Guiding Osteogenic Lineage Commitment: The Role of Mesenchymal Stem Cell Biology and the Mechanical Microenvironment

Today, bone is the second most implanted tissue in the body and although current technologies have the ability to restore function, there are inherent limitations with many complications. Advancements in the field of bone tissue engineering would alleviate the limitations of modern bone replacement technologies and enable a better quality of life for patients needing craniofacial reconstruction, segmental void replacement, spinal fusions and total joint replacements. Bone is an exceptionally mechanosensitive tissue, with both development and health adult tissue homeostasis requiring mechanical loading; however, the role of the mechanical microenvironment in controlling osteogenic differentiation and the molecular mechanisms responsible for transducing a physical signal into a cell fate commitment are currently unknown. The purpose of this dissertation is to broaden the current understanding of mesenchymal stem cell (MSC) biology and the role of the mechanical microenvironment in guiding osteogenic lineage commitment. The first study of this dissertation shows that periosteal derived progenitor cells and homogeneous populations of fibroblasts have a similar potential to commit to the osteogenic and adipogenic lineages suggesting that fibroblasts may be a more plastic cell than previously thought, given the proper environmental cues. The second study demonstrates that oscillatory fluid flow has the potential to induce osteogenic differentiation via Runx2 upregulation, and furthermore activates the small GTPase RhoA as well as its effector, ROCK. RhoA and flow have an additive effect on Runx2 expression, which is similar to their additive effect on actin fibril organization. Additionally, I show that a dynamic actin cytoskeletal under tension is necessary for the transduction of a physical signal into altered gene expression. The third study demonstrates that RhoA activation is dependent on Wnt5a signaling via the tyrosine receptor, ROR2. Additionally, this study shows that flow also induces beta-catenin signaling, which may be mediated by adherens junctions. Both Wnt5a signaling and beta-catenin signaling are necessary for mechanically induced osteogenic differentiation, indicating multiple pathways must act as switches to initiate cell fate. Finally, the last study investigates the relationship between epigenetic and genetic programming as it relates to osteogenic differentiation. This study shows that a mechanical stimulus is a potent signal capable of epigenetically modifying the chromatin via DNA methylation, which is the only modification that is inherited by future progeny. Furthermore, the decrease in methylation correlates with an increase in Osteopontin gene expression suggesting that the mechanical microenvironment of MSCs has the potential to induce stable alterations in gene expression resulting in osteogenic lineage commitment. Understanding MSC fate potential, the role of the mechanical microenvironment on osteogenic differentiation, and the molecular mechanisms involved in fate commitment will enhance the field of tissue engineering and promote novel approaches in bone regeneration.