The Relevance of DNA Methylation and Histone Modification in Periodontitis: A Scoping Review

Background: Periodontitis is a chronic inflammatory disease involving an interplay between bacteria, inflammation, host response genes, and environmental factors. The manifestation of epigenetic factors during periodontitis pathogenesis and periodontal inflammation is still not well understood, with limited reviews on histone modification with periodontitis management. This scoping review aims to evaluate current evidence of global and specific DNA methylation and histone modification in periodontitis and discuss the gaps and implications for future research and clinical practice. Methods: A scoping literature search of three electronic databases was performed in SCOPUS, MEDLINE (PubMed) and EMBASE. As epigenetics in periodontitis is an emerging research field, a scoping review was conducted to identify the extent of studies available and describe the overall context and applicability of these results. Results: Overall, 30 studies were evaluated, and the findings confirmed that epigenetic changes in periodontitis comprise specific modifications to DNA methylation patterns and histone proteins modification, which can either dampen or promote the inflammatory response to bacterial challenge. Conclusions: The plasticity of epigenetic modifications has implications for the future development of targeted epi-drugs and diagnostic tools in periodontitis. Such advances could be invaluable for the early detection and monitoring of susceptible individuals.


Introduction
Periodontitis is characterised by a disruption to the homeostatic integrity of the subgingival microbiota and host response, which promotes a self-sustained and reciprocal cycle of inflammation-driven tissue destruction and enhanced microbial dysbiosis [1][2][3][4]. This perturbation in tissue homeostasis and resulting periodontal destruction is facilitated by a variety of pro-inflammatory mediators, comprising interleukin (IL)1, IL6, IL8, interferongamma (IFNγ), tissue inhibitors of MMPs (TIMPs), and tumour necrosis factor-alpha (TNFα) [5,6]. A large body of evidence details the involvement of these cytokines in the innate immune response to bacterial challenge and tissue destruction.
It is well established that plaque biofilm is the key aetiological requirement for the initiation and progression of periodontitis [1,2,6,7], although in itself is insufficient for tissue destruction to occur, with a susceptible patient also necessary. Given the known effects of the environment and systemic health status on the epigenome and the established role of environmental exposures as determinants of disease susceptibility [7][8][9], epigenetic factors are particularly relevant in the pathobiology of periodontitis [10,11]. The term 'epigenetics', first coined in 1942, describes the reversible changes in gene expression patterns or cellular phenotypes that are not encoded in the DNA sequence [12]. Principal modes of epigenetic function include small non-coding RNAs, DNA methylation and histone modification [12,13]. For the purpose of this review, epigenetic modifications of DNA and histone proteins will be explored in the context of periodontitis. The role of epigenetic factors in the context of periodontitis diagnosis is an emerging field [17][18][19][20][21][22][23], and the unique traits of epigenetic biomarkers could provide invaluable diagnostic and therapeutic insights into more predictable, precise and personalised disease management. The epigenetic factors, HDACs, DNA methylation and circular RNA, are shown to express in periodontal tissues [22], oral fluids such as saliva [20], and circulating small extracellular vesicles (sEVs) [17][18][19][20]. sEVs are a type of biological nanoparticles secreted by all cell types that are circulating in extracellular space (i.e., biofluid or cell culture media), and containing genetic and epigenetic materials, protein, lipids and other biology molecules from their parental cells [17,19]. To date, few review papers outline the epigenetic mechanisms related to periodontitis, most of which are heavily focused on DNA methylation or non-coding RNA changes [9,22,[24][25][26][27][28]. There are relatively limited reviews summarising HDACs in periodontitis pathogenesis and the use of HDAC inhibitors (HDACi) as adjunctive therapies for periodontitis treatment. Additionally, there is a lack of knowledge outlining the application of sEVs-associated epigenetics in periodontitis. Histone modification can occur during post-translation stages through acetylation, deacetylation and methylation at amino acid tails (Figure 1b), directly altering selected regions on the chromatin structure [10]. Histone acetylation facilitated by histone acetyltransferases (HATs) involves the transfer of acetyl groups to lysine residues on the N-terminal tails of histones. This results in an open chromatin conformation, promoting transcription factor binding and increased gene transcription. In contrast, histone deacetylases (HDACs) remove acetyl groups from histones and condenses the chromatin structure into a closed conformation, repressing gene transcription [14]. In humans, there are 18 HDAC enzymes grouped into four main classes, of which Classes I and II are the most studied. Class I enzymes comprise HDACs1, 2, 3 and 8 which are confined to the nucleus, whilst Class II enzymes include two sub-groups HDACs 4, 5, 7 and 9 (class IIa) and HDACs 6 and 10 (class IIb), which can shuttle between the nucleus and cytoplasm, Class III enzymes include Sirtuins 1 to 7 and Class IV contains only HDAC11 [16]. The integrity of immune homeostasis relies on the delicate balance of acetylation and deacetylation activity, to prevent aberrant transcription of key immunomodulatory genes [10]. Lastly, histone methylation by lysine methyltransferases (KMTs) and lysine demethylases (KDMs) can activate or suppress gene expression. Such methylation events have an important role in the maintenance of cell cycle, DNA damage and stress response; however, their interaction with the inflammatory response is poorly understood [12].
The role of epigenetic factors in the context of periodontitis diagnosis is an emerging field [17][18][19][20][21][22][23], and the unique traits of epigenetic biomarkers could provide invaluable diagnostic and therapeutic insights into more predictable, precise and personalised disease management. The epigenetic factors, HDACs, DNA methylation and circular RNA, are shown to express in periodontal tissues [22], oral fluids such as saliva [20], and circulating small extracellular vesicles (sEVs) [17][18][19][20]. sEVs are a type of biological nanoparticles secreted by all cell types that are circulating in extracellular space (i.e., biofluid or cell culture media), and containing genetic and epigenetic materials, protein, lipids and other biology molecules from their parental cells [17,19]. To date, few review papers outline the epigenetic mechanisms related to periodontitis, most of which are heavily focused on DNA methylation or non-coding RNA changes [9,22,[24][25][26][27][28]. There are relatively limited reviews summarising HDACs in periodontitis pathogenesis and the use of HDAC inhibitors (HDACi) as adjunctive therapies for periodontitis treatment. Additionally, there is a lack of knowledge outlining the application of sEVs-associated epigenetics in periodontitis. Therefore, further review of the recent body of evidence is warranted, detailing the mediation of DNA, histone and sEVs epigenetic modulators in periodontal inflammation, and current developments in the utilization of histone deacetylase inhibitors in periodontitis management. This scoping review aimed to evaluate recent selective evidence of the epigenetic changes related to chemical modifications of DNA and chromatin structure in periodontitis and periodontal cells/tissues as well as potential histone epi-drugs for periodontitis treatment. Specifically, this review sought to answer the following questions: In periodontally diseased tissues or oral biofluids, what are the observed alterations in sEVs, global DNA methylation or specific DNA methylation patterns of pro-inflammatory cytokines genes?
What is the current research regarding histone modification and its relevance in periodontal inflammation and dysbiosis?

