Examination of age-related changes in the submandibular glands of male mice.

Salivary gland hypofunction adversely affects the oral environment and daily life by causing dry mouth (xerostomia). Senescence-related atrophy of salivary gland tissues is one cause of xerostomia, and it is particularly common among the elderly. However, the underlying mechanism is poorly understood, and no treatment has been established. Therefore, we examined age-related changes in senescence-associated secretory phenotype (SASP) factors, which regulate stemness and cellular senescence, in mouse submandibular glands. We analyzed the submandibular glands of 6-week-old (young group, n = 6) and 82-week-old mice (aged group, n = 6). We performed salivary flow rate measurements, histological analysis including immunohistochemistry, and quantitative real-time PCR. The salivary flow rate was significantly lower in the aged group than in the young group. In addition, immunostaining and quantitative real-time PCR illustrated that aquaporin-5 and α-amylase expressions were significantly decreased in aged mice, indicating salivary gland hypofunction. c-Kit and cytokeratin 5 expressions were also significantly decreased in this group, suggesting that the regenerative abilities of the submandibular glands were reduced because of decreased stem and progenitor cell counts. Furthermore, the levels of p16INK4a and p21 (the senescence markers) and TGF-ß1 and IL-6 (SASP factors) were significantly increased in mice, suggesting that senescence had been promoted. The decreased numbers of stem and progenitor cells and increased levels of SASP factors might be associated with age-related changes in mouse submandibular glands. These results might facilitate the development of treatments for senescence-related submandibular gland hypofunction.

Awareness of Complications of Dental Treatment in Patients Treated with Drugs Affecting the Immune System : A Nationwide Questionnaire Survey of Dental Practitioners in Japan.

The aim of this study was to investigate the awareness and experience, among dental practitioners, of adverse events resulting from dental treatment of patients undergoing therapy with drugs that affect the immune system [angiogenesis inhibitors, biological agents, immunosuppressants, and disease-modifying anti-rheumatic drugs (DMARDs)]. For this purpose, a nationwide questionnaire survey was conducted. Questionnaires were sent to 2,050 dentists, of which 206 (10.1%) were completed and returned. The results showed that most dentists were aware of complications associated with dental treatment of patients treated with drugs that affect the immune system, and about half had actually experienced such complications. Delayed wound healing, osteonecrosis of the jaw (ONJ), and postoperative infections were reported. Whereas approximately 50% of dentists did not discontinue the drugs during dental treatment, about 18% did. During temporary drug discontinuation, some patients experienced aggravation of the primary disease, such as worsening of rheumatism, growth of tumors, and rejection reactions of transplanted organs. As for medical cooperation, only less than half of the dentists were asked for oral hygiene management by a physician prior to starting the drug treatment. Prospective studies are needed because evidence for dental treatments in patients treated with these drugs remains limited.

Effects of the prevention of medication-related osteonecrosis of the jaw by local administration of a dental pulp stem cell-conditioned medium to the rat tooth extraction socket.

The onset of osteonecrosis of the jaw, which is a side effect of bisphosphonates, often develops after tooth extraction; measures for its prevention have not yet been established. While treatment with systemic administration of bone marrow stem cell-derived conditioned medium for medication-related osteonecrosis of the jaw (MRONJ) has been reported, its preventive effects have not been clarified yet, and the high degree of invasiveness of bone marrow fluid collection remains an issue. Therefore, we created a rat model of MRONJ using BP zoledronic acid, used a dental pulp stem cell-conditioned medium (DPSC-CM), which can be collected relatively easily, and locally applied it to the tooth extraction socket with atelocollagen and gelatin sponges. The preventive effect on the onset of MRONJ was subsequently examined. The results demonstrated that the bone exposure width of the extraction socket was reduced, and the mucosal covering was promoted in the atelocollagen + DPSC-CM group as compared with the other groups. Furthermore, histological results indicated a decrease in the number of empty bone lacunae, whereas immunohistochemical staining revealed the presence of many vascular endothelial growth factor (VEGF)-positive cells. Moreover, the results of the investigation of the sustained release of atelocollagen using VEGF indicated the release of VEGF over time. Our results suggest that local administration of DPSC-CM using atelocollagen may be a useful method for the prevention of MRONJ triggered by tooth extraction.

Relationship between masticatory performance using a gummy jelly and food intake ability in Japanese complete denture wearers.

