Kenji Fujisawa

Genetic analyses in patients with familial isolated hyperparathyroidism and hyperparathyroidism–jaw tumour syndrome

Background  A subset of familial isolated primary hyperparathyroidism (FIHP) is a variant of hyperparathyroidism–jaw tumour syndrome (HPT‐JT).

Effects of low crystalline carbonate apatite on proliferation and osteoblastic differentiation of human bone marrow cells

Carbonated apatite (CO3Ap) is the inorganic component of bone. We have proposed a new method for the fabrication of CO3Ap blocks based on a dissolution-precipitation method using a synthetic precursor. The aim of this study is to examine the effects of low crystalline CO3Ap on initial cell attachment, proliferation and osteoblastic differentiation of human bone marrow cells (hBMCs) using sintered hydroxyapatite and tissue culture plates as controls. Initial cell attachment and proliferation were assessed with a MTT assay. Expression of osteoblastic markers was examined by reverse transcription-polymerase chain reaction. XRD and FT-IR results showed formation of B-type carbonate apatite with lower crystallinity. No difference was observed for initial cell attachment between HAp and CO3Ap discs. hBMSC attached more significantly on tissue culture plate than on HAp and CO3Ap discs. The number of cells on HAp was higher than that on CO3Ap until day 7, after which the number of cells was similar. hBMSC proliferated more significantly on tissue culture plate than on HAp and CO3Ap discs. In contrast, hBMCs incubated on CO3Ap demonstrated much higher expression of osteoblastic markers of differentiation, such as type I collagen, alkaline phosphatase, osteopontin and osteocalcin, than hBMCs on HAp. On the tissue culture plate, they were not any change throughout the culture period. These results demonstrated that low crystalline CO3Ap exhibit higher osteoinductivity than HAp.

Osteogenesis by human osteoblastic cells in diffusion chamber In vivo

Osteogenic potential of osteoblastic cells isolated from human bone was evaluated by a diffusion chamber method. Cells placed in diffusion chambers were implanted intraperitcneally into the athymic mice. The diffusion chambers cultured in vivo were harvested and examined after implantation for 6–8 weeks. The content of the chamber was proved by a soft roentogenogram to contain the radioopaque area. Light microscopic study revealed that this area was made up of bone-like nodule. Within the diffusion chambers, the cell layers were observed alongside the interior surface of the membrane filters, and mineralized nodules were formed among the cell layers. The mineralized nodules were confirmed by staining with the von Kossa technique for calcium mineral deposits. In electron microscopic study, the osteoblastic cells had a relatively large nuclei, and an abundant rough endoplasmic reticulum produced numerous extracellular matrix. In the bone-like nodules, the osteoblastic cells were surrounded by a heavily mineralized matrix with nonmineralized matrix separating the osteoblastic cells from the mineralized matrix. The mineralized matrix contained well-banded collagen fibrils. Matrix vesicles and collagen fibrils which were closely associated with mineral deposition were observed at the mineralizing front. These results together with our previous report [13] indicate that isolated human osteoblastic cells possessed osteogenic potential in vivo as well as mineralization activity in vitro.

Bortezomib-enhanced radiosensitization through the suppression of radiation-induced nuclear factor‑κB activity in human oral cancer cells

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells.

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

Orthostatic Dysregulation during Postural Change on the Dental Chair and Intraoperative Monitoring by Heart Rate Variability Analysis

This is the first case report of orthostatic dysregulation (OD) manifested during postural change on the dental chair and intraoperatively monitored by heart rate variability (HRV) analysis. OD-associated autonomic dysfunction is induced by postural changes and easily leads to disturbance in circulatory dynamics; however, most dental practices have not yet realized the importance of managing OD. We measured autonomic activity in a patient with OD during dental therapy and assessed the clinical significance of HRV analysis for OD. The patient was a 17-year-old Japanese female. She was diagnosed with impacted wisdom teeth and had no previous history of a distinct systemic disease. A surgical procedure to extract the teeth was safely performed under both local anesthesia and sedation with nitrous oxide and midazolam. After the surgery, her postural change to sitting induced orthostatic hypotension. HRV variables showed parasympathetic dominance due to the upright position. Subsequently, her posture was returned to supine, and atropine sulfate administration for the immediate treatment of OD returned her blood pressure to normal levels. HRV variables showed relative sympathetic dominance due to an atropine-derived parasympathetic blockade. HRV analysis revealed OD-associated autonomic dysfunction and should become a standard tool for safe and secure dental management of OD.

