Noncoding RNA networks in endodontic regeneration: Current evidence and future directions

Abstract

Aim:

This narrative review provides a consolidated existing literature on regulatory functions of noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long ncRNAs, and circular RNAs (circRNAs) in pulp–dentin complex regeneration.

Methods:

A background search was implemented utilizing data from PubMed, Scopus, and Web of Science to determine research conducted between January 2010 and April 2026. In particular, studies focused on experimental in vitro studies, animal models, clinical molecular investigations, and transcriptomic analyses related to dental pulp stem cells and regenerative studies. Relevant conceptual reviews were also used to help explain mechanisms.

Results:

Research indicates that ncRNAs act as master regulators influencing stem cell differentiation, new blood vessels formation and generating a layer of hard tissue such as dentin, primarily through the competing endogenous RNA networks of epigenetic changes and posttranscriptional regulation of genes. The development of transcriptomics of individual cells has revealed cellular diversity and complex regulatory interactions during pulp injury and recovery. Recent animal studies and clinical molecular investigations have revealed that miRNAs bound to exosomes and circRNA-mediated regulatory circuits can improve pulp–dentin regeneration and also function as diagnostic biomarkers and therapeutic objective.

Conclusion:

ncRNAs serve as an essential regulatory tier in regenerative endodontics that connects molecular biology into clinical translational. The combination of multiomics analyses along with biomaterial-based delivery systems can lead to precision, mechanism-driven regenerative therapies and may also transform future endodontic treatment modalities.

INTRODUCTION

Conventional endodontic therapy, which aims to eliminate the endodontic infection and promote the healing of the periapical tissues is still the standard treatment of choice for periapical pathosis. However, it does not regenerate the functioning of the pulp and pulp–dentin complex.[1] Regenerative endodontic procedures (REPs) are biological therapies that aim to regenerate the pulp–dentin complex and restore vitality in necrotic teeth. Although regenerative endodontic procedures (REPs) for immature teeth with open apices have shown successful clinical and radiographic results, systematic reviews have revealed that REPs demonstrate a success rate comparable to nonsurgical endodontic treatment in mature permanent teeth.[2] Stem cell-based regenerative approaches, either transplantation or cell homing, have been proven to be capable of organized pulp–dentin regeneration. However, these techniques have not been applied extensively in clinical practice due to the complicated procedures involved, regulatory and ethical issues, as well as the high costs.[3] Besides clinical differences, different scaffold materials and inconsistent results are also connected to different outcome assessment criteria.[4]

Hence, it is of paramount importance to know the molecular mechanisms in pulp regeneration to make it more predictable and to ensure clinical application. Dental pulp stem cells (DPSCs) represent a cell population capable concerning differentiating into multiple cell types, and they can also promote angiogenesis and repair function. A recent molecular study shows that noncoding RNAs (ncRNAs) are the primary regulators of DPSC proliferation, differentiation, and inflammatory signaling.[5] ncRNAs comprise a diverse class of RNA transcripts that generally lack protein-coding capacity and vary widely in their size, structure, and biological roles. Key categories include microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs), all of which are known to modulate a range of physiological processes, particularly those related to inflammation and immune regulation. In the context of pulpitis, ncRNAs are involved in the regulation of multiple cellular and molecular mechanisms, including apoptosis, cell proliferation, differentiation, oxidative stress, autophagy, ferroptosis, as well as inflammatory and immune responses.[6]

One of the biggest problems in regenerative endodontics is the natural diversity of mesenchymal stem cell (MSC) groups. Conventional bulk transcriptomic methods only give the standard gene expression levels of dissimilar cell types. Hence, the lineage-specific regulatory mechanisms cannot be comprehended fully. Single-cell RNA sequencing (scRNA-seq) has revolutionized the field by enabling a very fine description of pulp cell organization and their differentiation pathways.[7] Current literature suggests that there are distinct DPSC subpopulations that are transcriptionally active and linked to new blood vessel formation and regenerative potential, such as PDGFRβ+ cell populations.[8] Most investigation was based on transcriptome profiles of dental stem cells for regeneration. Nevertheless, these studies use bulk RNA and must not contemplate cell-level heterogeneity. Hence, transcriptome comparisons have revealed even more lineage-specific differences between DPSCs and other dental stem cell types.[9] The complex cellular composition of both teeth and periodontium helps in understanding the new cell type and tracing the lineage profile for each cell type. scRNA-seq scrutinies have shown that the cellular constituent ratio remodels homeostasis or tissue repair.[10] Even the transcriptional dissimilarity between the freshly isolated and monolayer-cultured DPSCs was evaluated, revealing that monolayer culture proceeded in a substantial cellular composition swap in contrast to the freshly isolated DPSCs. Such discoveries underscore the critical role of cell-specific regulatory networks in tissue regeneration.[11]

This narrative review combines the existing information on ncRNA-driven regulation of pulp regeneration with cutting-edge transcriptomic tools, highlighting the translational importance and potential future clinical applications.

