Hitoshi Aizawa

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.

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.

A case of peri-implantitis and osteoradionecrosis arising around dental implants placed before radiation therapy

A little is known about the effect of radiotherapy on the dental implants that have previously been osseointegrated and charged. Here, we reported a case of osteoradionecrosis which arose around dental implants placed before radiation therapy.

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.

Progression level of extracapsular spread and tumor budding for cervical lymph node metastasis of OSCC

ObjectivesThe progression level of extracapsular spread (ECS) for cervical lymph node metastasis of oral squamous cell carcinoma (OSCC) was previously divided into three types, and their relationships with the prognosis of patients were re-examined.Patients and methodsThe Kaplan-Meier method was used to examine overall survival (OS) and relapse-free survival (RFS) curves. Prognosis factor for recurrence was analyzed with univariate and multivariate analysis.ResultsECS was detected in 216 cases of OSCC and analyzed. The 5-year overall survival and RFS rates of patients with type C, which was microscopically defined as tumor invasion to perinodal fat or muscle tissue, were significantly poor at 40.6 and 37.8%, respectively. The results of a univariate analysis suggested that the prognosis of ECS in OSCC patients is associated with its progression level, particularly type C. The 5-year RFS rate of type C with tumor budding was significantly poor at 31.5%. Type C with tumor budding correlated with local and regional recurrence as well as distant metastasis. In a multivariate analysis, tumor budding was identified as an independent prognostic factor.ConclusionsThese results suggest that the progression level of ECS and tumor budding are useful prognostic factors in OSCC patients.Clinical relevanceThis study indicated that the progression level and tumor budding of ECS for cervical lymph node metastasis were useful prognostic factors in OSCC patients.

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.