Study Eligibility Criteria
Titles and abstracts from all retrieved records were screened for potentially eligible studies by one reviewer (A.L.). For inclusion in the review, the studies were required to meet the following eligibility criteria:

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Patients with periodontitis; • Gingival biopsies, gingival crevicular fluid or saliva samples; • Retrospective and prospective studies of the observational, case-control, cohort, or randomised controlled trial design; • English language.
The exclusion criteria were as follows: • In vitro and in vivo studies (for DNA methylation-refer to 2.3); • Case reports; • Literature reviews; • Diagnoses of aggressive periodontitis; • Human blood or mouth rinse samples.
The search was conducted using three electronic databases: SCOPUS, MEDLINE (PubMed) and EMBASE. No filter for the date was placed to ensure that all relevant articles were included. In addition, a manual search for relevant selective studies from the past ten years was performed for five selected journals: Journal of Clinical Periodontology, Journal of Periodontology, Journal of Dental Research, Journal of Periodontal Research and Periodontology 2000.

Study Selection Strategy
Following a screening of titles and abstracts, potentially relevant studies were retrieved, as well as the full text for studies where eligibility was uncertain based on the title and abstract alone. All full-text studies were independently assessed in detail to determine whether they fulfilled the pre-specified inclusion/exclusion criteria. Studies that met the selection criteria were processed for data extraction. The reference lists of these selected studies were also hand-searched for any additional relevant articles. To address the clinical context of the first review question, clinical studies of specific and global DNA methylation in humans were exclusively selected. For the second review question, only one human study describing histone modification was retrieved from the preliminary search; therefore, to broaden the discussion for this focus area only, the inclusion criterion was revised to selectively include in vivo and in vitro studies.

