PRRX1+MSCs Enhance Mandibular Regeneration during Distraction Osteogenesis

W D Jiang; P Q Zhu; T Zhang; F C Liao; P P Jiang; N Zhou; X D Wang; X P Huang

doi:10.1177/00220345231176522

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.

Authors

W D Jiang^{1

2

3

4

5

6

7

8

9}, P Q Zhu^{1

2

3}, T Zhang^{1

2

3}, F C Liao^{1

2

3}, P P Jiang⁹, N Zhou^{1

2

3}, X D Wang^{4

5

6

7

8

9}, X P Huang^{1

2

3}

Affiliations

¹ Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Guangxi Medical University, Nanning, P. R. China.
² Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction, Nanning, P. R. China.
³ Guangxi Key Laboratory of Oral and Maxillofacial Surgery Disease Treatment, Nanning, P. R. China.
⁴ Department of Oral and Cranio-Maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China.
⁵ College of Stomatology, Shanghai Jiao Tong University, Shanghai, P. R. China.
⁶ National Center for Stomatology, Shanghai, P. R. China.
⁷ National Clinical Research Center for Oral Diseases, Shanghai, P. R. China.
⁸ Shanghai Key Laboratory of Stomatology, Shanghai, P. R. China.
⁹ Shenzhen Rare Disease Engineering Research Center of Metabolomics in Precision Medicine, Shenzhen, P. R. China.

PMID: 37387366
DOI: 10.1177/00220345231176522

Abstract

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 PRRX1^KO 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