Immunohistochemical localization of components of the insulin-like growth factor system in human permanent teeth

There is growing evidence that the insulin-like growth factor (IGF) system plays an important role in the biology of oro-dento-facial tissues and organs, including the development, homeostasis and regeneration of the periodontium. To obtain basic data on the occurrence and distribution of IGF components in human permanent teeth we immunohistochemically investigated 25 extracted, decalcified and paraffin-embedded teeth using mono and polyclonal antibodies against the ligands IGF-I and -II, the IGF1 receptor (IGF1R) and all six IGF binding proteins (IGFBP-1 to -6). In the extracellular matrix (ECM) of the adhering periodontal ligament (PDL), immunoreactivity for IGF-I, -II and IGFBP-1 and -6 was observed. PDL fibroblasts showed immunostaining for the IGF1R. For the cementum, in the acellular cementum only IGF-II could be detected, while outer cementum layers with inserting Sharpey's fibers reacted with all antibodies applied except for IGFBP-4 and -6. In the pulp, mainly fibrotic areas and areas around denticles were immunoreactive for IGF-I, IGFBP-1, -3, -5 and -6. Predentin and odontoblastic processes were stained for IGF-I and IGFBP-3. The spatially oriented occurrence of components of the IGF system in human permanent teeth indicates that specific functions of the IGFs may be localized in particular tissue compartments. In the cementum, several IGF components were found indicating roles in tissue homeostasis or attachment. The PDL may function as a reservoir for IGFs probably bound to ECM components. PDL fibroblasts could then respond in a paracrine manner. In the pulp, the IGF system may be involved in odontoblast biology, fibrosis and denticle formation.     

Role of the epithelial cell rests of Malassez in the development,maintenance and regeneration of periodontal ligament tissues

Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth‐supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal‐regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem‐cell populations that demonstrate similar properties to mesenchymal stromal/stem cells, both functionally and phenotypically. Therefore, the epithelial cell rests of Malassez, rather than being ‘cell rests’, as indicated by their name, are an important source of stem cells that might play a pivotal role in periodontal regeneration.     

Immunohistochemical localization of epidermal growth factor in cat paradental tissues during tooth movement.

Epidermal growth factor enhances proliferation and differentiation of cells during growth, maturation, and tissue healing. The objectives were to localize the epidermal growth factor in paradental cells and to determine the effect of orthodontic treatment on its concentrations in periodontal ligament fibroblasts, alveolar bone surface lining cells, and epithelial rests of Malassez. Sixty male cats, 1 year old, were divided into 2 groups: active and sham, and further divided into 10 time groups. In the active group, 1 maxillary canine was retracted by 80 g force; in the sham group, the animals received an inactive appliance. Sagittal sections of each half maxilla were stained for epidermal growth factor; staining intensity was measured microphotometrically in 10 periodontal ligament fibroblasts, alveolar bone surface lining cells, and epithelial rests of Malassez cells in sites of periodontal ligament tension and compression, and in corresponding sites near control and sham canines. The overall mean staining intensity of the cells of the active group animals was 30.47%, whereas that of the sham group was 21.78% (P <.0001). In all 3 types, cells near the actively treated canines stained significantly darker (P <.0001) than cells near the sham or control canines, particularly between 12 hours and 7 days. These results demonstrate that orthodontic forces increase epidermal growth factor concentrations in paradental cells, suggesting that epidermal growth factor participates in the tissue remodeling that facilitates tooth movement.     

In vitro characterization of the cytokine profile of the epithelial cell rests of Malassez

