# Epigenetic Signatures of Dental Stem Cells: Insights into DNA Methylation and Noncoding RNAs
## 4 Discussion
This functional divergence between hypomethyllated (poised or active loci) and hypermethylated (silenced) regions supports models in which epigenetic plasticity underlies stem cell multipotency and lineage commitment. These findings reinforce the notion that DNA methylation profiles and the related biological pathways play a significant role in defining the identity and functional activity of dental stem cells and the role of epigenetic regulation in cell fate determination. This may provide significant insights into odontogenesis, as well as into orthodontic context. For example, pathways involved in ECM-receptor interaction and actin cytoskeleton regulation--enriched in several clusters--are directly implicated in mechanotransduction, a process highly relevant in orthodontics. In this context, the response of periodontal ligament and surrounding tissues to mechanical force is fundamental for tooth movement. Epigenetic changes affecting focal adhesion and cytoskeletal genes may influence this response, potentially offering biomarkers for individual variability in treatment outcomes. Further, methylation-mediated modulation of TGF-\(\beta\) and Wnt signaling--key pathways in craniofacial development and remodeling--suggests that these profiles could be leveraged to understand susceptibility to root resorption, a common side effect in orthodontic therapy. Moreover, Wnt and BMP pathways are activated during tooth movement [1; 37]; therefore, the methylation profile of these pathways might help predict cellular responsiveness. This study also focused on shared epigenetic features among all three cell types, evaluating probes that were consistently hypermethylated (\(\beta\geq 0.8\)) or hypomethyllated (\(\beta\leq 0.2\)) across all samples. The analysis of the distribution of methylation probes across genomic regions revealed that hypomethyllated probes were significantly enriched in CpG islands and shores, supporting the idea that active promoters are generally unmethylated to permit transcription initiation [38; 39]. In contrast, hypermethylated probes were enriched in shelf regions, typically linked to repressive chromatin states. This distribution supports the idea that DNA methylation contributes to defining active versus silent regulatory domains,shaping cell-specific gene expression programs in DPSCs, DFPCs, and PDLSCs and are consistent with previous work showing the epigenetic plasticity of dental mesenchymal stem cells and their odontogenic potential [40; 41]. The identification of consistently hyper- or hypomethylylated CpG sites across all three stem cell types provides a useful resource for selecting stable epigenetic markers or potential targets for regenerative strategies, particularly in pulp-dentin complex regeneration and periodontal tissue engineering. Although the profiles were derived from early-passage cells, existing evidence suggests that DNA methylation in mesenchymal stem cells is highly dynamic and responsive to differentiation cues. Studies have shown that osteogenic or odontogenic induction protocols can lead to demethylation or hypermethylation of lineage-specific regulators enhancing their expression during differentiation. Therefore, the patterns observed may represent baseline epigenetic states that predispose each cell type to specific differentiation trajectories. The analysis was also extended to a class of genomic elements that are usually not fully considered in methylation analysis due to annotation limits, i.e., noncoding RNAs. Using an integrated functional annotation [15] based on dedicated databases (i.e., LNCaP and ENCODE), the occurrence of several probes in noncoding related elements was revealed. Notably, among those, some noncoding RNAs (DANCR, MIR22 HG, ZFAS1, and MALAT1) that are consistently hyper- or hypomethylulated across all three dental-derived stem cell lines in their promoter regions and have been previously implicated in tooth development, periodontal regeneration, and alveolar bone remodeling were identified [22].