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Chemical Digestion-Assisted Proteomic Analysis of Extracellular Matrix Profile of Human Periodontal Ligament

ABSTRACT: The extracellular matrix (ECM) plays a crucial role in tissue structure and function and serves as an integral component of diverse biological systems. However, the comprehensive characterization of ECM components is challenging because of the insoluble nature of highly cross-linked and glycosylated ECM proteins. This study introduces a chemical digestion-assisted proteomic approach to overcome challenges in ECM profiling, offering an in-depth analysis of the human periodontal ligament (PDL), a tissue critical for oral function and regeneration. Furthermore, we investigated alterations in the ECM composition of cultured PDL-derived cells to provide insights relevant to tissue engineering applications. Our protocol combined chemical digestion and deglycosylation to enhance the identification of ECM proteins.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Masaru Kaku

PROVIDER: PXD060345 | JPOST Repository | Sun Jul 12 00:00:00 GMT+01:00 2026

REPOSITORIES: jPOST

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	20230227_TT5600_D-BP-MK-009%233.wiff	Wiff
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	20230227_TT5600_D-BP-MK-009%234.wiff	Wiff
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Chemical Digestion-Assisted Proteomic Analysis of Extracellular Matrix Profile of Mouse Periodontal Ligament Cell Sheet

Project description:The extracellular matrix (ECM) plays a crucial role in tissue structure and function and serves as an integral component of diverse biological systems. However, the comprehensive characterization of ECM components is challenging because of the insoluble nature of highly cross-linked and glycosylated ECM proteins. This study established a chemical digestion-assisted proteomic approach to overcome these limitations and provide an in-depth analysis of the ECM of the mouse periodontal ligament cells.

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Differentially expressed long noncoding RNA expression between periodontal ligament stem cells from healthy periodontal tissue and periodontal ligament stem cells from inflammatory periodontal tissue.

Project description:Periodontitis can impair the osteogenic differentiation of human periodontal mesenchymal stem cells, but the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) have been demonstrated to play significant roles under both physiologic and pathological conditions. We performed comprehensive lncRNAs profiling by lncRNA microarray to identify differentially expressed long noncoding RNA expression between Periodontal ligament stem cells from healthy Periodontal tissue and periodontal ligament stem cells from inflammatory periodontal tissue. Our analysis identified 233 lncRNAs and 423 mRNAs that were differently expressed (fold change >2.0, p-value < 0.05) between the two groups of cells. The GO analysis revealed that the significantly down-regulated biological processes included multicellular organismal process, developmental process and multicellular organismal development and the significantly up-regulated biological processes included cellular process, biological regulation and response to stimulus in periodontal ligament stem cells from inflammatory periodontal tissue. The Pathway analysis revealed that the differentially expressed mRNAs may involved in Focal adhesion, ECM-receptor interaction, Bacterial invasion of epithelial cells, Long-term depression, Circadian entrainment and HIF-1 signaling pathway. Two-condition experiment, periodontal ligament stem cells from healthy periodontal tissue (hPDLSCs) vs. periodontal ligament stem cells from inflammatory periodontal tissue (pPDLSCs), Biological replicates: 3 control replicates (hPDLSCs), 3 testing replicates (pPDLSCs).

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Project description:Periodontal disease is a chronic inflammatory condition that destroys tooth-supporting tissues, particularly alveolar bone and the periodontal ligament, and effective regenerative therapies remain limited. While the role of metabolic-epigenomic crosstalk in determining cell fate is well established, the specific mechanism by which a tricarboxylic acid (TCA) cycle metabolite can modulate chromatin regulation to promote periodontal regeneration remains to be elucidated. The impact of one TCA-cycle metabolite, alpha-ketoglutarate (α-KG), was examined in human periodontal ligament fibroblasts cultured under osteogenic induction and profiled by ALP assays, RT-qPCR, analyses of multiple histone modifications, ATAC-seq, and RNA-seq. α-KG increased ALP activity and up-regulated genes associated with osteogenesis and the extracellular matrix (ECM). ATAC-seq revealed minimal genome-wide accessibility changes, whereas histone analyses showed reduced H3K27me3, consistent with an epigenetic mechanism that does not require extensive chromatin opening. The RNA-seq identified α-KG-induced 14 genes upregulated, including multiple components of the OGN-OMD-PLAP1/ASPN-ECM2 locus, supporting an osteogenic/ECM transcriptional program. In a mouse periodontal regeneration model, oral administration of α-KG enhanced alveolar bone regeneration and reduced H3K27me3 signals and collagen-rich tissue organization within the periodontal ligament space. These findings identify α-KG as a metabolite-driven epigenetic modulator that alleviates H3K27me3-mediated repression and supports periodontal regeneration.

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H3K27me3Alpha-Ketoglutarate Drives an Osteogenic and Extracellular Matrix Gene Program in Periodontal Ligament Fibroblasts via Selective Reduction of H3K27me3 [RNA-Seq]

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Project description:Single cell analysis of PDL cells in ligature-induced periodontitis: 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 fibrogenesis 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 towards the inflammatory response. We also observed that cultured COL6 deficient fibroblasts from the PDL exhibited decreased expression of genes related to collagen fiber metabolism and ECM organization, and migration and proliferation. Our findings suggest that COL6 plays a crucial role in the PDL, influencing fibroblast function in fibrogenesis and affecting the immune response in periodontitis. These insights could advance our understanding of the molecular mechanisms underlying PDL maturation and periodontal disease.

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