Twenty patients wearing complete dentures were asked to chew a gummy jelly, and their glucose extraction was measured. Two types of masticatory scores (MS1 and MS2) were calculated using a food intake questionnaire. The relationship between masticatory scores and glucose extraction was investigated. The values of masticatory scores were large when the amount of glucose extraction was also large, and there were significantly positive correlations between the two. There was also a positive correlation between MS1 and MS2. It was suggested that the food intake ability was high when the masticatory performance using a gummy jelly was high.

Intracellular Signaling Pathways Involved in the Regulation of Gene Expression by Pilocarpine.

OBJECTIVES: Pilocarpine is commonly used clinically to treat dry mouth. The long-term administration of pilocarpine reportedly improves salivary secretion more effectively than short-term administration. Therefore, we hypothesized that pilocarpine alters gene expression in salivary glands via muscarinic receptor stimulation. This study aimed to investigate the effects of pilocarpine use on gene expression mediated by mitogen-activated protein kinase (MAPK) activity. METHODS: The effects of pilocarpine on gene expression were investigated in rats and human salivary gland (HSY) cells using several inhibitors of intracellular signaling pathways. Gene expression in the rat submandibular gland and HSY cells was determined using reverse transcription-quantitative polymerase chain reaction analysis of total RNA. RESULTS: In animal experiments, at 7 days after pilocarpine stimulation, Ctgf and Sgk1 expressions were increased in the submandibular gland. In cell culture experiments, pilocarpine increased Ctgf expression in HSY cells. The mitogen-activated protein kinase kinase inhibitor trametinib, the Src inhibitor PP2, and the muscarinic acetylcholine receptor antagonist atropine suppressed the effect of pilocarpine on gene expression. CONCLUSIONS: Pilocarpine enhances Ctgf and Sgk1 expressions by activating Src-mediated MAPK activity. Although further studies are required to fully understand the roles of Ctgf and Sgk1, changes in gene expression may play an important role in improving salivary secretions.

The ECAS system can superselectively administer anticancer drugs to numerous feeding arteries from the superficial temporal artery: A case report and literature review.

Superselective intra-arterial chemoradiotherapy (SSIACRT) is one of the curative treatments for advanced oral cancer. SSIACRT can reportedly treat cervical lymph node metastases in the level I-IIA area by super selectively catheterizing the facial artery (FA) and infusing drugs. However, since advanced oral cancer lesions involve a number of feeding vessels, retrograde treatment requires the placement of catheters from the superficial temporal artery (STA) and occipital artery (OA). Furthermore, in the case of level IIB lymph node metastasis, the catheter must be changed because it is necessary to administer anticancer drugs to more than three routes, including the OA, when the feeding arteries of the primary tumor are combined. The external carotid artery sheath (ECAS) system used in the present study involves the insertion of a microcatheter or steering catheter from one route of the STA, allowing selection of numerous feeding vessels, including the OA. The ECAS system can facilitate the administration of chemotherapy via the STA simultaneously to the maxillary artery, lingual artery, FA and OA. The present study describes cases of maxillary gingival cancer and tongue cancer with cervical lymph node metastasis, which were treated with the ECAS system via the STA; the treatment successfully controlled both the primary tumor and cervical lymph node metastasis. In the two cases described in the present study, metastatic lymph nodes were found in the level ⅠB and ⅡB region, but were successfully treated by administering cisplatin via the OA, in addition to the primary lesion. To date, to the best of our knowledge, there is no case report clearly referring to the treatment of lymph node metastasis using the ECAS system. In conclusion, SSIACRT using ECAS may be considered a useful treatment for oral cancer with cervical lymph node metastasis.

Medication-related osteonecrosis of the jaw in a patient with multiple myeloma: an unusual case with tumor in the surgical specimen.

