Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells.

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14+ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1ß, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14+ cells are responsible, at least in part, for the production of IL-1ß, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14+ cells play an essential role in the occurrence of APRs following N-BP administration.

Maintaining a protective state for human periodontal tissue.

Periodontitis, caused by infection with periodontal pathogens, is primarily characterized by inflammatory bone resorption and destruction of connective tissue. Simply describing periodontitis as a specific bacterial infection cannot completely explain the various periodontal tissue destruction patterns observed. Periodontal tissue damage is thought to be caused by various factors. In recent years, research goals for periodontal pathogens have shifted from searching for specific pathogens to investigating mechanisms that damage periodontal tissues. Bacteria interact directly with the host in several ways, influencing expression and activity of molecules that evade host defenses, and destroying local tissues and inhibiting their repair. The host's innate and acquired immune systems are important defense mechanisms that protect periodontal tissues from attack and invasion of periodontal pathogens, thus preventing infection. Innate and acquired immunity have evolved to confront the microbial challenge, forming a seamless defense network in periodontal tissues. In the innate immune response, host cells quickly detect, via specialized receptors, macromolecules and nucleic acids present on bacterial cell walls, and this triggers a protective, inflammatory response. The work of this subsystem of host immunity is performed mainly by phagocytes, beta-defensin, and the complement system. In addition, the first line of defense in oral innate immunity is the junctional epithelium, which acts as a physical barrier to the entry of oral bacteria and other nonself substances. In the presence of a normal flora, junctional epithelial cells differentiate actively and proliferate apically, with concomitant increase in chemotactic factor expression recruiting neutrophils. These immune cells play an important role in maintaining homeostasis and the protective state in periodontal tissue because they eliminate unwanted bacteria over time. Previous studies indicate a mechanism for attracting immune cells to periodontal tissue with the purpose of maintaining a protective state; although this mechanism can function without bacteria, it is enhanced by the normal flora. A better understanding of the relationship between the protective state and its disruption in periodontal disease could lead to the development of new treatment strategies for periodontal disease.

Comparison of protein profiles of the pellicle, gingival crevicular fluid, and saliva: possible origin of pellicle proteins.

BACKGROUND: The pellicle, the acellular organic material deposited on the surface of tooth enamel, has been thought to be derived from saliva. In this study, protein compositions of the pellicle, gingival crevicular fluid, and saliva collected from healthy adults were compared to elucidate the origin of pellicle proteins. RESULTS: The pellicle, gingival crevicular fluid, and saliva from the parotid gland or mixed gland were collected; subsequently, protein expression in samples from the respective individual was compared by SDS-PAGE and mass spectrometry. Following SDS-PAGE, proteins in the major bands were identified by mass spectrometry. The band pattern of pellicle proteins appeared different from those of gingival crevicular fluid, or saliva samples. Using mass spectrometry, 13 proteins in these samples were identified. The relative abundance of the proteins was quantitatively analyzed using mass spectrometry coupled with stable isotope labeling and by western blot. Cystatin S and α-amylase detected in pellicle were enriched in saliva samples, but not in gingival crevicular fluid, by western blot, and their abundance ratios were high in saliva and low in gingival crevicular fluid when analyzed by stable isotope labeling. Serotransferrin, however, was found only in the pellicle and gingival crevicular fluid by western blot and its abundance ratio was low in saliva. CONCLUSIONS: Our study revealed that the gingival crevicular fluid appears to contribute to pellicle formation in addition to saliva.

Cdc42 regulates cranial suture morphogenesis and ossification.

Cdc42 (cell division cycle 42) is ubiquitously expressed small GTPases belonging to the Rho family of proteins. Previously, we generated limb bud mesenchyme-specific Cdc42 inactivated mice (Cdc42 conditional knockout mice; Cdc42 fl/fl; Prx1-Cre), which showed short limbs and cranial bone deformities, though the mechanism related to the cranium phenotype was unclear. In the present study, we investigated the role of Cdc42 in cranial bone development. Our results showed that loss of Cdc42 caused a defect of intramembranous ossification in cranial bone tissues which is related to decreased expressions of cranial suture morphogenesis genes, including Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs). These findings demonstrate that Cdc42 plays a crucial role in cranial osteogenesis, and is controlled by Ihh- and BMP-mediated signaling during cranium development.