Literature Search and Screening Process
The initial electronic and manual search yielded a total of 864 abstracts. In the preliminary stage of the study selection process, 422 records were excluded after removing duplicates, and a further 389 studies were excluded after the evaluation of titles and abstracts ( Figure 2). The complete full-text articles of the remaining 53 studies were further assessed for eligibility according to pre-specified inclusion and exclusion criteria. A total of 30 studies were deemed eligible for inclusion in this scoping review ( Figure 2).

Study Characteristics
A summary of included studies' characteristics is described in Tables 1-3. Briefly, 15 studies were of cross-sectional design, 14 studies of in vitro experimentation, and 1 study involved a case-control sample. All studies were published in English. Thirteen studies assessed the epigenetic modifications using gingival biopsies, two studies assessed alveolar bone and soft tissue in mice, two studies analysed oral biofluid including whole unstimulated saliva and gingival crevicular fluid, and the remaining studies cultured cells in vitro, including gingival epithelial cells, PDL stem cells, keratinocytes, monocytes and osteoclast progenitors. Cells 2022, 11, x FOR PEER REVIEW 5 of 28
De Souza and co-workers conducted a high-throughput methylation microarray (Illumina) on gingival tissues from 12 periodontitis patients and 11 age-matched healthy individuals [37]. The results showed that significant differences in methylation variation in pro-inflammatory genes exist between healthy and periodontitis tissues, including a wide range of cytokines, cytokine receptors, growth factors, transcription factors and cell membrane proteins. These methylation changes are inversely correlated with their respective levels of mRNA expression. More recently, Han and colleagues observed a significantly higher methylation status of global 5-methylcytosine and N6-Methyladenosine in salivary extracellular vesicles in periodontitis patients compared with health [19].

Changes in DNA Methylation Patterns of Cytokine-Encoding Genes
The comparison of human gingival biopsies from periodontitis and healthy tissues have demonstrated distinct hypermethylation patterns of TIMP1 [35] and TNFα [31] promoter regions in periodontitis. Moreover, TIMP1 hypermethylation correlated with disease severity, with positive associations with probing depths and negative correlations with bleeding on probing [35].
Zhang et al. [33] reported a hypomethylated profile of IFNγ promoter regions, concomitant with a 1.96-fold increase in IFNγ levels in periodontally diseased tissues compared with their healthy counterpart. In contrast, Asa'ad et al. [29] and Viana et al. [42] reported no significant differences in methylation status of IFNγ and IL10 gene promoter regions in periodontitis versus healthy tissues.
Other studies have explored the methylation levels of other potential targets, such as IL6 [34] and tet-methylcytosine dioxygenase 2 (TET2) enzymes [36], and observed partial methylation of periodontitis tissues with increased levels of gene expression.

Changes in DNA Methylation Patterns of Cellular Signalling Genes
Genes related to cellular signalling are another focused target of studies on DNA methylation in periodontitis. Differential DNA methylation at the promoter regions of 12 genes (DCC, KCNA3, KCNA2, RIMS2, HOXB7, PNOC, IRX1, JSRP1, TBX1, OPCML, CECR1, SCN4B) has been detected between periodontitis and clinically healthy tissues [41].
Cyclooxygenase-2 (COX-2) gene promoter (PTGS2), a key regulatory gate for prostaglandin E2 (PGE2), appears to be hypermethylated in periodontitis gingival tissues [29,32], with methylation levels of up to 5-fold higher than healthy tissue. This trend was inversely correlated with decreased PTGS2 gene transcription and may serve as an intrinsic protective mechanism to prevent the uncontrolled breakdown of the periodontium.
A study conducted by de Faria Amormino and co-workers [39] observed hypermethylated TLR2 gene profiles in gingival biopsies of chronic periodontitis patients, correlated with probing depth parameters and quantity of inflammatory cells. This was associated with repressed TLR2 gene expression which may facilitate a chronic persistence of bacterial perturbation within the periodontal tissues. Similar findings were reported by De Oliveira and colleagues [38], observing trends for hypermethylation in TLR2 genes and significant hypomethylation of TLR4 gene profiles in periodontitis tissues compared with healthy samples. These effects on TLR2 and TLR4 gene expression may have significant implications on the integrity of the host-microbe crosstalk and in part, may contribute to dysbiosis exacerbation.
More recently, Azevedo and co-workers [40] screened a selection of targets and showed hypomethylation of signal transducer and activator of transcription 5 (STAT5) genes in periodontitis gingival tissues compared with their healthy counterparts. This suggested that loss of methylation in the promoter region of the STAT5 gene may occur during the development of periodontitis. This hypomethylation pattern has been associated with upregulation in its respective gene expression, which may promote the induction and survival of specific cell lines in periodontal lesions, regulate T-cell differentiation, and regulate the inflammatory environment within the periodontal pocket [43].

