Alpha-Ketoglutarate Drives an Osteogenic and Extracellular Matrix Gene Program in Periodontal Ligament Fibroblasts via Selective Reduction of H3K27me3

TL;DR

Alpha-ketoglutarate promotes periodontal tissue regeneration by selectively reducing a repressive gene signal in human and mouse models.

Contribution

Alpha-ketoglutarate is shown to drive an osteogenic and extracellular matrix gene program via selective H3K27me3 reduction.

Findings

Alpha-ketoglutarate increased ALP activity and upregulated osteogenesis and ECM-related genes in human periodontal ligament cells.

Oral alpha-ketoglutarate enhanced alveolar bone regeneration and collagen-rich tissue formation in a mouse model.

Alpha-ketoglutarate reduced H3K27me3 without broadly altering chromatin accessibility, indicating selective gene regulation.

Abstract

Periodontal disease damages the tissues that support teeth and can ultimately lead to tooth loss, yet effective treatments to regenerate these tissues are still limited. Recent studies have shown that substances produced during normal cellular metabolism can influence how genes are regulated, but their role in periodontal regeneration has not been fully clarified. In this study, we investigated whether alpha-ketoglutarate, a naturally occurring metabolite involved in energy production, could promote periodontal tissue regeneration. We found that alpha-ketoglutarate enhanced bone-related and extracellular matrix-related gene expression in human periodontal ligament cells by reducing a repressive gene-regulatory signal that normally suppresses these genes. Importantly, alpha-ketoglutarate did not broadly alter chromatin accessibility, indicating that its effects were mediated through selective gene regulation. Furthermore, oral administration of alpha-ketoglutarate promoted alveolar bone regeneration and collagen-rich tissue formation in a mouse model of periodontal disease. Because alpha-ketoglutarate is a naturally occurring molecule in the body, these findings suggest that metabolite-based regulation of gene activity may represent a promising and safe approach for periodontal tissue regeneration.

Periodontal disease is a chronic inflammatory condition that destroys tooth-supporting tissues, particularly the 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 upregulated 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 14 upregulated α-KG-induced genes, including multiple components of the OGN-OMD-PLAP1/ASPN-ECM2 loci, 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.

Figures

B,C). Measured values were expressed as the mean ± standard deviation (SD). Each experiment was performed at least three independent times. Statistical significance was set at *p* < 0.05.

Full text

1. Introduction

Periodontal disease is an inflammatory condition caused by periodontal pathogens and leads to the destruction of periodontal tissues, including the alveolar bone, periodontal ligament, cementum, and gingiva. Early symptoms include gingival swelling and bleeding, and progression results in tooth mobility and eventual tooth loss, severely affecting masticatory function and quality of life [1]. In addition, accumulating evidence further suggests an association between periodontal disease and systemic disorders, such as diabetes, atherosclerosis, cardiovascular disease, and preterm and low birth weight infants, highlighting its significance as a condition that extends beyond the oral cavity [2,3].

Current periodontal therapy primarily aims to suppress inflammation by controlling bacterial infection. However, once periodontal tissues—particularly the alveolar bone—are destroyed, spontaneous regeneration is largely unattainable [4]. Even after successful treatment, healing generally results in reduced bone levels and gingival recession. Although regenerative approaches, such as guided tissue regeneration [5], enamel matrix derivatives [6], and fibroblast growth factor-2 (FGF-2) [7], have shown favorable outcomes, their indications remain limited, and no effective therapy exists for extensive or horizontal bone defects. Therefore, the development of novel regenerative strategies based on mechanisms distinct from current approaches is essential.

Epigenetic regulation, including DNA methylation and histone modifications, has recently attracted attention for its roles in stem cell differentiation and tissue regeneration [8,9]. Moreover, metabolic–epigenomic crosstalk has emerged as a concept linking cellular metabolism to transcriptional control [10], with metabolites such as S-adenosylmethionine (SAM), NAD^+^, fumarate, and succinate affecting epigenetic enzymes [11]. Alpha-ketoglutarate (α-KG), a key metabolite in the tricarboxylic acid (TCA) cycle, participates in energy metabolism, amino acid turnover, antioxidant pathways, and collagen production [12,13], and serves as a cofactor for histone and DNA demethylases that regulate gene expression [11,14]. Notably, α-KG supplementation has been reported to enhance osteoblast differentiation and increase bone mass, suggesting potential applications in periodontal tissue regeneration [15].

