Peptide Discovery for Chondrogenesis

软骨形成肽的发现

• 批准号: 10594547
• 负责人: Michael S. Detamore
• 金额: $ 16.74万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594547
• 关键词: 3-Dimensional; Accounting; Adhesions; Adult; Affinity; Amino Acids; BMP6 gene; BMP7 gene; Binding; Biocompatible Materials; Biomechanics; Bone Morphogenetic Proteins; Cartilage; Cartilage Matrix; Cartilage injury; Cells; Chondrogenesis; Collagen; Custom; Defect; Degenerative polyarthritis; Devices; Diagnosis; Encapsulated; Environment; Future; Gene Expression; Goals; Growth Factor; Growth Factor Interaction; Hyaline Cartilage; Hyaluronic Acid; Hydrogels; Implant; In Vitro; Inferior; Ligands; Membrane; Methods; Microarray Analysis; Natural regeneration; Nature; Osteogenesis; Outcome; Pathway interactions; Patients; Peptides; Persons; Phage Display; Pharmaceutical Preparations; Printing; Production; Protein Fragment; Proteins; Rattus; Regenerative Medicine; Reporting; Reproducibility; Research Personnel; Shapes; Signal Transduction; Slide; Source; System; TGFBR2 gene; Technology; Testing; Time; Tissue Engineering; Tissues; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Translations; Traumatic Arthropathy; United States; Up-Regulation; bone imaging; bone marrow mesenchymal stem cell; bone morphogenetic protein receptor type I; cartilage cell; cartilage regeneration; cost; crosslink; efficacy evaluation; improved; in vivo; interest; prevent; protein aminoacid sequence; receptor; release factor; repaired; screening; self assembly; stem cells; success; synergism; tissue regeneration; tool; transforming growth factor beta3; translational potential

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.

项目概要 在治疗局灶性软骨损伤的患者时，最大的挑战是防止病情进展 骨关节炎正在实现真正的功能性透明软骨，因此该 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，通过关节镜植入，并且 无需昂贵的生长因子或外源细胞即可再生透明样软骨的秘密。 软骨诱导生物材料的成功在于肽，它可以重复合成 并与生物材料结合以指导内源性骨髓间充质细胞的分化 干细胞（BMSC）缺乏严格的、系统的和可重复的方法来鉴定新的肽。 在这个空白中，我们为再生医学引入了一种新的肽发现策略。 我们的方法采用肽微阵列，它的劳动强度较小，成本较低，而且速度比 传统方法（例如噬菌体展示）快速迭代大量肽序列。 在我们的初步研究中，我们使用肽微阵列方法检查了 TGF-β3，以确定八种独特的 新的候选肽，我们很高兴地报告，其中三种新发现的肽导致 大鼠 BMSC 中胶原蛋白 II 基因表达的显着上调 我们现在有令人兴奋的机会 将此技术扩展到其他生长因子，例如 BMP-6 和 BMP-7 已显示出强大的功能。 因此，本提案的目的是评估 TGF-β 驱动的软骨形成的有效放大。 我们最近鉴定的 TGF-β3 肽以及鉴定的有前途的新肽的软骨诱导性 通过肽微阵列方法从 BMP-6 或 BMP-7 中提取，然后以 3D 形式评估先导肽 用于 BMSC 软骨形成的水凝胶 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，将通过以下具体目标进行测试：1）发现其他新肽 肽微阵列序列，2) 基于软骨诱导性高通量筛选肽 细胞球体，以及 3) 评估精制肽在 3D 软骨组织工程中的功效。 目标 1 将鉴定来自 BMP-6 和 BMP-7 的肽，以与上述 TGF-β3 启发的肽协同作用 Aim 2 利用细胞球作为高通量工具来筛选肽。 软骨诱导并验证肽的作用机制是否与其宿主蛋白一致。 在 Aim 2 筛选步骤中，Aim 3 将使用快速评估方法评估 3D 水凝胶系统中的主要候选肽。 该项目的预期成果是交联戊烯酸酯改性透明质酸（PHA）生物材料。 PHA 水凝胶与共轭软骨诱导肽的有效组合，用于未来的探索 R21 项目的成功完成为体内软骨缺损修复提供了一种新的肽发现工具。 其他人可以轻松适应的再生医学领域，改变再生医学研究人员的方式 世界各地开发自己的生物活性材料来引导人体自身的干细胞再生组织。