BACKGROUND: The epithelial cell rests of Malassez (ERM) are an integral part of the periodontal ligament and are considered to play an important role in dental pathology. Surprisingly, this cell type is poorly described and is often disregarded in the context of periodontal research. The aim of this study was to establish primary cell cultures of human ERM, characterize the cytokine profile, and compare it to other periodontal cell entities. METHODS: ERM-derived epithelial cells were isolated from the periodontal ligament of three subjects. A cytokine antibody array, including 120 cytokines in two membranes, was used to determine the cytokine profile of conditioned medium from the ERM-derived epithelial cells. The results were compared to those of gingival epithelial cells and periodontal ligament fibroblasts. RESULTS: ERM-derived epithelial cells expressed 29 of 120 cytokines in significant amounts, including cytokines, chemokines, growth factors, and related proteins, such as interleukin (IL)-1, -6, -8, and -10; granulocyte macrophage-colony stimulating factor; monocyte chemoattractant protein (MCP)-1, -2, and -3; amphiregulin; glial-derived neurotrophic factor; vascular endothelial growth factor; and insulin-like growth factor binding protein-2. The cytokine profile of ERM cells was similar to that of gingival epithelial cells but strikingly different from the profile of periodontal ligament fibroblasts. CONCLUSIONS: The results indicated that, via paracrine secretion of a variety of soluble factors, the ERM cells actively take part in the homeostasis of the periodontium. Therefore, future research on the pathophysiology of periodontal tissue should include this often overlooked cell type.     

Neuroendocrine cells in Malassez epithelium and gingiva of the cat

Malassez epithelium has been designated as epithelial cell rests, the biological significance of which is still under debate. This study was designed to analyze Malassez epithelium for the presence of neuroendocrine cells. Gingival tissue was included as a positive control. Using immunohistochemistry, confocal and light microscopy, Malassez epithelium and gingival epithelium from mature cats (n = 5) were examined for cells containing the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Both Malassez epithelium and the basal epithelial cell layers in gingival rete pegs regularly displayed cells immunoreactive to CGRP, SP, and VIP. The immunopositive cells were most frequently present in the epithelial cell clusters and strands of Malassez located in the cervical half of thc periodontal ligament. Double immunolabeling revealed cellular co-expression of CGRP or SP with VIP, and the neuropeptides were co-localized in the cellular compartments. Labeled cells in both epithelia were occasionally supported by immunoreactive nerve fibers. This study shows that cells immunoreactive to CGRP, SP, and VIP arc located within the cat Malassez epithelium. The localization of neuroendocrine cells verifies the diversity of this epithelium and confirms that Malassez epithelium is composed of different cell types, in common with epithelia from other locations. The presence of neuroendocrine cells in Malassez epithelium strongly suggests biological functions of this tissue, and the neuropeptide content may thus indicate endocrine functions of the cells.     

Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament

Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309–316. © 2009 John Wiley & Sons A/S Background and Objective: MMP‐2 can degrade type IV collagen and MMP‐14 can activate pro MMP‐2. The present study was undertaken to examine the expression of MMP‐2 and MMP‐14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum‐free medium. The distribution and expression of MMP‐2 and MMP‐14 were analysed using immunohistochemistry, in situ hybridization and RT‐PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP‐2. Results: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP‐2 and MMP‐14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP‐2 mRNA while putative epithelial rests of Malassez cells expressed MMP‐14 mRNA at the interface. The RT‐PCR analysis showed that the expression of MMP‐2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP‐2 was detected at higher levels in the conditioned medium of the co‐cultured cells. Conclusion: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP‐2 in human periodontal ligament fibroblasts. Up‐regulated proMMP‐2 bound by MMP‐14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.     

Neuroendocrine cells in Malassez epithelium and gingiva of the cat

Malassez epithelium has been designated as epithelial cell rests, the biological significance of which is still under debate. This study was designed to analyze Malassez epithelium for the presence of neuroendocrine cells. Gingival tissue was included as a positive control. Using immunohistochemistry, confocal and light microscopy, Malassez epithelium and gingival epithelium from mature cats (n=5) were examined for cells containing the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Both Malassez epithelium and the basal epithelial cell layers in gingival rete pegs regularly displayed cells immunoreactive to CGRP, SP, and VIP. The immunopositive cells were most frequently present in the epithelial cell clusters and strands of Malassez located in the cervical half of the periodontal ligament. Double immunolabeling revealed cellular co-expression of CGRP or SP with VIP, and the neuropeptides were co-localized in the cellular compartments. Labeled cells in both epithelia were occasionally supported by immunoreactive nerve fibers. This study shows that cells immunoreactive to CGRP, SP, and VIP are located within the cat Malassez epithelium. The localization of neuroendocrine cells verifies the diversity of this epithelium and confirms that Malassez epithelium is composed of different cell types, in common with epithelia from other locations. The presence of neuroendocrine cells in Malassez epithelium strongly suggests biological functions of this tissue, and the neuropeptide content may thus indicate endocrine functions of the cells.     

Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars

BACKGROUND AND OBJECTIVES: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis. METHODS: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy. RESULTS: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles--images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez. CONCLUSIONS: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.     

Reactions of periodontal ligament epithelial cell clusters and OX6-immunopositive cells to experimental tooth movement and periodontitis

Tadokoro O, Kawahara I, Vandevska‐Radunovic V. Reactions of periodontal ligament epithelial cell clusters and OX6‐immunopositive cells to experimental tooth movement and periodontitis. J Periodont Res 2011; 46: 584–591.©2011 John Wiley & Sons A/S Background and Objective: The aim of this study was to investigate reactions of periodontal ligament epithelial cell clusters and major histocompatibility complex class II (OX6)‐immunopositive cells to simultaneously induced tooth movement and periodontitis employing Waldo’s method. Material and Methods: Elastic gums were inserted between the right upper first and second molars of rats. Animals were killed by intracardiac perfusion on days 1, 3, 7 and 14 after the experimental procedures, and maxillary molars were decalcified and processed for OCT compound. Cytokeratin and OX6 antibodies to detect epithelial and immunocompetent cells were used for double‐fluorescence immunohistochemistry. Immunostained sections of rat upper molar regions were examined with a fluorescence microscope. Results: Large periodontal ligament epithelial cell clusters appeared and became contiguous with each other, and OX6‐immunopositive cells surrounded the clusters over time in the periodontal ligament near the gum insertion site. In the periodontal ligament distant from the gum insertion site, epithelial cell clusters and OX6‐immunopositive cells were scattered. After 14 d, thickened epithelium and elongated rete pegs were found close to large epithelial cell clusters in the periodontal ligament near the gum insertion site. Conclusion: These findings suggest proliferation and/or aggregation of periodontal ligament epithelial cells, and interaction between OX6‐immunopositive cells and the periodontal ligament epithelial cells, in response to tooth movement and periodontal inflammation. This method may be a useful experimental model to elucidate the relationship between rete pegs and periodontal ligament epithelial cell clusters in inflammatory conditions.     

Epithelial–mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro

Epithelial–mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase‐20 (MMP‐20), and kallikrein‐4 (KLK‐4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum‐free medium. Both the epithelial cells and fibroblasts were co‐cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in‐situ hybridization and RT‐PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP‐20 and KLK‐4 in epithelial cells of Malassez co‐cultured with periodontal ligament fibroblasts. in‐situ hybridization and RT‐PCR confirmed significant mRNA expression of these factors in co‐cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial–mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.     

Cell death and quantitative reduction of rests of Malassez according to age

Background and Objective:  Rests of Malassez are clusters of epithelial cells that remain in the periodontal ligament throughout life. However, it has been reported that the number of these structures decreases with age, and some epithelial cells undergo apoptosis in rests of Malassez of young and adult rats. Therefore, the purpose of the present study was to investigate the incidence of epithelial cell death and the quantitative changes in the rests of Malassez in rat molars of different ages.
Material and Methods:  Fragments containing the upper molars of rats aged 29, 45 and 120 d were fixed, decalcified and embedded for analysis by light microscopy. In the sections stained by hematoxylin and eosin, the number of rests of Malassez and the number of nuclei of these epithelial structures were obtained. Moreover, the nuclei exhibiting typical features of cell death were also counted in each rest of Malassez. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method for detection of cell death was also carried out.
Results:  In all groups examined, some rests of Malassez exhibited epithelial cell nuclei with typical features of apoptosis and some of them were also TUNEL positive. From 29 to 120 d of age in rats, the quantitative analysis showed a significant decrease in the total number of rests of Malassez in the cervical, middle and furcation regions of the periodontal ligament. Moreover, a significant decrease of epithelial cell nuclei was concomitant to an increase in the frequency of cell death in the oldest rats.
Conclusion:  These results suggest that epithelial cell death by apoptosis may be, at least in part, responsible for the reduction in the number of rests of Malassez according to age.     

