Biological mechanisms underlying the inflammatory radicular cyst formation-focus on epithelial proliferation: a systematic review of experimental cell and tissue models
Article information
Abstract
Objectives
This study aimed to assess the molecular and cellular mechanisms involved in the epithelial proliferation that leads to the transformation of periapical granulomas (PGs) into inflammatory radicular cysts (IRCs).
Methods
A comprehensive search was conducted in three databases. Experimental, observational, or descriptive studies using human or animal tissue samples, or epithelial cell cultures that assessed the molecular and/or cellular mechanisms driving the proliferation of epithelial rests of Malassez and their role in the transformation of PGs into IRCs were included. The risk of bias and applicability of the included studies were assessed using the QUADAS-2.
Results
Fourteen studies (including 399 samples) met the inclusion criteria for qualitative synthesis. The studies highlight the role of pro-inflammatory cytokines (IL-1β, IL-6), growth factors (EGF, KGF, TGF-β, and IGF), and signaling pathways (NF-κB, MAPK/ERK, PI3K/AKT, and Smad) in the progression of PG to IRC. Biomarkers of epithelial proliferation, such as Ki-67, PCNA, and CD34, are consistently associated with this process, while MMP-13 emerges as a key regulator of epithelial behavior and matrix remodeling.
Conclusions
IRC development arises from a transition from homeostatic to pathological signaling, in which pro-inflammatory mediator levels inside the periapical chronic inflammation override regulatory checkpoints.
INTRODUCTION
Inflammatory radicular cysts (IRCs) are the most prevalent cystic lesions in the jaws, accounting for more than two-thirds of all odontogenic cysts [1,2]. IRCs, considered a reactive aftermath to periapical granulomas (PGs), have long been defined as the last stage of a chronic inflammatory continuum, clinically referred to as apical periodontitis (AP) [1,3]. However, this widely accepted model, which assumes a linear, passive progression from PG to IRC, fails to explain a basic paradox: why do only a small proportion of PGs proceed into IRCs, despite sharing similar antigenic triggers and inflammatory microenvironments [1,2]
IRCs arise from epithelial rests of Malassez (ERM), with dormant proliferative capabilities that continue in the periodontal ligament (PDL) following complete radicular development [2,4]. ERMs are arranged in cord-like, net-like, or isolated island structures next to the radicular cementum and consist of pale epithelial-like cells interconnected by desmosomes, with few cytoplasmic organelles, enclosed by a basal lamina, and suspended in the interphase of the cell cycle [2,4,5].
Emerging evidence suggests that ERMs actively contribute to PDL homeostasis by preventing ankylosis and root resorption, thereby preserving PDL space, supporting neural components, and facilitating cementum healing [2,6]. Furthermore, ovine models have identified clonogenic epithelial stem cell populations within ERMs that share phenotypic and functional properties with mesenchymal stem/stromal cells [7,8]. What remains contentious is the exact molecular pathways involved in the reactivation and pathological proliferation of ERMs in the context of chronic AP, which leads to the transition of PG into IRC, and more significantly, why this reactivation occurs selectively [2].
Although epithelial cell rests are detected in approximately 45% of PGs, only about half of these lesions undergo cystic transformation [2,9]. The molecular and cellular mechanisms driving epithelial proliferation and the subsequent formation of IRC remain poorly elucidated and significantly underestimated. This knowledge gap holds significant clinical relevance, as the histopathological nature of AP fundamentally influences clinical decision-making, timely treatment planning, and prognosis [2,3]. Notably, it has been proposed that some cystic lesions may only respond predictably to surgical intervention [2].
Bacterial endotoxins are considered important triggers of the IRC’s proliferative stage, as they exhibit high mitogenic activity on epithelial cells and activate cytokine-producing cells, thereby increasing epithelial proliferation [1,2,10]. Both epithelial and endothelial cells in IRC tissues exhibit increased expression of cytokines, including transforming growth factor alpha (TGF-α), keratinocyte growth factor (KGF), epidermal growth factor (EGF), tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, IL-6, among others [1,2,10-12]. This suggests that these cytokines may play a key role in the early stages of epithelial proliferation, facilitating the transition from a PG to IRC [1,2,10-12]. Furthermore, it has been proposed that additional molecular mechanisms, such as an increase in intracellular cyclic adenosine monophosphate induced by prostaglandin E2, contribute to the development and proliferation of ERMs [2,10,12]. Notably, whereas the tissues of origin for IRCs are well known, the particular molecular and cellular mechanisms that drive a transition from PG to IRC are poorly understood. Furthermore, the scientific literature on this subject has not been adequately summarized to identify molecular patterns.
The molecular mechanisms and intracellular signaling pathways that drive ERM proliferation likely represent a critical inflexion point in the histopathological transition of AP, notably from PG to IRC. A thorough understanding of these mechanisms would significantly advance our knowledge of AP pathogenesis, facilitate the identification of reliable molecular markers for molecular diagnosis, and aid in the development of targeted therapeutic strategies to halt or reverse cystic transformation, thereby improving endodontic therapy outcomes. To date, these regulatory and pathological mechanisms have not been comprehensively summarized in the literature. To the best of our knowledge, this systematic review is the first to integrate and synthesize the best available experimental evidence on the biological pathways (molecular and cellular) underlying epithelial proliferation and the transition of PGs into IRCs.
METHODS
Protocol and registration
A detailed protocol was registered in the PROSPERO database (CRD420251062194). This systematic review follows the recommendations of the Cochrane Handbook of Systematic Reviews of Interventions and the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement [13,14].
PICO question
Using the PICO strategy, the focused research question and the inclusion criteria were systematically formulated:
Population (P): Histopathological samples of IRCs, PGs, and ERM obtained from periap-ical tissues.
Intervention (I): Evaluation of biological mechanisms underlying epithelial proliferation, specifically molecular and cellular signaling pathways, assessed through techniques such as histopathology, histochemical stains, immunohistochemistry (IHC), western blotting, in situ hybridization, double immunofluorescence assays, and in vitro cell culture or proliferation assays.
Comparison (C): Histological or cell samples from periapical lesions or ERMs without evidence of epithelial/cell proliferation or exhibiting low/absent expression of epithelial proliferation markers.
Outcome (O): Qualitative, semiquantitative, or quantitative identification of molecular and cellular pathways involved in epithelial proliferation within endodontic periapical lesions.
Focused question
What molecular and cellular mechanisms are involved in the epithelial proliferation that leads to the transformation of PGs into IRCs?
