Tetsu Shimane

Cultured epithelial grafting using human amniotic membrane: the potential for using human amniotic epithelial cells as a cultured oral epithelium sheet.

OBJECTIVE Human amniotic cells are a valuable source of functional cells that can be used in various fields, including regenerative medicine and tissue engineering. The aim of this study was to investigate the utility of human amniotic epithelial (hAE) cells as a new cell source for culturing stratified epithelium sheets for intraoral grafting. METHODS Enzymatically isolated hAE cells were submerged in a serum-free, low-calcium-supplemented MCDB 153 medium without a feeder layer. The hAE cells were seeded onto a Millicell cell culture plate insert and cultured while submerged in a high-calcium medium for 4 days. Then, they were cultured at an air-liquid interface for 3 weeks. Cultures of hAE cells proliferated at the air-liquid interface. RESULTS After 3 weeks, the hAE cells cultivated using the air-liquid interface method lead to almost 10 continuous layers of stratified epithelium without parakeratinization or keratinization. It confirmed immunohistochemically that the presence of CK10/13 and Ki-67 positive cells were spread throughout almost all the epithelial layer, and that CK19 positive cells were expressed throughout the entire epithelial layer in the cultured hAE cell sheets. Cultured hAE cells sheets showed a staining pattern similar to that of uncultured oral mucosa: ZO-1 and occludin were located in the intercellular junctions throughout all the epithelial layers. It was suggested that the hAE sheets consisted of highly-active proliferating cells and undifferentiated cells, and had a barrier function. CONCLUSIONS These results suggested that hAE cells may be a promising cell source for the development of stratified epithelium allograft sheets using a human cell strain.

Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. We recently showed that GRHL2 also controls normal epithelial cell proliferation and differentiation. In this study, we investigated the role of GRHL2 in oral carcinogenesis and the underlying mechanism. GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness and epithelial phenotype of oral cancer cells. GRHL2 loss also inhibited oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness, indicating that miR-200 genes in part mediate the functional effects of GRHL2. Taken together, this study demonstrates a novel connection between GRHL2 and miR-200, and supports protumorigenic effect of GRHL2 on OSCCs.

Oral cancer intraoperative detection by topically spraying a γ-glutamyl transpeptidase-activated fluorescent probe

Surgical resection of the primary tumor with adequate margins is an essential component of the treatment strategy for oral squamous cell carcinoma (OSCC). It is important to make sure the free margin [1]. Real-time cancer identification using fluorescence probes currently represents one of the most active research fields. There are very few techniques that are applicable in clinical practice. Amino peptidases are expressed at high levels in many cancers, such as hepatic cancer and glioma, and exhibit increased activity compared to normal tissue. Among them, c-glutamyl transpeptidase (GGT), a cell surface-associated enzyme involved in cellular glutathione homeostasis, has been reported to be overexpressed in several human cancers. A novel concept probe, c-glutamyl hydroxymethyl rhodamine green (g-Glu-HMRG), can detect enzymatic activity of GGT in human cancer cells in real time. g-Glu-HMRG activation occurs by a one-step enzymatic reaction in the presence of GGT to yield a highly fluorescent product (Fig. 1A). Instant activation of the probe makes it possible to recognize tumor particles very rapidly [2]. In this paper, we introduced this method to detect oral cancer tissue using a topically applied enzymatically activatable probe (g-Glu-HMRG) that fluoresces in the presence of GGT, an enzyme associated with cancer. OSCC tissue was stained using g-Glu-HMRG and fluorescence activity was assessed.

Effectiveness of vital staining with iodine solution in reducing local recurrence after resection of dysplastic or malignant oral mucosa

The purpose of this retrospective study was to assess the effect of vital staining with iodine solution in reducing local recurrence after resection of dysplastic or malignant oral mucosa. The historical control group had dysplastic or malignant mucosal lesions resected solely on the evidence of direct inspection and palpation. In the vital staining group tissue was resected only after vital staining with iodine solution. Seven of 25 patients in the conventional group developed recurrent dysplastic or cancerous oral mucosa around the primary site, while no patient among 23 reported recurrence in the vital staining group (p<0.01). Kaplan-Meier assessment showed that the 5-year primary control rate was 100% in the vital staining group and 75% in the conventional group. Although this retrospective study has some limitations, the results suggest that vital staining with iodine may be useful in reducing the incidence of recurrence of dysplastic or cancerous epithelium at a primary site. Further well-controlled study is essential.

Clinical significance of apoptosis-associated speck-like protein containing a caspase recruitment domain in oral squamous cell carcinoma

OBJECTIVES To assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression in oral squamous cell carcinoma (OSCC) and analyze its clinical and pathological significance. STUDY DESIGN ASC expression was studied using immunohistochemistry in 119 OSCCs patients. The relationships between ASC expression and clinical and pathological parameters were statistically analyzed. In addition, the relationships between ASC expression and cell differentiation [IVL (involcrin) expression] and apoptosis (TUNEL [TdT-mediated dUTP nick end labeling] positive cell number) were investigated. RESULTS ASC expression showed significant correlations with parameters including clinical tumor stage, mode of invasion, and histological differentiation, and had a significant impact on survival of OSCC. The distribution of ASC correlated well with that of IVL. ASC expression was significantly correlated with the TUNEL-positive cell number. CONCLUSIONS Lower ASC expression correlates with clinical and pathological malignancy and, consequently, poor prognosis of OSCC. ASC has a close association with cell differentiation and apoptosis.