Insulin-like growth factor system components in the periodontium during tooth root resorption and early repair processes in the rat

There is evidence that growth factors, such as the insulin-like growth factors (IGFs), are involved in biological and pathological processes in oro-dento-facial tissues. To investigate their roles in tooth movement, root resorption, and repair, the occurrence of components of the IGF system, including the ligands IGF-I and -II, the IGF receptor 1 (IGF1R) and six IGF-binding proteins (IGFBP-1 to -6), was investigated by immunohistochemistry on sections from rat maxillae where the first molar had been moved mesially by means of an orthodontic appliance for 9 d to induce root resorption. After force deactivation on day 0, early repair was studied after a further 5, 7, 10, 12, 14, and 17 d. The immunostaining pattern in the periodontal ligament, cementum, and bone of control animals showed similarities known from studies in human teeth. Increased immunostaining for nearly all components in pressure sides and resorption lacunae indicated an involvement in resorption processes and clastic activities. During early stages of repair, the occurrence of several components (e.g. IGF-II, IGFBP-5 or -6) within lacunae and in cementoblasts showed an involvement in the resorption-repair sequence, which is considered to be a coupling process as known from bone.     

Enhanced proliferation, attachment and osteopontin expression by porcine periodontal cells exposed to Emdogain

Emdogain^® (EMD) is an enamel matrix derivative extracted from developing porcine teeth with demonstrated periodontal regenerative potential. EMD has been shown to influence a number of properties of periodontal ligament cells including proliferation, cell attachment and matrix synthesis. To date, the effect of EMD on the epithelial cell rests of Malassez (ERM) is unknown.

In this study, periodontal ligament fibroblasts, ERM, alveolar bone cells and gingival fibroblasts were obtained from porcine periodontal ligament, alveolar bone and gingiva. This study investigated, in vitro, the effect of EMD at three concentrations on proliferation, cell attachment and expression of mRNA for two mineralised tissue-related proteins (osteopontin and bone sialoprotein).

As for other periodontal cells, the ERM proliferative response was enhanced by EMD. Attachment assays revealed a highly significant increase for ERM and gingival fibroblasts after EMD treatment at all concentrations. This study has also shown that EMD stimulated expression of osteopontin mRNA by ERM and alveolar bone cells.

The results from this study provide evidence that EMD enhanced cellular events related with proliferation, attachment and osteopontin mRNA expression by porcine periodontal cells, in a manner consistent with its role in periodontal regenerative therapy.     

Immunoshistochemical localization of epidermal growth factor receptors in human gingival epithelia

Epidermal growth factor (EGF) is a small molecular weight polypeptide which is thought to have important functions in epithelial growth and differentiation and in wound healing. EGF exerts its action on cells through binding to a cell surface receptor. Using immunohistochemistry and a monoclonal antibody (mAb) directed against the EGF receptor, we have examined gingival specimens of periodontally healthy individuals and patients with adult adult (AP) and juvenile periodontitis (JP), as well as epithelial cell rests of Malassez. EGF receptors were expressed at high levels on the cell surface of basal cell layers of gingival epithelium. In normal junctional epithelium, on the other hand, specific labeling was faint or negative, indicating that receptors are poorly expressed or absent in these cells. No differences were detected between uninflamed gingival specimens of periodontally healthy subjects and of patients with JP. Instead, in biopsies of inflamed tissue from AP patients, an intense cell surface labeling was revealed in proliferating epithelial cells. Moreover, the epithelial cell rests of Malassez bound the antibody intensely. The results suggest that EGF is involved in control of epithelial growth and differentiation in periodontal tissues.     

Patterns of keratin-expression in rests of Malassez and periapical lesions

Using immunocytochemistry and a panel of monoclonal antibodies directed against various keratin polypeptides we examined specimens of normal periodontal ligament, periapical granulomas and inflammatory dental cysts. Epithelial elements with the appearance of rests of Malassez were identified in 6 specimens of normal periodontium and 10 periapical granulomas. Altered epithelium was present in 16 periapical granulomas and a lining epithelium in 10 inflammatory dental cysts. The patterns of binding of antibodies by these epithelia indicated that (a) keratin 19 was expressed by all epithelia, and (b) rests of Malassez also expressed keratin 5 but not large amounts of other keratins and (c) epithelial proliferation in periapical lesions was associated with increased expression of keratin 14, a marker of stratifying epithelia, new expression of keratins 4 and 13, differentiation markers for non-cornifying epithelia and variable, low levels of keratins 8 and 18, markers of simple epithelia. Proliferation of the epithelial rests of Malassez to form the lining of inflammatory dental cysts thus appears to be associated with a change from an unusual epithelial phenotype to that of a stratified non-cornifying epithelium in which some simple epithelial keratins are coexpressed.     