Inclusion criteria
Experimental (in vitro, in vivo, or ex vivo), observational, or descriptive studies using human or animal tissue samples, or epithelial cell cultures, that assessed the molecular and/or cellular mechanisms driving the proliferation of ERM and their role in the transformation of PGs into IRCs were included in this systematic review. Eligible studies had to report qualitative, semiquantitative, or quantitative data on the expression, regulation, or functional role of cytokines, growth factors, receptors, biomarkers, co-stimulatory molecules, or intracellular signaling path-ways involved in epithelial proliferation within the context of AP. Techniques such as histopathology, IHC, in situ hybridization, western blotting, double immunofluorescence, or epithelial cell proliferation assays were considered. Studies were required to compare proliferative versus non-proliferative tissues or cell samples, and to offer mechanistic insights into the biological transition from PG to IRC.
Case reports, editorials, expert opinions, letters to the editor, and studies unrelated to endodontic periapical lesions (PGs or IRCs), lacking molecular or cellular data on epithelial proliferation, or focused primarily on non-inflammatory odontogenic cysts or non-epithelial mechanisms of lesion progression were excluded.
Information sources
The literature search was conducted following the methodological standards outlined by the Cochrane Collaboration. A comprehensive search strategy was developed utilizing Medical Subject Headings (MeSH), Emtree terms, Descriptores en Ciencias de la Salud (DeCS), and relevant text words. The following electronic databases were systematically searched from their inception to August 2025: PubMed, Scopus, ScienceDirect, and Web of Science. To enhance the comprehensiveness of the review and ensure literature saturation, additional sources were explored, including reference lists of pertinent studies, academic conference proceedings, thesis repositories, OpenGrey, Google Scholar, and ClinicalTrials.gov. No language restrictions were applied in the selection of studies (Appendix 1).
Screening and data collection
Initially, two independent reviewers screened the titles and abstracts of all retrieved studies. Full-text articles were then assessed for eligibility based on pre-established inclusion criteria. Any discrepancies were resolved through discussion and consensus; when necessary, a third reviewer was consulted to reach a final decision. Data extraction was performed in duplicate using a standardized data collection form, which captured the following information: Relevant data were independently extracted in duplicate using a standardized data collection form, which included the following variables: authors, year of publication, title, study design, geographic location, objectives, inclusion and exclusion criteria, sample size, study duration, outcome definitions, reported outcomes, measures of association, funding sources, study limitations, recommendations, and conclusions in correspondence with the main objective, as well as mechanistic insights into the biological processes underlying the transition from PG to IRC.
Synthesis of results
The included studies were evaluated for methodological homogeneity to assess the feasibility of conducting a meta-analysis. However, substantial heterogeneity across studies—particularly regarding the experimental models used and the diverse objectives evaluated in each study—precluded the performance of a quantitative synthesis.
Risk of bias and applicability assessment
Two independent reviewers evaluated the risk of bias and applicability of the included studies using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool [15]. This tool involves a four-phase process: (i) formulation of a focused review question; (ii) adaptation of a review-specific guidance document if needed; (iii) examination or reconstruction of the study flow diagram; and (iv) domain-level assessment of risk of bias and applicability concerns. Risk of bias was assessed across four key domains: (i) patient selection, (ii) index test, (iii) reference standard, and (iv) flow and timing. Each domain includes two to four signaling questions with possible responses of “Yes,” “No,” or “Unclear.” A “Yes” indicates low risk of bias, “No” indicates high risk, and “Unclear” reflects insufficient information. Domains where all questions are answered “Yes” are rated as low risk. If any question is answered “No,” or if multiple questions are “Unclear,” the domain is rated as high risk. A single “Unclear” response results in an overall “unclear” risk for that domain. Applicability concerns were assessed in three domains: (i) patient selection, (ii) index test, and (iii) reference standard. This evaluation measures the degree to which each study aligns with the review’s focused question. Applicability is rated as “low,” “high,” or “unclear,” with the latter applied when reporting is insufficient [15].
Although QUADAS-2 was originally designed for diagnostic accuracy studies, its structured domains were suitable for this review because they allowed for a systematic evaluation of key methodological aspects such as sample selection, molecular and histological assay application, reference comparisons, and study flow. Furthermore, the tool’s emphasis on risk of bias and application guaranteed that the experimental and observational models included were not only methodologically sound but also relevant to the biological topic at hand [15]. This provides a transparent and reproducible methodology for evaluating the diverse findings on epithelial proliferation and cystic transformation.
RESULTS
Study selection
We initially identified 218 references with the search strategy. Additionally, four records were identified through other sources. After removing 102 duplicates, we screened 120 titles/abstracts. Finally, 14 studies met the inclusion criteria for qualitative synthesis [5,16–28] (Figure 1).
Flow chart of included studies, based on PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines.
Characteristics of included studies
The general characteristics of the studies included in this systematic review are presented in Table 1. Methods and outcomes are presented in Table 2. The studies were conducted in Germany [5,22], the United States [17,21], Japan [20,25,26], Chile [19], Canada [18], Italy [23,27], India [24], and Romania [16]. All 14 included studies employed case-control designs and were published between 1996 and 2024 [5,16–28]. These studies examined diverse molecular mechanisms related to epithelial proliferation (growth factors, cytokines, intracellular signaling pathways, molecular biomarkers, and co-stimulatory proliferation mediators), either in the context of the physiological regulation of ERM functions or in the pathological progression of PGs into IRCs mediated by epithelial proliferation. All the studies offer a biological explanation for why the investigated mechanisms may be associated with epithelial proliferation linked with the progression of PGs into IRCs. They underscore their decisive contribution to epithelial activation, sustained proliferation, and the pathological reprogramming that drives cystic transformation [5,16-28]. A total of 399 tissue samples were analyzed, comprising 168 PGs, 141 IRCs, 8 dental follicles, 40 dentigerous cysts, 1 lateral cyst, and 1 odontogenic keratocyst [16–18,21–24,26–28]. Furthermore, one study analyzed epithelialized (n = 8) and non-epithelialized (n = 7) apical lesions of endodontic origin but did not provide a differential diagnosis [19]. One study employed 25 deciduous molars, which were processed for immunohistochemical analysis [5]. Three studies were based on experimental cell models [20,25,26]. Multiple methodologies, including histopathology, IHC, in situ hybridization, western blotting, double immunofluorescence, and epithelial cell proliferation assays, were employed to investigate the biological mechanisms underlying epithelial proliferation in periapical endodontic lesions. These mechanisms were subsequently assessed through quantitative, semiquantitative, or qualitative approaches according to defined evaluation criteria [5,16–28].
General characteristics of the studies included
Methods and outcomes of the studies included
Data synthesis
Of the 14 studies included, three evaluated growth factors—insulin-like growth factor (IGF), fibroblast growth factor-7 (FGF-7)/KGF, and TGF—concerning the regulatory mechanisms of epithelial proliferation [5,25,26]. Three studies investigated growth factors, including EGF and FGF-7/KGF, associated with pathological mechanisms of epithelial proliferation [17,18,21]. Four studies assessed cytokines (IL-6, IL-1β, and interleukins related to macrophage polarization) linked to pathological mechanisms of epithelial proliferation [5,22,25,26]. Two studies examined co-stimulatory molecules, such as matrix metalloproteinase 13 (MMP-13), involved in epithelial proliferation, promoting the transition from PG to IRC [23,24]. Finally, three studies reported biomarkers of epithelial cell proliferation, including toll-like receptors (TLRs), proliferating cell nuclear antigen (PCNA), Ki-67, and cluster of differentiation (CD) 34 [16,27,28].