Changes in DNA Methylation Patterns following Periodontal Therapy
Asa'ad and colleagues [29] explored the methylation levels and gene expression pattern of TNFα, IFNγ and COX-2 in gingival biopsies over two months following non-surgical periodontal therapy. They concluded that treatment could reset and significantly reduce COX-2 methylation to a level comparable to health; however, this had little effect on TNFα and IFNγ. The authors postulated that for such mediators, their epigenetic modifications might be locally sustained even after the reduction in periodontal inflammation.
Similarly, a study by Andia et al. reported after 3 months of periodontal treatment and resolution of inflammation, the methylation profiles of SOCS1, SOCS3 and LINE-1 return to a level that is comparable with health [30].

Changes of sEVs-Associated Epigenetic Markers in Periodontitis
Small extracellular vesicles (sEVs) are a new class of potential liquid biopsy that are biolayered lipid nanoparticles carrying a cargo of protein, epigenetic/genetic material, lipids and metabolites from their parent cells [21,44]. The use of sEVs from oral biofluids in diagnosing periodontitis is an emerging field and has been recently reviewed by the authors [17]. Our pilot studies showed that periodontitis salivary sEVs contain DNA methylation of pro-inflammatory cytokines genes in gingivitis [23], sEVs-microRNAs [18] and global DNA methylation [19]. Of note, compared with whole unstimulated saliva, salivary sEVs-associated global 5mC hypermethyaltion and microRNAs (hsa-miR-140-5p, hsa-miR-146a-5p and hsa-miR-628-5p) can distinguish periodontitis and healthy patients [18,19]. Furthermore, sEVs also contain circular RNA (i.e., subtype of non-coding RNAs) that are involved in periodontitis pathogenesis as recently reviewed by the authors [22]). However, this requires more studies in the field to further explore the sEVs epigenetic markers in diagnosing and treating periodontitis.

Conclusions from the Included Studies
Overall, the findings from these studies highlight the local alterations of DNA methylation patterns in periodontally diseased tissues versus health. These epigenetic modifications to gene promoter regions and their resulting effects on gene expression can work to activate a series of downstream mechanisms that minimise uncontrolled periodontal tissue destruction.

Histone Modification and Its Relevance in Periodontal Inflammation
This review identified ten relevant studies analysing post-translational histone modifications implicated in chronic periodontitis, and seven relevant studies focusing on HDACi for treating periodontitis. Of these studies, there was only one cross-sectional study [45], two in vivo studies [46,47], and the remaining publications were in vitro [48][49][50][51][52][53][54][55][56][57][58][59]. All publications in Tables 2 and 3 were critically assessed to explore correlations between histone modification and the periodontal inflammatory response, along with the effects of various novel HDACi on human in vitro cells.

