Hatsuhiko Maeda

Prognostic factors for keratocystic odontogenic tumor (odontogenic keratocyst): analysis of clinico-pathologic and immunohistochemical findings in cysts treated by enucleation

BACKGROUND The purpose of this study was to determine prognostic factors for the recurrence of keratocystic odontogenic tumors (KCOTs) following simple enucleation by examining clinico-pathologic and immunohistochemical findings. METHODS Following enucleation, the frequency of recurrence among 32 subjects diagnosed with KCOT was analyzed for tumor site, radiographic and histologic features, and immunopositivity for Ki-67 and p53. RESULTS Keratocystic odontogenic tumors in four out of 32 subjects (12.5%) recurred during the follow-up period (median: 33 months, range: 7-114 months). Three out of four subjects (75.0%) among recurrent group showed high expression of Ki-67 (LI >10%) in basal layer and four (4/28; 14.3%) among non-recurrence group (P = 0.025). Expression of p53 among non-recurrent group was observed in 11 subjects (11/28; 39.3%), and in three subjects (3/4; 75.0%) among the recurrent group (P = 0.295). Hazard risk for the recurrence of KCOT was 4.02 (95% CI 1.42-18.14) for high Ki-67 expression in the basal layer by the Cox proportional hazard model (P = 0.009). In our study, none of the other clinico-pathologic variables were associated with the recurrence of KCOT. CONCLUSION The results suggested that the evaluation of Ki-67 expression in KCOT at the time of pathological diagnosis might be helpful for consideration of appropriate adjunctive surgical procedures to avoid a recurrence and may serve as a prognostic marker.

Human papillomavirus type 38 infection in oral squamous cell carcinomas.

In this study, 53 paraffin-embedded oral squamous cell carcinoma (OSCC) biopsy specimens were used. Human papillomavirus type 38 (HPV-38) infection was demonstrated in OSCCs using the PCR technique, DNA sequencing analysis, in situ hybridization, and immunohistochemical techniques. Additionally, the correlation between HPV-38 infection and expressions of proliferating cell nuclear antigens (PCNA) or p53 protein was analyzed immunohistochemically. Using consensus primers for the L1 region (L1-PCR), we identified 35 of 53 specimens (66%) as positive for HPV-38 DNA. Furthermore, specimens from patients over 60 years of age revealed a lower prevalence for the HPV-38 (56.7%) than did those below that age (78.3%). Immunohistochemically, positive stainings for PCNA and p53 protein were more frequently detected in HPV-38 positive OSCCs than HPV negative ones. These results indicate that HPV-38 positive OSCCs were higher in proliferative cellular activity than HPV negative ones. Moreover, the findings suggest that HPV-38 infection may cause malignant transformation of the oral mucosal epithelium.

Human papillomaviruses in the normal oral cavity of children in Japan

The purpose of this study was to determine the frequency of human papillomavirus (HPV) infections in the normal oral cavity of children in Japan. Oral squamous cell specimens were collected from 77 children (44 boys and 33 girls), aged 3 and 5 years. Extracted DNA was evaluated for HPV infections by polymerase chain reaction (PCR) methods, using consensus primers for the L1 region, specific primers, and direct DNA sequencing analysis. Thirty-seven of 77 specimens (48.1%) were positive for HPV DNA. Positive rates of boys and girls in all specimens were 28.3 (22/77) and 19.5 (15/77)%, respectively. The positive rate in 3-year-old children was 45.2 (14/31)%, and positive rates in boys and girls were 52.6 (10/19) and 33.3 (4/12)%, respectively. The positive rate in 5-year-old children was 50.0 (23/46)%, and positive rates in boys and girls were 48.0 (12/25) and 52.4 (11/21)%, respectively. HPV types were determined by specific PCR and direct DNA sequencing analysis. Frequent HPV types in the specimens of all children were HPV-16 (11/37; 29.7%),-1 (6/37; 16.2%),-2 (6/37; 16.2%),-75 (6/37; 16.2%). The results of the present investigation indicate that many HPVs, including HPV-16 (a high-risk type for cancer), are present in the oral cavity of 3- and 5-year-old children. It is suggested, therefore, that the oral cavity is already a reservoir of HPVs in childhood where later HPV-associated diseases, such as oral cancer and other oral lesions, may develop.

