Peptide Discovery for Chondrogenesis

项目总结 在治疗局灶性软骨损伤患者时，防止进展到 骨关节炎正在实现真正的功能性透明软骨。因此，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，可以在关节镜下植入，以及 再生透明样软骨，不需要昂贵的生长因子或外源细胞。秘诀是 成功获得软骨诱导性生物材料的关键在于可重复合成的多肽。 并与生物材料结合诱导内源性骨髓间充质细胞分化 干细胞(BMSCs)。缺乏严格的、系统的和可重复的方法来鉴定新的多肽 用于软骨再生。在这一空白中，我们将一种新的肽发现策略引入再生医学。 我们的方法采用了多肽微阵列，它比传统的多肽芯片更节省人力、成本和速度 传统的方法(例如，噬菌体展示)快速迭代大量的多肽序列。 在我们的初步研究中，我们用多肽微阵列方法检测了转化生长因子-β3，以鉴定出八种独特的 新的候选肽，我们很高兴地报告，这些新发现的三个肽导致了 显著上调大鼠骨髓间充质干细胞II型胶原基因表达。我们现在有一个令人兴奋的机会 将这项技术推广到其他增长因素。例如，BMP-6和BMP-7已经证明了一种强大的 并有效放大转化生长因子-β驱动的软骨形成。因此，这项提案目的是评价 我们最近鉴定的转化生长因子-β-3多肽和有希望的新多肽的软骨诱导活性 通过多肽微阵列方法从BMP-6或BMP-7中提取，然后在3D中评估先导肽 用于骨髓间充质干细胞软骨形成的水凝胶。主要的假设是，软骨诱导肽的作用将超过转化生长因子-2。 β3在软骨形成中的作用，将通过以下特定目的进行测试：1)发现更多的新肽 来自多肽芯片的序列，2)高通量筛选基于软骨诱导活性的多肽 细胞球体；3)评价精制肽在三维软骨组织工程中的效果。 Aim 1将鉴定BMP-6和BMP-7的多肽，以与上述转化生长因子-β3启发的多肽协同作用 来自我们初步研究的多肽。AIM 2利用细胞球体作为高通量工具来筛选 并验证多肽的作用机制是否与其宿主蛋白一致。之后 目标2筛选步骤，目标3将评估3D水凝胶系统中的领先候选多肽，具有快速- 交联型戊烯酸改性透明质酸(PHA)生物材料。这个项目的预期结果将是 一种含有有效结合共轭软骨诱导肽的PHA水凝胶 体内修复软骨缺损区。该R21项目的成功完成为多肽的发现提供了新的工具 在其他人可以很容易适应的再生医学中，改变了再生医学的研究人员 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.