Regulatory mechanism of epithelial proliferation
Yamanaka et al. [25] investigated the KGF/FGF-7 and its receptor (FGFR2-IIIb) in epithelial cells from PGs and IRCs. Both PGs and IRCs were positive for KGF and FGFR2-IIIb, with stronger staining in cystic epithelium. IHC, PCR-Southern blot, RPA, and western blotting confirmed that PDL fibroblasts (PLFs) express FGF-7/KGF messenger RNA (mRNA) and peptide. These results suggest that FGF-7/KGF mediates epithelial–mesenchymal interactions with PLFs, contributing to the maintenance of PDL structure and function [25].
Götz et al. [5] assessed the IGF system in human ERMs from 25 deciduous teeth through IHC, light, and electron microscopy. The results showed weak immunoreactivity for IGF-II, minimal or absent expression of IGF-I and its receptors, and consistent cytoplasmic detection of high-affinity IGF-binding protein (IGF-BP) 6. IGF-BP6, due to its high affinity for IGF-II, may function as an autocrine anti-proliferative mechanism that maintains ERMs in a quiescent state. This control could be disrupted under inflammatory conditions [5].
Nagano et al. [26] showed that TGF-β1 produced by PLFs suppresses the proliferative activity of odontogenic epithelial cells, as evidenced by reduced Ki-67 expression in co-culture. Inhibition of TGF-β signaling restored proliferation, indicating that PLF-derived TGF-βs, via Smad2/3 signaling, maintain ERMs in a quiescent state under physiological conditions, while this regulation may be disrupted by inflammatory signaling [26].
Pathological mechanisms of epithelial proliferation
1) Growth factors
Lin et al. [17] examined the presence of EGF in inflammatory periapical lesions by IHC and 125I-EGF binding assays. Lesions without epithelial proliferation displayed weak or absent 125I-EGF staining, whereas lesions with active epithelial proliferation and cystic transformation exhibited strong immunoreactivity and significantly higher 125I-EGF binding. These findings indicate that EGF expression in odontogenic epithelial cells is upregulated in association with inflammation, suggesting that EGF signaling may drive epithelial proliferation and contribute to cyst transformation [17].
Nickolaychuk et al. [18] assessed extracellular signal-regulated kinase 1 (ERK1) and activated ERK1/2 (phosphorylated ERK [pERK] 1/2]) in dentigerous cysts and IRCs compared with dental follicles using IHC. ERK1/pERK1/2 staining was significantly higher in cyst epithelia than in follicles and concentrated in differentiating and highly proliferative epithelial compartments. Stimulation with EGF increased pERK, whereas MEK inhibition (PD98059) reduced it, confirming that epithelial proliferation in odontogenic cysts is mediated through EGF-dependent mitogen-activated protein kinase (MAPK)–ERK signaling, which may contribute to cystic path-ogenesis [18].
Gao et al. [21] investigated KGF expression in periapical lesions. While KGF mRNA was absent in normal PDL and PGs with minimal inflammation, strong expression was detected in stromal fibroblasts adjacent to inflammatory infiltrates and proliferating epithelium in PGs and IRCs. RT-PCR confirmed elevated KGF in lesion samples. These findings indicate that KGF, upregulated by inflammatory cytokines such as IL-1, TNF-α, and EGF, acts as a paracrine mediator of ERM activation and may drive epithelial proliferation and cyst formation [21].
2) Citokynes
Sako et al. [20] examined the effect of IL-6 on porcine ERM cultures. IL-6 significantly enhanced ERM proliferation and migration, effects abolished by IL-6 neutralizing antibody. IL-6 also induced integrin α3 redistribution, indicating activation of migratory pathways. IL-6 directly stimulates ERM activation, linking inflammation to epithelial proliferation in AP [20].
Schweitzer et al. [19] demonstrated IL-6 expression in the epithelial lining and stromal cells of radicular cysts. IL-6, through binding to IL-6R and gp130, can activate both classic signaling and trans-signaling pathways, thereby promoting epithelial proliferation and sustaining the inflammatory microenvironment. These findings implicate IL-6 as a central mediator linking chronic inflammation with histopathologic transformation of AP from PG to IRC [19].
Nagano et al. [26] demonstrated that TGF-β1/β2 derived from PLFs suppresses the proliferation of odontogenic epithelial cells through Smad2 signaling, thereby maintaining ERMs in a quiescent state. However, they also showed that IL-1β can activate p65 signaling, which interferes with TGF-β–Smad2 pathways and restores epithelial proliferative activity. While TGF-β exerts a homeostatic inhibitory effect, IL-1β–p65 signaling overrides this control during exacerbated inflammation, driving pathological epithelial proliferation [26].
Weber et al. [22] compared macrophage polarization using immunohistochemical markers (CD68, CD11c, CD163, CD206). IRC exhibited the highest degree of M1 macrophage polarization, while PGs showed a predominance of M2 macrophages. M1 polarization in IRCs was associated with pro-inflammatory cytokines such as IL-1 and IL-6, which can stimulate ERM proliferation and perpetuate epithelial activation. Progression from PG to IRC is linked to a shift toward M1 polarization. M1-derived cytokines act as potential drivers of cyst transformation in AP [22].
Biomarkers of epithelial cell proliferation
Roi et al. [16] demonstrated high Ki-67 expression (86.5%) and strong CD34 positivity in PGs, with co-expression of CD34 and Ki-67 in endothelial cells and evidence of intussusceptive angiogenesis. The persistent inflammation was shown to promote proliferative activity and abnormal angiogenesis. CD34+/Ki-67+ proliferating vessels in PGs highlight a mechanism by which angiogenesis may support epithelial proliferation and encourage progression towards IRC [16].
Leonardi et al. [27] evaluated TLR4 expression in PGs and IRCs. In PGs, TLR4 was strongly expressed in almost all ERM cells, while in IRCs it was weaker and limited to basal/parabasal layers. This suggests that TLR4–nuclear factor kappa-B (NF-κB) signaling supports ERM activation in PGs and maintains epithelial lining in IRCs. It also helps explain why lesions may regress after endodontic therapy when inflammatory stimuli decrease [27].
Tripi et al. [28] performed an immunohistochemical evaluation of PCNA, Ki-67, CD3, and p53 in periapical lesions. Ki-67 was positive in all lesions, primarily in the epithelial cells of the cystic linings. PCNA was positive in 22/24 cases, indicating active DNA synthesis and repair. CD3 positivity highlighted abundant T-lymphocytes, suggesting their role in sustaining chronic inflammation. No p53 expression was detected. These findings confirm that upregulated levels of pro-inflammatory mediators in chronic inflammation drive epithelial proliferation in periapical lesions, with Ki-67 and PCNA serving as key indicators of proliferative activity [28].