To define how α-KG influences regenerative gene regulation in periodontal cells, the effects of α-KG on histone modifications, chromatin accessibility, and osteogenic/periodontal regeneration-related gene expression were examined in human periodontal ligament fibroblasts (hPDLFs) under osteogenic induction. The present study aimed to clarify epigenetic mechanisms through which α-KG promotes periodontal regeneration.

2. Materials and Methods

2.1. Reagents

α-KG (K1128) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Cell Culture

hPDLFs were purchased from Lonza Inc. (Walkersville, MD, USA). Cells were cultured in low-glucose DMEM (Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% FBS (Biosera, Kansas City, MO, USA). Culture conditions were maintained at 37 °C in a humidified incubator, with 5% CO_2_ and 95% ambient air. Osteogenic induction medium (OIM) (low-glucose DMEM containing 100 µg/mL ascorbic acid and 10 mM β-glycerophosphate) was used for osteogenic induction, with medium changes every 3 days. In α-KG addition experiments, α-KG was dissolved in OIM. Experiments were performed using two independent human periodontal ligament fibroblast cell lines.

2.3. Cell Proliferation Assay

hPDLFs were cultured in OIM containing various concentrations of α-KG, and cell proliferation was evaluated after 3 days using the WST-8 assay (Dojindo, Kumamoto, Japan) [16].

2.4. Alkaline Phosphatase (ALP) Activity Measurement

ALP activity was measured as previously described [17]. It was normalized to the number of cells obtained from parallel cell cultures and quantified using the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan).

2.5. Quantitative PCR (qPCR) Analysis

Total RNA purification, cDNA preparation, and qPCR were conducted as previously described [18]. Human HPRT was used as an internal reference gene. PCR primer sequences for the target genes are listed in Table 1.

2.6. Histone Purification

hPDLFs were cultured for 3 days in α-KG-supplemented OIM. Total histone proteins were extracted using the EpiQuik Total Histone Extraction Kit (Epigentek, Farmingdale, NY, USA).

2.7. Immunoblotting

Immunodetection was conducted as previously described [19] with anti-H3K27ac (ab4729, Abcam, Cambridge, UK, 1:1000), anti-H3K4me3 (ab8580, Abcam, Cambridge, UK, 1:2000), anti-H3K27me3 (ab192985, Abcam, Cambridge, UK, 1:1000), and anti-histone H3 (17168-1-AP, ProteinTech, Rosemont, IL, USA, 1:10,000) as primary antibodies, and HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA, 1:5000) as the secondary antibody.

2.8. ATAC-Seq

After six days of osteogenic induction, hPDLFs were processed for ATAC-seq using the ATAC-seq Kit (#53150; Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The prepared libraries were sequenced on a NovaSeq platform using 150-bp paired-end reads [20]. Reads were processed as follows: trimming with Trimmomatic [21], the removal of PCR duplicates using MarkDuplicates, alignment to a reference genome (hg38) using Bowtie2 version 2.5.1 [22], and duplication comparisons using deepTools [23]. The identification of significantly enriched chromatin peaks using HOMER [24] was previously described [25]. Regarding visualization in the UCSC Genome Browser, BAM files were converted to bigwig files using ‘bamCoverage’ in deeptools with -binsize = 10, minMappingQuality 10 [26].

2.9. RNA-Seq

To analyze genome-wide gene expression changes in cultured hPDLFs, DNase-treated total RNA was prepared [19]. RNA-seq data processing involved adapter trimming using Trim Galore (ver. 0.6.6; Babraham Bioinformatics, Cambridge, UK) with default settings, followed by mapping to the reference genome (hg38) using HISAT2 (v2.2.1) [27]. Transcript expression at the exons was quantified using the “analyzeRepeast.pl” command in HOMER with “-strand both” and “-count exons” for α-KG-treated hPDLFs as the target and non-treated hPDLFs as the background. The expression ratio of all transcripts was calculated by dividing the expression level in α-KG-treated hPDLFs by non-treated hPDLFs. Transcripts with a ratio greater than 2 were identified as upregulated genes, while those with a ratio less than 0.5 were identified as downregulated genes by α-KG.

