Development of a synthetically modified fibronectin fragment as a building block for recyclable biomaterials
Embargo Date
2024-05-23
OA Version
Citation
Abstract
Over the past few decades, the fields of tissue engineering and biomaterials have increasingly intersected to develop therapies designed for interaction with cells. While biomaterials with tunable mechanical and biochemical properties have been developed to direct cell behavior, the cell-material interface progressively changes as cells deposit new extracellular matrix (ECM) or degrade materials via hydrolytic or enzymatic digestion. One major challenge is that cells cannot actively remodel preexisting biomaterials into nascent tissue, thus signals that direct cell behaviors are gradually lost. Fibronectin (FN), a key player in the regulation of cellular processes and adsorption onto material surfaces, is readily remodeled during cell-mediated assembly of a fibrillar network during wound healing. Given new insights into the cell’s ability to recycle preexisting FN, we explored the development of synthetically modified fibronectin, a class of synthetic biomaterials that can be delivered to wound environments and directly integrated into native ECM during remodeling. This thesis aims to (1) demonstrate proof-of-principle that synthetic materials can be integrated within newly formed ECM when conjugated to FN, (2) explore functional domains of FN and how they contribute to FN recycling, and (3) assess biofunctionality of synthetic materials after integration into newly formed ECM. We found that PEGylated FN can be recycled and deposited in a new provisional matrix by fibroblasts, although at a lower efficiency than full length FN labeled with AF488. To better understand recycling and how specific functional domains within FN contribute to it, experiments were conducted with single domain FN fragments generated by proteolytic cleavage and with chimeric FN mimetics containing both cell binding and matrix-binding domains. While proteolytically cleaved fragments were degraded by cells, chimeric FN mimetics underwent limited recycling, suggesting that more than one domain may be necessary for recycling and that interactions with both ECM proteins and cells are required. Informed by these studies, new chimeric fibronectin fragments composed of ECM and cell binding domains were designed, cloned and inserted into a GST fusion vector to generate recombinant FN proteins with a higher efficiency of recycling. Finally, we demonstrated that fibroblasts recycle and integrate biotinylated FN in newly formed tissue, and biotinylated FN retains its bioactive sites for binding partners after undergoing recycling. Together, our studies suggest that coupling biomaterials to fibronectin is a promising strategy to integrate materials in native tissues.