Establishment of mouse gingival junctional epithelial cell line using a bioengineered tooth system.

Junctional epithelium (JE), one of the constituents of periodontal tissue, has several unique features to prevent bacterial infection. However, the molecular mechanisms of these cells remain to be completely elucidated because there has been no JE cell line to date. We have succeeded in isolating JE cells expressing green fluorescent protein (GFP) by using a bioengineered tooth technique in mice. The gene expressions of GFP-positive JE cells, isolated from around the erupted bioengineered teeth using flow cytometry, were analyzed by RNA sequencing. GFP-positive cells derived from the bioengineered tooth germs showed similar gene expression patterns to primary JE cells. The isolated GFP-positive JE cells were immortalized by transducing the simian virus 40 large T antigen using lentiviral vectors. The established GFP-positive JE cells maintained proliferative activity for more than 20 passages, and did not show cellular senescence as demonstrated by ß-galactosidase assay. These cells also expressed similar gene expression patterns to primary JE cells. The established cell lines may prove useful for future investigation of JE characteristics in vitro.

Quantitative proteomic analysis of gingival crevicular fluids from deciduous and permanent teeth.

AIM: Gingivitis commonly progresses to periodontitis in permanent dentition but rarely in deciduous teeth. Little is known about the biochemical differences between gingiva of deciduous and permanent teeth. Here, we compared the protein profiles of gingival crevicular fluids (GCF) from the gingiva of deciduous and permanent teeth. MATERIALS AND METHODS: Forty children with mixed dentition (Hellman's dental age IIIA) were selected and GCF samples were collected from deciduous cuspids and central incisors in the maxilla. Pairs of GCF samples were labelled using isobaric tags to permit quantitative comparison of protein abundance in the samples using liquid chromatography-electron spray ionization-tandem mass spectrometry. RESULTS: Sixty-two proteins were upregulated in deciduous teeth GCF and 54 in permanent teeth GCF. In particular, neutrophil-derived proteins, including myeloperoxidase and lactoferrin, were repeatedly higher in deciduous teeth GCF than in permanent teeth GCF. These differences were verified using ELISA (p < 0.01). In contrast, immunoglobulin components were upregulated in permanent teeth GCF. CONCLUSIONS: Neutrophil-related proteins were enriched in deciduous teeth GCF and immunoglobulins in permanent teeth GCF. This suggests that neutrophil accumulation plays a protective role in innate immunity against bacterial infection in gingival tissue of deciduous teeth.

Analysis of root surface properties by fluorescence/Raman intensity ratio.

The aim of this study is to evaluate the existence of residual calculus on root surfaces by determining the fluorescence/Raman intensity ratio. Thirty-two extracted human teeth, partially covered with calculus on the root surface, were evaluated by using a portable Raman spectrophotometer, and a 785-nm, 100-mW laser was applied for fluorescence/Raman excitation. The collected spectra were normalized to the hydroxyapatite Raman band intensity at 960 cm-1. Raman spectra were recorded from the same point after changing the focal distance of the laser and the target radiating angle. In seven teeth, the condition of calculus, cementum, and dentin were evaluated. In 25 teeth, we determined the fluorescence/Raman intensity ratio following three strokes of debridement. Raman spectra collected from the dentin, cementum, and calculus were different. After normalization, spectra values were constant. The fluorescence/Raman intensity ratio of calculus region showed significant differences compared to the cementum and dentin (p < 0.05). The fluorescence/Raman intensity ratio decreased with calculus debridement. For this analysis, the delta value was defined as the difference between the values before and after three strokes, with the final 2 delta values close to zero, indicating a gradual asymptotic curve and the change in intensity ratio approximating that of individual constants. Fluorescence/Raman intensity ratio was effectively used to cancel the angle- and distance-dependent fluctuations of fluorescence collection efficiency during measurement. Changes in the fluorescence/Raman intensity ratio near zero suggested that cementum or dentin was exposed, and calculus removed.

Removal of Oral Biofilm on an Implant Fixture by a Cavitating Jet.

