Rational design to control multipotent stromal cell migration for applications in bone tissue engineering and injury repair

Abstract

Multipotent stromal cells derived from bone marrow hold great potential for tissue engineering applications because of their ability to home to injury sites and to differentiate along mesodermal lineages to become osteocytes, chondrocytes, and adipocytes to aid in tissue repair and regeneration. One key challenge, however, is the scarcity of MSC numbers isolated from in vivo, suggesting a role for biomimetic scaffolds in the cells' ex vivo expansion before reintegration into target tissue. Toward this end, immobilized epidermal growth factor (tEGF) has recently been found to promote MSC survival and proliferation and is a prime candidate to be incorporated into scaffolds to control MSC behavior. To rationally and effectively design scaffolds to drive MSC responses of survival, proliferation, migration, and differentiation, we must first understand these responses and the underlying protein signaling pathways that mediate them. While our knowledge of MSC behavior is limited as a field, MSC migration is particularly less studied despite being critical for tissue and scaffold infiltration. In this thesis, we quantitatively investigate the effects of tEGF and extracellular matrix (ECM) on MSC migration response and signaling. We take a systems level computational view to show a combined biomaterials and small molecule approach to control MSC migration. Cell migration is a delicately integrated biophysical process involving polarization and protrusions at the cell front, adhesion and translocation of the cell body through contractile forces, followed by disassembly of adhesion complexes at the cell rear to allow detachment and productive motility. This process is mediated by a multitude of crosstalking signaling pathways downstream of integrin and growth factor activation. Using a poly(methyl methacrylate)-grafted-poly(ethylene oxide) (PMMA-g-PEO) copolymer base, we modify the PEO sidechains with immobilized epidermal growth factor (tEGF) as a model system for biomimetic scaffolds. We systematically adsorb fibronectin, vitronectin, and collagen ECM proteins to alter surface adhesiveness and measure MSC migration responses of speed and directional persistence alongside intracellular activities of EGFR, ERK, Akt, and FAK phosphoproteins. While tEGF and ECM proteins differentially affected signaling and migration, univariate correlations between signals and responses were not informative, prompting the need for multivariate modeling to identify key patterns. Using decision tree "signal-response" modeling, we predicted that inhibiting ERK on collagen-adsorbed tEGF polymer surfaces would increase cell mean free path (MFP) by increasing directional persistence. We confirmed this experimentally, successfully demonstrating a two-layer approach-"coarse" biomaterials followed by small molecules "fine-tuning"-to precisely and differentially control MSC migration speed and persistence, setting the stage for combination therapies for bone tissue engineering.