Co-stimulatory molecules
Leonardi et al. [23] and Bhalla et al. [24] immunohistochemically evaluated the expression of MMP-13 in PGs IRCs. Both studies demonstrated widespread immunopositivity, with stronger expression in the epithelium and fibroblasts of epithelialized PGs and IRCs compared to non-epithelialized PGs. MMP-13 contributes to extracellular matrix degradation and tissue remodeling, facilitating epithelial migration, invasion, and the transition from PG to IRC. Its expression is driven by pro-inflammatory cytokines such as IL-1, IL-6, linking chronic inflammation to epithelial activation and bone resorption [23,24].
Risk of bias assessment
The risk of bias analysis of the included studies reveals that none of the 14 studies received a low risk of bias rating across all four evaluated domains. Two studies [25,26] were rated as low risk of bias in three out of the four domains. In contrast, three studies [5,19,20] were assessed as having an “unclear” or “high” risk of bias across all domains (Table 3).
Methodological quality graph of included studies (QUADAS-2)
When analyzing the evaluation by domains, the domain with the most significant issues was “patient selection,” as six studies [16–18,20,21,26] were assessed as having a high risk of bias, and seven studies [5,19,22–24,27,28] were rated as “unclear” risk of bias. This was mainly because all studies relied on a case-control design, and in most cases, the method of patient selection—whether consecutive or random—was not clearly reported. The second most problematic domain was “flow and timing,” as 12 of the 14 evaluated studies [5,16–25,27,28] did not report or describe a patient selection flowchart. They also failed to describe each of the patients included in the study who were evaluated with both the index test and the reference standard test. Additionally, they did not report the time interval between the index test and the reference standard (Table 3 and Figure 2).
Proportion of studies with low, high, or unclear risk of bias.
In the other two domains, the majority of the studies were rated as having a low risk of bias. In the “Index Test” domain, the few studies [5,19,20] that received an ‘unclear” risk rating did so because it was not clear whether the index test results were interpreted without knowledge of the reference standard results. Similarly, in the “reference Standard” domain, only a few studies [5,19,20,28] were rated as “unclear” risk of bias due to a lack of information on who interpreted the tests and whether any blinding was performed (Table 3 and Figure 2).
Applicability assessment
In the applicability analysis, all studies were assessed as having a low risk of bias across the three evaluated domains (“patient selection,” “index test,” and “reference standard”). This was because no issues were identified concerning discrepancies between the included patients and the review question, the conduct or interpretation of the index test, or the condition defined by the reference standard differing from the review question (Table 3 and Figure 3).
Proportion of studies with low, high, or unclear concerns regarding applicability.
DISCUSSION
This systematic review aims to synthesize the best available experimental evidence on the biological pathways underlying epithelial proliferation leading to the transition of PGs into IRCs. Cell and tissue experimental models provide a powerful framework to unravel the intracellular signaling pathways and molecular mediators that govern epithelial activation. These models not only validate the biological relevance of experimental findings but also reveal how molecular alterations translate into structural and functional changes. This dual perspective strengthens the causal link between cellular signaling and lesion development. Findings from this systematic review suggest that biological mechanisms linked to the IGF, FGF-7/KGF, and TGF-β are involved in the regulatory mechanisms of epithelial proliferation within AP under physiological conditions. Under conditions of elevated pro-inflammatory mediators and exacerbated AP, specific growth factors, including EGF and FGF-7/KGF [17,18,21], along with cytokines such as IL-6 and IL-1β, can disrupt homeostatic regulatory pathways, inducing ERM activation and pro-liferation, thereby facilitating the histopathological progression of PGs into IRC [5,25,26]. Furthermore, macrophage M1 polarization, characterized by Th1-associated cytokines such as IL-1β, IL-6, and TNF-α, drives inflammation, ERM proliferation, and migration, thereby facilitating ERM-driven cyst formation [22]. Additionally, co-stimulatory molecules such as MMP-13 promote epithelial cell migration, proliferation, and stratification within AP, while simultaneously mediating extracellular matrix degradation [23,24]. Finally, several biomarkers of epithelial cell proliferation, including TLRs, PCNA, Ki-67, and CD34, were linked to PG/IRC transition [16,27,28]. The methodological appraisal (QUADAS-2) indicated an overall low-to-moderate risk of bias, with the main concerns arising from patient selection and flow/timing. Several studies lacked clearly defined inclusion criteria or did not provide a flowchart describing patient selection, while others failed to specify whether all patients underwent both the index test and the reference standard. In addition, the time interval between the index test and the reference standard was often not reported, further limiting methodological transparency. In contrast, the validity of index tests (mostly IHC) and reference standards was typically satisfactory, with few questions about application. Thus, the overall strength of evidence of this systematic review can be regarded as moderate, sufficient to support mechanistic hypotheses concerning the transition from PG to IRC, but still insufficient for direct clinical extrapolation due to methodological heterogeneity, small sample sizes, and the predominantly case-control design of the included studies, which limits causal inference and the establishment of temporal relationships.
Regulatory mechanism of epithelial proliferation
FGF-7/KGF is a mesenchyme-derived paracrine mediator with potent mitogenic activity for epithelial cells but not for fibroblasts or endothelial cells [25,29]. FGF-7/KGF exerts its biological effects by binding to specific tyrosine kinase receptors (FGFRs) on the cell surface [25,30]. Immunohistochemical and molecular studies have demonstrated that proliferating human PLFs express and secrete FGF-7/KGF. Expression peaks during the proliferative phase and declines with differentiation, as indicated by increased alkaline phosphatase activity. FGF-7/KGF protein is also detectable in PLF-conditioned medium [25]. When epithelial cells derived from ERMs were cultured in serum-free conditions, their proliferation was selectively stimulated by FGF-7/KGF, following initial PLF outgrowth from the same tissue explants. Notably, ERMs express only the FGFR2-IIIb isoform, a high-affinity receptor for FGF-7/KGF. This isoform-specific expression suggests that ERMs are physiologically responsive to stromal-derived FGF-7/KGF, which regulates their proliferation and differentiation. FGF-7/KGF binding to FGFR2-IIIb induces receptor dimerization and autophosphorylation, activating intracellular signaling pathways such as the MAPK (Ras–Raf–MEK–ERK) cascade, which promotes cyclin D1 expression and G1–S cell cycle progression [31]. Simultaneously, the PI3K–AKT pathway is activated, enhancing epithelial cell survival by phosphorylating pro-apoptotic factors, such as BAD, and suppressing caspase activation [32]. Together, these pathways support epithelial homeostasis by balancing proliferation, differentiation, and survival within the PDL epithelial compartment. However, persistent inflammation, dysregulated FGF-7/KGF expression, or aberrant FGFR2-IIIb activation may disrupt this balance, promoting uncontrolled ERM proliferation and contributing to pathological epithelial proliferation in PGs, leading to transformation into IRCs [21,33].
