Type VI Collagen Deficiency Causes Enhanced Periodontal Tissue Destruction

Abstract

The periodontal ligament (PDL) is a fibrillar connective tissue that lies between the alveolar bone and the tooth and is composed of highly specialized extracellular matrix (ECM) molecules and a heterogeneous population of cells that are responsible for collagen formation, immune response, bone formation, and chewing force sensation. Type VI collagen (COL6), a widely distributed ECM molecule, plays a critical role in the structural integrity and mechanical properties of various tissues including muscle, tendon, bone, cartilage, and skin. However, its role in the PDL remains largely unknown. Our study shows that deficiency of COL6 impairs PDL fibrillogenesis and exacerbates tissue destruction in ligature-induced periodontitis (LIP). We found that COL6-deficient mice exhibited increased bone loss and degraded PDL in LIP and that fibroblasts expressing high levels of Col6α2 are pivotal in ECM organization and cell-ECM interactions. Moreover, COL6 deficiency in the PDL led to an increased number of fibroblasts geared toward the inflammatory response. We also observed that cultured COL6-deficient fibroblasts from the PDL exhibited decreased expression of genes related to collagen fiber turnover and ECM organization as well as migration and proliferation. Our findings suggest that COL6 plays a crucial role in the PDL, influencing fibroblast function in fibrillogenesis and affecting the immune response in periodontitis. These insights advance our understanding of the molecular mechanisms underlying PDL maturation and periodontal disease.

Keywords: biomechanics; bone loss; extracellular matrix; inflammation; periodontal ligament; single-cell RNAseq.

Figure 1.

Alterations in PDL fibers and mechanical properties in Col6α2-KO mice. (A) Second hormonic generation microscopy images of coronal sections of mandibles from 3-month-old WT and Col6α2-KO mice. Following the images of the entire tooth, magnified images of the areas outlined in boxes are shown. Red arrowheads show PDL. Scale bars: 200 µm (entire tooth), 50 µm (magnified images). (B) Quantification of detected fibers using CT-Fire. (WT: n = 3, Col6α2-KO: n = 5). Individual collagen fiber metrics were quantified in pixels. Thick fiber: pixels >7, medium fiber: pixels >5, pixels <7, thin fiber: pixels <5. (C–F) Quantitative measurements of dynamic mechanical analysis (DMA) from mandibles from 8-wk-old mice. (n = 8/genotype) *P < 0.05. **P < 0.01. ***P < 0.001. AB, alveolar bone; C, cementum; KO, knockout; ns, not significant; PDL, periodontal ligament; WT, wild type.

Figure 2.

Increased bone loss in COL6-deficient PDL by induced periodontitis. (A) Quantitative analysis of bone loss induced by LIP (1 day: n = 4/genotype, 3 days: n = 10/genotype, 5 days: n = 9/WT, n = 10/Col6α2-KO). (B) Immunofluorescent staining for COL6 in WT PDL 1 and 3 days post-LIP. (C) H&E staining for 3 days post-LIP in WT and Col6α2-KO. (D) Representative image of TRAP staining for 3 days post-LIP. (E) Quantitative number of osteoclasts from TRAP staining at 3 days post-LIP (n = 5/WT, n = 6/Col6α2-KO). (F) CHP staining at 3 days post-LIP in the upper panel and the representative image of PSR staining at 3 days post-LIP in the lower panel. Yellow arrowheads indicate degraded collagens. (G) Quantitative data of CHP staining (n = 4/genotype). Scale bars: (B), (C), (F): 25 µm; (D): 50 µm. *P < 0.05. ****P < 0.001. AB, alveolar bone; CHP, collagen hybridizing peptide; H&E, hematoxylin and eosin; KO, knockout; LIP, ligature-induced periodontitis; ns, not significant; PDL, periodontal ligament; PSR, picrosirius red; TRAP, tartrate-resistant acid phosphatase; WT, wild type.

Figure 3.

Functional analysis of fibroblasts expressing high levels of Col6α2. (A) Schematic of the single-cell collection for analysis. (B) Single-cell RNA-seq data obtained from 31,470 cells. Clustering analysis with 8 cell types is shown in UMAP. (C) A dot plot of the expression of representative genes for each cluster. (D) The UMAP shows WT fibroblasts identified from the total cell population, and the UMAP distinguishes the cells collected at 1 day and 3 days post-LIP. (E, F) The UMAP shows the fibroblasts distinguished by the expression level of Col6α2. (G) Gene set enrichment analysis in the BP and Kyoto Encyclopedia of Genes and Genomes pathway. BioRender.com was used to create the schematic image. BP, Biological Process; LIP, ligature-induced periodontitis; WT, wild type.

Figure 4.

Altered fibroblast subtypes in COL6-deficient periodontal ligament (PDL). (A) UMAP shows the WT and Col6α2-KO fibroblasts identified from the total cell population. (B) Violin plot showing the expression of Col6α2. (C) UMAP of fibroblasts separately showing the genotype and samples collected at 1 day and 3 days post-LIP. (D) The ratio of the population of fibroblasts. (E) Terms of ORA in Biological Process. KO, knockout; LIP, ligature-induced periodontitis; ORA, overrepresentation analysis; WT, wild type.

Figure 5.

Altered function in COL6-deficient fibroblasts. (A) Schematic of the fibroblast collection for the bulk RNAseq analysis. (B) Volcano plot showing 144 DEGs (WT versus Col6α2-KO, FDR-adjusted P value <0.05, logFC > 1, logFC < −1). (C) Pathways identified by IPA. (D) Heat map of gene expression associated with the listed pathways in (C). (E) Heat map showing the expression of genes associated with the ECM. (F, G) Quantification data of scratch assay and BrdU. *P < 0.05. BioRender.com was used to create the schematic image. DEGs, differentially expressed genes; ECM, extracellular matrix; FDR, False Discovery Rate; IPA, Ingenuity Pathway Analysis; KO, knockout; WT, wild type.