Plastic Surgery Research Council

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Mechanoresponsive Skeletal Stem Cells Enact Bone Regeneration in Mandibular Distraction Osteogenesis
Ryan C. Ransom, B.A., Ankit Salhotra, B.S., Ava C. Carter, B.A., Matthew P. Murphy, M.D., Ruth E. Jones, M.D., Deshka S. Foster, M.D., Howard Y. Chang, M.D., Ph.D., Derrick C. Wan, M.D., Michael T. Longaker, M.D., M.B.A..
Stanford University, Stanford, CA, USA.

PURPOSE: Distraction osteogenesis (DO) offers the ability to promote endogenous bone formation across a mechanically controlled environment in a clinical setting, providing anatomical and functional replacement of deficient tissue. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration can shed light on how mechanical forces are transduced in regenerative processes.
METHODS: Here we employ a mandibular distraction model in mice for detailed examination of the fundamental principles regulating de novo bone formation during this regenerative process. To define the clonal characteristics and contribution of local skeletal stem/progenitor cells to regeneration in mandibular DO (Fig. 1a), we performed DO on the mandibles of skeleton-specific rainbow mice (Sox9::CreERT2;R26Rainbow). Mandible calluses were whole mounted for imaging analysis at the mid-point (post-operative day 10, n=5) (Fig. 1c) and late (POD 12, n=5) (Fig. 1d) phases of distraction. Confocal micrographs of whole mounted tissues were obtained for clonal analysis of the regenerate over time. To determine the effect of distraction on the relative frequencies of skeletal stem and progenitor cells (Fig. 2a), distracted bone calluses were dissected across multiple time points for FACS isolation of cell subtypes and compared to control calluses which were fractured without distraction (Fig. 2b). We performed ATAC-seq on skeletal stem and progenitor subsets isolated from uninjured, fractured, and distracted mandibles at POD 10 to capture the chromatin landscape during the process of regeneration (Fig. 2c). To probe the changes in chromatin state that underlie drastic functional differences in bone healing, we directly compared subsets from fracture to distraction (Fig. 2d,e).
RESULTS: Confocal microscopy of micro-dissected tissues revealed single-colored clones of large size (n = 50 clones; 16-151 cells per clone) within the periosteum of the mandible, demonstrating the presence of a stem/progenitor cell population within the periosteum (Fig. 1b). Cells within the periosteum expand into large clones with a linear alignment (n=5) at POD 10 continuing into (n=5) POD 12 in response to mandibular distraction. At the cellular level, mandibular distraction elicited an expansion of skeletal stem cells (SSC), bone cartilage and stromal progenitors (BCSP), and osteoprogenitors (OP) that was not seen under fracture conditions (n=5 per time-point for each condition). At the chromatin level, we found that the cell types clustered into three distinct primary groups.
CONCLUSIONS: We have developed a comprehensive, genetically-dissectible model for distraction osteogenesis in the mouse mandible. We use this model to profile the process of bone regeneration in the jaw from the tissue level to the chromatin level in order to understand how controlled physical separation of bones leads to proliferation, migration, and reorganization of tissue to lengthen the mandible.


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