Multiple myeloma is characterized by a neoplastic proliferation of plasma cells primarily in the bone marrow. Neoplastic plasma cells stimulated osteoclasts, and destroy bone tissue, causing bone pain, pathological fractures, paralysis due to spinal cord compression, and hypercalcemia. Bisphosphonates are used as supportive therapy in the management of multiple myeloma. Medication-related osteonecrosis of the jaw (MRONJ) is a well-known complication of treatment with bisphosphonates, denosumab, and other drugs, such as anti-angiogenic agents and novel anti-cancer drugs. We report MRONJ in a patient with multiple myeloma, especially an unusual case with tumor in the surgical specimen. A 73-year-old woman presented with pain on the left side of the mandible within 3 months. On clinical examination, an exposed bone without purulent drainage presented on the left side of the mandible. Before 2 years, she received chemotherapy of zoledronate for multiple myeloma at another hospital. Panoramic imaging showed radiopacities of bone in the left side of the mandibular molar area. Multidetector computed tomography (MDCT) with axial, multiplanar reformation (MPR) and three-dimensional (3D) images showed the sequestrum without periosteal reaction. She was diagnosed as MRONJ, and underwent surgery. Finally, the surgical specimen was diagnosed as multiple myeloma in the sequestrum. This case suggests that the evaluation of the surgical specimen of MRONJ could be essential for detection of primary tumor.

Questionnaire survey of angiogenesis inhibitor-related oral complications based on a nation-wide study in Japan.

BACKGROUND/PURPOSE: The prevalence of oral adverse events and dental treatments related complications during the molecular targeted drugs therapy remains unclear. The purpose of this study was to investigate the contents of dental treatment-related complications in Japanese patients during molecular targeted therapy. MATERIALS AND METHODS: The nation-wide survey of dental treatment related complications was performed by the Japanese Society of Dentistry for Medically Compromised Patient as retrospective cohort study. RESULTS: Among 212 dentists, 87.3% recognized the possibility of dental treatments related complications in patients with angiogenesis inhibitors. The oral adverse events including dental treatment-related complications associated with angiogenesis inhibitors were 79 cases. In patients with angiogenesis inhibitors, 73.4% of patients were administrated with bevacizumab. The average administration period of angiogenesis inhibitors was 14.0 ±â€¯10.0 months. As the dental treatment related complications, delayed wound healing was most commonly seen in 45 patients (57.0%), followed by osteonecrosis of the jaw in 25 (31.6%). The triggered factor of complications was tooth extraction in 51 cases (96.2%). CONCLUSION: The extraction of the tooth with pre-existing inflammation may be suggested as one of risk factors for angiogenesis inhibitor-related dental treatment complications.

Investigating the effect of antiseptic solution on the release of interleukin-6 and transforming growth factor beta 1 from human gingival fibroblasts using wound healing assays.

This in vitro study evaluated the effect of different antiseptics and different concentrations thereof in a model of wound healing using human gingival fibroblasts. The fibroblasts were rinsed with four different antiseptic solutions: sodium hypochlorite (HYP), hydrogen peroxide (H2O2), chlorhexidine digluconate (CHX), and benzalkonium chloride (BC). The effect on the release of interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-ß1) was investigated using enzyme-linked immunosorbent assays (ELISAs). In addition, the effects of the antiseptics on wound healing at 1, 12, 24, and 48 h were assessed through a wound healing assay. The viability of the fibroblasts rinsed with antiseptics was investigated with respect to the concentrations inhibiting cell growth by 50% (IC50), 25% (IC25), and ≤2% (IC2). A statistically significant increased release of IL-6 was obtained with BC IC25 and IC2 after 12, 24, and 48 h (P < 0.01). For TGF-ß1, no significant release was found for CHX IC2 after 24 and 48 h or for IC50 and IC25 after 12 h. There was no significant effect on wound healing capacity for CHX or for BC IC25 and IC2. This study demonstrated that antiseptic rinses of human gingival fibroblasts alter the release of IL-6 and TGF-ß1 and impact wound healing capacity, with both BC and CHX conferring neutral effects.

Tumor-specific cytotoxicity and type of cell death induced by naphtho[2,3-b]furan-4,9-diones and related compounds in human tumor cell lines: relationship to electronic structure.