LITERATURE SEARCH STRATEGY AND METHODOLOGY

A detailed investigation was implemented in the electronic databases PubMed, Web of Science and Scopus, which were systematically searched for articles published from January 2010 to April 2026 to find studies that focus on the role of ncRNAs in the regeneration of the pulp–dentin complex and regenerative endodontics. Besides that, a manual investigation for reference lists of selected articles was carried out. Human and animal experimental, translational, and clinical studies involving ncRNA-linked pulp–dentin regeneration processes published in the English language were included in the study. Papers unrelated to dental pulp or endodontic regeneration, conference abstracts without full-text accessibility, and nonpeer-reviewed sources. The research for this narrative review was performed using systematic review methodology to make it more transparent and reproducible. The selection of the literature was done based on the principles adapted from the Cochrane Collaboration pulling framework for structured evidence synthesis, including search strategies and eligibility criteria.

RESULTS

Study selection

The structured critical review search across PubMed, Scopus, and Web of Science identified (n = 455) records related to ncRNAs in dental pulp biology and regenerative endodontics. Duplicate records (n = 95) were removed, and (n = 360) articles abided for title and abstract screening. Based on predetermined inclusion and exclusion benchmarks, (n = 250) articles were prohibited due to lack of relevance to pulp–dentin regeneration or ncRNA-mediated regulatory mechanisms. Full-text assessment was conducted for (n = 110) studies, of which (n = 81) articles were eliminated due to the absence of dental pulp-specific outcomes. Finally, (n = 26) studies were included for qualitative synthesis.

Characteristics of included studies

The included literature consisted primarily of experimental and preclinical investigations, with limited clinical studies directly evaluating ncRNA-guided regenerative strategies.

The distribution of study models was as follows:

• In vitro studies using DPSCs: (n = 10)

• Animal studies investigating pulp regeneration: (n = 04)

• Transcriptomic and multiomics studies: (n = 08)

• Translational biomaterial or exosome-based studies: (n = 03)

• Clinical studies related to regenerative endodontic procedures: (n = 01).

Recent transcriptomic and functional investigations together reveal that ncRNAs are critical regulators in pulp–dentin regeneration via control of stem cell differentiation and microenvironmental signaling. For example, Liu et al. establish that several lncRNAs, including SNHG7, are differentially expressed during odontogenic differentiation of human DPSCs (hDPSCs). Pathway enrichment analysis showed involvement of Phosphoinositide 3-kinase/protein kinase B (PI3K-Akt), transforming growth factor (TGF-β), and Wnt signaling pathways, indicating possible molecular targets for regenerative pulp therapies.[12] Similarly, Li et al. also found altered lncRNA expression during the induction of angiogenesis in DPSCs and emphasized the functional significance of ncRNA-mediated regulation of vasculature as an indispensable part of pulp regeneration.[13] Supporting these results, Chen et al. discovered circRNA-related competing endogenous RNA (ceRNA) networks that control odontogenic differentiation of cells through Wnt and TGF-signalling pathways and their results gave additional support to the idea that combined ncRNA regulatory networks dictate reparative dentinogenesis.[14] Mechanistic assessments of Zhong et al. (lncRNA H19 study) showed that lncRNA H19 enhances odontoblastic differentiation via the miR-140-5p/bone morphogenetic protein (BMP)-2/FGF9 pathway, hence directly influencing mineralization pathways.[15]

Altogether, these investigations suggest that ncRNAs are pivotal upstream molecular regulators that connect differentiation, angiogenesis, and signaling pathway activation in pulp–dentin regeneration. Despite being largely at the in vitro and experimental model stage, their results align well with the advent of regenerative endodontic concepts that revolve mainly around the molecular modulation of DPSCs. Recent functional studies are, in fact, providing more persuasive mechanistic support for the translational feasibility of ncRNA-targeted therapies in contrast to earlier descriptive transcriptomic analyses. In one clinical investigation, de-Jesus-Soares et al. assessed regenerative endodontic treatments in immature permanent teeth and found not only a marked reduction in microbes but also molecular alterations related to tissue healing and root development, lending support to the idea that the pulp microenvironment is modulated during clinical regeneration.[4] Nonetheless, there is still a scarcity of extensive clinical validation of ncRNA-specific pathways.

