Identification of a Force-Induced Sox9+Acan+ Transitional Subpopulation Linked to FGF2-FGFR2-ERK Signaling in Orthodontic Bone Remodeling

Abstract

Orthodontic tooth movement (OTM) under excessive force is often accompanied by orthodontically induced inflammatory root resorption (OIIRR). Multiple cell types and pathways contribute, yet the heterogeneity of mesenchymal lineage cells remains poorly defined. Murine models of OTM are established, and single-cell RNA sequencing (scRNA-seq) is performed to profile force-induced transcriptional dynamics. Mesenchymal lineage cells are resolved into five subsets, including mesenchymal stem cells, cementoblasts, osteoblasts, fibroblasts, and a previously unrecognized cluster co-expressing Sox9 and Acan. Functional validation by RNAscope, multiplex immunohistochemistry, and mechanostimulation confirms the localization and activity of this subpopulation. scRNA-seq also identifies 14 additional cell types, including immune and mesenchymal populations. Reclustering of macrophages reveals gene programs associated with bone resorption. Sox9⁺Acan⁺ cells exemplify a mechanosensitive transitional population that integrates biomechanical stress with osteoimmune regulation, a paradigm relevant to skeletal mechanobiology. These cells interact with Mmp14⁺ macrophages to activate FGF2-FGFR2-ERK signaling, thereby enhancing osteoclast differentiation and bone resorption. A GelMA@siRNA hydrogel system for localized delivery of Sox9-targeting siRNA silences Sox9 expression in vivo, suppresses osteoclast activity, reduces root resorption, and modulates tooth movement. Together, these findings identify Sox9⁺Acan⁺ cells as a force-sensitive regulatory node in skeletal biology and propose a therapeutic strategy to mitigate OIIRR.

Keywords: FGF signaling; crosstalk; mesenchymal lineage cell; orthodontic tooth movement; single‐cell RNA sequencing.