Epigenetics and Stem Cells Applications in Periodontal Therapy

Abstract

While periodontitis is closely linked with pathogen outgrowth, many patients have the risk of recurrence after therapy. Variations of inflammatory genes are associated with an increased susceptibility of periodontitis. Epigenetics can regulate these gene expression. In this chapter, we will highlight on the potential role of epigenetic changes in different facets, more particularly in genes involved in inflammation. Epigenetics act through remodeling of chromatin and can selectively activate or inactivate genes, determining their expression. Epigenetics could play an essential role in understanding the mechanism of gene-environment interactions, and the factors which stimulate periodontitis and reduce its response to therapy are now the subject of many studies. Also, mesenchymal stem cells (MSCs) are a promising source to regenerate periodontal tissues. They could be a good alternative to the adopted therapies, ignoring the artificial biomaterial limitations. They could be considered as a natural process for periodontium regeneration and has an immunomodulatory role to resolute the infection. For this reason, it is necessary to investigate and evaluate MSCs applicability in humans, and their clinical approach involved in regeneration of periodontal tissues.

1. Introduction

Periodontitis is a chronic inflammatory disease caused by a gram-negative bacterial plaque located on gingiva and teeth surfaces. It is induced by immune imbalance between the plaque and host inflammatory response toward the alveolar bone [1]. It is frequently encountered in the oral cavity affecting the structures that support teeth, leading to periodontal pocket, loss of attachment, and alveolar remodeling [2, 3].

Periodontitis is a complex disease caused by multiple factors, extrinsic (modifiable), and intrinsic (nonmodifiable) factors [4]. Although its mechanisms are still unclear, outgrowth of multiple opportunistic microbes mainly contributes to inflammation, but it is not solely sufficient factor leading to this condition. Numerous risk factors can impede the immune response and periodontitis prevalence [5], like environmental factors, systemic inflammation diseases such as diabetes [6], cardiovascular diseases [7], metabolic syndrome [8], and pneumonia [9], lifestyle-related factors, such as smoking and diet, as well as oral hygiene [10], and genetic variables [11].

Although most of the periodontal studies have focused on microbial pathogens and oral environments, inflammation susceptibility association with intrinsic factors such as genetics is also determined. Some people are more disease-susceptible or treatment resistant, which emphasizes that fact.

In this chapter, we summarize the most important genetic factors in periodontitis progression and focus on epigenetic modifications involved in the etiology of periodontitis, which can be considered as mediators between environmental and genetic factors. This information could be useful guidelines for new biomarker that will help in periodontitis diagnosis and treatment.

2. Periodontitis-related genetic variation

2.1 SNPs in inflammatory genes

Genetic factors are very important in periodontitis development, but we can consider genetic variations as a risk factor only when combined with external agents and physical activities. Genetic variations associated with multifactorial diseases cannot be able to define easily.

Genetic polymorphisms can be caused by a single nucleotide substitution in a DNA sequence with another one, deletion or insertion one or more nucleotides, or repetitive sequences insertion [12]. The polymorphism leads to a change in the coded protein structure if it occurs in an exon, the part of the gene that codes the protein, while it can lead to a change in gene function if it comes in the non-coding sequence (intron). Changes in the transcription-regulating sequence (promoter) can affect gene expression [12].

Single nucleotide polymorphism (SNP) in a gene is used as a genetic marker when it can express a special phenotype [4]. Periodontitis susceptibility can be detected with a SNP. Many studies have focused on the description of genetic polymorphism effect in inflammatory proteins and gene expression in several populations. They have shown that susceptibility is clearly affected by multiple allelic variants [13].

Defensins (DEFB) are important components of innate immunity and extensively exist in the gingival tissue. SNPs in defensin gene have been detected to correlate with gene expression and with the risk of periodontitis [14]. Pro-inflammatory cytokines have a critical role in periodontitis pathogenesis, and their gene SNPs may influence the degree of host response to microbial infection and then the disease severity [13, 15].

The most periodontitis-related SNPs have been studied in the Toll-like receptor-2 (TLR-2) [16], TLR-4 [17], Fc-γ receptor (FCGR2A) [18], interleukin-1 (IL-1) [19, 20, 21], IL-4 [22], IL-6 [23, 24, 25], IL-10 [23, 26], IL-18 [27, 28], vitamin D receptor [11, 29], tumor necrosis factor alpha (TNFA) [30, 31, 32], matrix metalloproteinase (MMP) [33, 34], and cyclo-oxygenase-2 (COX-2) [35].