Animal studies were performed to overcome this clinical limitation. In this study by Li and Ge, in an in vivo investigation on mice, it was demonstrated that exosomes derived from DPSC enriched with lncRNA-Ankrd26 promoted dental pulp regeneration by regulating the lncRNA-miR-150/Toll-like receptor signal transduction in MSCs.[16] Among other effects, exosomal lncRNA-Ankrd26 greatly increased in vitro MSC migration and osteogenic differentiation, and in an animal model of pulp injury, it improved tissue repair. The study also provided mechanistic insights into how specific ncRNA cargo can regulate cell behavior and reparative outcomes.[16] On the other hand, Shi et al., after evaluation of MSC-derived exosomes, demonstrated that DPSC activity was more broadly affected by triple functional changes, including increased migration, proliferation, and odontogenic differentiation. This was achieved through the pathway of exosomal CD73-mediated adenosine receptor activation of AKT and ERK signaling. In the rat pulp defect model, MSC exosomes enhanced dentin matrix protein expression and formation of dentin-like and bridge-like structures, with the regenerative performance being equal to that of mineral trioxide aggregate controls. Moreover, this research revealed that MSC exosomes are a viable means by which recellularized pulp–dentin tissue can be formed in human premolar root canals that have been implanted subcutaneously in mice.[17]

Two main transcriptomic and multiomics studies mapped the ncRNA regulatory networks involved in odontogenic differentiation of hDPSCs. Chen et al. did genome-wide expression profiling and based on which, differentially expressed lncRNAs, miRNAs, and messenger RNAs (mRNAs) were identified, and eRNA networks related to odontogenic pathways were built.[18] Furthermore, a complementary circRNA profiling study by Chen et al. showed regulatory interactions of circRNA, miRNA, and mRNA involved in DPSC differentiation.[14] In translational studies, Wei et al. have developed a chemically modified miRNA delivery method using DNA tetrahedral nanostructures that increased angiogenesis and odontogenic differentiation in vitro and led to better regeneration outcomes in vivo, thus pointing to the therapeutic potential of biomaterial-assisted ncRNA delivery.[5]

Ganesh et al. explored the regenerative potential of DPSC-derived exosomes in growth and angiogenic differentiation conditions and found that exosomes substantially increased cell proliferation, migration, and angiogenic differentiation.[19] Further analysis of gene expression showed upregulated levels of angiogenic markers, namely vascular endothelial growth factor (VEGF) A, FLT1, and PECAM1, whereas the profiling of exosomal miRNA uncovered some regulators that are linked to cell homing and vascularization. These results align with the notion that exosome-mediated ncRNA transfer is one of the ways exosomes modify the microenvironment, leading to pulp regeneration. Overall, the research shows that identifying ncRNA regulatory networks at the transcriptomic level and employing exosome-based delivery systems for translational purposes are two other approaches that can significantly contribute to regenerative endodontic therapies.[20]

OVERVIEW OF NONCODING RNAS

MicroRNAs

miRNAs are a category of small ncRNAs that impact gene expression at the posttranscriptional level by interacting with the complementary sequences of specific target mRNAs. Such interaction results in translation inhibition or mRNA decay. Besides, a single miRNA can target multiple genes, thus allowing highly coordinated regulation of the complex biological processes involved in tissue development, immune regulation, and regeneration. Accordingly, in the context of dental pulp biology, miRNAs are indispensable players in shaping the DPSC function-proliferation, differentiation, and cellular responses to injury.[21] As per a comprehensive study analyzing the role of miRNAs in the control of human dental pulp-derived MSCs, several critical regulators of odontogenic and osteogenic differentiation were discovered. In this respect, hsa-miR-140-5p, hsa-miR-143 family members, and hsa-miR-218 were most commonly identified as repression factors for lineage-specific differentiation. Moreover, the MAPK, PI3K-Akt, and FoxO signalling pathways were found to play a role in the regulation of these processes as per the pathway analysis. This points toward the capability of precise miRNA-based interventions to influence stem cell decisions during regeneration.[22] In addition to differentiation, miRNAs closely interact with inflammation that is inherent to pulp tissues. Several miRNAs with altered expressions in pulpitis have been identified, with both upregulated and downregulated miRNAs targeting cytokine signaling, apoptosis, and cellular migration.[23] This indicates that miRNAs are probably involved to balance inflammatory responses and reparative mechanisms within the pulp microenvironment.

Experimental data confirm the therapeutic possible role of individual miRNAs. For example, miR-200c is substantially downregulated in inflamed pulp tissues, whereas its overexpression diminishes the production of inflammatory cytokines and, at the same time, increases the expression of odontogenic markers in human dental pulp cells. Delivery of miR-200c in pulpitis models in vivo has promoted dentin regeneration, thus hinting at a future use of miRNA-based molecular therapies in regenerative endodontics.[24] Overall, the body of current research considers miRNAs as major regulators of DPSC activity and pulp healing. It also points to their growing translational potential, both as diagnostic biomarkers and therapeutic targets.