Promotion of functioning of human periodontal ligament cells and human endothelial cells by nerve growth factor

BACKGROUND: We have previously shown that cultured human periodontal ligament (HPL) cells produce nerve growth factor (NGF) and express mRNA of tyrosine kinase receptor (trkA), a high-affinity receptor of NGF. These findings suggest that NGF modulates the differentiation and proliferation of the periodontal ligament cells by paracrine and autocrine functions in vivo. Endothelial cells also express NGF and trkA. Therefore, NGF may regulate functions of periodontal ligament cells and endothelial cells during periodontal tissue regeneration. METHODS: Effects of NGF on expressions of bone/cementum-related proteins (osteocalcin [OC], bone sialoprotein [BSP], bone morphogenetic protein [BMP-7], core binding factor alpha [Cbfa-1], and type I collagen), calcification in HPL cells, and proliferation and mRNA expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen, in human microvascular endothelial cells (HMVECs) were examined. RESULTS: NGF elevated mRNA levels of OC, BSP, BMP-7, Cbfa-1, and type I collagen and enhanced mineral deposition in cultures of HPL cells. Furthermore, NGF stimulated mRNA expressions of VEGF-A and VEGF-B and cell proliferation in HMVEC. CONCLUSION: These findings suggest that the functional regulation of periodontal ligament cells and endothelial cells by NGF might result in the acceleration of periodontal tissue regeneration in vivo.     

Patterns of keratin-expression in rests of Malassez and periapical lesions

Using immunocytochemistry and a panel of monoclonal antibodies directed against various keratin polypeptides we examined specimens of normal periodontal ligament, periapical granulomas and inflammatory dental cysts. Epithelial elements with the appearance of rests of Malassez were identified in 6 specimens of normal periodontium and 10 periapical granulomas. Altered epithelium was present in 16 periapical granulomas and a lining epithelium in 10 inflammatory dental cysts. The patterns of binding of antibodies by these epithelia indicated that (a) keratin 19 was expressed by all epithelia, and (b) rests of Malassez also expressed keratin 5 but not large amounts of other keratins and (c) epithelial proliferation in periapical lesions was associated with increased expression of keratin 14, a marker of stratifying epithelia, new expression of keratins 4 and 13, differentiation markers for non-cornifying epithelia and variable, low levels of keratins 8 and 18, markers of simple epithelia. Proliferation of the epithelial rests of Malassez to form the lining of inflammatory dental cysts thus appears to be associated with a change from an unusual epithelial phenotype to that of a stratified non-cornifying epithelium in which some simple epithelial keratins are coexpressed.     

Cellular turnover in epithelial rests of Malassez in the periodontal ligament of the mouse molar

Fragments of Hertwig's epithelial root sheath persist in the periodontal ligament (PDL) in small clusters known as epithelial rests of Malassez (ERM). It is generally agreed that ERM are maintained as a quiescent and exclusively dental epithelial cluster in PDL. However, we speculate that homeostasis and cellular turnover underlies cluster maintenance. We also hypothesize that the fate of ERM clusters – diminishing or remaining – might be regulated via the presence or absence of epithelial stem cells therein. Histological analysis of aging mouse molar PDL showed that ERM clusters gradually increase in size with increasing age. Immunocytochemistry and cell culture revealed that ERM clusters contained Ki67‐positive cells and were able to expand when brought in culture. The TdT‐mediated biotin–dUTP nick‐end labeling (TUNEL) procedure also detected signs of apoptosis. Finally, we identified putative epithelial stem cells in the clusters by 5‐bromo‐2′‐deoxyuridine (BrdU) pulse–chase experiments and immunohistochemistry, using the stem‐cell marker leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (Lgr5). The results suggest that ERM clusters are maintained in the PDL, via cellular turnover, throughout life.