TGF-β isoforms, particularly TGF-β1 and TGF-β2, are pleiotropic cytokines predominantly released by activated immune cells, platelets, and PLFs [26,34]. TGF-β plays a vital role in maintaining tissue homeostasis by regulating cellular differentiation, inflammation, and proliferation [26,34]. The TGF-β ligand binds to the type II receptor (TGFβ-R2), which then recruits and phosphorylates the type I receptor (TGFβ-R1), thereby activating the intracellular Smad pathway. Specifically, Smad2 and Smad3 become phosphorylated and form a complex with Smad4 that translocates into the nucleus to regulate gene expression [26,35,36]. In epithelial cells, this mechanism has an anti-proliferative effect, partly by upregulating transcription factors like ATF3 and repressing proliferative genes such as Id1. TGF-β signaling helps to keep ERMs in a quiescent state [26,35,36]. Co-culture studies involving human PLFs and odontogenic epithelial cells have demonstrated a reduction in Ki-67 expression, a marker of proliferation, in epithelial cells, indicating growth inhibition [26]. Moreover, blocking TGF-β signaling—through receptor inhibition and small interfering RNA-mediated knockdown of TGF-β1 and TGF-β2—restores proliferative activity in these epithelial cells. These findings suggest that PLF-derived TGF-β, acting via Smad2/3 signaling, serves as a natural control of ERM proliferation. However, under exaggerated inflammatory conditions, this regulatory balance may be disturbed, potentially allowing the abnormal epithelial proliferation that contributes to IRC formation [26].
The IGF system is a pivotal regulator of cell proliferation in diverse cell types, including ERM [5]. IGF ligands, IGF-I and IGF-II, secreted primarily by stromal fibroblasts, exert pleiotropic actions encompassing growth, differentiation, proliferation, and metabolic regulation [5,10]. Signal transduction is mainly mediated by the ubiquitously expressed IGF-1 receptor (IGF-1R) [5,37], whereas the IGF-2 receptor functions predominantly in the sequestration and lysosomal degradation of IGF-II [5].
High-affinity IGF-BPs act as carrier molecules in biological fluids, regulating IGF bioavailability, prolonging their half-life, and modulating receptor interactions [5,38]. IGF-BP6 exhibits the greatest specificity for IGF-II. Immunohistochemical studies have demonstrated IGF-BP6 localization within the cytoplasm and secretory vesicles of ERMs, suggesting an autocrine regu-latory loop. PLFs may constitute an additional source of IGF-BP6 [5]. In ERMs, IGF-BP6 immunoreactivity co-occurs with weak IGF-II and minimal to absent IGF-1R expression, implying negligible sensitivity to IGF-I and the absence of autocrine IGF-I signaling [5]. The preferential binding of IGF-BP6 to IGF-II—up to 100-fold greater than to its receptor—positions IGF-BP6 as a potent inhibitor of IGF-II-mediated mitogenesis [39]. Under physiological conditions, this interaction may restrain ERM proliferation and preserve homeostasis [40].
Inflammatory microenvironments, such as those observed in AP, alter this regulatory balance. A cathepsin D-like protease has been implicated in the proteolytic cleavage of IGF-BP6, liberating bioactive IGF-II within ERMs [5,40]. This IGF-II release may act in an autocrine manner to drive uncontrolled ERM proliferation. Additionally, pro-inflammatory cytokines (IL-1, IL-6) and growth factors such as EGF, TGF-α, and KGF—some of which can be produced by ERMs—may further compromise IGF-BP6-mediated inhibition, thereby facilitating proliferative responses [2,5,25,41].
Pathological mechanisms of epithelial proliferation
Cytokines
IL-1β, a pro-inflammatory cytokine mainly produced by macrophages during the progression of AP, activates NF-κB signaling, with p65 as its key subunit regulating gene transcription and cell proliferation [26,42,43]. In IRCs, elevated IL-1β and p65 expression have been linked to lesion progression. IL-1β also suppresses TGF-β1 expression in PDL cells, disrupting ERM homeostasis [26,44,45]. An immunohistochemical study comparing IRCs and non-inflammatory dentigerous cysts found nuclear and/or cytoplasmic p65 expression in 84.6% of IRCs, significantly higher than in dentigerous cysts (p < 0.01). Smad2/3 was consistently expressed in dentigerous cysts but detected in only 25.0% of IRCs. Moreover, Ki-67 positivity was higher in p65-positive IRCs (77.3%) than in p65-negative ones (37.5%), suggesting an inflammation-associated proliferative phenotype [26].
In vitro, SF2 odontogenic epithelial cells stimulated with TGF-β1 showed time-dependent Smad2 phosphorylation and reduced proliferation. Conversely, IL-1β promoted p65 nuclear translocation, induced inducible nitric oxide synthase expression (a direct NF-κB target), and suppressed TGF-β1–induced Smad2 phosphorylation. IL-1β reversed TGF-β1–mediated growth inhibition and proliferation. These findings suggest that IL-1β/p65 signaling promotes epithelial proliferation by antagonizing TGF-β/Smad2 activity, contributing to the histological progression from PG to IRC [26,44,45].
In periapical tissues, IL-6 is secreted by local immune cells—primarily monocytes/macrophages, Th1/Th2 cells, B lymphocytes, and PMNs—in response to endodontic bacterial components like lipopolysaccharides (LPS) [2,19,20,46]. Non-immune cells, such as epithelial, endothelial, and stromal cells, also contribute to IL-6 production under inflammatory stimuli [20]. Elevated IL-6 levels contribute to IRC pathogenesis by enhancing ERM proliferation and migration and sustaining inflammation [2,20].
IL-6 signals via two main pathways: classic signaling, through membrane-bound receptor (mIL-6R), and trans-signaling, through the soluble receptor (sIL-6R) bound to IL-6, which engages gp130 on cells lacking IL-6R [47]. While classic signaling supports tissue homeostasis, trans-signaling mediates pro-inflammatory responses [19,48]. In AP, trans-signaling dominates, as IL-6R is absent in healthy PDL but highly expressed in inflamed lesions. sIL-6R is released by infiltrating mononuclear cells and mediates IL-6 effects on PDL fibroblasts [19,49]. Immature epithelia may respond to IL-6 via trans-signaling from inflammatory or autocrine epithelial sources, while mature lining cells appear responsive to both pathways. Thus, IL-6 may promote proliferation and inflammation via trans-signaling in early lesions (PGs), and migration, regeneration, or apoptosis resistance via classic signaling in IRCs [19,50,51].
