Kenji Kakudo

Effects of Compressive Loading on Human Synovium-derived Cells

Compressive stress may be involved in temporomandibular joint (TMJ) synovitis, but its mechanism has not been fully elucidated. We hypothesized that mechanical stress to the synovial cells of the TMJ potentially causes degenerative changes in temporomandibular joint disease. We examined the effect of cyclic compressive loading on three-dimensionally engineered constructs using human TMJ synovium-derived cells in vitro. Human TMJ synovium-derived cells were cultured onto collagen scaffolds, resulting in three-dimensional constructs. Cyclic compression loading was applied to the constructs by means of a custom-designed apparatus. DNA amount, apoptotic cells, and mRNA levels for inflammatory cytokines were analyzed. The protein expression and activity of MMPs were examined. DNA amount or apoptotic cell number was unchanged by loading. MMP-2, -3, and IL-8 mRNA expression was up-regulated by the compression, and both MMP-1 and -3 protein expression and MMP-2 activity were detected. Thus, compression of human TMJ synovium-derived cells appears to modulate inflammatory cytokines.

NANOG expression correlates with differentiation, metastasis and resistance to preoperative adjuvant therapy in oral squamous cell carcinoma

NANOG protein, a transcription factor expressed in embryonic stem cells, is overexpressed in tumor development. Although studies investigating the function of NANOG in cancer have shown that it plays several roles, such as in cell proliferation, invasion and metastasis, the overall function of NANOG in cancer cells has remained elusive. In the present study, NANOG expression in oral squamous cell carcinoma (OSCC) was examined to determine its potential clinical significance. The expression of NANOG protein was assessed in 60 patients with OSCC by immunohistochemistry, and its correlation with clinicopathological factors and metastasis was evaluated. NANOG protein levels in human OSCC cell lines were determined by western blotting and immunofluorescence staining. NANOG protein expression was identified in 52 cases (86.7%) and expression levels were higher in primary foci of poorly differentiated OSCC than in those of well-differentiated OSCC, indicating that NANOG expression is associated with OSCC differentiation. Regardless of the differentiation levels of primary foci, NANOG expression levels in metastatic foci were extremely high. In addition, NANOG expression in metastatic foci was maintained at high levels following preoperative adjuvant therapy. Furthermore, NANOG protein was detected at an identical level in human OSCC cell lines. These data indicate that NANOG-expressing OSCC cells tend to metastasize and that metastatic tumors expressing NANOG may be resistant to preoperative adjuvant therapy, including chemoradiation. Thus, assessment of NANOG expression may assist the strategy for treatment of OSCC metastasis.

Effects of epidermal growth factor on the invasion activity of the oral cancer cell lines HSC3 and SAS

Epidermal growth factor (EGF) is excreted in a high concentration in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer growth and invasion, we analyzed the Matrigel invasion activity of the cultured oral cancer cell line. Cells grown under the influence of EGF were subjected to Matrigel invasion assays and cells grown in the absence of EGF were used as controls. Gelatin-zymography and Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay (CAT assay) determined the EGF stimulation of matrix metalloproteinase (MMP) expression. EGF increased the number of cells penetrating a Matrigel membrane. Gelatin-zymography and Northern blot analysis revealed that MMP9 and Ets1 expressions correlated with EGF but MMP2 was not changed. a transient transfection assay revealed that EGF increased the promoter activities of the MMP9 genes in HSC3 and SAS cells. These results suggest that EGF increases the invasion activity of oral cancer cells partly by increasing MMP9.

