R. Jones1, A. Salhotra1, C. Ransom1, D. Foster1, T. Chari1, K. Robertson1, D. Wan1, M. T. Longaker1 1Hagey Laboratory for Pediatric Regenerative Medicine,Division Of Plastic And Reconstructive Surgery, Department Of Surgery,Palo Alto, CA, USA
Introduction:
We have previously established a mouse model of inferior alveolar nerve (IAN) disruption, and demonstrated via fluorescence active cell sorting that IAN denervation impairs skeletal stem cell (SSC) recruitment function after fracture injury.However, the impact of denervation on healing morphology and tissue-specific progenitor activity has yet to be examined.
Methods:
Wild-typemice were subjected to surgical disruption of the right IAN, and two weeks were allowed for complete nerve degeneration. This model of denervation has been validated in our previous studies (data not shown). Using innervated mandibles as a control, mice underwent unicortical osteotomy on the buccal surface of the right mandible (Fig 1a). Hemimandibles were harvested 2 weeks after fracture, then sectioned and stained for analysis. The same surgical protocol was enacted on Actin-CreERT2::Rosa26-VT2/GK3 mice, or actin rainbow mice, which house a multicolor Cre-dependent reporter. Upon Cre recombinase activation by tamoxifen, individual cells randomly and permanently express one of four fluorochromes: eGFP (cytoplasmic green), mCerulean (membrane blue), mCherry (membrane red), and mOrange (membrane yellow). Selection of the ubiquitous actin Cre driver permits visualization of clonal expansion of cells, with clones defined as continuous regions of a single color. Denervated and innervated mandibles from actin rainbow mice were harvested two weeks postoperatively after unicortical fracture and mounted whole. They were examined with stereoscopic microscopy and corrected fluorescence intensity of fracture area was computed in ImageJ.
Results:
Denervated fractures healed with gross morphological abnormalities (Fig 1b). Compared to the compact callus formed in innervated bone healing, denervated tissue exhibits large gaps in bone tissue repair. Fluorochrome intensity was measured and compared between innervated and denervated fracture sites in actin rainbow mandibles. The denervated tissue exhibited significantly less fluorescent signal than the innervated samples in green, red, and merge channels (p= 0.0349*, 0.0079**, and 0.0097**, respectively) (Fig 1c). Images of fluorochrome signals are exhibited (Fig 1d), consistent with a striking fault in clonal expansion of denervated fracture healing tissue.
Conclusion:
With our novel mouse model of mandibular denervation, we show that bony healing of denervated mandibular fracture is morphologically aberrant. This defect is paired with lack of clonal expansion into the zone of healing, indicating that impaired stem/progenitor activity is responsible for deficient injury repair in denervated healing. Our data suggest that mandibular fracture healing is enacted via stem/progenitor cells which exhibit a high degree of nerve dependency.
