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Denervation During Mandibular Distraction Osteogenesis Results In Impaired Osteogenesis
Ruth Tevlin, MB BAO BCh MRCSI MD, Michelle Griffin, MD, Kellen Chen, PhD, Derrick Wan, MD, Michael Longaker, MD FACS.
Stanford University, Stanford, CA, USA.

PURPOSE: Craniofacial distraction osteogenesis (DO) is a powerful regenerative technique. It continues however to be marred by complications, often necessitating surgical revision. Mandibular DO is mediated by skeletal stem cells (SSCs) in mice, which enact bone regeneration via neural crest re-activation (1). As peripheral nerves are essential to progenitor function during development and in response to injury (2,3), we question if denervation impairs mandibular DO in both mice and humans.
METHODS: Eight-week old C57Bl6 mice were divided into two groups: DO with inferior alveolar nerve (IAN) denervation ("DO-Den") and DO with IAN intact ("DO-Inn"). An IAN segmental defect was created in the DO-Den cohort following mandibular osteotomy (Figure 1A, B). The IAN was protected in Do-Inn. Following a latency period, all mice underwent gradual DO. Specimens were harvested at POD 10, 15, 23 for analysis of the stem and progenitor cell populations using fluorescence-activated cell sorting (FACS) and at POD 43 for microCT and histological analysis.
In addition, a 13 year old patient with a past medical history of micrognathia underwent bilateral mandibular DO and was noted post-operatively to have a new unilateral IAN clinical deficit. This allowed a unique opportunity to question if our mouse model findings were conserved in humans. Panorex CT was used to assess bone formation. Bone was then harvested from the bilateral mandible at the time of distractor removal for assessment by histology and FACS analysis of human SSCs.
Single cell RNA sequencing was performed on mouse and human Do-Inn and Do-Den SSCs.
RESULTS: DO-Den demonstrated reduced histological osteogenesis relative to DO-Inn, which was evident on microCT at POD 43 (*p=0.018) (Figure 1C). Furthermore, DO-Den regenerate had significantly reduced mSSCs present at POD 10, 15, 23 relative to DO-Inn (*p=0.0032, *p=0.0099, *p=0.0111 respectively) (Figure 1D, E). Single cell RNA sequencing analysis of mouse SSCs revealed that there was a predominance of innervated SSCs in clusters dominated by pathways related to bone formation. Intriguingly, the same pattern of impaired histological and radiological DO in the setting of IAN denervation was also conserved in a rare human mandible clinical specimen three months following completion of distraction. In our human patient specimen, we demonstrated that there was notable differential transcription of Osteopontin and RUNX2 between Den DO SSC and Inn DO SSC, suggesting that mandible denervation prior to DO results in delayed osteogenesis via DO.
CONCLUSION:
Together these data suggest that peripheral nerves maintain a salient role in DO by promoting non-neuronal tissue repair, a pattern conserved in mice and humans. Harnessing the underlying biology of nerve-dependent DO may aid clinical innovation in craniofacial skeletal regeneration. 1. Ransom, R. C. et al. Nature 2018;563:514-521.2. Cao, J. et al. Int J Med Sci 2019;16:831-837.3. Jones, R. E., et al. Cell Rep 2019;28:2757-2766.e2755.


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