Long noncoding RNAs

lncRNAs are RNA transcripts that are greater than 200 nucleotides in length and do not encode proteins. However, they regulate gene expression at epigenetic, transcriptional, and posttranscriptional levels. Unlike miRNAs, lncRNAs have a wider range of functions, including chromatin remodeling, transcriptional regulation, and interactions with regulatory RNA networks. Their expression patterns that are specific to tissues make them particularly important in developmental and regenerative processes. In dental pulp biology, lncRNAs have been a subject of research, with studies describing their roles in regulating inflammation and stem cell differentiation. Through differential expression analyses of inflamed pulp tissues, several lncRNAs associated with immune signaling pathways and ceRNA networks have been identified. This indicates that they are playing roles in the progression of pulpitis and reparative responses. On the other hand, lncRNAs have also been proven through research to impact the differentiation of DPSCs into different cell types. Hence, lncRNA SNHG1 increases the differentiation of odontogenic cells by interacting with miRNA networks and activating Wnt/β-catenin signalling pathways. In addition, angiogenesis induction transcriptomic profiling of PDSCs has unveiled major changes in the expression of lncRNA responsible for the extracellular matrix organisation and vascular differentiation.[25,26] Besides that, immune-related lncRNAs such as MIR181A2HG and LINC00426 are proposed as potential markers of pulp inflammation state. Furthermore, genome-wide analyses of developing dental tissues indicate that lncRNAs are cell-specific regulatory markers participating in odontogenesis through transcriptional and epigenetic modulation.[27]

These findings indicate that lncRNAs act as higher-order regulatory molecules integrating inflammatory and regenerative signalling within the pulp microenvironment.

Circular RNAs in pulp regeneration

circRNAs constitute a type of ncRNA naturally present in cells that are characterized by the formation of covalently closed loop structures as a result of back-splicing events. This circular nature makes them more stable than linear RNAs because they are less likely to be degraded by exonucleases. circRNAs mainly modulate gene expression through their binding to miRNAs and modulating downstream signaling pathways. Recent transcriptome analyses have revealed the changes in circRNA expression profiles in the dental pulp tissues exposed to inflammation, thus indicating their participation in the regulatory circuits related to pulp damage and healing.[6] Through the construction of circRNA miRNA mRNA interaction networks, it is revealed that circRNAs regulate cellular pathways involved in inflammation, oxidative stress, and odontogenic differentiation. Laboratory testing points out that circRNA-mediated regulatory networks modulate DPSC growth, differentiation, and inflammatory reactions through connections with signaling pathways such as nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Wnt signaling pathways.[28] Combined circRNA and miRNA sequencing, along with bioinformatic analysis provide solid evidence for their engagement in pulp regeneration and also expose their potential as molecular markers and therapeutic targets.[29] Thanks to the continuous improvement of transcriptomics and multiomics approaches, circRNAs are becoming more and more appreciated as gene expression regulatory players that add to the intricacy of the networks controlling dental pulp regeneration.

Mechanisms of long noncoding RNA-mediated regulation

lncRNAs are involved in controlling the regeneration activities at various molecular levels, even beyond the traditional transcriptional modulation. For the biology of DPSCs and reparative dentinogenesis, lncRNAs act as regulatory hubs that combine epigenetic control, transcriptional regulation, and posttranscriptional signaling networks. Transcriptomic and functional analyses reveal that lncRNA-mediated regulation affects lineage determination, inflammatory reactions, and microenvironment-triggered regenerative signaling. Recent findings emphasize three major modes of regulation: ceRNA interactions, epigenetic changes and transcriptional regulation, along with posttranscriptional events such as alternative splicing [Figure 1].

Figure 1.

Regulatory network in pulp regeneration. ncRNA: Noncoding RNA, ceRNA: Competing endogenous RNA, lncRNA: Long ncRNA, TNF-α: Tumor necrosis factor-alpha, IL: Interleukin

Competing endogenous RNA interactions

ceRNA interactions involving lncRNAs are one of the most well-studied ways in which lncRNAs function. In this scenario, lncRNAs act as molecular sponges that sequester certain miRNAs and as a consequence, reduce the impact of miRNA-induced suppression of the genes targeted downstream. Regulating indirectly in this way, lncRNAs are capable of modulating the signalling pathways related to odontogenic differentiation as well as pulp regeneration. For instance, lncRNA SNHG1 is capable of promoting odontogenic differentiation of hDPSCs by the mechanism of acting as a ceRNA and also by modulation of Wnt/β-catenin signalling through the interaction with the specific miRNAs.[30] Similarly, in silico and wet lab studies have identified pulpitis-related lncRNAs to inflammatory signaling pathways via ceRNA-mediated regulation, indicating their role in linking inflammatory and regenerative responses.[31,32] Collectively, these results signify that ceRNA networks help in gene regulation during pulp repair by coupling microenvironmental stimuli with downstream transcriptional activity.