These studies have determined the relationship of genetic polymorphisms at multiple loci with periodontitis, but it is still unclear. It should be noted that this relation is not always strong due to differences between populations and periodontitis subtypes.

2.2 Genome-wide analysis of genetic variation

Recently, it has used important “omics” techniques such as genomics, proteomics, host metabolomics, and oral microbiota metagenomics to well understand the progression of periodontitis [36]. Genome-wide association studies (GWAS) have been applied to detect genetic variations, from a large set of SNPs overall DNA, that are correlated with periodontitis [37, 38]. Many novel genes have been identified by GWAS studies for susceptibility of periodontitis, including NIN, NPY, and WNT5A in severe chronic type, NCR2 and EMR1 in moderate chronic [39], and GLT6D1 in aggressive type [40].

GWAS has provided new insights into periodontitis etiology at the genome level to answer how pathogenesis is affected by the identified susceptibility genes. Genome-wide genetic variation analysis includes total gene transcript, which referred to as transcriptomics. It has been carried on periodontal tissues [41, 42]. Proteomics and metabolomics have also been conducted on gingival crevicular fluids or saliva to study the influence of proteins and metabolites on immune mechanisms [43, 44]. It is remarkable that not only the genetic programming can be reflected by transcripts, proteins, and metabolites levels, but also disease progression and the results of response to environmental factors. Gene expression is regulated by chemical modification on DNA and its associated proteins (histones), which referred to epigenetics.

Now, it is accepted that heritable changes can be caused by the environment. In other words, it is known that neoplastic changes, normal differentiation, and changes caused by infections and inflammation are all results of epigenetic modifications.

Epigenetics is described as chemical changes in gene expression patterns, without changes in the DNA sequence. These modifications are consequences of environmental response, thereby remodeling the chromatin and selectively activating or inactivating genes, and determining their expression during development [45].

3. Epigenetic modifications

3.1 DNA methylation

Nucleosome is a repeating structure in which the chromatin is coordinated around and tightly packaged in the nucleus. It consists of an octamer of (H2A, H2B, H3, and H4) histones [46]. N-terminal tail of every histone and the C-terminal tail of histone H2A stick out of nucleosomal structure facilitating post-translational modifications. These epigenetic changes play a critical role in condensing and decondensing the DNA according to gene activity, regulating gene transcription in disease onset and progression, and in maintaining the integrity of chromosomes in all cell cycle phases [46]. The nucleosomes are fairly distributed over the chromosomes, and recently there are advanced techniques that can predict where their positions are accurately. Significantly, post-translational modifications occur in histones at defined positions and strongly correlate to DNA activities [4]. For instance, the addition of trimethyl at lysine 4 in histone H3 (H3K4) activates gene transcription, while trimethylation at H3K9 and H3K27 represses the transcription [4].

The most well-distinguished epigenetic modification is DNA methylation, which is an active process catalyzed by DNA methyltransferase (DNMT) enzymes. These enzymes add methyl group at the 5th carbon of cytosine in CpG dinucleotides known as CpG islands, which are CpG-rich areas of the DNA. They exist mostly in the promoter region of genes. Approximately, these CpG islands are noticed in 50% of human genes, and most of them are unmethylated in normal tissue [45].

Unmethylated CpGs are linked to transcriptionally active regions, whereas methylated DNA is promoted to interact with histone deacetylase enzymes (HDACs). HDAC enzyme removes acetyl groups of histones leading to compact the chromatin (heterochromatin) and then gene silencing because of prevention of transcription factor binding (Figure 1) [45]. Therefore, genes are not expressed. DNA methylation could regulate many cellular functions, including DNA repair and expression of inflammatory gene [47].

Figure 1.

represents DNA methylation on cytosine by DNA methyltransferase (DNMT) enzymes, leading to chromatin compaction (heterochromatin) and silencing gene expression.

DNA methylation is maintained by DNMT1, and DNMT3a and DNMT3b mediate de novo methylation [48]. DNTM1 and HDAC activity are associated by making up a transcriptional repression complex. According to the role of DNMT1 and HDAC, their inhibitors could be therapeutic drugs in chronic inflammation [45].

