Peptide Discovery for Chondrogenesis

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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.