The Role of Histone Deacetylases, Methylases and Demethylases in Periodontal Inflammation
Most of the studies investigating histone modification stimulated human epithelial or monocytic cells in vitro to P. gingivalis and/or Fusobacterium nucleatum LPS. This perturbation to the balance of histone acetylation/deacetylation and methylation/demethylation results in disruption of epigenetic regulation of key regulatory genes and differential expression of pro-inflammatory mediators in periodontitis.
Histone methylation serves to maintain both active and suppressed states of gene expression, depending on the sites of methylation. Yin and colleagues [48] assessed the methylation of Lys4 of histone 3 (H3K4), which is associated with transcriptionally active chromatin. When gingival epithelial cells were exposed to P. gingivalis or F. nucleatum for 24 h, there were decreased protein concentrations and differential induction of H3K4 methylation compared with unstimulated cells. Similarly, when PDL stem cells were exposed to P. gingivalis LPS, H3K27me3 and H3K4me3 expression were increased on extracellular matrix and osteogenesis lineage genes, and inflammatory response genes, respectively [53].
There is growing evidence supporting the role of dysregulated epigenetic modulation of histone demethylases (KDMs) in the inflammatory response, particularly in cancer and viral infections. In the context of periodontitis, substrates of KDMs have been linked to the regulation of inflammatory and osteoclastic processes. Kirkpatrick et al. observed a significant increase in both KDM4B and KDM4E abundance in periodontally diseased connective tissue compared with healthy tissue [52]. Other subtypes of KDMs including KDM3C [54] and PHF8 [55] have demonstrated protective effects in regulating pro-inflammatory cytokine signalling and differentiation of osteogenic precursors, which appears to be suppressed with exposure to P. gingivalis LPS.
Histone acetylation and deacetylation have been implicated in epigenetic modifications of gene expression in periodontitis. Inflammation in the periodontal microenvironment stimulated downregulation of histone acetyltransferase GCN5, resulting in decreased osteogenic differentiation of PDL stem cells [49]. In experimental models where oral epithelial cells were exposed to LPS from P. gingivalis and F. nucleatum, abrupt, shortlived acetylation of histone H3 and accumulation of p300/CBP was observed [50]. The authors suggested that targeting the disruption to p300/CBP could restore tissue homeostasis through regulation of pro-inflammatory cytokine activity, and cellular differentiation, proliferation, and apoptosis.
Histone deacetylases are epigenetic markers of increasing interest, due to their potential for therapeutic targets in bone destructive diseases. Currently, there is a paucity of data on the alterations in HDAC expression in periodontal inflammatory conditions. In [48], significantly decreased HDAC1 and HDAC2 gene expression was observed in gingival epithelial cells and keratinocytes challenged by P. gingivalis and F. nucleatum. In monocytes stimulated by TNFα, Algate et al. observed increased expression in Class I and II HDACs [59]. HDAC9 RNA expression levels appear to be significantly increased in PDL stem cells under inflammatory conditions driven by periodontitis or exposure to TNFα, which impaired osteogenic capacity in vitro [51].

Changes in Histone Deacetylase Expression in Periodontitis versus Health
Cantley and co-workers [45] compared gingival biopsies of chronic periodontitis patients with their healthy counterparts. An elevated mRNA expression of Class I HDAC1 and 8 and Class II HDAC5 and 9 was found in periodontally diseased samples. Moreover, immunohistochemical staining demonstrated expression of HDAC1 by CD3-and TNF-α positive cells, indicating that such cells may be important additional targets to suppress inflammation, suggesting that HDACs modification may be involved in periodontitis pathogenesis. However, the implications of these higher levels of expression in periodontitis are unclear. More studies are required to warrant the role of HDACs in periodontal tissues and oral biofluids.

The Effects of Histone Deacetylase Inhibitors on the Periodontal Inflammatory Response
The modulation of HDACs using inhibitors is an emerging treatment for cancer, neurodegenerative diseases, autoimmune disorders and viral infections. This is largely related to its ability to upregulate cell-cycle inhibitors, downregulate immune stimulators, and suppress pro-inflammatory cytokines and osteoclast activity. Novel types of HDACi have progressed to clinical trials, with the vast majority targeting multiple HDACs in both classes I and II (i.e., 'pan' inhibitors). In the context of periodontitis, their application is limited to in vitro and in vivo experimental models of inflammatory bone loss.
Algate et al. investigated novel selective inhibitors of specific HDACs classes, utilising the stimulatory effects of TNF to induce the inflammatory response in human monocytic cells [59]. Treatment with Class I HDAC1i and HDAC2i demonstrated an anti-inflammatory effect through a dose-dependent suppression of IL-1β, TNF, MIP-1α and MCP-1 mRNA expression. Moreover, inhibition of osteoclastogenesis was observed through the downregulation of TRAF-6 and NFATc1, which subsequently reduced the expression of Cath K, DC-STAMP and TRAP osteoclast genes. Interestingly, no effects were seen with Class II HDAC5i and HDAC7i [59]; however, HDAC-9i restored the osteogenic differentiation capacity of inflamed PDL stem cells to a level comparable with their healthy counterparts [51].
Although these studies highlight that HDACi targeting single classes could reduce osteoclastic bone resorption, Cantley and colleagues [56] explored the efficacy of a broader spectrum HDACi (targeting Class I and II), known as 1179.4b. In their initial in vitro study, 1179.4b produced a strong suppression of osteoclast transcription factors NFATc1 and OSCAR and a substantial reduction in CTR and TRAP gene expression. Such changes corresponded with a marked inhibition of bone resorption in vitro. These bone-protective effects were further confirmed in periodontitis-induced mice treated with 1mg/kg/d of 1179.4b, observing significant reductions in osteoclasts and alveolar bone loss [46]. However, interestingly, no reductions in inflammation of the gingival tissues occurred. This likely suggests that the 1179.4b exerts its mechanistic effects directly on bone tissue rather than indirectly through suppression of inflammation. When comparing the bone resorption effects of 1179.4b against a selective inhibitor of HDAC Class I (MS-275), MS-275 appeared to reduce gingival inflammation; however, it had no observed effect on bone loss.
Further investigations of combined Class I and II HDACi compounds, such as Trichostatin A (TSA), were conducted by Huynh and colleagues [47,57]. When pre-treating PDL stem cells with TSA, osteogenic differentiation of PDL stem cells were induced and osteoblast-related gene expression increased. This subsequently enhanced mineral deposition and bone regeneration in vivo of mouse calvaria defects.
Lagosz et al. detailed the effects of treating healthy and periodontally diseased gingival fibroblasts with pan-HDACi and selective HDAC3/6i. Treatment of both healthy and periodontally diseased gingival fibroblasts with pan-HDACi or HDAC3/6i prior to P. gingivalis infection significantly reduced the induction of a subset of inflammatory mediators (CCL2, CCL5, CXCL10, IL1B, COX2 and MMP3) implicated in periodontitis [58].

