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.

Collagen gel droplet-embedded culture drug sensitivity testing in squamous cell carcinoma cell lines derived from human oral cancers: Optimal contact concentrations of cisplatin and fluorouracil.

The collagen gel droplet-embedded culture drug sensitivity test (CD-DST) is an anticancer drug sensitivity test that uses a method of three-dimensional culture of extremely small samples, and it is suited to primary cultures of human cancer cells. It is a useful method for oral squamous cell carcinoma (OSCC), in which the cancer tissues available for testing are limited. However, since the optimal contact concentrations of anticancer drugs have yet to be established in OSCC, CD-DST for detecting drug sensitivities of OSCC is currently performed by applying the optimal contact concentrations for stomach cancer. In the present study, squamous carcinoma cell lines from human oral cancer were used to investigate the optimal contact concentrations of cisplatin (CDDP) and fluorouracil (5-FU) during CD-DST for OSCC. CD-DST was performed in 7 squamous cell carcinoma cell lines derived from human oral cancers (Ca9-22, HSC-3, HSC-4, HO-1-N-1, KON, OSC-19 and SAS) using CDDP (0.15, 0.3, 1.25, 2.5, 5.0 and 10.0 µg/ml) and 5-FU (0.4, 0.9, 1.8, 3.8, 7.5, 15.0 and 30.0 µg/ml), and the optimal contact concentrations were calculated from the clinical response rate of OSCC to single-drug treatment and the in vitro efficacy rate curve. The optimal concentrations were 0.5 µg/ml for CDDP and 0.7 µg/ml for 5-FU. The antitumor efficacy of CDDP at this optimal contact concentration in CD-DST was compared to the antitumor efficacy in the nude mouse method. The T/C values, which were calculated as the ratio of the colony volume of the treatment group and the colony volume of the control group, at the optimal contact concentration of CDDP and of the nude mouse method were almost in agreement (P<0.05) and predicted clinical efficacy, indicating that the calculated optimal contact concentration is valid. Therefore, chemotherapy for OSCC based on anticancer drug sensitivity tests offers patients a greater freedom of choice and is likely to assume a greater importance in the selection of treatment from the perspectives of function preservation and quality of life, as well as representing a treatment option for unresectable, intractable or recurrent cases.

Anti-inflammatory effects of astaxanthin in the human gingival keratinocyte line NDUSD-1.

Oral lichen planus is a chronic inflammatory disease that affects the mucous membrane of the oral cavity and can contribute to the development of other diseases. Inflammation in oral lichen planus is a T-cell-mediated autoimmune disease that acts through cytotoxic CD8(+) T cells to trigger apoptosis of keratinocytes. However, the specific cause of oral lichen planus remains unknown and no effective medical treatment has yet been established. Astaxanthin is a carotenoid pigment with capacity for anti-inflammatory and anti-oxidant activities. In this study, we evaluated whether astaxanthin could be used to improve the pathology of oral lichen planus by reducing inflammation. In particular, the anti-inflammatory effects of astaxanthin on the chronic inflammation caused by lipopolysaccharide derived from Escherichia coli O55 in human gingival keratinocytes (NDUSD-1) were evaluated. Following astaxanthin treatment, localization of nuclear factor κB/p65 and the level of inflammatory cytokines (interleukin-6, tumor necrosis factor-α) tended to decrease, and cell proliferation significantly increased in vitro. These results suggest that astaxanthin could be useful for improving chronic inflammation such as that associated with oral lichen planus.

Establishment and characterization of human lingual squamous cell carcinoma cell lines designated Nialym derived from metastatic foci of lymph node, and Nialymx derived from transplanted tumor of Nialym cells.

The squamous cell carcinoma cell lines Nialym was successfully established from metastatic foci of lymph nodes from a 48-year-old male Japanese patient with squamous cell carcinoma of the tongue. In addition, the Nialymx cell line was established from a transplanted tumor of Nialym cells in SCID mice. Nialym cells were angular, with neoplastic and pleomorphic features. Two types of Nialym cell were observed by electron microscopy; light cells and dark cells. The dark cells had a number of waved tonofilaments in the cytoplasm, while light cells showed poorly developed organelles. The population doubling times for Nialym and Nialymx cells were approximately, 46 and 42 h at the 10th passage. Nialym cells secreted 4.8 ng/ml VEGF and 5.9 ng/ml HGF, Nialymx cells also secreted 6.7 ng/ml VEGF and 4.3 ng/ml HGF at the 10th passage for 3 days of culture. Histopathological aspects of Nialym and Nialymx cell lines were similar. We believe that these cell lines are valuable tools for elucidating the mechanisms of cancer metastasis and developing immunotherapy and chemotherapy regimens.