Prolonged matrix metalloproteinase-3 high expression after cyclic compressive load on human synovial cells in three-dimensional cultured tissue

Excessive mechanical stress is thought to be a factor in the development of joint disorders through the expression of matrix metalloproteinases (MMPs) and related cytokines. Although studies revealed that mechanical stress on the synovium induces MMP expression, it is still not known which MMPs prolonged high level expression. The authors focused on MMP-3, which is one of the major factors in joint disorders such as rheumatism and temporomandibular joint disorders. They examined mRNA and protein levels of MMP-3, other MMPs and related cytokines after loading stress. Human synovial cells were seeded onto a collagen scaffold and different magnitudes of cyclic compressive load were applied for 1h. Time-dependent mRNA and protein levels for catabolic genes were examined after loading. mRNA expressions of MMP-1, MMP-3, MMP-9, IL-6, IL-8 and IL-1β increased after excessive compression. In particular, only mRNA of MMP-3 was up-regulated and maintained at a high level for 24h after excessive loading. The concentrations of MMP-3, IL-6 and IL-8 in culture media after loading increased with excessive compression. These results may account for the pathomechanism of MMP-3 induced by cyclic load on synovial cells in joint disorders.

Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer

CRM197, a nontoxic mutant of diphtheria toxin, is a specific inhibitor of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family that has been implicated in the increased progression, proliferation, and metastasis of oral cancer. In this study, we analyzed the antitumor effects of CRM197, which represent possible chemotherapeutic agents for oral cancer. In the experiment, we used the oral squamous cell carcinoma cell lines HSC3 and SAS. Cells treated with CRM197 were analyzed based on cell viability, MTT assay, invasion assay, Western blot, and zymography. HSC3 cells were injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age. CRM197 and/or CDDP were injected intraperitoneally into tumor-bearing mice, and tumor volume was measured over time. HB-EGF expression in HSC3 and SAS cells treated with CRM197 was significantly reduced and cell proliferation was inhibited. The invasiveness of CRM197-treated cells was relatively low. MMP-9 and VEGF were suppressed in HSC3 treated with CRM197 on zymography and Western blot. Further, tumor growth in xenografted mice was suppressed by CRM197 or CDDP at 1 mg/kg/day. Also, the coadministration of CDDP and CRM197 at 1 mg/kg/day completely inhibited tumor formation. These results suggest that HB-EGF is a target for oral cancer and that CRM197 is effective in oral cancer therapy.

Calcifying Odontogenic Cysts: Co-Expression of Intermediate Filament Proteins, and Immunohistochemical Distribution of Keratins, Involucrin, and Filaggrin

The epithelia lining the cyst of five cases of calcifying odontogenic cyst (COC) were evaluated immunohistochemically with the use of monoclonal antibodies (MoAbs) against keratin (PKK1, KL1, K4.62, K8.12) and vimentin, and polyclonal antisera agonist involucrin and filaggrin. Epithelial lining of COC was classified into 1) thin squamous-cell epithelium, 2) ameloblastoma-like, and 3) thin or 4) thick calcifying odontogenic epithelium. Foci consisting of ghost cells or calcified cells were categorized as calcifying epithelial odontogenic tumor (CEOT). Thin squamous-cell epithelium reacted with PKK1, KL1, K4.62, K8.12, and anti-vimentin MoAbs, thus demonstrating the co-expression of keratin and vimentin. Ameloblastoma-like cells showed positive staining with PKK1, KL1, and sometimes with anti-vimentin. Thick calcifying odontogenic epithelial lining showed stratification of cell layers, and the most strikingly reactive zone was the upper intermediate layer, which showed the presence of keratin, involucrin, and a small amount of filaggrin. Cells of this layer might be the most differentiated type of cells in COC. Undifferentiated odontogenic cells of COC masses were characterized by co-expression of keratin and vimentin, and by the absence of involucrin and filaggrin. All ghost cells were devoid of any immunostaining except for filaggrin, which was rarely positive, but eosinophilic or basophilic cells surrounding the ghost cells showed intense staining for all keratin proteins except vimentin.