The Effect of Bromodomain and Extra-Terminal Proteins Inhibitors in Periodontitis
Recent studies in the field of epi-drugs have evaluated small molecule inhibitors known as bromodomain and extra-terminal motif proteins inhibitors (BETi) that reversibly bind the bromodomains and prevent protein-protein interaction between BET proteins and acetylated histones [60]. For example, BETi, I-BET151 and JQ1, were shown to suppress inflammatory gene expression (IL6, IL1β and CCL2) in gingival fibroblasts and telomeraseimmortalized gingival keratinocyte cell line (GEC line TIGK) after P. gingivalis stimulation [61]. Systemic administration of BETi-JQ1 in a murine periodontitis model showed a significantly inhibited inflammatory cytokine expression in diseased gingival tissues and alleviated alveolar bone loss through osteoclast reduction in periodontal tissues [62]. Moreover, the use of Apabetalone inhibited P. gingivalis LPS-induced macrophage M1 polarization via regulating miR-130a/STAT3 axis [63]. Overall, current evidence highlights the potential of BETi to regulate key inflammatory mediators in periodontitis and warrants further clinical translation to evaluate their efficacy as periodontal treatment adjuncts.

Conclusions from the Included Studies
It is difficult to translate the current in vitro and in vivo findings into the context of human periodontitis, as existing models fail to address that periodontitis is associated with a complex community of oral bacterial species, including commensals, rather than a single dominant species. Therefore, the role of various KDMs, histone acetylases and HDACs in the response to bacterial challenge may ultimately differ depending on the individual specific pathogenicity of their microbial profile. Nonetheless, one cannot refute the evidence of epigenetic effects on histone modification in the periodontal inflammatory response, which has pioneered the advancement of precision medicine and 'epi-drugs'.
The focus of HDACi within periodontitis, albeit confined to animal and experimental studies, has mostly shown that such compounds can dampen the exaggerated inflammatory response to plaque biofilm, and potentially suppress osteoclast activity. Furthermore, there may be some redundancy in targeting specific HDACs in periodontitis, as a combination of HDAC classes may need to be inhibited for more potent effects. Nonetheless, these compounds show promising clinical benefits as an adjunct to conventional periodontal therapy.

Discussion and Conclusions
This review identified the current evidence relating to epigenetic modifications in periodontitis, comprising specific modifications to DNA methylation patterns and histone proteins. Hypermethylation and hypomethylation of various gene promoter regions associated with pro-inflammatory cytokines, and upregulation and downregulation of HDAC activity in gingival tissues, GCF, saliva sEVs and cultured periodontal cells under inflammatory conditions have been well demonstrated. These epigenetic alterations can either dampen or promote the inflammatory response to bacterial challenge (see Figure 3).

