The Regenerative Role of Skeletal Stem Cells in Homeostasis and Trauma

Abstract

More than six million fractures occur in North America annually, which can be complicated by age, infection, and degenerative disease. Skeletal regeneration is known to be mediated by skeletal stem cells (SSCs), which play a central role in the maintenance and repair of bone. Our group recently identified a novel self-renewing and multipotent population of SSCs in the growth plate and periosteum of postnatal mice. This previous work focussed on population-level kinetics, and thus far, the potential of postnatal SSCs at the single cell level and their role in fracture repair has not been established. Moreover, no attempts to model SSC proliferation kinetics exist. Therefore, the aims of this work were to (1) characterize SSCs at the single cell level in vivo, (2) determine SSC-mediated regeneration following fracture, and (3) develop a mathematical model of SSC population kinetics. Firstly, lineage tracing is performed on individual SSC clones using a multicolour mouse model. Herein, the existence of multipotent SSC-derived clones in the postnatal skeleton is shown at the single cell level, which persist and retain bipotency 48 days post-labelling. These clones are shown to have broad proliferative variability, demonstrated by contrasting clonal analysis of quiescent and proliferative clones. Secondly, the role of SSCs in fracture repair is quantified using a novel blunt force trauma model, capable of generating closed fractures stabilized by intramedullary fixation. Dual lineage tracing and imaging cytometry quantifies SSC-mediated regeneration across entire, intact mid-diaphyseal femoral bone sections and demonstrates that postnatal SSCs participate in fracture repair. Thirdly, a novel, predictive SSC kinetic model is presented which accounts for self-renewal, genetic marker expression, proliferative capacity, and cell turnover through a unique domain partitioning strategy. The model is validated against biological data and is applied to an aging model, offering meaningful insight into age-dependent fluctuations in regenerative capacity. Ultimately, this work represents a significant advancement in the characterization of postnatal SSCs as we strive to develop novel therapeutic strategies targeting fracture regeneration and the prevention of degenerative bone diseases.