Epigenetic and transcriptional regulation

lncRNAs also regulate gene expression via direct binding to chromatin-modifying complexes and transcriptional regulators. lncRNAs change chromatin flexibility and transcriptional levels of lineage-specific genes by targeting histone methylating, acetylating enzymes, or chromatin-remodeling complexes to particular genomic regions.[33] lncRNA-driven epigenetic regulation of dental pulp cells has been correlated with up-regulation of different odontogenic markers such as dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2). Besides that, these markers regulate dentin matrix formation and mineralization.[34] Not only differentiation-related pathways, but also immune-related lncRNAs obtained from pulpitis samples depict correlation with inflammatory gene expression, which means epigenetic modifications can be a way to coordinate inflammatory signals with regenerative activities.[35,36,37] The triggering of well-known signal transduction pathways such as Wnt/β-catenin and TGF-/BMP cascades has been identified as lncRNA-driven transcriptional regulation during odontoblast differentiation.[38] These findings imply that lncRNAs act as master regulators of epigenetic and transcriptional programs during pulp–dentin regeneration.

Posttranscriptional regulation and alternative splicing

Besides regulating transcription, lncRNAs also have a role in posttranscriptional control as a result of their associations with RNA-binding proteins and components of the spliceosome. For example, by controlling the inclusion or exclusion of exons within lineage-specific transcripts, lncRNAs influence protein isoform diversity during stem cell differentiation. While this process is still rarely described in dental pulp tissues, transcriptomic analyses of MSCs have shown that lncRNAs physically associate with splicing regulators that are implicated in osteogenic and odontogenic differentiation pathways.[39] It is expected that such regulatory switches will affect the production of extracellular matrix, mineralization ability, and differentiation paths. New multiomics data are also pointing out that alternative splicing is probably one of the ways through which signals from the regenerative microenvironment are integrated to produce functional cellular responses. Developments in high-throughput transcriptomic technologies, especially scRNA-seq, reveal that the regulatory mechanisms mediated by lncRNAs vary between different cell types and are changing throughout the process of pulp injury and repair.[9] On top of that, preclinical research is looking into the potential of ncRNA-targeted delivery systems (e. g., scaffolds exosome mediated) to improve pulp regeneration.

Altogether, these mechanisms not only explain how lncRNAs can regulate multiple processes but also place them at the core of the pulp–dentin regeneration machinery. In this context, they integrate inflammatory signaling, pathways of stem cell activation, as well as epigenetic controls of cell differentiation. Nevertheless, it should be noted that the majority of the mechanistic understandings came from in vitro set-ups or analyses of the entire tissue transcriptome. In-depth studies that utilize single-cell level technologies and preclinical models with high clinical relevance will be required to map the cell-specific regulatory networks and further regeneration of endodontic therapies.

Integrated noncoding RNA regulatory networks in endodontic regeneration

New transcriptomic studies of inflamed dental pulp tissues have unravelled intricate regulatory circuits between circRNAs, miRNAs, and mRNAs that together control inflammatory signaling and odontogenic differentiation. These integrated networks of ncRNAs point out that the same ncRNAs are involved in the transition from inflammatory responses to reparative processes during pulp healing.[40] Besides working at the level of individual molecular interactions, ncRNAs actually operate in large-scale regulatory frameworks that govern stem cell differentiation and tissue remodelling. For instance, transcriptomic analyses have shown that ncRNA-involved posttranscriptional regulation is one of the factors driving lineage commitment and extracellular matrix arrangement in reparative dentinogenesis. While these control mechanisms are still not fully understood in dental pulp tissues, the latest stem cell studies highlight their implication in adjusting the protein expression profiles that are key to mineralization and regeneration. Another level of intercellular signaling in the regenerative microenvironment is provided by extracellular vesicles, especially exosomes. For example, exosomes released from DPSCs and loaded with miRNAs have been found to potentiate angiogenesis, ameliorate inflammatory signaling, and stimulate odontogenic differentiation in different experimental models. To a certain extent, the administration of DPSC exosomes could be seen as a replacement for stem cell therapy. It is a cell-free therapeutic approach still at the preclinical stage, but it already shows various advantages, such as better safety and lower immunogenicity.[41]

With the progress in biomaterial engineering and nanotechnology, it is now possible to deliver the ncRNAs locally using various types of scaffolds, injectable hydrogels, and nanoparticle-based carriers. Experimental findings show that regulated delivery of miRNA mimics or inhibitors could improve pulp–dentin regeneration by influencing inflammatory and differentiation pathways. Translational challenges. However, there still exist issues, such as maximizing delivery efficiency, reducing off-target effects, and regulatory standardization. Combining ncRNA profiling with single-cell transcriptomics and biomaterial-based delivery platforms could pave the way for the development of precision regenerative endodontic approaches.

Immunomodulatory functions

Inflammation regulation is a major factor in determining a successful pulp–dentin regeneration. ncRNAs have a key role in changing the inflammatory signaling pathways that control the balance between tissue destruction and tissue repair in the pulp microenvironment. Two of the most well-known regulators are miR-146a and miR-155, which have been the focus of the majority of studies on the modulation of the innate immune system through regulation of NF-kB-associated signalling and cytokine expression miR-146a mainly serves as a negative feedback regulator by targeting adaptor molecules like IRAK1 and TRAF6 while, on the contrary, miR-155 is linked to the worsening of the inflammatory responses in disease conditions. Experimental work also shows that influencing these miRNAs changes macrophage polarization and cytokine expression patterns, which leads to an immune environment that is not only regenerative but also supports pulp healing.[42] New preclinical experiments have also shown that using immunoregulatory miRNAs delivered by scaffolds or nanoparticle form may improve regenerative effects by lessening extreme inflammation while at the same time maintaining reparative signaling pathways. This evidence reveals the practical side of ncRNA-based immunoregulation in regenerative endodontics.