3.2 Epigenetics of histone proteins

Epigenetic changes to the histones are caused by post-translational covalent modifications. Histone acetylation is catalyzed by histone acetyltransferase (HAT) enzymes which hold an acetyl group from acetyl-CoA to amino groups of lysine in histones. Covalent acetylation of histone leads to an open chromatin (euchromatin) which facilitates gene transcription by increasing transcription factor binding [49].

On the contrary, histone deacetylases (HDACs) remove acetyl groups from histones to form heterochromatin conformation which represses gene transcription [49]. In inflammatory, euchromatin activates genes of innate immune, whereas conformation of heterochromatin could suppress these genes. Hence, processes of the acetylation (by HAT) and deacetylation (by HDAC) must be matched to preserve immune homeostasis by the prevention of abnormal transcriptional activation or repression [49, 50].

Histone methylation is catalyzed by methyltransferase enzymes. Methylation of H3 histone is the most principal variation that includes H3 lysine 4 (H3K4), H3K9, H3K27, H3K36, H3K79, and H4K20. H3K4me1 presents enhancer functions, and H3K27me3 is correlated with gene repression. These methylation processes may affect the disease by mediating regulation of cell cycle, DNA damage and development, and differentiation [51]. Histone methylation in aging tissue may lead to stem cell dysfunction. This may explain why chronic inflammations are popular in older people. Differential histone methylation could influence T cell inflammatory responses [52, 53].

For instance, in the presence of H3K27m3, interleukin-4 (IL4) and IL17 genes in Th1 cells and interferon-gamma (IFNG) gene in Th2 cells have been silenced. Histone methylation presents an important effect in controlling allogenic immune responses in graft-versus-host-disease (GVHD) [45].

Demethylating H3K27me3 at jumonji domain by JMJD3 histone demethylase regulates gene expression. Several studies have indicated the important roles of JMJD3 in numerous human diseases, such as infections, immune diseases, developmental diseases and cancer [54], and enhancing STAT6 gene expression upon IL-4 treatment [55].

H2A and B histones are changed by ubiquitination in cells and play an essential role in stem cell homeostasis by controlling gene pluripotency and differentiation, DNA repair, and X-chromosome deactivation [56].

Phosphorylation is another histone modification important in gene regulation. Histones are phosphorylated in cell cycle regulation in the physical state of chromatin maintaining. Researches presented that phosphorylation of histone H3 at Ser 10, 28, and Thr 11 activates transcription. Inflammation could activate tyrosine kinase, Janus kinase 2 (JAK2) which can directly phosphorylate Tyr 41 on H3 to stimulate specific genes transcription. That links histone phosphorylation to immune signaling [45].

3.3 RNA modifications

Among post-transcriptional modifications that regulate gene expression of an immune response, there are three mechanisms, RNA splicing, mRNA polyadenylation, and regulatory molecules, which include microRNA and long non-coding RNAs. The first mechanism is mRNA transcript modification when DNA is transcribed into mRNA. This process is called splicing, which removes non-coding sequences of mRNA (introns). Splicing patterns can be altered by bacterial products resulting in different protein isoforms [57].

After splicing, at the terminal end of the transcript, mRNA gains a sequence of adenosine bases called a poly(A)tail. This poly(A) tail length detects mRNA stability and the amount of mRNA transferred to the cytoplasm. It is also a way to maintain silenced mRNA within cell cytoplasm. In bacterial infection, cells can response rapidly by using silenced mRNA to induce gene expression without any need to retranscript DNA in the nucleus. This was also advocated as a mechanism to bind immune response to prevent chronic inflammatory inducing [57].

MicroRNA (miRNA) is a small non-coding molecule of RNA obtained from cleavage of longer RNA transcripts. It could regulate and fine-tune gene expression by a sequence-pairing homology to mRNA. When it binds to mRNA, it could repress the expression. Bacterial products and inflammatory cytokines can modify miRNA expression [57].