Implications of the Findings for Research
Most existing studies utilise a cross-sectional design, providing a snapshot of dis activity at a single time point. Given that periodontitis is a dynamic inflammatory dis fuelled by dysbiosis, with periods of remission and exacerbation, cross-sectional ep netic studies provide limited insight into disease onset and progression over time f the host perspective [64]. Rather, future longitudinal studies are warranted to mon epigenetic modifications in periodontitis over time and identify susceptible and non sponding individuals. Large-scale epidemiological epigenome-wide association stu may also have merit, preferably through widespread screening of DNA methyla markers to reduce data extraction errors [35].
Whilst addressing the second review question, a gap in the literature was identi with no longitudinal human studies conducted on histone modification changes in periodontal tissues. Preliminary evidence from current in vitro and in vivo studies demonstrated marked histone modulatory changes in response to periodontopathic terial challenge (see Figure 3). However, their use of single pathogen-induced LPS mo inherently has severe limitations in applicability to humans; therefore, future rese needs to be carried out in the clinical setting. This could entail the long-term follow-u histone modifications in treated and untreated patients with various disease severi Such study designs could broaden our current understanding of periodontitis aetio and susceptibility.

Implications of the Findings for Clinical Practice
Research into epigenetic biomarkers may facilitate a more detailed understandin the interactions between an individual, their genes and the environment, which pre pose them to periodontitis. This forms the basis for precision medicine, where epigen could be used as a reliable tool to identify why certain patients with the same clin phenotype do not respond to conventional periodontal treatment. Most of the exis human studies exploring this topic have collected gingival biopsies from periodon

Implications of the Findings for Research
Most existing studies utilise a cross-sectional design, providing a snapshot of disease activity at a single time point. Given that periodontitis is a dynamic inflammatory disease fuelled by dysbiosis, with periods of remission and exacerbation, cross-sectional epigenetic studies provide limited insight into disease onset and progression over time from the host perspective [64]. Rather, future longitudinal studies are warranted to monitor epigenetic modifications in periodontitis over time and identify susceptible and non-responding individuals. Large-scale epidemiological epigenome-wide association studies may also have merit, preferably through widespread screening of DNA methylation markers to reduce data extraction errors [35].
Whilst addressing the second review question, a gap in the literature was identified, with no longitudinal human studies conducted on histone modification changes in the periodontal tissues. Preliminary evidence from current in vitro and in vivo studies has demonstrated marked histone modulatory changes in response to periodontopathic bacterial challenge (see Figure 3). However, their use of single pathogen-induced LPS models inherently has severe limitations in applicability to humans; therefore, future research needs to be carried out in the clinical setting. This could entail the long-term follow-up of histone modifications in treated and untreated patients with various disease severities. Such study designs could broaden our current understanding of periodontitis aetiology and susceptibility.

Implications of the Findings for Clinical Practice
Research into epigenetic biomarkers may facilitate a more detailed understanding of the interactions between an individual, their genes and the environment, which predispose them to periodontitis. This forms the basis for precision medicine, where epigenetics could be used as a reliable tool to identify why certain patients with the same clinical phenotype do not respond to conventional periodontal treatment. Most of the existing human studies exploring this topic have collected gingival biopsies from periodontitis patients; however, this may deter patient acceptance and invalidate sample reproducibility [65]. Other less invasive and easily accessible DNA sources, such as gingival crevicular fluid and saliva, could be considered clinically feasible diagnostic tools for targeted epigenetic analysis [66]. It is worth noting that applying DNA methylation and histone modification in the clinic is still at its infancy, due to limited understandings of epigenetic mechanisms in periodontitis pathogenesis. More studies are required to validate the role of epigenetics in periodontitis disease management.
Although still an emerging field, understanding periodontitis-induced epigenetic changes in DNA methylation and/or histone modifications could point towards new targeted drug developments in periodontal management. Furthermore, the plasticity of epigenetic modifications has driven increased interest in the development of targeted epidrugs which could take advantage of this reversible state to ultimately restore a normal epigenetic landscape [33]. Recent studies evaluating HDACi have demonstrated promising results in the field of medicine, with implications for periodontitis treatment using 1179.4b, TSA, HDAC3/6i, pan-HDACi, HDAC1i and HDAC2i (see Figure 3). With its unique ability to target bone remodelling, HDACi therapeutics could serve as a significant advance in personalised periodontal treatment approaches.