Fabrication and Physical Evaluation of Gelatin-Coated Carbonate Apatite Foam

Carbonate apatite (CO3Ap) foam has gained much attention in recent years because of its ability to rapidly replace bone. However, its mechanical strength is extremely low for clinical use. In this study, to understand the potential of gelatin-reinforced CO3Ap foam for bone replacement, CO3Ap foam was reinforced with gelatin and the resulting physical characteristics were evaluated. The mechanical strength increased significantly with the gelatin reinforcement. The compressive strength of gelatin-free CO3Ap foam was 74 kPa whereas that of the gelatin-reinforced CO3Ap foam, fabricated using 30 mass % gelatin solution, was approximately 3 MPa. Heat treatment for crosslinking gelatin had little effect on the mechanical strength of the foam. The gelatin-reinforced foam did not maintain its shape when immersed in a saline solution as this promoted swelling of the gelatin; however, in the same conditions, the heat-treated gelatin-reinforced foam proved to be stable. It is concluded, therefore, that heat treatment is the key to the fabrication of stable gelatin-reinforced CO3Ap foam.

Antitumour effect of valproic acid against salivary gland cancer in vitro and in vivo

Salivary gland cancer (SGC) has a comparatively poor prognosis and is prone to frequent recurrence and metastases. Therefore, the development of more effective chemotherapy against SGC is desirable. The aim of the present study was to investigate the antitumour effects of valproic acid (VPA) against SGC in vitro and in vivo. Two human SGC cell lines (HSY and HSG cells) were used in the present study. The effects of VPA on the proliferation of SGC cells in vitro were assessed by MTT assay. Cancer cells treated with VPA were subjected to cell cycle analysis by flow cytometry. In addition, the expression levels of p21 and p27 were examined by real-time RT-PCR to identify the mechanisms of the antitumour effect of VPA on SGC. The effects of VPA on cancer growth in vivo were evaluated in a xenograft model. VPA inhibited the proliferation of SGC cells in a dose-dependent manner in vitro. Degenerated cancer cells were observed at high concentrations of VPA. In the cell cycle analysis, VPA induced cell-growth inhibition and G1 arrest of cell cycle progression in both cancer cell lines in a time- and dose-dependent manner. VPA markedly upregulated the mRNA expression levels of both p21 and p27 in both SGC cell lines in a time-dependent manner. In the xenograft model experiment, VPA treatment markedly inhibited the growth of salivary gland tumours when compared with the growth of the untreated controls. VPA may be a valuable drug in the development of better therapeutic regimens for SGC.

Abstract 4954: The expression of X-linked inhibitor of apoptosis in human oral squamous cell carcinoma and its relationship with clinical factors

[Background] X-linked inhibitor of apoptosis (XIAP) is a member of the inhibitor of apoptosis protein family, which is associated with cell survival by blocking caspase-mediated apoptosis. XIAP is expressed in various malignant tumors. The overexpression of XIAP has been reported to be a poorer prognostic factor in various malignancies. However, the prognostic value of XIAP expression in patients with oral cancer is not still cleared. [Purpose] The aims of present study were to evaluate the expression of XIAP protein in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the XIAP expression, clinical stages, histological differentiation and classification of invasion mode. [Materials and Methods] Four human OSCC cell lines, B88, CAL27, HNt, and HSC3 cells were used in this study. Normal gingival epithelial cells served as control. XIAP expression of cultured cells was detected by western blot. Tissue specimens were obtains from 85 patients with OSCC after surgery or biopsy. XIAP expression was detected by an immunohistochemical method. [Results] The expression of XIAP was detected in all cancer cells, but not in normal cells. Immunohistochemical analysis of 85 cases of OSCC showed that 73 (86%) cases expressed XIAP. There was no relationship between XIAP expression and clinical stages, or classification of invasion mode. There were significant differences between XIAP expression and histological differentiation. Most of non-staining and weakly staining of cancer was well differentiated. In contrast, intense and extensive staining was frequently found in poorly differentiated cancer. [Conclusions] These findings suggested that the expression of XIAP in OSCC could be related to histological differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4954. doi:1538-7445.AM2012-4954

Fabrication and evaluation of carbonate apatite-coated calcium carbonate bone substitutes for bone tissue engineering

Carbonate apatite‐coated calcium carbonate (CO3Ap/CaCO3) was fabricated through a dissolution–precipitation reaction using CaCO3 granules as a precursor to accelerate bone replacement based on superior osteoconductivity of the CO3Ap shell, along with Ca2+ release from the CaCO3 core and quicker resorption of the CaCO3 core. In the present study, CaCO3, 10% CO3Ap/CaCO3, 30% CO3Ap/CaCO3, and CO3Ap granules were fabricated and examined histologically to evaluate their potential as bone substitutes. Larger contents of CaCO3 in the granules resulted in higher Ca2+ release and promoted cell proliferation of murine preosteoblasts at 6 days compared with CO3Ap. Interestingly, in a rabbit femur defect model, 10% CO3Ap/CaCO3 induced significantly higher new bone formation and higher material resorption compared with CO3Ap at 8 weeks. Nevertheless, CO3Ap showed a superior osteoconductive potential compared with 10% CO3Ap/CaCO3 at 8 weeks. All tested granules were most likely resorbed by cell mediation including multinucleated giant cell functions. Therefore, we conclude that CO3Ap/CaCO3 has a positive potential for bone tissue engineering based on well‐controlled calcium release, bone formation, and material resorption.