Recently, the long non-coding RNAs (lncRNAs) were discovered as another class of non-coding RNAs. lncRNAs involve in many biological roles. They bind to chromatin regulatory proteins, adjusting binding of these proteins to enhancer regions in the DNA, thereby regulating gene expression [57]. These RNAs function in the immune response involve host response regulation, including innate immunity since virus infection modifies lncRNAs expression. Chronic inflammations, such as periodontitis, have particular target tissues to destroy. Epigenetic patterns differ in each gene depending on the cell type, resulting in local and systemic gene expression [57].

This presents a site-specific change in the immune response to external stimuli, which may vary between patients and increase disease susceptibility. At last, we can say that studying immune genes and their susceptibility to epigenetic and genetic modification could define the vulnerability of periodontitis development.

4. Epigenetics in complex diseases

Continuously, there is new evidence showing the valuable role of epigenetic profiles in disease development and thus in diagnosis and treatment. GWAS have demonstrated that epigenetic alterations in tissues exposed to stress and extrinsic environmental factors give insight into cell response mechanism [58]. These studies also provide information about cell dealing with these alterations, what the risk factors involved, and whether they can be treated, or lead to cell apoptosis. GWAS has detected association between genetic and epigenetics modification and the susceptibility and progression of the disease [58]. Extensive analysis has been performed to determine gene expression profiles and/or loci modified epigenetically to identify which are associated with a specific disease [59]. Epigenetic marks and clinical outcomes combination enhances the vision about the prediction of the disease and therapeutic target determination. And yet it is more a perception than a reality in medicine nowadays.

5. Epigenetics in environmental response

Each tissue including embryonic stem cells has a specific epigenetic pattern, and upon processes of development and regeneration this pattern modifies. Epigenetic pattern modifications are influenced by external factors that involve in differentiation, like hormones [60]. So we can say that these patterns are reflection of the tissue activity in defined time and its gene expression profile.

Experiments on induced pluripotent stem cells have proved that treatment could generate a specific epigenetic profile. Microbiota could trigger an immune response by excreting signals that are able to change that profile. In gingival tissues, microbial signals combined with external risk factors from food intake and smoking could make a similar effect on epigenetic profile [61].

6. Epigenetics in periodontitis

Epigenetics is a relatively fresh thought in periodontitis studies. This concept could reveal the lost relationship between disease, genetics, and environment. It is clear that periodontitis is most common in the elderly, but recently researches have presented that some types of this disease like gingivitis, may develop at any age [62].

Each tissue can have a special epigenetic pattern, and alterations depend on regenerative and progression processes. For instance, there is certain indication that the epigenetic profile of embryonic stem cells changes upon differentiation [63]. Continuously, the oral mucosa is exposed to pathogens and microflora. Gingival cells must distinguish both of them and prompt inflammatory responses only to pathogens. It is already well proved that periodontium inflammation is induced by the microbial biofilm [62]. Its continuous fluster causes to constant apoptosis, thereby tissue regeneration process that may enforce unique epigenetic alterations resulting in a different phenotype. External environmental factors, growth factors, and hormones that regulate differentiation may lead to these epigenetic modifications [64].

Oral hygiene is certainly a key factor to healthy gingiva and decreases the inflammatory condition. Lifestyle of smoking, malnutrition, lack of physical activity, and drug use heavily affect the epigenetic profile leading to many diseases, including periodontal disease. Chronic periodontitis needs elevated production of gingiva cells such as epithelial cells, fibroblasts, and osteoblasts to compensate for cell death. Cell proliferating imperfection may cause abnormal response, which in turn leads to a motley phenotype [65].

In fact, periodontal disease is identified by site susceptibility with certain areas showing the majority of the disease and others being healthy. Epigenetic modifications could happen at specific regions or affect most of the periodontium. Accumulation of site plaque may explain site prediction, but still after treatment and with high hygiene these sites' susceptibility remains, and epigenetics explain their inflammatory responsiveness alteration [45].

It has been pointed out that the inflammatory response in periodontitis is similar to other inflammatory diseases, where transcription factors upregulation and signal transducer and activator of transcription (STAT), and chromatin changes occur that might induce epigenetic modifications [66].

6.1 DNA methylation in periodontal disease

In periodontitis, as we already mentioned that the epigenome differs between inflamed and unflamed periodontal tissues in the same person. In addition, different methylation profiles correlated with regulating apoptosis, cell differentiation, lipopolysaccharide-mediated signaling, and oncogenesis pathways were presented in inflamed sites compared to healthy ones.