Recent Developments of Functional Scaffolds for Craniomaxillofacial Bone Tissue Engineering Applications

Autogenous bone grafting remains a gold standard for the reconstruction critical-sized bone defects in the craniomaxillofacial region. Nevertheless, this graft procedure has several disadvantages such as restricted availability, donor-site morbidity, and limitations in regard to fully restoring the complicated three-dimensional structures in the craniomaxillofacial bone. The ultimate goal of craniomaxillofacial bone reconstruction is the regeneration of the physiological bone that simultaneously fulfills both morphological and functional restorations. Developments of tissue engineering in the last two decades have brought such a goal closer to reality. In bone tissue engineering, the scaffolds are fundamental, elemental and mesenchymal stem cells/osteoprogenitor cells and bioactive factors. A variety of scaffolds have been developed and used as spacemakers, biodegradable bone substitutes for transplanting to the new bone, matrices of drug delivery system, or supporting structures enhancing adhesion, proliferation, and matrix production of seeded cells according to the circumstances of the bone defects. However, scaffolds to be clinically completely satisfied have not been developed yet. Development of more functional scaffolds is required to be applied widely to cranio-maxillofacial bone defects. This paper reviews recent trends of scaffolds for crania-maxillofacial bone tissue engineering, including our studies.

Epithelial cell proliferation in oral lichen planus.

Abstract. Although the pathogenesis of oral lichen planus (OLP) is not clear, a small proportion of cases with OLP are reported to transform to cancer. We examined the epithelial cell proliferation status of OLP to relate the labelling index to microscopic features surveyed routinely in pathology. Mucosal biopsies obtained from 44 cases diagnosed with OLP with an intact oral epithelium and 10 normal control specimens from Japanese subjects were immunohistochemically stained with MIB and p53 antibodies. The Ki67 labelling index (LI) was significantly higher in OLP compared with normal controls. A particularly large number of OLP lesions (64%) were p53 positive. No association was, however, found with p53 expression and the Ki67 LI. Atrophic and flat epithelia had a quantitatively higher LI, which did not significantly differ from acanthotic biopsies. Increased cell proliferation in OLP is likely to be a secondary phenomenon due to the damage inflicted on keratinocytes by infiltrating mononuclear cells in the submucosa.

DNA Vaccine against Hamster Oral Papillomavirus-associated Oral Cancer

Previously we developed a carcinogenesis model involving the combination of 9, 10-dimethyl-1, 2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer.

Osteoblast Mechanoresponses on Ti with Different Surface Topographies

During implant healing, mechanical force is transmitted to osteogenic cells via implant surfaces with various topographies. This study tested a hypothesis that osteoblasts respond to mechanical stimulation differently on titanium with different surface topographies. Rat bone-marrow-derived osteoblastic cells were cultured on titanium disks with machined or acid-etched surfaces. A loading session consisted of a 3-minute application of a 10- or 20-μm-amplitude vibration. Alkaline phosphatase activity and gene expression increased only when the cells were loaded in 3 sessions/day on machined surfaces, regardless of the vibration amplitude, whereas they were increased with 1 loading session/day on the acid-etched surface. The loading did not affect the osteoblast proliferation on either surface, but selectively enhanced the cell spreading on the machined surface. Analysis of the data suggests that osteoblastic differentiation is promoted by mechanical stimulation on titanium, and that the promotion is disproportionate, depending on the titanium surface topography. The frequency of mechanical stimulation, rather than its amplitude, seemed to have a key role.

N-Acetyl cysteine (NAC) inhibits proliferation, collagen gene transcription, and redox stress in rat palatal mucosal cells