2.10. Animal Experiments

All animal experiments in the present study were conducted in accordance with the “Regulations on Animal Experiments at Tohoku University” and under the review and approval of the Tohoku University Facility Animal Management and Use Committee (Approval Number: 2024 Shidou-009-04). Twelve-week-old male specific pathogen-free C57BL/6J mice were purchased from SLC Inc. (Shizuoka, Japan). Mice were housed under a 12-h light-dark cycle (lights on at 8:00 AM, lights off at 8:00 PM). In administration experiments, mice received drinking water containing 1.0% α-KG (pH 7.4), following dosing strategies reported in previous studies [28]. Oral administration began 14 days prior to ligation. Each group was allowed access to drinking water until the endpoint, with water being changed every two days. Based on the observation that α-KG exhibited a slow-onset effect in vitro, a 14-day pretreatment period was established prior to the 14-day stimulation phase. In the mouse periodontal tissue regeneration model, the area around the maxillary second molar was ligated with 5-0 silk suture under anesthesia. Fourteen days after suture removal, under anesthesia, the tissue was perfused with PBS to remove circulating blood and was then perfused and fixed with 4% paraformaldehyde. The maxillary bone was subsequently excised, collected, and fixed for an additional 24 h.

2.11. Alveolar Bone Level Quantification

The fixed maxillary bone was immersed in PBS and placed in a 15-mL centrifuge tube. Imaging was performed using micro-CT (ScanXmate-E090, Comscantecno Co., Ltd., Yokohama, Japan) with an isotropic resolution of 50 µm. Acquired images were reconstructed using coneCTexpressI software (ver. 1.59, WhiteRabbit Co., Ltd., Tokyo, Japan) and TRI/3D-BON software (ver. R2.00.06.0-H-64, Ratoc System Engineering Co., Ltd., Tokyo, Japan). After reconstruction, two-dimensional images were created with the standardized buccal-lingual and crown-apical orientation, and the occlusal surface was adjusted horizontally.

2.12. Histology

Fixed maxillary bone was demineralized by immersion in demineralizing solution B (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 4 °C for 3 days. It was then dehydrated using a graded ethanol series, cleared with xylene, and embedded in paraffin. Four-micrometer-thick paraffin sections were prepared, deparaffinized, and subjected to both Masson’s trichrome (MT) and immunofluorescence staining. Immunofluorescence staining was performed using the Alexa Fluor™ 488 Tyramide SuperBoost™ kit (B40922, Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s protocol. An anti-H3K27me3 antibody (ab192985, Abcam, Cambridge, UK, 1 µg/mL) and rabbit anti-IgG antibody (Cell Signaling Technology, Danvers, MA, USA, 1 µg/mL) were used as primary antibodies. Fluorescent signals were observed using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan), and analyses were performed using dedicated software (BZ-X800 Analyzer, ver. 1.1.30; Keyence, Osaka, Japan). Fluorescence intensity was quantified in the periodontal ligament space within the coronal half of the linear distance from the alveolar crest to the root apex. Detailed definitions of the regions of interest (ROIs) and original uncropped images are provided in the Supplementary Materials. An optical microscope (ECLIPSE 80i, Nikon, Tokyo, Japan) was used to observe MT-stained specimens.

2.13. Statistical Analysis

Statistical analyses were performed using one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (Figure 1A), Tukey’s post hoc test (Figure 1B), and a two-tailed unpaired Student’s t-test (Figure 5B,C). Measured values were expressed as the mean ± standard deviation (SD). Each experiment was performed at least three independent times. Statistical significance was set at p < 0.05.

2.14. Use of Generative Artificial Intelligence

Generative artificial intelligence was used to assist with English language editing. The authors reviewed and verified all content.

3. Results

3.1. Effects of α-KG on hPDLF Proliferation and ALP Activity

Different concentrations of α-KG were added to OIM to examine its effects on hPDLF proliferation using the WST-8 assay. Proliferation was significantly lower in the 16 mM α-KG-supplemented group than in the control group (Figure 1A). Based on these results, the maximum non-inhibitory concentration of α-KG for subsequent experiments was set at 8.0 mM. To examine the effects of α-KG on the osteogenic differentiation of hPDLFs, 0, 4.0, and 8.0 mM of α-KG were added to OIM. ALP activity was then measured. Increased ALP activity is indicative of early-stage osteogenic differentiation, reflecting osteogenic commitment and matrix maturation rather than terminal mineralization. ALP activity increased beginning on day 6, with significantly higher values being observed at each time point in the α-KG-supplemented groups than in the control group (Figure 1B). To assess whether the observed increase in ALP activity was reproducible, the same analyses were performed using an independent hPDLF cell line. Similar increases in ALP activity were observed in this cell line in a concentration-dependent manner during osteogenic induction (Figure S1).