PURPOSE: To demonstrate the effectiveness of the cavitating jet in removing biofilms from the rough surface of 3-dimensional structures. MATERIALS AND METHODS: The optimal nozzle dimensions and injection conditions were identified by cavitation impact measurements. Biofilm was grown intraorally for 72 hours by 4 volunteers. The stained fixtures were assigned to different experimental groups. One comparison was performed between the cavitating jet and the water jet at 60 seconds. Additional comparisons were conducted among the time course experiments at 30, 60, and 180 seconds. After injection, the residual plaque biofilm (RPB) area was measured using a digital microscope. RESULTS: The total RPB of the cavitating jet was significantly lower than that of the water jet. Although there were no significant differences between the total RPB at 30 and 60 seconds, a significant difference was detected between 60 and 180 seconds. The RPB on the root sector was significantly lower than that on the crest sector at 60 and 180 seconds. CONCLUSION: The cavitating jet can effectively clean the biofilm formed on the rough surface of the implant screw, especially on the root sector.

Enhanced and suppressed mineralization by acetoacetate and ß-hydroxybutyrate in osteoblast cultures.

It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and ß-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and ß-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, ß-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by ß-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or ß-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

Rho GTPase protein Cdc42 is critical for postnatal cartilage development.

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 (fl/fl); Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 (fl/fl)) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages.

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms.

CONTEXT: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail. OBJECTIVE: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms. MATERIALS AND METHODS: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were -13 and 8 mV, respectively, and both had a mean particle size of approximately 180 nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy. RESULTS: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms. DISCUSSION AND CONCLUSION: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.

Development of a Cavitation Generator Mimicking Pistol Shrimp.

Pistol shrimp generate cavitation bubbles. Cavitation impacts due to bubble collapses are harmful phenomena, as they cause severe damage to hydraulic machinery such as pumps and valves. However, cavitation impacts can be utilized for mechanical surface treatment to improve the fatigue strength of metallic materials, which is called "cavitation peening". Through conventional cavitation peening, cavitation is generated by a submerged water jet, i.e., a cavitating jet or a pulsed laser. The fatigue strength of magnesium alloy when treated by the pulsed laser is larger than that of the jet. In order to drastically increase the processing efficiency of cavitation peening, the mechanism of pistol shrimp (specifically when used to create a cavitation bubble), i.e., Alpheus randalli, was quantitatively investigated. It was found that a pulsed water jet generates a cavitation bubble when a shrimp snaps its claws. Furthermore, two types of cavitation generators were developed, namely, one that uses a pulsed laser and one that uses a piezo actuator, and this was achieved by mimicking a pistol shrimp. The generation of cavitation bubbles was demonstrated by using both types of cavitation generators: the pulsed laser and the piezo actuator.

The etiological consideration of oxidized low-density lipoprotein in periodontitis.

BACKGROUND: Epidemiologically, correlations between periodontal disease activity and CVD/serum lipid-related condition have been reported. Known mediators of these links include triglycerides, oxidized LDL (oxLDL) and inflammatory cytokines such as TNF-α supplied by adipocytes as well as oxidative degeneration products of these lipids. In this review, we focused on oxidized LDL and considered the relationship between periodontal disease and systemic conditions. HIGHLIGHT: The degree of oxidation in the periodontal pocket can be evaluated by analyzing the Gingival Cervicular Fluid (GCF), which can be easily collected with paperpoint. The oxLDL/LDL ratio in GCF has been shown to be 17 times as high as that in blood, and IL-8 and IL-1ß were also abundantly found in GCF. Periodontal treatment significantly lowers oxLDL levels in not only GCF but also plasma. In addition, there has been growing body of evidence that periodontal infections by periodontopathic bacteria affect arteriosclerosis. On the other hands, neutrophil extracellular traps (NETs), a form of innate immune responses, reportedly play a role as a defense mechanism in the periodontal pockets. However, the regulatory mechanism of NETs in periodontal pocket is still unknown. Recently, NETs induced by oxidized cholesterol have been reported to be involved in inflammatory damage to vascular endothelial cells. CONCLUSION: Further understanding of the newly discovered roles of oxLDL in the defense and destruction of periodontal tissues are anticipated.

