ABSTRACT
Heparin-binding insulin-like growth factor 1 (HB-IGF-1) is a fusion protein of IGF-1 with the HB domain of heparin-binding epidermal growth factor-like growth factor. A single dose of HB-IGF-1 has been shown to bind specifically to cartilage and to promote sustained upregulation of proteoglycan synthesis in cartilage explants. Achieving strong integration between native cartilage and tissue-engineered cartilage remains challenging. We hypothesize that if a growth factor delivered by the tissue engineering scaffold could stimulate enhanced matrix synthesis by both the cells within the scaffold and the adjacent native cartilage, integration could be enhanced. In this work, we investigated methods for adsorbing HB-IGF-1 to self-assembling peptide hydrogels to deliver the growth factor to encapsulated chondrocytes and cartilage explants cultured with growth factor-loaded hydrogels. We tested multiple methods for adsorbing HB-IGF-1 in self-assembling peptide hydrogels, including adsorption prior to peptide assembly, following peptide assembly, and with/without heparan sulfate (HS, a potential linker between peptide molecules and HB-IGF-1). We found that HB-IGF-1 and HS were retained in the peptide for all tested conditions. A subset of these conditions was then studied for their ability to stimulate increased matrix production by gel-encapsulated chondrocytes and by chondrocytes within adjacent native cartilage. Adsorbing HB-IGF-1 or IGF-1 prior to peptide assembly was found to stimulate increased sulfated glycosaminoglycan per DNA and hydroxyproline content of chondrocyte-seeded hydrogels compared with basal controls at day 10. Cartilage explants cultured adjacent to functionalized hydrogels had increased proteoglycan synthesis at day 10 when HB-IGF-1 was adsorbed, but not IGF-1. We conclude that delivery of HB-IGF-1 to focal defects in cartilage using self-assembling peptide hydrogels is a promising technique that could aid cartilage repair via enhanced matrix production and integration with native tissue.
Subject(s)
Chondrocytes/cytology , Chondrocytes/physiology , Delayed-Action Preparations/chemistry , Heparin-binding EGF-like Growth Factor/administration & dosage , Insulin-Like Growth Factor I/administration & dosage , Peptides/chemistry , Adsorption , Animals , Cattle , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Chondrocytes/drug effects , Crystallization/methods , Delayed-Action Preparations/administration & dosage , Diffusion , Drug Design , Heparin-binding EGF-like Growth Factor/chemistry , Heparin-binding EGF-like Growth Factor/genetics , Hydrogels/chemical synthesis , Insulin-Like Growth Factor I/chemistry , Insulin-Like Growth Factor I/genetics , Materials Testing , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
OBJECTIVE: Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anti-catabolic effects in cartilage tissue engineering systems, though the mechanisms by which these effects are mediated are not well understood. We tested the hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine bone marrow stromal cells (BMSCs) cultured in self-assembling peptide and agarose hydrogels. DESIGN: We cultured young bovine and adult human BMSCs in (RADA)4 self-assembling peptide and agarose hydrogels in medium containing TGF-ß1±Dex and analyzed extracellular matrix composition, aggrecan cleavage products, and the effects of the glucocorticoid receptor antagonist RU-486 on proteoglycan content, synthesis, and catabolic processing. RESULTS: Dex improved proteoglycan synthesis and retention in agarose hydrogels seeded with young bovine cells, but decreased proteoglycan accumulation in peptide scaffolds. These effects were mediated by the glucocorticoid receptor. Adult human BMSCs showed minimal matrix accumulation in agarose, but accumulated ~50% as much proteoglycan and collagen as young bovine BMSCs in peptide hydrogels. Dex reduced aggrecanase activity in (RADA)4 and agarose hydrogels, as measured by anti-NITEGE Western blotting, for both bovine and human BMSC-seeded gels. CONCLUSIONS: The effects of Dex on matrix production are dependent on cell source and hydrogel identity. This is the first report of Dex reducing aggrecanase activity in a tissue engineering culture system.
