Bio-Functionalized PEG Hydrogels to Study and Direct Mesenchymal Stem Cell Migration and Differentiation

摘要

The in vivo microenvironment niche is a dynamic structure that locally presents a multitude of biophysical and biochemical signals that regulate cell behavior. Reciprocally, cells remodel this local microenvironment through altering the physical structure and biochemical composition of this heterogeneous milieu. These cell-matrix interactions play an integral role in directing mesenchymal stem cell (MSC) behavior (e.g., migration, proliferation, differentiation) during bone regeneration. The focus of this thesis is to exploit synthetic PEG-based hydrogels and bio-click conjugation reactions to functionalize relevant bio-molecules (e.g., mimetic peptide, proteins) to recapitulate important facets of the native extracellular matrix (ECM) to study their role in regulating MSC behavior (e.g., migration, differentiation, proliferation) and elucidate aspects of how MSCs interact and remodel their local niche.;First, peptide-functionalized thiol-ene hydrogels are utilized to tune the biophysical (e.g., crosslinking density) and biochemical (e.g., adhesive ligand density) nature of the MSC microenvironment and observe their effect on 3D MSC spreading and migration. Next, microrheological techniques are exploited to elucidate the dynamic cell-mediated remodeling of the local hydrogel structure during 3D MSC migration. Both of these studies provide insight and characterization of cell-matrix interactions within enzymatically degradable hydrogels. Thiol-ene photoconjugation is then employed to functionalize PEG hydrogel scaffolds with full-length proteins (e.g., stromal derived factor 1alpha, bone morphogenetic protein 2) to manipulate MSC behavior in vitro. Subsequently, this platform is introduced into a critical-sized bone defect model to study the effect of immobilized protein signals on cellular invasion and mineralized tissue formation during bone regeneration in vivo. Finally, to probe the singular and synergistic effects of multiple protein signals on MSC migration, differentiation or proliferation a series of bio-click reactions (e.g., thiol-ene, thiol-yne, strain-promoted azide alkyne cycloaddition, inverse electron demand Diels-Alder) are exploited to spatiotemporally conjugate proteins to hydrogel scaffolds in biologically complex serum solutions. The knowledge garnered from these studies will contribute to better engineering of synthetic hydrogels as in vitro cell culture substrates, drug delivery vehicles, and cell carrier or recruitment platforms for tissue engineering applications.