PROJECT SUMMARY The long-term goal is to achieve a chondroinductive biomaterial implant for regenerating injured cartilage. In treating a patient with a focal cartilage injury, the greatest challenge in preventing the progression to osteoarthritis is achieving true functional hyaline cartilage. Peptides have emerged as an exciting option to empower a translational class of reproducible biomaterials that may be capable of chondroinduction without the need for growth factors. An entirely different approach to peptide discovery was taken by asking the question of whether two different chondroinductive molecules?aggrecan and TGF-?3?may have anything in common. Through a comparison of aggrecan?s core protein sequence and the TGF-?3 proprotein, we identified a single novel peptide sequence: SPPEPS. This peptide has outperformed negative controls in chondrogenesis of rat bone marrow- derived cells (rBMSCs) in our preliminary studies. Moreover, upregulation of genes associated with chondrogenesis was observed, with preliminary proteomic analyses suggesting some pathways in common with TGF-? upregulation. Interestingly, we discovered that RGD and SPPEPS had a powerful synergistic effect with an order of magnitude greater collagen II gene expression by MSCs compared to either peptide alone. In our first in vivo attempt, pentenoate-modified hyaluronic acid (PHA) hydrogels with SPPEPS led to collagen II immunostaining 12 weeks after implantation in rabbit medial femoral condyle defects, with visible columns of chondrocyte-like cells in lacunae. The objective of this proposal is thus to test the chief hypothesis that acellular PHA with the chondroinductive peptide SPPEPS will lead to hyaline-like cartilage that is functionally comparable to healthy native cartilage, with the following specific aims: 1) Enhance synergistic chondroinductivity of SPPEPS with a BMSC-binding peptide, and 2) Regenerate functional hyaline cartilage in rabbit knees with the PHA- SPPEPS hydrogel. Aim 1 will explore SPPEPS chondroinduction on rBMSC spheroids, including a mechanistic TGF-? pathway analysis, followed by an investigation of rBMSCs seeded or encapsulated in PHA hydrogels with SPPEPS and a BMSC-binding peptide. Aim 2 will leverage the findings of Aim 1 to implant PHA hydrogels in rabbit medial femoral condylar cartilage defects, including a comparison between males and females with functional analysis of the structure and mechanical performance of regenerated cartilage. Successful completion of this R21 project offers a paradigm shift in cartilage regeneration, with the potential clinical impact of introducing a reproducible, low-cost, material-only device capable of regenerating hyaline cartilage for patients suffering from knee cartilage injuries, the biomaterials field impact of a flagship chondroinductive peptide that others can easily incorporate into their own novel materials, and a lasting impact by opening a new avenue of investigation worldwide in peptide discovery approaches for chondrogenesis.

R21AR077800

2021-09-24

2023-08-31