Optimizing chondrogenic factors and protein delivery methods for cartilage repair

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Joint injuries are common and often result in damage to cartilage, which has a limited ability to repair itself. Tissue engineering is a promising approach for improving cartilage healing in which biomaterials and chemical factors are supplied to direct cells to create a new tissue. The objective of this thesis was to optimize cartilage-like extracellular matrix production by investigating the effects of Dexamethasone (Dex) and HB-IGF- I (heparin-binding insulin-like growth factor-1) on cells encapsulated in the self-assembling peptide RAD and agarose hydrogels. Dex is a synthetic corticosteroid that has been shown to improve cartilage-like tissue production by bone marrow stromal cells (BMSCs), but the mechanisms underlying BMSC response to Dex are not understood. The hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine BMSCs in RAD and agarose hydrogels was tested. The effects of Dex were dependent on the hydrogel material and the species/age of the BMSCs. Importantly, Dex reduced aggrecanase-mediated degradation of matrix in both agarose and RAD hydrogels and for both young bovine and adult human BMSCs. HB-IGF-1, a fusion protein of the heparin binding domain of HB-EGF and IGF-1, can be retained in cartilage matrix and stimulate proteoglycan synthesis with a single dose, whereas unmodified IGF-1 easily diffuses out of cartilage tissue. The RAD peptide was used as a scaffold for retaining growth factor to stimulate encapsulated chondrocytes and adjacent cartilage tissue. RAD was modified by adsorption of HB-IGF-1 before and after RAD assembly, as well as adsorption of heparan sulfate (HS) and IGF-1. The RAD material retained HS adsorbed pre-assembly and HB-IGF-1 delivered in both adsorption methods. Adsorbed HB-IGF-1 and IGF-1 led to increased aggrecan content regardless of the method of adsorption. A trend was found for increased proteoglycan synthesis in adjacent explants as well. RAD self-assembling hydrogels are a promising material for culturing BMSCs undergoing chondrogenesis, retaining, and delivering HB-IGF-1. Dex decreases aggrecanase activity of differentiating BMSCs and adsorbed HB-IGF-1 appears to enhance aggrecan production by encapsulating chondrocytes and adjacent tissue. These findings show potential for improving cartilage repair in vivo.by Emily Marie Florine.Ph.D

Effects of Dexamethasone on Mesenchymal Stromal Cell Chondrogenesis and Aggrecanase Activity: Comparison of Agarose and Self-Assembling Peptide Scaffolds

Objective: Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anticatabolic 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)[subscript 4] self-assembling peptide and agarose hydrogels in medium containing TGF-β1±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)[subscript 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.National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (Grant EB003805

Delivering Heparin-Binding Insulin-Like Growth Factor 1 with Self-Assembling Peptide Hydrogels

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.National Science Foundation (U.S.). Graduate Research Fellowship ProgramNational Institutes of Health (U.S.) (Grant EB003805)National Institutes of Health (U.S.) (Grant AR060331)Whitaker Health Sciences Fund FellowshipMassachusetts Life Sciences CenterBiomeasure, Inc