A total of thirty-nine naphtho[2,3-b]furan-4,9-diones and related compounds were tested for their cytotoxicity against three human normal oral cells (gingival fibroblast, HGF, pulp cell, HPC, periodontal ligament fibroblast, HPLF) and four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). 2-Acetylnaphtho[2,3-b]furan-4,9-dione [1] was highly cytotoxic to both normal and tumor cells, yielding low tumor-specificity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan [4], the 2-(3-furanoyl) benzoic acids [5, 6] and the 1,4-naphthoquinones [7, 8] showed much reduced cytototoxicity and low tumor-specificity. The introduction of phenoxy [18], isopropylamino [23] or 2-methylpiperidino [33] groups to the 2-position of naphtho[2,3-b]furan-4,9-dione yielded compounds that showed the greatest tumor-specificity. These compounds, at twice or four times higher concentrations than CC50, induced the activation of caspase-3, caspase-8 and caspase-9 in the HSC-2 and HL-60 cells, but not so apparently in the HSC-4 cells. However, they did not induce internucleosomal DNA fragmentation in the HSC-2 and HSC-4 cells even after 24 hours incubation and only slightly induced DNA fragmentation in the HL-60 cells. Compound [18] induced the production of annexin-positive cells, but did not induce microtubule-associated protein light chain 3 (LC3) accumulation in autophagosomes in LC3-green fluorescent protein (GFP)-transfected HSC-2 cells. These data suggested that naphtho[2,3-b]furan-4,9-diones may induce the early apoptotic marker, without induction of caspase activation and DNA fragmentation in oral squamous cell carcinoma cell lines. Quantitative structure-activity relationship (QSAR) analysis suggests the applicability of the theoretical calculations such as frontier molecular orbital, dipole moments and hydrophobicity in predicting their cytotoxic activity.

Three-dimensional bone formation including vascular networks derived from dental pulp stem cells in vitro.

The regeneration of bone tissue is an active area of research, and future clinical applications are expected. Here, to establish new bone graft materials and an experimental bone tissue model, we generated united compact and spongy bone tissues containing vascular networks from human dental pulp stem cells in vitro. We applied the cell bead and cell sheet methods to construct three-dimensional bone tissue, which was cultured using a circumfusion apparatus for 30 days. Using micro-computed tomography, we assessed structural differences between compact and spongy bone. Histological examinations revealed the presence of bone lacunae containing osteocytes, Haversian canal-like structures, and extensive vascularization. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining-positive osteoclast-like cells were also observed. Thus, the bone tissue generated using this method closely resembles native bone tissue and may possess bone remodeling ability. We successfully generated bone tissue containing blood vessel networks in vitro using this method. The generated bone tissue will likely be highly applicable to medical care, the study of osteogenesis, drug-screening assays, and drug development for bone tissue.

Correction to: Three-dimensional bone formation including vascular networks derived from dental pulp stem cells in vitro.

The authors would like to correct the error in the publication of the original article.

Establishment and characterization of human gingival squamous cell carcinoma cell line NOCS-1.

The NOCS-1 cell line was established from the left gingiva tumor in an 86-year-old Japanese man. Histopathological diagnosis of the original tumor was well-differentiated squamous cell carcinoma. NOCS-1 cells were adhesive epithelial cells with neoplastic or pleomorphic features and grew without contact inhibition. It has been subcultured 70 times during the past 26 months. From passage 3, melanin-containing cells began to be observed in the NOCS-1 cell line. The plating efficiencies were 25% and 23%, doubling times were 29 and 26 h, and saturation densities were 6.9 × 104/cm2 and 8.7 × 104/cm2, at passage 12 and 30, respectively. When NOCS-1 cells were xenotransplanted subcutaneously into SCID mice, they produced tumors that histopathologically resembled the original tumor. In addition, NOCS-1-XG cells derived from the xenotransplanted tumor were similar to NOCS-1 cells. We believe that this cell line may be a valuable tool to develop immunotherapy and chemotherapy regimens.

Section showing minimal intra-individual variations in masticatory movement.

PURPOSE: To clarify the section showing minimal intraindividual variations in the movement of the mandibular incisal point during mastication of softened chewing gum. METHODS: Twenty healthy subjects were asked to chew softened chewing gum on the habitual side for 20 seconds. The change in the spatial parameters (gape and masticatory width) and temporal parameter (cycle time) were investigated for 20 cycles from the first cycle. The coefficients of variation of these parameters were investigated for each of 10 consecutive cycles (first to eleventh series). RESULTS: The spatial and temporal parameters were maximal at the first cycle, decreased progressively until the fourth or fifth cycle, and then remained almost unchanged thereafter. The coefficients of variation of the parameters were maximal during the first series, decreased progressively until the fourth to sixth series, and then tended to increase gradually thereafter. Minimal coefficients of variation were observed during the fifth and sixth series for the gape, during the fifth series for the width, and during the fourth series for the cycle time. CONCLUSION: These results suggest that the ten cycles after the fourth to the sixth cycle was the section showing minimal intra-individual variations in the masticatory movement during the chewing of softened chewing gum.

Elastin­derived peptides are involved in the processes of human temporomandibular disorder by inducing inflammatory responses in synovial cells.

