The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1–18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.
Background and Objectives
Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+]i) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin‐induced increase in [Ca2+]i in human gingival fibroblasts (HGFs).
Methods
Effects of 100 μM phenytoin on [Ca2+]i in HGFs were examined at the single‐cell level using fluorescence images of fura‐2 captured by an imaging system consisting of an EM‐CCD camera coupled to an inverted fluorescence microscope at room temperature.
Results
Exposure of HGFs to 100 μM phenytoin induced a transient increase in [Ca2+]i in the absence of extracellular Ca2+, indicating that the phenytoin‐induced increase in [Ca2+]i does not require an influx of extracellular Ca2+. In addition, phenytoin increased [Ca2+]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin‐induced [Ca2+]i elevation was reduced to 18.8% in the absence of extracellular Na+, and [Ca2+]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+]i of HGFs by suppressing the Na+/Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single‐cell level showed that stimulation with 1 μM ATP or 3 μM histamine increased [Ca2+]i in 20–50% of cells, and [Ca2+]i increased in many unresponsive cells in the presence of phenytoin.
Conclusion
Our findings demonstrate that phenytoin induced increase in [Ca2+]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug‐induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.
The epithelial cell rests of Malassez (ERM) play a pivotal role in preventing ankylosis between the alveolar bone and the tooth. Although several functions of ERM has been reported, the mechanism behind preventing dentoalveolar ankylosis remains unclear. In this study, 18 clones were isolated from ERM (CRUDE) using the single-cell limiting dilution method. Among them, ERM-2 and -3, which exhibited the highest and lowest proliferation rates, respectively, were selected. ERM-2, ERM-3, and CRUDE ERM were stained with epithelial markers, including cytokeratin-wide and cytokeratin-19, via immunofluorescence. The qRT-PCR analysis revealed increased expression levels of p75 (ameloblast marker), amelogenin, and sfrp5 (inner enamel epithelial cell marker) in the ERM-2 cells. Alternatively, ameloblastin and ck-14 (outer enamel epithelial cell marker) were highly expressed in ERM-3 cells. The mineralization of human periodontal ligament fibroblast (HPDLF) was inhibited when co-cultured with ERM-2, ERM-3, and CRUDE ERM cells. The addition of an anti-amelogenin antibody restored the mineralization of HPDLF cells. Transplanted rat molar cultured in ERM-2 (high amelogenin secretive clone) cell-derived supernatant resulted in significantly smaller bone formation than those cultured in the CRUDE ERM and ERM-3 cell-derived supernatants. These findings indicate that amelogenin produced by ERM cells might be involved in preventing dentoalveolar ankylosis.
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