PRRX1+MSCs Enhance Mandibular Regeneration during Distraction Osteogenesis

J Dent Res. 2023 Aug;102(9):1058-1068. doi: 10.1177/00220345231176522. Epub 2023 Jun 30.


Bone defect (BD) caused by trauma, infection, congenital defects, or neoplasia is a major cause of physical limitation. Distraction osteogenesis (DO) is a highly effective procedure for bone regeneration, while the concrete mechanism remains unknown. In this study, canine DO and BD models of the mandible were established. The results of micro-computed tomography and histological staining revealed that DO led to an increased mineralized volume fraction and robust new bone formation; in contrast, BD demonstrated incomplete bone union. Mesenchymal stem cells (MSCs) from DO and BD calluses were isolated and identified. Compared with BD-MSCs, DO-MSCs were found to have a stronger osteogenic capability. Single-cell RNA sequencing analysis was further performed to comprehensively define cell differences between mandibular DO and BD calluses. Twenty-six clusters of cells representing 6 major cell populations were identified, including paired related homeobox 1-expressing MSCs (PRRX1+MSCs), endothelial cells (ECs), T cells, B cells, neutrophils, and macrophages. Interestingly, 2 subpopulations in PRRX1+MSCs in the DO group were found to express the marker of neural crest cells (NCCs) and were associated with the process of epithelial-mesenchymal transition. The immunofluorescence assay was performed to further corroborate these results in vivo and in vitro, experimentally validating that continuous distraction maintained the PRRX1+MSCs in an embryonic-like state. Finally, we used CRISPR/Cas9 to knock out (KO) PRRX1 in the context of DO, which significantly blunted the capability of jawbone regeneration, resulting in a diminished NCC-like program and reduction of new bone volume. In addition, the ability of osteogenesis, cell migration, and proliferation in cultured PRRX1KO MSCs was inhibited. Taken together, this study provides a novel, comprehensive atlas of the cell fates in the context of DO regeneration, and PRRX1+MSCs act essential roles.

Keywords: CRISPR-Cas9 system; bone regeneration; jaw; mechanotransduction; mesenchymal stem cells; single-cell RNA sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bone Regeneration
  • Cell Differentiation
  • Endothelial Cells
  • Mandible / surgery
  • Mesenchymal Stem Cells*
  • Osteogenesis / genetics
  • Osteogenesis, Distraction* / methods
  • X-Ray Microtomography