Temporomandibular joint dysfunction (TMD) is a collection of clinical symptoms that involve masticatory muscles and the temporomandibular joint (TMJ). Common symptoms include limited jaw motion and joint sound/pain, along with TMJ disc displacement. TMD is frequently associated with synovitis, a chronic inflammation of the synovium. Fibroblast­like synovial cells have been identified to produce several inflammatory mediators and may have an important role in the progression of TMJ inflammation. Degradation of the extracellular matrix molecule elastin may lead to the release of bioactive peptides. The present study aimed to explore the role of elastin­derived peptides (EDPs) in human temporomandibular disorders. Therefore, interleukin­6 (IL­6) expression in the synovial fluid obtained from patients with TMD correlated significantly with two clinical parameters, specifically TMJ locking and pain/jaw function on a visual analog scale (VAS). To the best of our knowledge, this is the first study to determine that the concentration of EDPs in synovial fluid from patients with TMD may also be significantly correlated with the duration of TMJ locking, the VAS score and IL­6 expression. In vitro, EDPs act on human TMJ synovial cells to promote upregulation of IL­6 and the elastin­degrading enzyme matrix metalloproteinase­12 (MMP­12). The upregulation of IL­6 and MMP­12 expression by EDPs may be mediated through elastin­binding proteins (EBP) and a protein kinase A signalling cascade. These findings suggest a model for inflammation in the TMJ where EDPs are generated by harmful mechanical stimuli, induce both a pro­inflammatory cascade and increase expression of MMP­12 through activation of the EBP signalling cascade. This may lead to further increases in EDP levels, establishing a positive feedback loop leading to chronic inflammation in the TMJ. Therefore, significantly elevated levels of EDPs and IL­6 in the synovial fluid of the TMJ may be indicators of the pathological conditions of the joint.

Induction and differentiation of adipose-derived stem cells from human buccal fat pads into salivary gland cells.

Atrophy or hypofunction of the salivary gland because of aging or disease leads to hyposalivation that affects patient quality of life by causing dry mouth, deterioration of mastication/deglutition, and poor oral hygiene status. Current therapy for atrophy or hypofunction of the salivary gland in clinical practice focuses on symptom relief using drugs and artificial saliva; therefore, there is still a need to develop new therapies. To investigate potential novel therapeutic targets, we induced the differentiation of salivary gland cells by co-culturing human adipose-derived stem cells isolated from buccal fat pads (hBFP-ASCs) with human salivary-gland-derived fibroblasts (hSG-fibros). We examined their potential for transplantation and tissue neogenesis. Following the culture of hBFP-ASCs and hSG-fibros, differentiated cells were transplanted into the submandibular glands of SCID mice, and their degree of differentiation in tissues was determined. We also examined their potential for functional tissue reconstitution using a three-dimensional (3D) culture system. Co-cultured cells expressed salivary-glandrelated markers and generated new tissues following transplantation in vivo. Moreover, cell reconstituted glandular structures in the 3D culture system. In conclusion, coculture of hSG-fibros with hBFP-ASCs led to successful differentiation into salivary gland cells that could be transplanted to generate new tissues.

Comparative effects of acupuncture in Japan and the United States on dental pain perception.

The effect of low frequency electrical acupunctural stimulation on the perception of induced dental pain were compared in two cultural settings. Twenty Japanese and 20 American subjects (consisting of 10 Caucasians and 10 second or third generation Japanese) were tested in two functionally identical laboratories, one at Tottori University in Yonago, Japan and the other at the University of Washington. Each subject underwent a control and an acupuncture session on separate days wtih subjects counterbalanced for carry-over order effects. Sensory decision theory (SDT) analysis demonstrated a significant reduction in perceptual capability and an increased bias against reporting stimuli as painful following the acupuncture as treatment which was performed bilaterally at traditional facial points. No significant differences between groups in alteration of perceptual capability, bias or pain threshold were demonstrated, indicating that the cultural and racial differences studied did not influence responses to acupuncture in a laboratory setting.

Randomized controlled trial comparing subcuticular absorbable suture with conventional interrupted suture for wound closure at elective operation of colon cancer.