Angiogenesis and neurogenesis

Functional regeneration of the pulp–dentin complex is a complex process that not only involves tissue growth but also requires vascularization and sensory nerve regeneration. Angiogenesis-involved miRNAs, especially miR-126 and miR-210, were recognized as factors controlling the endothelial cell differentiation and vascular network development by the regulation of VEGF signalling and hypoxia-responsive mechanisms. Based on the experimental results, these miRNAs are not only capable of promoting the angiogenic differentiation of DPSCs but are also crucial for the rapid establishment of microvascularization during pulp regeneration.[43] Besides angiogenesis, ncRNAs play a role in inducing neuronal differentiation probably by regulating the neurotrophic signalling pathways. Several miRNAs and lncRNAs altering expression of nerve growth factor and other components of neural regeneration were identified. Transcriptome-based research studies reveal that the two processes, angiogenesis and neural regeneration are functionally related and operate through both regulatory networks during the dental pulp tissue repair. Especially in the last few years, some studies have reported the effectiveness of ncRNA delivered via exosomes and encapsulated in hydrogel systems aimed at the regeneration of the vasculature and the neural tissue. These findings offer encouragement for the clinical use of nucleic acid-based ncRNA-directed tissue engineering of the pulp.[44]

Transcriptomic landscape of dental pulp stem cells

Bulk transcriptomic insights

Early transcriptomic studies using microarray and bulk RNA sequencing first elucidated the gene expression landscapes of stemness, odontogenic differentiation, and immune regulation in DPSCs. Such investigations helped to uncover essential signaling networks. For example, Wnt, TGF-/BMP, and MAPK, which are implicated in both pulp development and healing. Nevertheless, bulk transcriptomic techniques determine the average gene expression from mixed populations of cells, which hinders uncovering cell-type-specific gene regulatory mechanisms. More recent comparative studies illustrate that bulk RNA data sets fail to adequately reflect the dynamic changes in ncRNA expression that differentiate distinct pulp cell compartments upon injury and regeneration. This drawback of the previous methods underlines the necessity of employing transcriptomic approaches with better resolution that can accurately define cellular heterogeneity in the pulp.[45]

Single-cell RNA sequencing in pulp regeneration

scRNA-seq has considerably deepened the comprehension of dental pulp biology. It has done so by allowing transcriptomic profiling at the single-cell level and enabling the reconstruction of cellular differentiation trajectories. Recent scRNA-seq research has delineated transcriptionally different DPSC subpopulations with variable proliferative, angiogenic, and immunomodulatory properties, thus further defining the cellular make-up of pulp tissue. Trajectory inference analyses have shown that regulatory networks comprising both coding and ncRNAs are activated at different stages, and they control lineage commitment to odontoblast-like, endothelial-like, and neural-like phenotypes during regenerative processes. These results suggest that ncRNA expression is very dynamic and may be regulated by microenvironmental signals during pulp injury and repair. As the identification of functionally specialized stem cell subsets through scRNA-seq could be useful, from a translational point of view, in improving cell selection strategies for regenerative endodontic procedures and in supporting the development of precision-based regenerative protocols.[46]

Exosomal noncoding RNAs in dental pulp regeneration

Cell-to-cell communication via exosomes has been recognized as a key factor in dental pulp regeneration. Exosomes released from DPSCs carry a variety of biologically active molecules, such as miRNAs, lncRNAs, and proteins that regulate genes, which in turn impact processes such as the formation of new blood vessels, immune system regulation, and tissue repair. This happens as a result of changes brought about in the gene expression of cells that take up these exosomes. Research shows that some DPSC-derived exosomal miRNAs, such as miR-21, miR-26a, and miR-125a, not only promote the formation of dentin-producing cells but also increase the capability of cells to form new blood vessels by triggering pathways linked to cell division and vessel development.[47] In contrast to stem cell transplantation, exosome-based methods offer an innovative cell-free approach that may give advantages in terms of safety and ease of regulation. Latest preclinical studies reveal that the use of exosomes together with biomaterial scaffolds leads to better formation of pulp-like tissue and blood vessel development in animal models, thus providing support for their use in human regenerative endodontic therapies.[48]