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.

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.

[Virulence of Candida dubliniensis in comparison with Candida albicans using an experimental model of mouse oral candiddiasis].

Certain species of Candida are known as opportunistic fungal pathogens and Candida albicans has especially been isolated oral candidiasis patients at high frequency as a result of its strong pathogenicity. Recently C. dubliniensis is isolated mainly from immunocompromised patients, but is also detected from healthy persons. C. dubliniensis has similar cell morphology and molecular biological properties to C. albicans. Thus, in order to clarify the pathogenicity of C. dubliniensis, the activities of two extracellular enzymes, phospholipase (PL) and proteinase (PT), were measured, and pathological features were compared using mice. PL activity was examined in the improved Price's PL activity assay. In brief, the white precipitation zone was detected by spraying NaCl on egg yold plates without NaCl after colonies had grown. PL activity was no detected in any of the 31 C. dubliniensis strains tested. On the other hand, PT acitivty of C. dubliniensis was almost equivalent to that of C. albicans. Although we attempted to make an experimental model of mouse oral candidiasis using C. dubliniensis in yeast form as an inoculum following the conventional method, oral candidiasis did not develop in any mice. Thrush was successfully developed after inoculation with mycelial form cells, and there was no significant difference in histopathological findings of the thrush in comparison with C. albicans. These results strongly suggest that the two enzymes, PT and PL, do not play a crusial role in the establishment of mouse oral experimental candidiasis by C. dubliniensis.

Heterogeneity of anticancer drug sensitivity in squamous cell carcinoma of the tongue.

Heterogeneity is known to be present to varying degrees in cancer cell groups. There have been no reports, however, of studies in which a single cell clone was prepared from a cancer cell group to examine heterogeneity with respect to anticancer drug sensitivity. Thus, the authors herein report an investigation into the heterogeneity of cancer cells within the same tumor with respect to anticancer drug sensitivity. Anticancer drug sensitivity was investigated in primary tumors, metastatic lymph node tumors, recurrent tumors and established cell lines obtained from four cases of tongue cancer using an oxygen electrode apparatus. As differences were observed in anticancer drug sensitivity from one case to another, even though all four were of the same pathological tissue type, the individual differences were apparently significant. Moreover, primary tumors and recurrent tumors demonstrated different sensitivities to the anticancer drugs even in the same patient. When single cell clones were prepared from primary tumors and anticancer drug sensitivity testing was carried out, sensitivity to anticancer drugs that was not seen in the primary tumors was observed. We performed RT-PCR on cell groups derived from this single cell using MDR1, MRP1, MRP2 and ERCC1, which are primary genes that are resistant to anticancer drugs. Expression of MDR and ERCC1 was not observed in single cell clones nos. 1-10. MRP1 and MRP2, on the other hand, were expressed in all of these single cell clones. Because cells with different sensitivity levels were initially present in the cancer cell groups, even when large numbers of cancer cells died in response to anticancer drug therapy, the results suggest the possibility that recurrence and metastasis occur based on cells with differing sensitivities. After examining anticancer drug sensitivity at the single cell level, we believe that anticancer drug-resistant genes may be involved in the heterogeneity of anticancer drug sensitivity with respect to cancer cell groups.

Clinical anatomy of the maxillary artery.

The Maxillary artery is a component of the terminal branch of external carotid artery and distributes the blood flow to upper and lower jawbones and to the deep facial portions. It is thus considered to be a blood vessel which supports both hard and soft tissues in the maxillofacial region. The maxillary artery is important for bleeding control during operation or superselective intra-arterial chemotherapy for head and neck cancers. The diagnosis and treatment for diseases appearing in the maxillary artery-dominating region are routinely performed based on image findings such as CT, MRI and angiography. However, validations of anatomical knowledge regarding the Maxillary artery to be used as a basis of image diagnosis are not yet adequate. In the present study, therefore, the running pattern of maxillary artery as well as the type of each branching pattern was observed by using 28 sides from 15 Japanese cadavers. In addition, we also took measurements of the distance between the bifurcation and the origin of the maxillary artery and the inner diameter of vessels. These findings thus obtained could contribute to knowledge of improved accuracy of image diagnosis as an index for embolization and for knowledge of an adequate superselective intra-arterial chemotherapy.