3.2. α-KG Increases Bone/ECM Gene Expression

The qPCR analysis was performed to assess the expression of genes related to osteogenesis. The results obtained showed the significant upregulation of ALPL, OMD, COL1A1, PLAP1/ASPN, OGN, and ECM2 on day 6. By day 12, the significant upregulation of multiple hard tissue formation-related genes, including RUNX2, was confirmed (Figure 2). These results suggest that the addition of α-KG promoted the expression of bone tissue formation and ECM-related genes in hPDLFs, thereby enhancing their osteogenic capacity. To further confirm the reproducibility of α-KG-induced osteogenic gene expression, qPCR analysis was performed using an independent hPDLF cell line. Similar increases in the expression of representative osteogenic and extracellular matrix-related genes, including ALPL, RUNX2, COL1A1, PLAP1/ASPN, and OMD, were observed on day 6 of osteogenic induction (Figure S2).

3.3. α-KG Reduces H3K27me3, a Repressive Histone Marker

To examine the effects of α-KG on histone modifications in hPDLFs, 0, 4.0, and 8.0 mM of α-KG were added to OIM. After three days of culture, total histones were extracted. The expression levels of H3K27ac, H3K4me3, and H3K27me3 were analyzed. The results obtained showed no significant changes in the expression levels of H3K27ac or H3K4me3. However, the expression level of H3K27me3 significantly decreased in a concentration-dependent manner (Figure 3). These results suggest that the addition of α-KG reduced H3K27me3 levels during osteogenic induction. The uncropped Western blot images used for this analysis are provided in Supplementary Figure S3.

3.4. α-KG Does Not Affect Chromatin Accessibility, but Increases the Expression of Genes Within Specific Gene Clusters

To investigate the effects of α-KG on chromatin accessibility during hPDLF osteogenic differentiation, hPDLFs were cultured in OIM supplemented with α-KG at concentrations of 0, 4.0, and 8.0 mM. On day 6, cells were harvested and subjected to ATAC-seq. The results obtained showed no characteristic changes in genome-wide chromatin accessibility induced by α-KG (Figure 4A). To analyze the changes induced in gene expression by the addition of α-KG, RNA was harvested and RNA-seq was performed. The addition of α-KG significantly upregulated 14 genes (Figure 4B), which are listed in Table 2. The expression of genes encoding the following proteoglycans was included among the 14 genes: OGN, PLAP1/ASPN, and ECM2. These genes, along with OMD, formed a gene cluster (OGN-OMD-PLAP1/ASPN-ECM2) in the 92.38–92.55 Mb region on chromosome 9 (chr9). Visualization of the chromatin peaks in this region using the UCSC Genome Browser (Figure 4C) revealed multiple open chromatin regions, suggesting the presence of distal regulatory regions. In this gene cluster, OMD was the only gene not identified as being upregulated by RNA-seq on day 6. However, a detailed analysis of expression changes using qPCR revealed that OMD expression significantly increased, similar to OGN, PLAP1/ASPN, and ECM2 (Figure 2). These results suggest that the addition of α-KG did not affect genome-wide chromatin accessibility, but rather enhanced transcriptional output and promoted osteogenic differentiation by activating enhancers within specific gene cluster regions.

3.5. α-KG Administration Reduces H3K27me3 and Promotes Alveolar Bone Regeneration

The effects of α-KG on periodontal tissue regeneration were examined using a mouse model. Experiments were conducted according to the timeline shown in Figure 5A. The micro-CT analysis revealed similar levels of alveolar bone resorption around the second molar on day 14 post-ligation in the α-KG non-drinking and drinking groups. On day 28 (14 days after ligature removal), significantly higher alveolar bone regeneration was observed in the α-KG drinking group (Figure 5B). MT and immunofluorescence staining were subsequently performed on day 28. An anti-H3K27me3 antibody was used for immunofluorescence staining, and fluorescence intensity was quantified. The results obtained showed that fluorescence intensity was significantly lower in the α-KG drinking group than in the control group (Figure 5C). The detailed definitions of the regions of interest (ROIs) and the uncropped immunofluorescence images corresponding to this analysis are provided in Supplementary Figure S4. These results suggest that the administration of α-KG promoted alveolar bone regeneration by reducing H3K27me3 levels.