Spatial transcriptomics and microenvironment mapping

While scRNA-seq offers a detailed view of cell-specific transcriptional profiles, one downside is that it does not retain spatial information about tissue organisation. Spatial transcriptomics is a new technique that complements single-cell RNA-seq by allowing visualization of gene expression patterns in tissue sections. For instance, application of spatial transcriptomic technology in dental tissues has revealed differentially expressed genes in odontoblast layers, vascular regions, and immune cell niches, thus showing the areas with distinct transcriptional activities.[49] This observation goes on to show that the regeneration signalling networks are set up to a large extent by the organisation of the pulp microenvironment. For instance, during pulpitis and reparative processes, various transcriptional patterns have been observed that are not only specific to the mineralization pathways of the odontoblast layer but also to the immune regulatory ones that are localized both in the pulp. Combining spatial transcriptomics with single-cell sequencing and ncRNA profiling will help to greatly enhance the knowledge of pulp regeneration mechanisms and facilitate the making of targeted regenerative therapies.

Integration of noncoding RNA networks and regenerative microenvironments

The pulp regeneration takes place in a dynamic microenvironment, which is regulated by the interactions of the following factors: scaffold materials, growth factors, mechanical stimuli, vascular supply, immune response, etc., These microenvironmental elements together impact stem cell activation, lineage commitment, and tissue remodelling in pulp–dentin regeneration. Bioactive endodontic substances and scaffold systems are capable of altering the expression of certain ncRNAs, such as miRNAs and lncRNAs, which play a role in odontogenic differentiation and structural organisation of the regenerated tissues, according to recent experimental and translational studies. Different types of materials, such as calcium silicate-based biomaterials, growth factor-releasing scaffolds, and mechanically responsive matrices, are known to trigger the activation of signalling pathways such as Wnt/β-catenin, TGF-/BMP, and MAPK, which are intimately linked with ncRNA-mediated regulatory networks in DPSCs.[50,51] Ruparel et al. conducted a study to find out the direct effects of most frequently used intracanal medicaments on the survival of stem cells of the apical papilla (SCAP), which are making regenerative endodontic procedures successful. Through their research, they proved that both the medicament type and concentration greatly change stem cell viability. Triple antibiotic paste, especially at higher concentrations, was harmful to SCAP, whereas calcium hydroxide was more biocompatible and maintained stem cell survival.[52] In addition to the choice of medicament, the delivery vehicle also plays a vital role in enhancing its efficacy. Enhanced penetration may contribute to better disinfection of the canal system, thereby potentially improving the outcomes of regenerative endodontic procedures.[53]

The findings are an endorsement of the ongoing transition to formulations of antibiotics with lower concentrations as well as strategies for intracanal medicaments that are compatible with stem cells, aimed at enhancing the features of regenerative endodontic therapy. Aside from laboratory tests, which generally represent a very simple and minutely controlled environment, animal studies and first clinical trials of regenerative endodontic interventions reveal that the ability of a biomaterial to influence the local microenvironment largely determines, among other things, the degree of regeneration of the blood supply, bone creation, and the body’s defence system. There is a growing body of evidence that extracellular vesicle-encapsulated ncRNAs and scaffold-based delivery systems are very promising as a clinically adaptable method for improving pulp regeneration, and at the same time, they help in reducing the limitations of direct stem cell transplantation. For a reasoned design of the next generation of regenerative endodontic therapies, it is fundamental to comprehend how regenerative biomaterials and ncRNA-regulated gene expression influence each other. By combining biomaterial engineering with transcriptomic profiling, it is possible to develop regenerative protocols that are precision-based and biologically predictable as well as have great potential for clinical translation.

Future directions

Despite significant advances in identifying regulatory roles of ncRNAs during pulp–dentin complex regeneration, there remain several scientific and translational issues which must be solved before ncRNA-based approaches can be part of the routine clinical practice of endodontics. Majorly, it is the lack of standardized methods for ncRNA isolation, sequencing normalization, and bioinformatic analysis that poses a significant limitation. Differences in sample preparation, sequencing equipment, and computational methods contribute to the variability of results obtained from different studies. To address this issue and make ways for experiments to be reproducible and results obtained from various studies to be reliable, a standardized way of conducting experiments and reference datasets related to dental pulp transcriptomics need to be set up. Transcriptome-based research has so far led to the discovery of many ncRNAs that play a role in pulp inflammation and regeneration. However, a single-layer molecular dataset is not adequate for a complete understanding of the complex regenerative signalling networks. To elucidate molecular pathways that lead to stem cell activation, immune modulation, and tissue repair, systems-level approach using multiple omics such as epigenomics, proteomics, and metabolomics in addition to transcriptomics will be necessary. New technologies in spatial transcriptomics and single-cell multiomics also make it possible to depict cell-type-specific regulatory interactions in the pulp microenvironment, thus giving profound knowledge of lineage dynamics and intercellular communication during regeneration.

