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Impact of Low-Intensity Ultrasound on Pediatric Dural Cells: Implications for Calvarial Repair. BACKGROUND AND OBJECTIVES: Pediatric patients frequently exhibit delayed bone flap resorption after autologous cranioplasty. The dura has been shown to be mechanosensitive and has been implicated in cranial regeneration, but an improved understanding of the mechanisms underlying this relationship may yield new strategies for reducing resorption after cranioplasty. Our objective was to examine the role of dural cells in cranial regeneration by investigating the presence of potential stem cell populations and their capacity for osteogenic differentiation, as well as the response of dural cells to mechanical stimulation by low-intensity ultrasound-derived acoustic radiation forces. METHODS: Rat-derived dural cells were isolated from 4 to 6-week-old Sprague Dawley rats and treated with low-intensity ultrasound administered at 150 mW/cm 2 for 20 minutes/d. Flow cytometry was used to investigate stem cell surface marker expression with or without ultrasound. Dural cells were also maintained in osteogenic media and assessed for markers of osteogenic differentiation. RNA-seq analysis was then performed to examine the effect of ultrasound treatment on the global mRNA expression of dural cells. RESULTS: Over 90% of dural cells expressed cell surface markers consistent with a stem cell population and were maintained after 28 days of culture, both with and without ultrasound treatment. Dural cells cultured in osteogenic media, however, demonstrated increased proliferation, decreased osteocalcin expression, and no evidence of mineralization. RNA-seq analysis demonstrated upregulation of genes related to cellular movement, cellular assembly and organization, and cell-cell signaling after ultrasound treatment. Both canonical and noncanonical Wnt signaling were upregulated, but downstream effectors of noncanonical Wnt signaling were downregulated. CONCLUSION: Dural cells express characteristic stem cell markers but do not undergo osteogenic differentiation after chemical or mechanical stimulation; however, ultrasound treatment upregulates elements of the Wnt paracrine signaling pathway, suggesting a novel mechanism that might be harnessed to improve outcomes after autologous cranioplasty.