Nephronectin expression is regulated by SMAD signaling in osteoblast-like MC3T3-E1 cells.

Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8ß1. We previously demonstrated that Npnt expression was suppressed by TGF-ß through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-ß type I receptor (TGF-ß R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-ß signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.

BMP2 differentially regulates the expression of Gremlin1 and Gremlin2, the negative regulators of BMP function, during osteoblast differentiation.

Bone morphogenetic proteins (BMPs) control the expressions of many genes involved in bone formation. On the basis of our hypothesis that BMP2 stimulation-regulated gene expression plays a critical role in osteoblast differentiation, we performed genome-wide screening of messenger RNA from BMP2-treated and -untreated C2C12 cells using a DNA microarray technique. We found that the expressions of Gremlin1 and Gremlin2, which are known BMP antagonists, were bidirectionally regulated by BMP2. Gremlin1 was down-regulated by BMP2, while Gremlin2 was up-regulated in both time- and dose-dependent manners. Ablation of Gremlin1 or Gremlin2 enhanced osteoblast differentiation induced by BMP2. On the other hand, treatment with recombinant Gremlin1 inhibited BMP2-induced osteoblast differentiation. Furthermore, treatment with Smad4 siRNA and the p38 MAPK inhibitor SB203580 suppressed BMP2-induced Gremlin2 gene expression. The differential regulation of Gremlin1 and Gremlin2 gene expressions by BMP2 may explain the critical function of these genes during osteoblast differentiation.

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia.

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.

Surface lipoprotein PpiA of Streptococcus mutans suppresses scavenger receptor MARCO-dependent phagocytosis by macrophages.

Streptococcus mutans is associated with the initiation and progression of human dental caries and is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. For the pathogen to survive in the infected host, surface lipoproteins of S. mutans are likely to play important roles in interactions with the innate immune system. To clarify the role that a putative lipoprotein, peptidyl-prolyl cis/trans-isomerase (PpiA), of S. mutans plays in the macrophage response, we investigated the response of THP-1-derived macrophages to S. mutans challenge. The deletion of the gene encoding Lgt eliminated PpiA on the cell surface of S. mutans, which implies that PpiA is a lipoprotein that is lipid anchored in the cell membrane by Lgt. Human and murine peritoneal macrophages both showed higher phagocytic activities for the ppiA and lgt mutants than the wild type, which indicates that the presence of PpiA reduces S. mutans phagocytosis. In addition, infection with S. mutans markedly induced mRNAs of macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A) in human macrophages. In particular, transcriptional and translational levels of MARCO in human macrophages infected with the ppiA mutant were higher than those in macrophages infected with the wild type. Phagocytosis of S. mutans by human macrophages markedly decreased after treatment with anti-MARCO IgG. These results demonstrate that the S. mutans lipoprotein PpiA contributes to suppression of MARCO-mediated phagocytosis of this bacterium by macrophages.

Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells.

Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E(2) (PGE(2)) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE(2)-producing enzymes, cyclooxygenase-2 and microsomal PGE(2) synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-κB) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-κB pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α.

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.

Structure of junctional epithelium is maintained by cell populations supplied from multiple stem cells.

The junctional epithelium (JE) is an epithelial component that attaches directly to the tooth surface and performs the unique function of protecting against bacterial infections; its destruction causes inflammation of the periodontal tissue and loss of alveolar bone. A recent study that used the single-color lineage tracing method reported that JE is maintained by its stem cells. However, the process by which individual stem cells form the entire JE around a whole tooth remains unclear. Using a 4-color lineage tracing method, we performed a detailed examination of the dynamics of individual stem cells that constitute the entire JE. The multicolor lineage tracing method showed that single-color areas, which were derived from each cell color, replaced all the constituent JE cells 168 d after the administration of tamoxifen. The horizontal section of the first molar showed that the single-color areas in the JE expanded widely. We detected putative stem cells at the external basal layer farthest from the enamel. In this study, JE cells that were supplied from different stem cells were visualized as individual monochromatic regions, and the JE around the first molar was maintained by several JE-specific stem cells. These findings indicated that the JE consisted of several cell populations that were supplied from their multiple stem cells and could help to explore the mechanisms involved in periodontal tissue homeostasis.