BACKGROUND: Subcuticular closure provides a superior cosmetic result in clean wounds. The aim of this work was to investigate the safety in terms of postoperative infection and cosmetic effectiveness of subcuticular wound closure after elective colon cancer surgery in clean-contaminated wounds. METHODS: Patients who underwent elective resection of colon cancer were randomized to interrupted subcuticular and interrupted transdermal suture groups. The large bowel was prepared by mechanical washout with polyethylene glycol. All patients received metronidazole and kanamycin orally and flomoxef sodium once parenterally for antimicrobial prophylaxis. The primary end point was the incidence of incisional surgical-site infections within 30 days after operation. We assessed noninferiority of subcuticular suture within a margin of 10%. Analysis was by intent-to-treat. Secondary objectives include comparison of wound closure time, comfort, and cosmesis of the scar and satisfaction of patients. This study was registered with UMIN-CTR, UMIN000003005. RESULTS: A total of 293 patients were randomized to the two groups. Incisional surgical-site infection rates were 11.0% (90% confidence interval 7.0-16.3%) for both groups. The relative risk of subcuticular suture was 1.00 (0.58-1.73, one-tail P = .57). Interrupted subcuticular suture was noninferior to interrupted transdermal suture (P = .0088). Throughout 6 months after operation, patients expressed a significant preference for the subcuticular suture technique, noting rapid relief from pain, decreased vascularity, and smaller width, although the procedure took twice as long. CONCLUSION: Subcuticular suture did not increase the incidence of wound complications in elective colon cancer operation. Patients preferred a technique of interrupted subcuticular closure, citing better cosmetic results, and less pain.

Functional transplantation of salivary gland cells differentiated from mouse early ES cells in vitro.

Atrophy or hypofunction of the salivary gland because of aging or disease causes hyposalivation and has an effect on the quality of life of patients, for example not only dry mouth but deterioration in mastication/deglutition disorder and the status of oral hygiene. Currently conducted therapies for atrophy or hypofunction of the salivary gland in clinical practice are only symptomatic treatments with drugs and artificial saliva, and therefore it is preferable to establish a radical therapy. At this time, as a fundamental investigation, by co-culturing mouse early ES (mEES-6) cells with human salivary gland-derived fibroblasts (hSG-fibro), differentiation of mEES-6 cells to salivary gland cells has been attempted. Also, the possibility of cell engraftment was examined. After identifying the cells which were co-cultured with GFP-transfected mEES-6 cells and hSG-fibro, the cells were transplanted into the submandibular gland of SCID mice, and the degree of differentiation into tissues was examined. The possibility of tissue functional reconstitution from co-cultured cells in a three-dimensional culture system was examined. Our results confirmed that the co-cultured cells expressed salivary gland-related markers and had an ability to generate neo-tissues by transplantation in vivo. Moreover, the cells could reconstitute gland structures in a three-dimensional culture system. By co-culture with hSG-fibro, mEES-6 cells were successfully differentiated into salivary gland cells which were transplantable and have tissue neogenetic ability.

Establishment and characterization of METON myoepithelioma cell line derived from human palatal myoepithelioma: apical reference to the diverse differentiation potential.

Myoepithelioma is an extremely rare condition that accounts for 1-1.5 % of salivary gland tumors. It was formerly regarded as a subtype of pleomorphic adenoma, in which myoepithelial structural components predominated, but was listed as a separate disease entity in the 1991 World Health Organization classification (Seifert in Histological typing of salivary gland tumours. Springer, Berlin, 1991). Its histology is highly varied and recurrence is frequent (El-Naggar et al. in J Larygol Otol 103:1192-1197, 1989), with cases of malignant transformation having been reported (Seifert in Histological typing of salivary gland tumours. Springer, Berlin, 1991; Barnes et al. in Pathology and Genetics of head and neck tumours. IARC Press, Lyon, 2005), making this a difficult tumor to control in many cases. This is thought to be due to the multiple differentiation potential of myoepithelial cells, but the details are unknown. There have been a number of reports of the establishment of cell lines (Shirasuna et al. Cancer. 45:297-305, 1980; Jaeger et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 84:663-667, 1997), but numerous points remain unclear. We established a myoepithelial cell line designated METON, and investigated its characteristics. METON consists of cells with two different morphologies: spindle-shaped cells and epithelial-like cells. Then. we also used single-cell cloning method to establish various subclones (epithelial-like, spindle-like, and mixed epithelial-like/spindle-like cell lines). Among these, pluripotency markers were expressed by the mixed epithelial-like/spindle-like cell lines. The newly established cell line expressing these pluripotency markers will be extremely useful for elucidating the diverse histologies of salivary gland tumors.