The Contribution of Meckel's Cartilage-Derived Type II Collagen-Positive Cells to the Jawbone Development and Repair

Abstract

Jawbones and long bones, despite their shared skeletal lineage, frequently exhibit distinct origins and developmental pathways. Identifying specific progenitor subsets for mandibular osteogenesis remains challenging. Type II collagen is conventionally associated with cartilaginous structures, yet our investigation has identified the presence of type II collagen positive (Col2⁺) cells within the jawbone development and regeneration. The role of Col2⁺ cells in jawbone morphogenesis and repair has remained enigmatic. In this study, we analyze single-cell RNA sequencing data from mice jawbone at embryonic day 10.5. Through fate-mapping experiments, we have elucidated that Col2⁺ cells and their progeny are instrumental in mandibular osteogenesis across both fetal and postnatal stages. Furthermore, lineage tracing with a tamoxifen-inducible CreER system has established the pivotal role of Col2⁺ cells, marked by Col2-CreER and originating from the primordial Meckel's cartilage, in jawbone formation. Moreover, our research explored models simulating jawbone defects and tooth extraction, which underscored the osteogenic differentiation capabilities of postnatal Col2⁺ cells during repair. This finding not only highlights the regenerative potential of Col2⁺ cells but also suggests their versatility in contributing to skeletal healing and regeneration. In conclusion, our findings position Col2⁺ cells as essential in orchestrating osteogenesis throughout the continuum of mandibular development and repair.

Keywords: Col2; jawbone; lineage tracing; mandibular osteogenesis; osteogenic differentiation; progenitor.

Figure 1.

scRNA-RNA sequencing analysis of mice jawbone embryonic progenitors. (A) Dimensional reduction (Uniform Manifold Approximation and Projection [UMAP]) of scRNA-seq results of E10.5 jawbone cells, colored according to the clusters. (B) Heatmap showing the representative markers for each cluster across cells in the six clusters. (C) Diagram of further clustering. Dimensional reduction (UMAP) of scRNA-seq results of neural crest (NC) cells, colored according to the clusters. (D) Feature plots showing normalized expression levels of the selected stem cells. (E) Violin plots showing normalized expression levels of the selected stem cells. (F) Single-cell trajectory obtained by unsupervised ordering based on a pseudo-temporal order from left to right, colored by cell clusters. Abbreviations: MC, Meckel’s cartilage.

Figure 2.

The distribution of Col2-Cre⁺ (Col2-Cre-labeled cells) cells in jawbone. (A to E) Representative immunofluorescence images from the lineage tracing of Col2-Cre⁺ cells in jawbone at different time points (E15.5, P3, 4W, 8W, and 1Y). (F) Percentage of tdTomato⁺ cells in the periosteum of jawbone (n=3). (G, H) Col2-CreER; R26R^tdTomato mice were administered tamoxifen at E15.5 and harvested at P0. Representative immunofluorescence images of Col2⁺Runx2⁺ cells in the jawbone of P0 mice. (I, J) Immunofluorescence images of tdTomato⁺ cells surround the Meckel’s cartilage (MC). Green: chondrocyte, red: tdTomato, blue: nucleus. Data are presented as the mean ± SD. ****p<0.0001. Bars A-E = 100 μm; bars G-J = 50 μm.

Figure 3.

Col2-Cre⁺ cells (Col2-Cre-labeled cells) exist in jawbone from embryonic to aged mice. (A to E) Lineage tracing analysis of Col2-Cre⁺ cells was performed using Col2-Cre; R26R^tdTomato. Representative immunofluorescence images of Col2⁺Runx2⁺ cells in the jawbone in E15.5, P3, 4W, 8W, and 1Y mice. Green: Runx2, red: tdTomato, blue: nucleus. (F to J) Representative immunofluorescence images of chondrocyte in the jawbone in E15.5, P3, 4W, 8W, and 1Y mice. Green: chondrocyte, red: tdTomato, blue: nucleus. (K) Percentage of tdTomato⁺Runx2⁺ cells in the jawbone (n=3). (L) Percentage of tdTomato⁺ cells in jawbone (n=3). (M) Percentage of chondrocytes in tdTomato⁺ cells (n=3). Data are presented as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bars A-J = 100 μm.

Figure 4.

Col2⁺ cells are responsive toward jawbone injury and tooth extraction. (A) Schematic diagram of bone defect model in mouse jawbone. (B) Illustration of the experimental design. This analysis was performed by injecting tamoxifen into mice at the first day after surgery and harvesting the tissues after 7 and 14 days of healing. (C) Percentage of tdTomato⁺ cells in jawbone callus (n=3). (D) Percentage of tdTomato⁺Runx2⁺ cells in the jawbone callus (n=3). (E to H) Representative immunofluorescence images of tdTomato⁺Runx2⁺ cells in the jawbone callus at 7 and 14 days after surgery in Col2-CreER; R26R^tdTomato mice. (I) Representative H&E image in the jawbone callus at 7 days after surgery. (J) Illustration of the extraction of the first molar in mouse jawbone. (K) Percentage of tdTomato⁺ cells in extraction sockets (n=3). (L) Percentage of tdTomato⁺Runx2⁺ cells in extraction sockets (n=3). (M to P) Representative immunofluorescence images of tdTomato⁺Runx2⁺ cells in the sockets at 7 and 14 days after extraction in Col2-Cre; R26R^tdTomato mice. Data are presented as the mean ± SD. *p<0.05. Bars E, G, I, M, O = 200 μm; bars F, H = 50 μm; bars N, P = 20 μm.