Many studies have searched for methylation of cytokine genes in periodontitis. Analysis of the IL-8 promoter suggests a tissue-specific profile in DNA methylation. In epithelial cells, the methylation frequency of IL-8 was altered in patients when compared with controls, but no difference was noticed in gingival cells or blood leukocytes. It was found that IL-6 promoter partially methylated in both controls and patients, with higher level of IL-6 expression in periodontitis subjects [67]. Increased IL-6 serum level in chronic periodontitis patients was correlated with hypomethylation of the −74 bp CpG site in the IL-6 promoter. This shows that single CpG site methylation may affect the production of cytokines and thus disease [68].

Studies have reported a lower methylation level of CpG sites in IFN-γ promoter in periodontitis compared to healthy tissues. This was associated with rising IFN-γ transcription [68]. The increase was independent of promoter methylation. Regarding the IL-10 gene, three CpG islands near to the promoter around the −1087 polymorphism were methylated in peripheral blood cells and gingival tissues. B cells treatment with 5-aza-deoxycytidine (5-aza), the inhibitor of DNA methylation, increased IL-10 mRNA whatever the genotype of the −1087 SNP [69].

Hypermethylation of two CpG islands in the TNF-α promoter was defined in chronic periodontitis. Treatment of ThP1 cells with the 5-aza regulated TNF-α transcription and raised TNF-α expression [57].

Not only cytokine methylation level has been investigated in periodontitis, but also some other genes linked to inflammation. The pattern of DNA methylation in the toll-like receptor-2 (TLR2) and TLR4 genes was estimated in gingival tissues. Their results showed major unmethylation of the TLR4 gene promoter, but the TLR2 gene promoter was found as a mosaic of methylated and unmethylated DNA in the majority of samples. Hypermethylation and a subsequent reduced expression of TLR2 in periodontitis tissues was reported. A positive association was found between periodontal probing depth and TLR2 methylation [70].

E-Cadherin and COX-2 genes methylation status was studied in breast cancer and chronic periodontitis patients and compared hypermethylation between both diseases and healthy people [71]. The thought that cancer and chronic inflammation may have an identical epigenetic profile was proved. This was shown that DNA methylation may be a connection between cancer and inflammation.

Zhang et al. study illustrates a mechanism on how gene expression may be affected by DNA methylation of a gene promoter. It has shown that inflammation causes hypermethylation of PTGS2 promoter which correlated with reduced levels of its gene transcription [72]. Interestingly, a highly methylated CpG island was found close to the binding site of transcription factor, suggesting that methylation status may prevent the binding of NFκB [72]. This is in accordance with other studies on methylation of cytokine promoters in which the CpG islands analyzed were in close proximity to transcription factors NFκB and Sp1 binding sites [73]. NFκB and Sp1 regulate gene expression of the immune response. They are activated by bacteria binding to TLRs and the subsequent activation of mitogen-activated protein kinase (MAPK) pathways. Binding of Sp1 and NFκB to adjacent sites influenced nucleosomes repositioning upstream and downstream of the factors, thereby inducing gene transcription. It has been proved that extracellular signal transduction pathways regulate methylation enzymes, such as the MAPK ERK kinase pathway [57].

Abnormal DNA methylation in tissues has been caused by inflammation. This mechanism has been a correlation between periodontitis and changes in methylation level in cancer. In infected tissues, eosinophils and neutrophils create materials that can halogenate cytosine. Halogenated cytosine and methylated DNA cannot be differentiated by DNMT1 enzyme, thereby de novo methylation increases in cells of inflamed tissues [74].

6.2 Histone modifications and periodontitis

Gene expression is regulated by post-translational histone alterations, such as acetylation/deacetylation, methylation, and phosphorylation, that have an epigenetic role in chromatin organization. Studies have presented a relationship between histone acetylation and cardiovascular diseases. Regarding inflammation, several genes of pro-inflammatory cytokines are managed by interacting with some transcription factors, such as NF-kB, SP1, and AP-1. As these factors are activated, they bind to particular response elements on the gene promoter and interact with co-activators with HAT activity to control gene transcriptional activity [75].