Discussion of clinical anatomy of the lingual nerves.

The tongue has various functions, such as gustation, pronunciation, mastication, and deglutition. The nerve fibers are complexly intermingled, and communications between the lingual nerve and the hypoglossal nerve have been reported. Fifteen Japanese heads (30 sides) donated to the 2nd-year students dissection course at the Nippon Dental University School of Life Dentistry, Niigata, were studied with regard to the following aspects: 1. relation of the bifurcation between the lingual and the inferior alveolar nerves close to the oval foramen; 2. distance between the oral foramen and the bifurcation of the lingual and inferior alveolar nerves; and 3. communication between the lingual and hypoglossal nerves. Three types of bifurcation were observed. The standard bifurcation was observed in 21 cases (70.1%). A high bifurcation was observed in 5 cases (16.6%), and a communicating bifurcation was observed in 4 cases (13.3%). The average distance between the oval foramen and the bifurcation of the lingual and inferior alveolar nerves was 8.7 +/- 4.2 mm (minimum: 0 mm/maximum: 14 mm). An anterior-type communication between the lingual and hypoglossal nerves was observed in 8 cases (26.6%), a posterior-type was observed in 17 cases (56.7%), and no communication was observed in 5 cases (17.7%).

Anticancer drug sensitivity testing using an oxygen electrode apparatus.

Conventional anticancer drug sensitivity testing methods, such as succinate dehydrogenase inhibition (SDI), histoculture drug-response assay (HDRA) and collagen gel droplet embedded culture drug sensitivity testing (CD-DST), all require primary culturing and are extremely complex tests that require considerable time for analysis. A major drawback of these methods is that if culturing is not performed properly, ambiguous results are produced. Therefore, we developed an oxygen electrode apparatus that uses cellular metabolism as an indicator of anticancer drug sensitivity and investigated its usefulness in 29 breast cancer patients with the following histopathological classifications: papillotubular carcinoma (n= 15); solid tubular carcinoma (n= 6); and scirrhous carcinoma (n= 8). Comparison of anticancer drug sensitivity testing results obtained using the conventional HDRA method and those obtained using the oxygen electrode apparatus showed significant reproducibility between the two methods. In addition, similar anticancer drug sensitivity testing results using the oxygen electrode apparatus were obtained for in vivo testing of nude mice transplanted with established cancer cell lines. These findings suggest that the oxygen electrode apparatus is a useful procedure in anticancer drug sensitivity testing that provides better reproducibility and that is faster, more convenient, and less expensive than other testing methods.

Establishment and characterization of the rhabdomyosarcoma cell line designated NUTOS derived from the human tongue sarcoma: Special reference to the susceptibility of anti-cancer drugs.

Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco's modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 microg of streptomycin, 50 U/mL of penicillin and 0.25 microg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, alpha-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.

Experimental study of antiangiogenic gene therapy targeting VEGF in oral cancer.

It is well known that tumor angiogenesis plays an important role in local growth and metastasis of oral cancer; therefore, inhibiting angiogenesis is considered to be effective for treating oral cancer. This study aimed to investigate the effectiveness of systemically available antiangiogenic gene therapy targeting vascular endothelial growth factor (VEGF), which is one of the most important angiogenesis accelerators. We administered a soluble form of VEGF receptor-expressing gene incorporated into adenovirus (AdVEGF-ExR) intraperitoneally to nude mice to which oral cancer cell lines (SAS, HSC-3, and Ca9-22) had been transplanted subcutaneously in vivo to inhibit angiogenesis and tumor proliferation. Then, we measured tumor volumes over time, and tumors were enucleated and examined histopathologically and immunohistologically at 28 days after AdVEGF-ExR administration. Compared to the controls to which we administered AdLacZ or saline, significant antiproliferative effects were observed (P < 0.05) in the AdVEGF-ExR administration group, and extensive tumor necrosis was found histopathologically. Immunohistochemical analysis with CD34 (NU-4A1) revealed tumor angiogenesis was suppressed significantly (P < 0.05), and that with ssDNA revealed apoptosis induction was significantly high (P < 0.05) in the AdVEGF-ExR group. However, analysis with Ki-67 (MIB-1) revealed tumor proliferative capacity was not significantly different between the groups. Consequently, we consider that AdVEGF-ExR administration achieved tumor growth suppression by inhibiting angiogenesis and inducing apoptosis, but not by inhibiting the proliferative capacity of tumor cells. Neither topical administration of a soluble form of VEGF receptor (sVEGFR) to the tumor nor a megadose was needed to achieve this inhibition effect. These results suggest gene therapy via sVEGFR would be an effective oral cancer therapy and benefit future clinical applications.

