identified TGF-β3 peptides alongside promising new peptides identified from BMP-6 or BMP-7 via the peptide microarray approach, and then to evaluate leading peptides in a 3D hydrogel for BMSC chondrogenesis. The chief hypothesis is that chondroinductive peptides will outperform TGF- β3 in chondrogenesis, to be tested by the following specific aims: 1) To discover additional new peptide sequences from peptide microarrays, 2) To screen peptides based on chondroinductivity with high-throughput cell spheroids, and 3) To evaluate refined peptides for efficacy in 3D cartilage tissue engineering. Aim 1 will identify peptides from BMP-6 and BMP-7 to synergize with the aforementioned TGF-β3-inspired peptides from our preliminary studies. Aim 2 leverages cell spheroids as a high-throughput tool to screen for chondroinduction and to verify whether peptide mechanisms of action are consistent with their host protein. After the Aim 2 screening step, Aim 3 will evaluate leading peptide candidates in a 3D hydrogel system with a fast- crosslinking pentenoate-modified hyaluronic acid (PHA) biomaterial. The intended outcome of this project will be a PHA hydrogel with a potent combination of conjugated chondroinductive peptides for future exploration in cartilage defect repair in vivo. Successful completion of this R21 project offers a new tool for peptide discovery in regenerative medicine that others can easily adapt, changing how investigators in regenerative medicine worldwide develop their own bioactive materials to guide the body’s own stem cells to regenerate tissues.