Heparin-binding epidermal growth factor-like growth factor is a potent regulator of invasion activity in oral squamous cell carcinoma

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) has been shown to stimulate the growth of various cell types in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors when compared with normal tissue, its contribution to cancer progression remains obscure. The objective of this study was to explore the effects of HB-EGF on proliferation, invasion activity and MMP-9 levels of an oral squamous cell carcinoma cell line, HSC3, in vitro. MTT assays, Matrigel invasion assays and RT-PCR in combination with RNA interference (RNAi) were used in this study. An RNAi-mediated decrease in HB-EGF expression reduced invasion activity and MMP-9 mRNA levels, but not proliferation, in HSC3 cells. The addition of purified HB-EGF to cell culture medium upregulated MMP-9 mRNA levels in HSC3 cells. Furthermore, the TACE inhibitor TAPI-2 or EGFR inhibitor AG1478 decreased MMP-9 mRNA levels in HSC3 cells. These data indicate that HB-EGF released from HSC3 cells by TACE stimulates EGFR in an autocrine manner, which in turn activates invasion activity via MMP-9 upregulation.

Calcification at the Interface Between Titanium Implants and Bone: Observation With Confocal Laser Scanning Microscopy

It has not been previously possible to observe bone formation in undecalcified sections with titanium implants at high magnification because of the difficulty in sectioning bone together with implants. A method for examining the bone-implant interface in undecalcified sections is described in which implants are left in situ and confocal laser scanning microscopy (CLSM) is used to examine both the implant surface and adjacent bone. Pulsing of animals at different times with the fluorescent dyes calcein and alizarin red permitted assessment of temporal patterns of bone formation by CLSM. Reflectivity of the polished implant surface permitted accurate assessment of the position of the implant relative to labeled bone. The analysis showed that bone first formed as thin processes towards and across the implant surface, followed by further bone formation behind these processes. The interface between calcified bone tissue and the implant surface was characterized by a 10-microm space. The CLSM technique enabled detailed observations of new bone formation at the titanium implant interface.

Cetuximab-resistant oral squamous cell carcinoma cells become sensitive in anchorage-independent culture conditions through the activation of the EGFR/AKT pathway

We have previously shown that growth of the oral squamous cell carcinoma cell line SAS, is resistant to cetuximab in monolayer culture conditions, even though epidermal growth factor receptor (EGFR) was phosphorylated, but the growth of SAS aggregates was sensitive to cetuximab. In the present study, we demonstrate differences in the EGFR signaling pathways utilized by SAS cells in monolayer and suspension cultures at the molecular level. Cetuximab treatment of SAS cells in monolayer cultures inhibits the phosphorylation of EGFR and ERK, and reduces the cell migratory potency, but not cell proliferation. AG1478 treatment reduces the phosphorylation of EGFR, ERK and AKT, and affects cell growth in monolayer cultures. The phosphorylation levels of EGFR and AKT are significantly higher in SAS cell aggregates compared to monolayer cultures. Treatment with cetuximab and AG1478 reduces the growth of SAS aggregates and eliminates the phosphorylation of EGFR and AKT. Furthermore, proliferation of SAS aggregates is also inhibited by LY294002 and MK2206, which are inhibitors of PI3K and AKT, respectively. In addition, treatment with the lipid raft disruptor filipin III reduced the phosphorylation levels of EGFR and Akt in SAS aggregates, but not in SAS monolayer culture. These results suggest the possibility that ligands in the serum stimulate the phosphorylation of EGFR localized in lipid rafts leading to PI3K-AKT activation, which results in the growth of SAS aggregates, therefore resulting in the sensitivity of SAS aggregates to cetuximab.

Biomechanical Properties of the Condyle Created by Osteodistraction

A new condyle can be reconstructed by osteodistraction, but the biomechanical properties of the neocondyle remain unknown. This study examined the hypothesis that the biomechanical properties of neocondylar cancellous bone could reach control levels 24 weeks after its creation by osteodistraction. The right mandibular condyles were removed and reconstructed by osteo-distraction in 16 adult goats. Their contralateral condyles served as controls. Microstructural and mechanical properties were examined by microcomputed tomography and mechanical testing. At 24 weeks after distraction, the neocondyle grew larger in size, but the shape and histological features were similar to those of the controls. The cancellous bone of the neocondyle even appeared to be more dense and stiffer in comparison with the control condyle. The results of this study suggest that the neocondyle created by osteodistraction develops nearly normal biomechanical properties for functional loading by 24 weeks after creation.