One main issue in translational research is the creation of delivery techniques for ncRNA-based therapeutics that can be used in clinical practice. The delivery systems that work well should keep the molecules intact, release them promptly, target specific tissues, and be compatible with the body. Some of the strategies that have been developed already include nanoparticle carriers, hydrogels that can be injected, delivery via scaffold, and transport through extracellular vesicles. For instance, in preclinical settings, it has been shown that using biomaterials to deliver miRNAs and lncRNA regulators can improve the differentiation of cells capable of producing dental tissue as well as the formation of new blood vessels; however, more studies are necessary to ensure that these new therapies are safe, identify the best doses, and meet the regulatory standards for clinical use. In fact, many ncRNA targets have been identified through sequencing on a large scale and in the laboratory; unfortunately, only a few have made their way into clinical use. Thus, creating models of the disease that closely mimic the biology of the pulp in humans will be a great help in testing the validated therapeutic targets and conditions that more closely resemble the physiological ones. Besides that, clinical trials that have a good design are needed in order to assess safety, effectiveness, and lasting benefits of ncRNA-based regenerative treatments. Strategies aiming at a combination of stem cell therapy, biomaterials, and molecular modulation represent what approaches that have potential are however standardization of methodology and regulatory compliance are the essentials to implement the clinical one. With the help of artificial intelligence, combining multi-omics data will soon facilitate the discovery of functional ncRNA networks and prediction biomarkers that are linked to the regenerative outcomes. Machine learning-based analysis of transcriptomic datasets may enable patient-specific regenerative planning and optimization of treatment protocols, representing an important future direction toward precision endodontics. A summary of translational ncRNA-based strategies in regenerative endodontics is presented in Table 1.

Table 1.

Translational noncoding RNAs strategies in regenerative endodontics

ncRNA strategy/model	Experimental platform	Key biological outcome	Translational stage
miRNA regulation of odontogenic differentiation (e.g., miR-148a, miR-200c)[6,23]	In vitro DPSCs	Enhanced odontogenic differentiation via Wnt/β-catenin signaling	Early preclinical (mechanistic)
lncRNA-mediated ceRNA networks (e.g., H19, SNHG1)[15,25]	In vitro DPSCs	Regulation of odontoblast differentiation and mineralization pathways	Early preclinical
circRNA–miRNA–mRNA regulatory networks[14,18]	In vitro transcriptomic profiling	Identification of ceRNA networks controlling differentiation	Discovery stage (omics-driven)
scRNA-seq transcriptomic mapping of pulp regeneration[8,10]	Multi-omics/single-cell studies	Identification of regenerative cell subpopulations and signalling pathways	Discovery-to-preclinical transition
Exosome-mediated ncRNA delivery (MSC/DPSC-derived EVs)[17,19]	In vitro+animal pulp injury models	Enhanced angiogenesis, migration, and dentin-like tissue formation	Advanced preclinical
Biomaterial-assisted miRNA delivery (DNA tetrahedron systems)[5]	In vitro+in vivo models	Controlled miRNA delivery improving pulp regeneration and vascularization	Advanced preclinical
Hydrogel-based regenerative scaffolds with molecular signalling support[21]	Animal regeneration models	Functional pulp-like tissue formation	Advanced preclinical
Clinical regenerative endodontic procedures with molecular microbiological evaluation[3]	Human immature teeth (clinical study)	Reduction in microbial load and molecular microenvironment changes supporting regeneration	Early clinical translation
Clinical outcome systematic evidence for regenerative endodontics[1,2]	Clinical systematic reviews/meta-analysis	Demonstrated clinical success and continued root development	Clinical validation (procedure-level, not molecular)

ncRNA: Noncoding RNA, DPSCs: Dental pulp stem cells, miRNA: Include microRNA, ceRNA: Competing endogenous RNA, lncRNA: Long ncRNA, circRNA: Circular RNA, mRNA: Messenger RNA, scRNA-seq: Single-cell RNA sequencing, MSC: Mesenchymal stem cell, EVs: Extracellular vesicles

CONCLUSION

ncRNAs represent a vital biological control layer in the regeneration of the pulp–dentin complex through a coordinated regulation of stem cell actions, inflammation, and tissue remodelling. Development of transcriptomic tools, especially single-cell sequencing, has greatly reshaped our understanding of cell heterogeneity and ncRNA-driven cellular regulatory events in the context of pulp injury and healing. Linking these molecular discoveries with translational models highlights a new perspective of ncRNAs, both as unique markers for diagnosis and agents for therapeutics in the field of regenerative endodontics. Ongoing integration of ncRNA research with the design of delivery platforms using biomaterials and profiling through multiomics is enabling a shift from mere molecular documentation to the formulation of regenerative strategies based on underlying mechanisms. The prospective implementation of molecular diagnostics, sophisticated biomaterials, and selective ncRNA targeting is likely to improve both the accuracy and the biological relevance of the outcomes of regenerative endodontic procedures. Nevertheless, there is a need for further translational studies, standardized protocols, and thoroughly planned clinical trials in order to connect laboratory discoveries with clinical practice regularly.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.