Difference in glycogen metabolism (glycogen synthesis and glycolysis) between normal and dysplastic/malignant oral epithelium

BACKGROUND The purpose of this study was to investigate a difference in glycogen metabolism (glycogen synthesis and glycolysis) between the iodine stained (normal non-keartinized) and the unstained (dysplasctic/malignant) oral epithelium. METHODS Twenty-one frozen tissue samples of iodine-stained and unstained mucosal tissue were obtained from 21 OSCC patients. Serial frozen sections were cut and examined with the hematoxylin-eosin and periodic acid-Schiff methods and immunohistochemical (IHC) staining for Ki67, P53, molecules associated with glycogenesis (i.e., glycogen synthase (GS) and phospho-glycogen synthase (PGS)), and molecules associated with glycogenolysis (i.e., glycogen phosphorylase isoenzyme BB (GPBB) examine the glycogen metabolism in OSCC. Additionally, in vitro study, the expression levels of GS and GPBB in the cultured cells were analyzed by immunofluorescent staining, Western blot analysis, and the real-time quantitative polymerase chain reaction (PCR). RESULTS There was no significant difference in GS and PGS immunoactivity between iodine stained and unstained area. On the other hand, significantly greater GPBB immunoreactivity was observed in the basal and parabasal layers of iodine-unstained epithelium, where higher positivity for p53 and Ki67 was also showed. Additionally, western blot analysis, immunofluorescent staining, and real-time quantitative PCR revealed that the oral squamous cancer cells exhibited greater expression of GPBB than normal epithelial cells. CONCLUSIONS The results of this study showed that GPBB expression, which resulted in up-regulation of glycogenolysis, is enhanced in oral dysplastic/malignant epithelium compared with non-keartinized normal epithelium, in spite of the fact that glycogenesis continues in both of them. Premalignant and malignant epithelial cells consume greater quantities of energy due to their increased proliferation, and hence, exhaust their glycogen stores, which resulting in negative stain reaction with iodine solution.

Iodine penetration and glycogen distribution in vital staining of oral mucosa with iodine solution

OBJECTIVE To assess and compare iodine penetration and glycogen distribution in a vital staining of oral mucosa with iodine solution. STUDY DESIGN Twenty samples were obtained including both iodine-stained and -unstained mucosa. Intraepithelial iodine was examined using frozen sections. Glycogen distribution was assessed by periodic acid-Schiff staining and transmission electron microscopy. RESULTS Iodine accumulation was observed mainly superficially and in the upper and middle thirds of prickle cell layers, with glycogen in almost the whole epithelium except for the para- and basal cell layers. The pattern of iodine and glycogen distribution was classified into 3 types (full-, surround-, and scatter-type). The iodine color was mainly derived from the cells with full- and surround-type distributed glycogen in the upper half of the oral epithelium. CONCLUSIONS The results of this study suggested that iodine penetrated into nonkeratinized oral epithelium and reacted mainly with intraepithelial glycogen homogeneously distributed in the cytoplasm.

Construction and characterization of human oral mucosa equivalent using hyper-dry amniotic membrane as a matrix

BACKGROUND Human amniotic membrane(HAM) as a graft material has been used in various fields. Hyper-dry amniotic membrane (HD-AM) is a novel dried amniotic membrane that is easy to handle and can be preserved at room temperature without time limitation. The purpose of this study was to investigate the useful properties of HD-AM in reconstruction of the oral mucosa. METHODS Human oral keratinocytes were isolated and seeded on HD-AM in serum-free culture system. Oral mucosa equivalent (OME) was developed and transplanted onto full-thickness wound on athymic mice. The wound healing was analyzed and the OME both before and after transplantation was analyzed with hematoxylin-eosin staining and immunohistochemical staining for Cytokines 10 (CK10), Cytokines 16 (CK16), and Ivolucrin (IVL). RESULTS Oral keratinocytes spread and proliferated well on HD-AM. Two weeks after air-lifting, OME had formed with good differentiation and morphology. We confirmed immunohistochemically that the expression of CK10 was positive in all suprabasal layers, as was CK16 in the upper layers, while IVL was present in all cell layers. Three weeks after transplantation to athymic mice, the newly generated tissue had survived well with the smallest contraction. The epithelial cells of newly generated tissue expressed CK10 throughout in all suprabasal layers, IVL was mainly in the granular layer, and CK16 positive cells were observed in all spinous layer and granular layer but were not expressed in the mouse skin, all of which were similar to native gingival mucosa. CONCLUSIONS The OME with HD-AM as a matrix revealed a good morphology and stable wound healing. This study demonstrates that HD-AM is a useful and feasible biomaterial for oral mucosa reconstruction.