Integrins, particularly α3β1, are key regulators of epithelial migration and IL-6 release [20,52]. ERMs express several integrins, including α3, β1, and β3 [20,53,54]. IL-6 stimulation increases ERM proliferation and migration, an effect abolished by IL-6 neutralization. IL-6 also drives spatial redistribution of integrin α3 to filopodia, supporting cytoskeletal reorganization linked to enhanced motility [20]. Cytoskeletal changes during ERM activation involve a shift in cytokeratin expression. Quiescent ERMs express keratins 5 and 19. Upon activation, keratin 14 (a stratifying marker) is upregulated, followed by keratins 13 and 4 (markers of non-keratinizing epithelia). Low levels of keratins 8 and 18 (simple epithelium markers) are also observed, sug-gesting partial epithelial plasticity during cystic transformation [55]. Collectively, IL-6 promotes ERM proliferation and migration through integrin α3 redistribution and cytoskeletal remodeling, contributing to epithelial plasticity and the transition from PG to IRC [19,20,55].
PGs and IRCs are characterized by dense infiltration of macrophages and lymphocytes. In PGs, these immune cells are diffusely distributed throughout the connective tissue, whereas in IRCs they localize primarily within the fibrous cyst wall, contributing to a sustained chronic inflammatory microenvironment [2]. Lymphocytes differentiate into Th1 or Th2 subsets depending on local cytokine cues [56–58], while macrophages polarize into pro-inflammatory M1 or anti-inflammatory M2 phenotypes, typically associated with Th1 and Th2 responses, respectively [56,59].
Immunohistochemical studies reveal that IRCs show a significantly higher prevalence of M1-polarized macrophages, whereas PGs are more enriched in M2 macrophages [22]. M1 polarization, associated with Th1 cytokines such as IL-1β, IL-6, and TNF-α, promotes inflammation, ERM proliferation and migration, and epithelial lining expansion [19,20,26]. In contrast, M2 macrophages, associated with Th2 responses, mediate tissue repair through the clearance of debris, angiogenesis, and the production of anti-inflammatory cytokines such as IL-10 and TGF-β. TGF-β also suppresses ERM proliferation under physiological conditions and contributes to the induction of regulatory T cells (Tregs), reinforcing resolution of inflammation [22,26,56].
Given the stem-like properties of ERMs and their sensitivity to macrophage-derived cytokines [22,60], increased M1 polarization may be a key driver of their activation and the histological transition from PG to IRC. However, the regulation of macrophage polarization in periapical lesions remains incompletely understood. Host factors such as genetic background, the intensity of the inflammatory response, and the composition of the root canal microbiota likely contribute [22,60,61].
In particular, bacterial LPS—abundant in gram-negative organisms—are potent inducers of M1 macrophage activation. IRC fluids often contain gram-negative bacteria, including Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Treponema denticola, Tannerella forsythia, and Campylobacter rectus. Notably, C. rectus and T. denticola induce robust IL-1β production, a hallmark of M1 activation [62]. These data support a model in which the microbial profile of endodontic infections modulates macrophage phenotype and promotes ERM-driven cyst formation via M1-associated inflammatory signaling [22].
Growth factors
EGF is a potent mitogen for epithelial, fibroblastic, and endothelial cells, acting through binding to its high-affinity receptor, EGF-R—a transmembrane protein with intrinsic tyrosine kinase activity [17,63,64]. This interaction triggers intracellular signaling cascades, including MAPK/ERK, leading to gene transcription, cell cycle progression, and epithelial proliferation—key events implicated in IRC pathogenesis [65–67].
EGF-R is abundantly expressed in the epithelial components of the tooth germ, ERMs, and IRC linings, particularly in proliferative epithelial cells [21,63,68]. In contrast, its expression is minimal in PGs [69]. This differential expression suggests that EGF-R upregulation may drive pathological epithelial proliferation in IRCs. EGF produced by stromal fibroblasts is hypothesized to activate ERMs under inflammatory conditions [10].
Inflamed tissues exhibit increased EGF binding and EGF-R expression. IHC and radio-labeled EGF assays have shown strong receptor expression and ligand binding in epithelial cells of IRCs and PGs with epithelial proliferation, but not in lesions lacking such activity [17,70]. Once EGF-R is activated, MAPK/ERK signaling is initiated: ERK1/2 translocates to the nucleus and activates transcription factors like Elk-1, c-Fos, and c-Myc, promoting mitogenic gene ex-pression [18,67]. In ERMs and IRC epithelium, pERK1/2 is associated with proliferation, cytoplasmic vacuolization, and squamous differentiation [18,71]. These findings suggest that ERK phosphorylation functions as a key trigger for reactivating quiescent ERMs, promoting their activation, differentiation, and proliferative response. This observation is consistent with a mechanistic model in which inflammatory mediators—specifically bacterial endotoxins and pro-inflammatory cytokines such as IL-1 and IL-6—induce MAPK pathway activation, thereby driving ERM proliferation and epithelial differentiation programs [18,72].
KGF/FGF-7 is a stromal-derived paracrine mediator that selectively stimulates epithelial cell proliferation via its high-affinity receptor, FGFR2-IIIb, which is expressed exclusively in epithelial cells [21,33,73]. In vitro, fibroblasts constitutively express KGF mRNA and peptide, with expression tightly regulated by epithelial-derived signals [74]. While baseline KGF expression in the PDL is low, AP can upregulate its production in stromal fibroblasts via inflammatory cytokines such as IL-1α/β, TNF-α, and platelet-derived growth factor [10,21].
This cytokine-driven increase in KGF promotes epithelial proliferation indirectly, as shown by elevated KGF expression in PGs and IRCs with active epithelial growth, particularly in stromal cells adjacent to inflammatory infiltrates [21]. In situ hybridization and RT-PCR analyses confirm that proliferative periapical lesions express significantly more KGF than normal PDL. KGF expression is notably stronger in early-stage IRCs, suggesting its role in the initial phases of cyst formation [63].
At the signaling level, KGF binding to FGFR2-IIIb activates the Ras–ERK/MAPK pathway, leading to increased cyclin D1 expression and transcriptional activation of c-Fos and c-Myc, driving epithelial cell cycle progression and hyperproliferation [31]. Chronic AP sustains this signaling loop via persistent cytokine stimulation, creating a microenvironment favoring epithelial activation and proliferation, ultimately contributing to IRC pathogenesis [19,26].