OBJECTIVES Control of hyperplastic and invasively growing gingival tissue is crucial for maintaining normal oral function and for successful bone regenerative therapy. We tested the hypothesis that materials containing N-acetyl cysteine (NAC), an antioxidant cysteine derivative, can control proliferation and function of oral mucosal cells. METHODS Oral mucosal cells derived from the rat palatal tissue were cultured with or without NAC at different concentrations (2.5-10.0mM). To simulate inflammatory conditions, cultures were treated with hydrogen peroxide. NAC was also applied via collagen materials in membrane and sponge forms to explore the clinical applicability. The redox balance inside the cells was evaluated by measuring the concentration of intracellular glutathione (GSH). RESULTS Adding NAC into cultures of oral mucosal cells reduced their proliferation, transcriptional expression, and collagen production in an NAC-concentration-dependent manner without cytotoxic effects. Furthermore, NAC substantially reduced the hydrogen peroxide-induced elevation of cellular proliferation and collagen production. The controlling effects of NAC were also demonstrated in cells cultured on NAC-containing collagen materials and were associated with an increase in intracellular glutathione (GSH) reserves and a decrease in the oxidized form of glutathione (GSSG). SIGNIFICANCE These results indicate that NAC may abrogate inflammation- or oxidative-stress-induced hyperfunction of oral mucosal cells and that it can be delivered effectively via biodegradable materials. This study provides a basis to explore NAC-containing biomaterials that are functionalized to control oral soft tissue growth and function without cytotoxicity.

Follicular dendritic cell‐secreted protein is decreased in experimental periodontitis concurrently with the increase of interleukin‐17 expression and the Rankl/Opg mRNA ratio

BACKGROUND AND OBJECTIVE T-helper type 17 (Th17) cells produce interleukin-17 (IL-17) and help to protect against inflammation and infection in periodontal disease. Furthermore, while follicular dendritic cell-secreted protein (FDC-SP) may be involved in the inflammation of periodontal tissue, the biological role of FDP-SP in periodontal disease is still unknown. The purpose of the present study was to clarify the expression of IL-17 and FDC-SP in experimental periodontitis in rats. MATERIAL AND METHODS Seven-week-old male Wistar rats were divided into baseline control, sham and test groups. Experimental periodontitis was induced by placing a ligature in the mesiopalatal area, and untreated rats served as a baseline control group. Morphological changes in alveolar bone were investigated 7, 14 and 28 d after treatment. Expression of the Rankl, osteoprotegerin (Opg) and Il17 genes was analyzed 5 and 7 d after the induction of experimental periodontitis. RESULTS Alveolar bone resorption progressed in the test group for 7 d, but not thereafter. At 5 d after the induction of periodontitis, the Rankl/Opg mRNA ratio and the expression of IL-17 in the test group were significantly increased compared with the respective values in the baseline control group; however, there were no significant differences between the test and control groups at 7 d. The expression of FDC-SP was significantly decreased in the test group compared with the baseline control group at 5 and 7 d after the induction of periodontitis, and this value had returned to normal levels at 14 and 28 d. CONCLUSION These results suggest that both IL-17 and FDC-SP could be involved in the inflammatory response, and FDC-SP in the junctional epithelium might play an important role in the Th17 cell-related immune response.

N-acetyl cysteine prevents polymethyl methacrylate bone cement extract-induced cell death and functional suppression of rat primary osteoblasts.

This study examines the cytotoxicity of bone cement extract to osteoblasts and the potential detoxification and restoration of osteoblastic function by an antioxidant amino acid, N-acetyl cysteine (NAC). The osteoblastic cells derived from rat femurs were cultured with extract from polymethyl methacrylate (PMMA)-based bone cement. The calcein and ethidium homodimer staining of the cells after 24-h incubation showed that 23.0% of the cells were dead in the culture with bone cement extract, while the addition of 5 mM NAC into the culture reduced the percentage to 4.3%. Annexin V and propidium iodide-based flow cytometric analysis also revealed that the apoptotic cells present at 15.8% in the culture with bone cement extract was reduced to 2.4% in the culture cotreated with bone cement extract and NAC. Severely suppressed alkaline phosphatase activity and matrix mineralization in the culture with bone cement extract (reduced to 10% and 5%, respectively, compared with the control culture) were restored to a normal level when treated with 5 mM NAC. The bone cement extract-induced, downregulated expression of osteoblastic genes, such as alkaline phosphatase, collagen I, and osteocalcin, was also restored to the baseline level by cotreatment with NAC. The data indicated that the addition of NAC into acrylic bone cement extract remarkably ameliorated the cytotoxicity to osteoblasts and restored their phenotype and function to a biologically significant degree, suggesting the potential usefulness of NAC in developing more biocompatible acrylic bone cement.