In inflammation, it has been shown that lysine residues acetylation on histone H4 increases gene expression of pro-inflammatory cytokines such as IL-1B and TNF. An experiment has applied by using trichostatin A (TSA) in airway epithelial cells which is able to inhibit HDACs. It has resulted in increased inflammatory signals, which confirms HDACs role in inflammatory gene repression [76].

Although very few observations, histone modifications are also implicated in periodontitis. Maintenance of histone acetylation of genes correlated to osteoclastogenesis has been found to be crucial to prevent bone loss in periodontitis. Treatment with HDACs inhibitor has improved bone levels [77].

When stimulated with P. gingivalis and F. nucleatum, gingival epithelial cells presented differential expression of HDAC1, HDAC2, DNMT1 genes, and H3 Lys 4 methylation. Studies have suggested that P. gingivalis and epigenetics interact together to regulate immune response, and a correlation between LPS stimulation and histone alteration has been found [78].

7. Therapeutic approach and future perspectives

Researches have investigated patient care benefit in relation to current therapeutics in inflammations. Indeed, clinicians could incorporate some of the advances in epigenetics and anti-inflammatory management procedures to improve diagnosis and treatment of periodontal diseases. Epigenetic alterations previously discussed could benefit in better diagnosis, and some of them are worth highlighted.

Epigenetic processes evolutionarily give an equilibrium in gene regulation. However, abnormal alterations of epigenetics may cause major problems in inflammations. The studies governing epigenetic modifications may answer to periodontitis initiation and progression. For example, methylation levels in genes and miRNA targeting may be utilized as possible biomarkers for the periodontitis severity. Clinicians may use, in addition to their diagnostic methods, identifying SNPs related with high disease susceptibility. The etiological similarities between periodontitis and other immune diseases have made it possible to develop potential new therapeutic approaches [79].

Actually, many studies aim to discover new targeted drugs regarding the exact inflammatory and genetic factors of periodontitis progression. Inhibitors of histone deacetylase (HDACs) and DNA methyltransferase (DNMTs) could affect periodontal therapy positively [79]. The main warning is that currently, the epigenetic reprogramming drugs are not targeted and site-specific, even so, 5-Aza-2′-deoxycytidine (decitabine) as a demethylation drug could shift methylated genes in the inflammatory system [80].

More experiments are necessary to confirm targeting drugs at a goal loci. Suberoylanilide hydroxamic acid (SAHA), approved HDAC inhibitor, represses class 1 and 2 of HDACs. SAHA increases histone acetylation, which unwinds the chromatin and induces gene expression. Histone methyltransferase inhibitor (HMTase) inhibits histone methyltransferase EZH2 and activates tumor suppressor gene expression including specific inflammatory genes [81].

Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulatory proteins that can read acetylated histone tails and contribute to gene transcription regulation. Recently, the BET inhibitor JQ1 was suggested to be a potential treatment model for periodontitis. It represses both an immune response and alveolar bone loss in periodontitis. Experiments have demonstrated that JQ1 decreases BET protein binding to the NFκB promoters, leading to reduce NFκB transcription [82].

Aspirin has a negative effect on allergic inflammation by regulating DNMT1 expression. Aspirin could prevent antigen from modifying HDAC3 and DNMT1 expression [83].

8. Stem cells and periodontitis

Recently, stem cells have been considered as a new tissue-engineering treatment approach. Various studies have demonstrated that these cells have regenerative and immunomodulatory properties make them applicable in inflammation treatment. The immunomodulatory properties are due to secretion of extracellular vesicles (EVs) that are membrane-bound compounds. EVs have the ability to transport DNA, peptides, and lipids that may influence target cell activity and non-coding RNA leading to gene expression modifications. EVs could offer a prolong therapy free of cells, to avoid immunogenicity reduction and tumorigenesis risk. Also, they play as a carrier of therapeutics [84].

Mesenchymal stem cells (MSCs) have several tissue sources, such as umbilical cord, bone marrow, or adipose tissue [85]. Furthermore, many stem cell subtypes have been isolated from gingival and ligament tissues called dental mesenchymal stem cells (DMSCs) [86]. DMSCs are pluripotent have the ability to renew and differentiate into numerous cell types. They could alter activity of immune-related cells depending on many factors secreted by immune cells [87]. Because of pathogens of periodontitis and inflammatory response, stem cell effects and their osteogenic differentiation are suppressed.

