R. C. Ransom1,2, A. C. Carter3, A. Salhotra1,2, T. Leavitt1, O. C. Marecic1,2, M. Lopez1,2, M. Murphy1,2, C. K. Chan1,2, D. C. Wan1, H. Y. Chang3, M. T. Longaker1,2 1Hagey Laboratory For Pediatric Regenerative Medicine,Department Of Surgery, Division Of Plastic And Reconstructive Surgery, Stanford University School Of Medicine,Stanford, CA, USA 2Institute For Stem Cell Biology And Regenerative Medicine,Stanford University,Stanford, CA, USA 3Center For Personal Dynamic Regulomes,Stanford University,Stanford, CA, USA
Introduction: Accumulated evidence indicates that mechanical cues, which include physical forces, alterations in extracellular matrix mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for tissue regeneration. Although mechanotransduction is thought to occur via integration of multiple signaling pathways, the precise mechanism leading to downstream cellular responses is not well understood. We have developed a mouse model of mandibular distraction osteogenesis (DO) which allows for tracing of cell fate and genetic dissection of mechanotransduction during bone formation.
Methods: We examined cell-type-specific responses to mechanical force within distinct subpopulations of the mouse skeletal stem cell (mSSC) hierarchy. After determining that bone, cartilage, and stromal tissue are clonally derived in mice from lineage-restricted stem and progenitor cells in vivo, we employed this strategy to purify specific skeletogenic populations during mandibular distraction osteogenesis by prospective isolation using FACS. We employed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to profile open chromatin landscapes in these cell populations to understand the epigenetic changes in response to distraction. To investigate the role of mechanotransduction via focal adhesion kinase (FAK) in distraction osteogenesis, we inhibited FAK using the small molecule PF57223, a specific and potent inhibitor of FAK signaling. Three-dimensional reconstruction of μCT images of gradually distracted specimens revealed disrupted bone formation under conditions of FAK inhibition. ATAC-seq was employed for determination of FAK-responsive regions of the epigenome within each subpopulation of the skeletal stem cell hierarchy.
Results: We show that mechanical force augments the numbers and function of multiple cell populations across the skeletal hierarchy, including mouse skeletal stem and progenitor cells and their differentiated subsets. Mechanistically, distraction induces robust cell-matrix interactions that are coupled to cell-specific transcriptional responses via epigenomic pathways and pharmacological inactivation of this pathway disrupts bone formation.
Conclusion: Here we employ a rigorous mandibular DO model in mice that is genetically dissectable, allowing for detailed examination of the fundamental principles regulating de novo bone formation. The identification of the cellular source of regeneration, the timeline for progenitor cell response, and determination of how these cells transduce physical stimuli to enact a regenerative response may provide new and effective strategies for reconstruction of the craniofacial skeleton.