4. Discussion

The present study revealed that α-KG reduced the inhibitory histone marker H3K27me3 in hPDLFs and was associated with increased expression of osteogenic and ECM-related genes, including the OGN–OMD–PLAP1/ASPN–ECM2 loci. These genes are implicated in periodontal regeneration and bone remodeling, suggesting that α-KG may support osteogenic differentiation by facilitating transcriptional programs involved in osteogenesis and ECM formation. The epigenomic analysis revealed that, although α-KG did not have a significant effect on H3K27ac or H3K4me3, it reduced H3K27me3 (Figure 3). This effect aligns with the established mechanism through which α-KG functions as a cofactor for Fe(II)-dependent JmjC domain-type histone demethylases (e.g., KDM6A/B), potentially alleviating repressive chromatin states [29,30,31,32]. H3K27me3 is a prevalent repressive marker on gene promoters; its reduction indicates the release of transcriptional repression. In contrast, α-KG did not affect genome-wide chromatin accessibility, as measured by ATAC-seq (Figure 4A). It is conceivable that these epigenetic changes may occur at a scale below the detection threshold of global ATAC-seq signals. The concept of bivalent domains is of critical importance [9,33,34]. As indicated by the data, in stem and progenitor cells, the promoters of genes that are essential for development and differentiation often simultaneously carry both H3K4me3, an activation marker, and H3K27me3, a repression marker [9,33]. The reduction in H3K27me3 observed in the present study may contribute to the resolution of this bivalent state. The reduction of the repressive marker and the retention of the active marker could enable rapid and efficient transcriptional induction. Some osteogenesis-related genes are found in bivalent regions where H3K4me3 and H3K27me3 coexist [33,34]. One possible interpretation of this finding is that α-KG promotes the activation of osteogenesis-related genes by favoring the resolution of bivalency towards an H3K4me3-dominant state in these regions. The results obtained are consistent with a model in which α-KG modulates transcription primarily through changes in histone modifications rather than broad alterations in chromatin accessibility. To assess whether this effect involves transcriptional regulation of H3K27me3 demethylases, we examined our RNA-seq data and found that α-KG treatment did not significantly alter the expression of KDM6 family demethylases, including KDM6A and KDM6B (Figure S5). These results suggest that the reduction in H3K27me3 is unlikely to be driven by transcriptional upregulation of these enzymes. Instead, given that KDM6 demethylases are α-KG-dependent, α-KG may enhance their catalytic activity without changing gene expression, which may explain the selective reduction of H3K27me3.

Furthermore, the present study revealed that the effects of α-KG were not uniform across the entire genome; they may be context-dependent and more pronounced at specific loci. Within the OGN–OMD–PLAP1/ASPN–ECM2 gene cluster, increases in the expression of the corresponding genes were observed during osteogenic induction (Figure 4B,C). These results support locus-level transcriptional upregulation in this region. Genome browser analysis further supported this locus-selective response: while the OGN–OMD–PLAP1/ASPN–ECM2 region, particularly the PLAP1/ASPN locus, exhibited relatively higher chromatin accessibility and enrichment of active histone marks, other differentiation-associated loci such as SPARCL1, DSPP, and DMP1 showed minimal ATAC-seq signals and weaker active histone marks (Figure S6). These genes are markers of cementum and dentin/bone differentiation and are not expected to be strongly induced at early stages of periodontal regeneration. However, locus-resolved chromatin assays would be required to directly test chromatin activation at the OGN–OMD–PLAP1/ASPN–ECM2 loci. Furthermore, the present results suggest that the effects of α-KG are influenced by the cell type-specific epigenetic landscape. In osteoblasts, osteogenic gene clusters are already activated, and α-KG predominantly enhances the transcriptional activity of existing pathways [35]. In contrast, hPDLFs retain undifferentiated characteristics, and the reduction of H3K27me3 by α-KG may facilitate transcriptional activation. However, it remains to be determined whether this is accompanied by chromatin remodeling at specific loci. Consequently, while osteoblasts undergo the amplification of pre-existing osteogenic programs, hPDLFs may undergo a reprogramming-like shift triggered by the release of an inhibitory epigenetic state. Collectively, these findings suggest that α-KG functions not only as a metabolic cofactor but also as a context-dependent epigenetic regulator that governs differentiation and may promote periodontal tissue regeneration.