Co-stimulatory molecules
MMP-13 exhibits broad substrate specificity [75]. It is produced by fibroblasts, epithelial cells, malignant squamous epithelium, and plasma cells associated with bone-destructive processes [24,75]. In endodontic periapical lesions, MMP-13 has been consistently detected and is considered a major mediator of bone matrix degradation and lesion progression [24,75]. Experimental models of periapical inflammation in rats further demonstrate increased MMP-13 expression during the early stages of lesion development, underscoring a temporally regulated role in extracellular matrix remodeling [76]. Importantly, immunohistochemical studies reveal high levels of MMP-13 immunoreactivity (80%–100%) across PGs and IRCs, with stronger staining in lesions containing epithelial tissue compared with non-epithelial PGs [23,24,77]. Within epithelial compartments, expression is accentuated in PGs with epithelium and IRCs, particularly in thinner epithelial strands indicative of proliferative activity, whereas thicker strands show only scattered positive cells [23,24]. These findings support the concept that ERMs, normally quiescent, can acquire proliferative potential under inflammatory conditions, and that MMP-13 contributes to epithelial migration, proliferation, and invasion. Consequently, MMP-3 is likely involved in the transition from PGs with epithelium to fully developed IRCs [23,24].
Biomarkers of epithelial cell proliferation
Ki-67 is a nuclear protein and an established marker of cellular proliferation [78]. Its upregulation is a hallmark of chronic inflammatory states, including AP. Inflammatory cytokines trigger stress responses that enhance Ki-67 expression in PGs [63,78]. Ki-67 immunoreactivity has been specifically noted in the nuclei of basal epithelial layers in PGs, reflecting epithelial proliferative activity and possible progression toward cyst formation [78].
CD34, a transmembrane sialomucin, is widely expressed in hematopoietic progenitor cells and vascular endothelial cells, as well as in fibrocytes, keratinocytes, and epithelial progenitors [78,79]. Beyond its role in hematopoiesis, CD34 is crucial for angiogenesis, promoting endothelial cell proliferation and migration. In periapical lesions, its expression correlates with areas of intense inflammatory infiltrate, suggesting a dual role in inflammatory modulation and vascular remodeling [78].
Combined immunohistochemical analyses of Ki-67 and CD34 in PGs have demonstrated that Ki-67 marks epithelial cell activation and proliferative potential, while CD34 highlights regions of neovascularization associated with inflammatory cell recruitment. Their co-expression supports a link between inflammation, microvessel density, endothelial proliferation, and epithelial remodeling—key features in the transition from granuloma to cystic pathology [78].
PCNA is a key regulatory protein involved in DNA replication and cell cycle control. Under inflammatory conditions, PCNA is upregulated in epithelial cells and serves as a sensitive marker of active proliferation [80]. In periapical lesions, particularly those containing epithelial components such as PGs and IRCs, PCNA immunoreactivity is prominently observed in epithelial linings and the ERMs [28,81]. This pattern reflects a proliferative epithelial response to persistent inflammatory stimuli, consistent with the dynamic interplay between tissue proliferation and degeneration in these lesions.
Proliferative activity in periapical lesions has been consistently demonstrated through immunohistochemical detection of PCNA and Ki-67. All proliferative lesions examined showed concurrent expression of both markers, confirming active cell cycle progression within the epithelial remnants exposed to chronic inflammation [28]. The co-expression of Ki-67 and PCNA provides strong evidence that inflammatory microenvironments stimulate epithelial proliferation, particularly of ERMs, thereby contributing to the pathogenesis of periapical lesions and facilitating their potential transformation into radicular cysts.
TLRs are transmembrane receptors found on immune cells such as macrophages and dendritic cells, but also often or inducibly expressed in epithelial cells, where they serve as primary microbial sensors for pathogen-associated molecular patterns. When ligands bind, TLRs trigger intracellular signaling pathways that lead to the transcription of pro-inflammatory cytokines and other immune mediators [82,83].
In the context of periapical lesions, TLR2 and TLR4 have been strongly implicated in epithelial and immune activation [27]. TLR4 is robustly expressed in epithelial strands and islands derived from ERMs within PGs, while in IRCs, TLR4 expression is predominantly confined to the basal and parabasal epithelial layers, with a patchy immunostaining pattern [27]. This distri-bution reflects TLR4’s roles in regulating epithelial survival, proliferation, and migration. In PGs, TLR4 expression may promote ERM activation, whereas in IRCs, it appears to maintain epithelial lining thickness by balancing proliferation and apoptosis.
Functionally, TLR4 activation leads to NF-κB translocation into the nucleus, driving transcription of genes involved in cell survival, proliferation, and inflammatory signaling [27,84]. In PGs, sustained TLR4 signaling in ERMs likely disrupts their quiescent state, inducing pathological epithelial expansion.
Importantly, this inflammation-driven proliferation is reversible. Following non-surgical endodontic therapy, the local decline in inflammatory cytokines and growth factors results in reduced TLR signaling. Consequently, proliferative activity in basal epithelial cells ceases, and differentiated squamous cells undergo apoptosis [10,85]. This regression mirrors the physiological turnover of oral epithelium and underscores the dependence of periapical epithelial structures on chronic inflammatory stimuli [27]. Ultimately, the dynamic balance between proliferation and apoptosis dictates the progression or regression of epithelialized periapical lesions [10,18,86].
Strengths and weaknesses
This systematic review was conducted in full accordance with the Cochrane Handbook for Systematic Reviews of Interventions and the PRISMA statement [13,14], with the protocol prospectively registered in PROSPERO. Only experimental studies (cell and tissue models) using validated assessment methodologies were included. Comprehensive searches were carried out across three major databases and grey literature sources without language restrictions, maximizing coverage and reducing the risk of publication bias. Risk of bias was assessed using QUADAS-2, with two independent reviewers conducting the literature search and data extraction; any disagreements were resolved by consensus. These methodological strengths ensured transparency, reproducibility, and a balanced appraisal of the available evidence, enabling a reliable identification of both strengths and limitations within the included studies.
Methodologically, although this systematic review was rigorously designed, the primary studies included present important limitations: most were small observational or case-control series with heterogeneous and poorly defined inclusion criteria, often lacking patient flowcharts and adequate reporting of the interval between index and reference tests. Sample sizes were generally limited and frequently derived from surgically obtained specimens, introducing potential selection bias. Considerable variability in immunohistochemical procedures and largely semiquantitative interpretation further restricted cross-study comparability. Furthermore, the lack of longitudinal designs and clinical outcome connections limits the findings’ external applicability. Collectively, these flaws limit the total strength of evidence, which should be classified as moderate.
CONCLUSIONS
The transition of PGs into IRCs is governed by a dynamic balance between regulatory and pathological molecular mechanisms acting on ERMs. Under physiological conditions, regulatory mediators such as TGF-β/Smad2/3 and IGF-BP6 maintain ERM quiescence, while stromal-derived KGF/FGF-7 supports controlled epithelial homeostasis. However, exacerbated inflammatory microenvironments disrupt these regulatory circuits: elevated levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), growth factors (EGF, KGF), and bacterial endotoxins activate intracellular pathways such as NF-κB, MAPK/ERK, and PI3K–AKT, reprogramming ERMs towards pathological proliferation, migration, and epithelial plasticity. In parallel, macrophage polarization towards the M1 phenotype amplifies this pathogenic loop through the release of IL-1β, IL-6, and TNF-α, sustaining epithelial activation and cystic expansion.