OCN and Runx2 genes involved in osteogenic have reduced expression in inflammatory tissues. Abnormal osteogenic differentiation in ligament stem cells is influenced by epigenetic alterations. Hypermethylation of bone development genes reduces their expression [88].

8.1 Stem cells and periodontal tissue regeneration

Alveolar bone resorption, a characteristic of periodontitis, needs regenerative therapy. It is caused by imbalance of bone homeostasis which upgrade osteoclastogenesis. Nuclear factor kappa light-chain enhancer of activated B cells (NFκB) has involved in this process. Stem cells could be a great pathway for regeneration and bone loss repression.

Experiments have presented that biomarks expressed by PDLSCs are correlated with osteoblasts and cementoblasts. Injection of MSCs derived from gingiva has reduced bone resorption in mice after 1 month [89]. Similarly, it is shown that BM-MSCs therapy could reduce osteoclastogenesis by repression of mRNA expression of the receptor activator of NFκB (RANKL) [90].

Another study demonstrated that treatment with MSCs in fibroin/chitosan hydrogel in rats induced alveolar bone gain [91]. Furthermore, new tissue formation can be promoted by mediators secreted from MSCs such as cytokines and growth factor, and EVs. Studies have proved that MSC-EVs associated with increased bone volume [92]. Numerous researches have proved the critical role of stem cells and their products in bone homeostasis modulation. For example, PDLSCs and GMSCs secrete exosomes that could disturb Wnt pathway expression, leading to induce osteogenic differentiation [93, 94].

Additionally, modulation of mesenchymal stem cells could promote EVs production with overexpressed materials. In periodontal experiment on mice, exosomes from bone marrow MSCs charged with miR-26a have the ability to induce differentiation of osteogenic and improve new bone formation [95].

Interestingly, studies have compared the potential role in osteogenic between PDLSCs, dental follicle progenitor cells, and dental pulp stem cells. Genes related to osteogenic were upregulated, and their osteogenic ability was higher [96]. Furthermore, it has presented that PDLSCs could improve osteogenic differentiation in other cells [84]. Additionally, other stem cell types could affect the infected microenvironment, like bone marrow stem cells that cause repression of T lymphocyte proliferation using IFN-γ. The transplantation of stimulated BMSCs repressed cytokine production, inflammatory tissue destruction, and new tissue formation [84]. The important role of stem cells is due to their proliferation capacity and ability to migrate and differentiate, and immunosuppressive properties in the inflamed microenvironment [97].

9. Conclusions

Although many questions remain, epigenetic modifications are important targets for developing new drugs of periodontitis. Experimental periodontitis in vivo can provide insights into understanding bacterial-induced disease-specific epigenetic changes in histone changes and/or DNA methylation that may indicate new therapeutic targets.

MSCs and their EVs have induced bone loss repression. EVs secreted from healthy stem cells are able to improve cell reformation properties in inflamed tissues. This promising role could be benefit in clinical application of periodontal therapy controlling the inflammation through non-surgical and surgical treatments. However, the therapeutic success needs many factors that include the microenvironment, stem cell sources, the experimental design, and the procedures and protocol of isolation and transplantation. Also, it should clarify the inclusion and exclusion criteria.

Acronyms and abbreviations

MSCs	Mesenchymal stem cells
SNP	Single nucleotide polymorphism
DEFB	Defensins
TLR-2	Toll-like receptor-2
FCGR2A	Fc-γ receptor
IL-1	Interleukin-1
TNFA	Tumor necrosis factor alpha
MMP	Matrix metalloproteinase
COX-2	cyclo-oxygenase-2
GWAS	Genome-wide association studies
DNMT	DNA methyltransferase
HDACs	Histone deacetylases
HATs	Histone acetyltransferases
IFNG	Interferon gamma
GVHD	Graft-versus-host-disease
JAK2	Janus kinase 2
miRNAs	microRNAs
lncRNAs	Long non-coding RNAs
STAT	Signal transducer and activator of transcription
LPS	Lipopolysaccharide
5-aza	5-aza-deoxycytidine
MAPK	Mitogen-activated protein kinase
EVs	Extracellular vesicles
DMSCs	Dental mesenchymal stem cells
NFκB	Nuclear factor kappa light-chain enhancer of activated B cells
RANKL	Receptor activator of NFκB