In vivo, α-KG was associated with reduced H3K27me3 signals and enhanced alveolar bone regeneration, consistent with osteogenic tissue-level outcomes (Figure 5B,C). A sample size of four mice per group was used in these experiments, which is consistent with previous mouse models of periodontal and alveolar bone regeneration, where similar group sizes (typically n = 4–6) have been commonly used to assess regenerative outcomes [36]. Previous studies have demonstrated that oral α-KG supplementation via drinking water (e.g., 2.0% w/v) significantly increases circulating α-KG levels, supporting the use of relatively higher concentrations for in vivo administration compared with in vitro experiments [37]. In contrast, in vitro, the α-KG concentration (8 mM) was determined based on previous studies [15] and preliminary dose–response experiments as the maximum non-inhibitory dose. This concentration exceeds reported physiological α-KG levels in serum and was therefore used as an experimental tool to probe α-KG-mediated cellular and epigenetic responses under controlled in vitro conditions. As an endogenous metabolite, α-KG has demonstrated potential for periodontal regeneration; however, challenges persist, including limited tissue specificity and clinical evidence. Moreover, while Masson’s trichrome staining suggested the presence of collagen-rich tissue within the periodontal ligament space, immunohistochemistry for specific periodontal ECM proteins and quantitative assessment of cementum formation and functional attachment were not performed in this study. In addition to the transcriptional regulation of collagen-related genes demonstrated in this study, α-KG is a well-established cofactor for prolyl hydroxylases that catalyze post-translational modifications essential for collagen stability and maturation [38]. The collagen-rich tissue formation observed in vivo may therefore reflect a combination of enhanced collagen gene expression and improved collagen protein maturation. Notably, while the present study provides evidence for epigenetic and transcriptional regulation, the contribution of post-translational mechanisms was not directly examined and warrants further investigation. Its distinctive mechanism also suggests the possibility of synergistic effects with EMD or FGF-2. We acknowledge that a direct gene-level correspondence between human and mouse tissues was not examined in this study. Importantly, our findings suggest that α-KG-mediated epigenetic modulation may contribute to periodontal tissue regeneration by promoting a permissive chromatin environment that supports matrix-rich tissue formation, rather than by inducing a single conserved gene expression program across species. However, we acknowledge that this study does not establish a direct causal relationship between H3K27me3 reduction and periodontal regeneration. Instead, our data support an associative and permissive role for H3K27me3 modulation in enabling regenerative transcriptional programs. Further studies incorporating locus-specific and functional analyses in vivo will be necessary to clarify the causal contribution of H3K27me3 modulation to periodontal regeneration.

In summary, α-KG has the potential to stimulate periodontal regeneration by modulating osteogenic and ECM-related gene expression, which is associated with a decrease in H3K27me3. This suggests that metabolite-based regenerative therapies may be a viable treatment option.

5. Conclusions

In the present study, alpha-ketoglutarate (α-KG) promoted osteogenic differentiation and the expression of extracellular matrix-related genes in human periodontal ligament fibroblasts. These effects were associated with a selective reduction in the repressive histone modification H3K27me3, without marked changes in global chromatin accessibility. Integrated epigenomic and transcriptomic analyses suggested that α-KG supports transcriptional activation of osteogenic gene programs, including the OGN–OMD–PLAP1/ASPN–ECM2 gene cluster, likely through the resolution of bivalent chromatin states.

In vivo, oral administration of α-KG enhanced alveolar bone regeneration and reduced H3K27me3 signals in a mouse periodontal regeneration model, supporting the relevance of metabolite-driven epigenetic regulation. Collectively, these findings suggest that α-KG functions as a context-dependent epigenetic modulator linking cellular metabolism to periodontal tissue regeneration. These results may provide a basis for future metabolite-based approaches in periodontal regenerative therapy.