Micro-CT analysis of experimental Candida osteoarthritis in rats.

Experimental osteoarthritis induced by Candida albicans in rats was studied using micro-computed tomography (micro-CT). When C. albicans cells at a nonlethal dose were intravenously injected into 40 rats, joint swelling was induced in 24 rats. Two or more joints were affected in 10 of the 24 rats. Tarsal regions of the hind paw were affected most frequently, followed by elbows of the fore paw. Micro-CT analysis in vivo showed that erosions of the affected tarsal joint bones were apparent several days after the onset of swelling. Thereafter, severe surface roughness and disintegration in the joint bones progressed during the development of arthritis. Three-dimensional (3D) trabecular microstructures and changes in 3D bone parameters were characterized ex vivo with calcanei from affected hind paws. Three-dimensional morphology showed coarsening of the trabecular distribution and weakening of the trabecular connectivity in arthritic bones. These morphological changes were quantitatively confirmed by changes in 3D bone parameters measured from consecutively scanned bone slices. Micro-CT has been shown to be useful for quantifying morphological changes occurring in Candida arthritic bones.

Clinical anatomy in the neck region--the position of external and internal carotid arteries may be reversed.

Knowledge of clinical anatomy in the neck region is useful for the diagnosis of primary tumors and metastatic lymph nodes. Arteries and nerves in the neck region of forty Japanese cadavers (80 cases), 18 males (36 cases) and 22 females (44 sides) were studied by dissection. We obtained the following results. Reverse of the location of the external and internal carotid arteries was found in 5 cases (6.3%). The course of the hypoglossal nerve made an acute curve and ran anterior-inferior in the neck region. In regard to the height of bifurcation of the common carotid artery (CC), high bifurcation was seen in 25 (31.2%), standard bifurcation in 46 (57.5%), and low bifurcation in 9 (11.3%) in a total of 80 cases. Furthermore, the facial artery had the largest inner diameter among the branches of the external carotid artery. Based on these findings, the facial artery will be one of the most beneficial arteries for transplantation as a recipient artery.

Enhancement of amphotericin B activity against Candida albicans by superoxide radical.

This study aimed to examine the involvement of oxidative damage in amphotericin B (AmB) activity against Candida albicans using the superoxide (O2-) generator paraquat (PQ). The effects of PQ on AmB activities against growth, viability, membrane permeability and respiration were examined in a wild-type parent strain (K) and a respiration-deficient mutant (KRD-19) since PQ-induced superoxide generation depends on respiration. In the parent strain, the minimal inhibitory concentration (MIC) of AmB, 0.25 microg/ml, tested with a liquid culture was lowered to 0.025 microg/ml by 1 mM PQ. Such a PQ-induced decrease in the MIC value of AmB was minimal in the mutant. Similar PQ-induced enhancement of AmB activity toward the parent strain was also observed with growth on an agar medium. In viability tests, when candidal cells were exposed to AmB (0.1 microg/ml) for I h, the lethality of AmB was enhanced by 1 mM PQ only in the parent strain. Exogenous superoxide dismutase and catalase failed to diminish the enhancing effect of PQ on the growth inhibitory activity of AmB in the parent strain, suggesting an interaction between superoxide and AmB in candidal cells. The enhancement of AmB activity by PQ, observed preferentially in the wild-type strain, can be explained by extensive superoxide generation depending on respiration. These results suggest that oxidative damage induced by superoxide is involved in AmB activity against C. albicans.

Retrieval and replacement of a malpositioned dental implant: a clinical report.

This clinical report describes the retrieval of a malpositioned mandibular implant with a severe lingual inclination. A replacement implant was inserted with an emphasis on its relationship with the maxillary antagonist, resulting in a buccal inclination of approximately 10 degrees. The treatment review highlights the importance of thorough communication among all members of the dental implant team.