Nerve-dependent Bone Regeneration In Mandibular Fracture Healing
Ruth Ellen Jones, M.D.1, Ryan Chase Ransom, B.A.1,2, Ankit Salhotra, B.S.1, Deshka S. Foster, M.D.1, Derrick C. Wan, M.D.1, Michael T. Longaker, M.D., M.B.A., F.A.C.S.1,2.
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
PURPOSE: Injury of the inferior alveolar nerve (IAN) in mandibular surgery is avoided due to the resulting sensory defect and morbidity for the patient. The diminished regenerative capacity of skeletal tissues in the setting of denervation has been profiled in the setting of epimorphic regeneration, seen in the axolotl limb and mouse digit tip. Our lab has previously described the role of skeletal stem cells (SSCs) in bone regeneration and sought to determine the role of the IAN during SSC-driven mandibular fracture repair. METHODS: Wild-type mice were allocated into four experimental groups: uninjured (n = 6), denervation of the IAN without fracture (n = 6 per time point), denervation of the IAN with mandible fracture (n = 6 per time point), mandible fracture only (n = 6 per time point), and sham-operated (n = 6 per time point). Denervation of the IAN was accomplished surgically and two weeks were allowed for degeneration to occur. The denervation model was confirmed grossly and histologically with PLP::CreERT2;R26mTomato (Fig. 1a) mice, which express red fluorescent protein (mTomato) in Schwann cells after induction with tamoxifen. Unicortical fractures were executed with a circular saw to create an osteotomy on body of the mandible. The mandibles were then harvested for fluorescent-activated cell sorting (FACS) analysis of SSCs at days 5, 10 and 15 post-fracture. RESULTS: Gross and histologic examination of denervated PLP::CreERT2;R26mTomato mice confirmed complete disruption of the IAN. Wild-type mice underwent FACS analysis (Fig. 1b) which revealed decreased SSC frequency (Fig. 1c,d) in denervated mice as compared to mice with intact IAN. This difference grew in magnitude after mandibular fracture and with increased time from the fracture. We then performed in vitro analysis of SSCs isolated from FACS. SSCs from denervated mice had decreased colony forming unit (CFU) capacity compared to SSCs from innervated mice. Again, this difference was exacerbated after bony injury, with very few CFUs produced from denervated SSC after fracture (Fig. 1e). CONCLUSIONS: We present a model of IAN denervation and mandibular fracture in mice, and show that denervation of the mandible disrupts the expansion of SSCs post-fracture. This alteration in SSC frequency and function is only observed after fracture and represents a significant deviation from regeneration in the setting of an intact nerve. This information provides valuable insight into the biology of the facial skeleton, highlighting that nerve injury after fracture or during elective mandible surgery may have significant effects on bone regeneration.
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