Co-stimulatory molecules, particularly MMP-13, promote epithelial cell migration, proliferation, and stratification within the lesion while driving extracellular matrix degradation. Its selective upregulation in PGs containing epithelium and in IRCs parallels zones of heightened epithelial activity, indicating that MMP-13 not only remodels the microenvironment but also facilitates invasive epithelial behavior. This dual role positions MMP-13 as a key mediator of the transition from granulomatous to cystic pathology.
Immunohistochemical and molecular biomarkers—including Ki-67, PCNA, CD34, and TLR4—highlight the functional pathways that sustain cystic transformation. Their expression patterns reflect a coordinated interplay of epithelial proliferation, angiogenesis, and immune-driven activation, indicating that epithelial–stromal crosstalk is not a bystander phenomenon but a central driver of lesion progression. Ki-67 and PCNA denote the persistence of epithelial mitotic activity, reinforcing the notion that chronic inflammation maintains ERMs in a proliferative state. CD34 expression underscores the angiogenic support required for epithelial expansion, linking vascular remodeling to proliferative demands. Meanwhile, TLR4 activation integrates microbial sensing with intracellular signaling cascades that potentiate inflammatory amplification, epithelial plasticity, and survival.
Collectively, these biomarkers illustrate that cystic transformation is governed by the convergence of proliferative and inflammatory circuits, sustained by angiogenic reinforcement. Their co-expression delineates a pathogenic signature that not only identifies lesions with higher proliferative potential but also provides a prognostic framework for anticipating cystic behavior. This positions them as candidate tools for molecular stratification of periapical lesions, offering potential clinical value in distinguishing lesions prone to regression from those likely to progress towards irreversible cystic pathology.
Nevertheless, the included studies revealed significant methodological heterogeneity, frequent reliance on case-control designs, limited sample sizes, and incomplete reporting of patient selection and timing, resulting in an overall moderate to high risk of bias. These limitations precluded quantitative synthesis and underscore the urgent need for standardized, longitudinal, and functional studies using advanced molecular tools.
In methodological terms, the predominance of high or unclear risk of bias in patient selection and flow/timing domains weakens the generalizability of current findings. Future investigations should adopt rigorous designs, integrate multiomics approaches, and validate candidate biomarkers in prospective clinical settings.
Collectively, this review emphasizes that IRC development arises from a transition from homeostatic to pathological signaling, in which pro-inflammatory mediator levels inside the periapical chronic inflammation override regulatory checkpoints. To find trustworthy biomarkers and create biologically based diagnostic and treatment approaches, a greater mechanistic knowledge of the biological pathways underlying the transition from PG to IRC is essential. With this information, cystic transformation may be predicted, prevented, or modulated.
Future perspectives
While classical pathways such as EGF, KGF, TGF, and IGF and pro-inflammatory cytokines such as IL-1 and IL-6 have been extensively studied, recent evidence indicates that non-canonical mechanisms also influence ERM behavior during chronic inflammation. The Hippo pathway and its effectors YAP and TAZ are particularly relevant, as their dysregulation promotes epithelial proliferation and survival and has been implicated in odontogenic tumors. Although not yet investigated in ERM, the conserved epithelial origin of periapical lesions suggests a similar contribution to abnormal proliferation during cystic transition [87,88]. Epigenetic regulators such as specific microRNAs (e.g., miR-155 targeting SEMA3A) and exosome-mediated miRNA transfer appear to shape the inflammatory milieu, while long non-coding RNAs such as PACER and THRIL are upregulated in IRCs, supporting their role in molecular reprogramming [1,89,90]. Altogether, these insights underscore that integrating transcriptomic, epigenetic, and systems biology approaches is essential to unravel ERM plasticity and to identify early molecular markers of cystic transformation. Such advances may ultimately guide the development of novel therapeutic strategies aimed at preventing or reversing the progression from PG to IRC.
Emerging molecular therapies
Recent advances in the molecular understanding of IRC pathogenesis have unveiled promising therapeutic avenues aimed at restraining ERM proliferation and even reversing early cystic transformation. Among the most compelling targets is IL-6 trans-signaling, mediated by the sIL-6R. Selective blockade with sgp130Fc has been shown to attenuate pathological ERM activation while preserving the beneficial homeostatic effects of classic IL-6 signaling [19]. Equally significant is the MAPK/ERK axis, where pERK1/2 drives epithelial proliferation. Inhibition of this pathway with MEK1/2 inhibitors (e.g., PD98059) could suppress aberrant epithelial growth and halt progression from PG to IRC [18]. Another innovative strategy involves exploiting the TRAIL–DR5 apoptotic axis, which is active in IRC linings; DR5 agonists may selectively eliminate hyperplastic epithelial cells while sparing surrounding tissues [91]. Moreover, the strong upregulation of ΔNp63 and Ki-67 underscores their role in sustaining epithelial stemness and proliferation. Although direct ΔNp63 inhibition remains experimental, epigenetic modulators and inflammatory microenvironment control represent feasible approaches to temper these pro-liferative signals [92]. Finally, the NLR family pyrin domain-containing 3 (NLRP3) inflammasome–IL-1β pathway emerges as a central driver linking inflammation, bone resorption, and epithelial activation. Targeted suppression using NLRP3 inhibitors (mcc950), IL-1 receptor antagonists (Anakinra ((Kineret®, Amgen Inc., Thousand Oaks, CA, USA)), or monoclonal antibodies against IL-1β (Canakinumab (Ilaris®, Novartis Pharma AG, Basel, Switzerland)) holds great translational promise [42]. Together, these molecular strategies herald the prospect of non-surgical modulation of IRC, shifting endodontics toward biologically guided interventions that can actively reprogram epithelial dynamics and redefine the therapeutic landscape of periapical disease.
Notes
CONFLICT OF INTEREST
No potential conflict of interest relevant to this article was reported.
FUNDING/SUPPORT
The authors have no financial relationships relevant to this article to disclose.
AUTHOR CONTRIBUTIONS
Conceptualization: Rios-Osorio N. Data curation: Briñez-Rodríguez S; Grajales M, Jiménez-Peña OM . Formal analysis: Grajales M, Jiménez-Peña OM. Investigation: Rios-Osorio N, Briñez-Rodríguez S, Betancur-Calle D, Guerrero-Torres M. Methodology: Rios-Osorio N, Grajales M, Jiménez-Peña OM. Project administration: Rios-Osorio N . Resources: Rios-Osorio N . Software: Jiménez-Peña OM. Supervision: Rios-Osorio N, Fernández-Grisales R . Validation: Rios-Osorio N, Fernández-Grisales R . Writing - original draft: Rios-Osorio N, Fernández-Grisales R Writing - review & editing: Rios-Osorio N.
DATA SHARING STATEMENT
The datasets are not publicly available but are available from the corresponding author upon reasonable request.
