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Ultrasound-mediated destruction of microbubbles has been proposed as an innovative non-invasive drug delivery system for cancer therapy. We developed a specific drug delivery system for squamous cell carcinoma that uses sonoporation with the anti-epidermal growth factor receptor (EGFR) antibody. Administration of a low dose of bleomycin (BLM) by sonoporation with the anti-EGFR antibody produced a marked growth inhibition of Ca9-22 cells in vitro. In addition, scanning electron microscopic analysis revealed apparent surface deformation of Ca9-22 cells treated with sonoporation in the presence of the antibody. Interestingly, the population of apoptotic cells was remarkably increased when a low dose of BLM was delivered using sonoporation with the Fab fragment of the anti-EGFR antibody. These findings indicate that sonoporation with the Fab fragment makes it possible to administer drugs into cells more efficiently and specifically, suggesting a novel application for chemotherapy and gene therapy treatments for oral squamous cell carcinoma.

Background: There is limited knowledge relating to the influence of mechanical stress on the temporomandibular joint (TMJ) and the degree of damage this stress can induce in the case of an altered occlusal plane on the extracellular matrix cartilage and chondrocytes. The aim of this study was to examine the histopathological influence of an altered occlusal plane on rabbit TMJ cartilage., Methods: Fifteen male Japanese white rabbits were used in this study. In 12 cases, the upper right and lower left teeth were ground to their cervical level so that the occlusal plane was tilted right side up. Their temporomandibular joints (TMJs) harvested at 3 days, 1, 2 and 4 weeks were analyzed immunocytochemically and immunohistochemically. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick endlabeling (TUNEL) method was used for immunocytochemical study to detect DNA fragmentation in chondrocytes. An immunohistochemical study was also performed to evaluate the metabolism of type 2 collagen in the extracellular matrix in the TMJ cartilage., Results: The number of apoptotic cells increased in all experimental animals. More apoptotic cells were observed in the articular eminences than in the condyles. The type 2 collagen-positive area in the condyles was reduced at the beginning but recovered within 4 weeks, and remained small throughout this experimental period in the articular eminences., Conclusions: These findings suggest that histopathological changes occurred in response to alteration of the occlusal plane. The influence decreased as the occlusal plane recovered in the condyles, but the immunocytochemical and immunohistochemical influence continued in the articular eminences.

Background: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) staining is a widely accepted method for the detection of DNA fragmentation in nuclei of apoptotic cells. Tumor necrosis factor (TNF)-alpha is closely associated with changes in condylar cartilage and modulates apoptosis in various tissues including cartilage. The aim of this study was to investigate the relationship between apoptotic chondrocytes and TNF-alpha in a rabbit model of arthritis., Method: Unilateral temporomandibular joint (TMJ) arthritis was induced in 20 adult New Zealand White rabbits. From 1 day to 6 weeks after the induction of arthritis, immunohistochemical analysis for TNF-alpha and TUNEL was performed., Results: In condylar cartilage, TNF-alpha-positive cells and TUNEL-positive cells were localized together. TNF-alpha-positive chondrocytes seemed to precede TUNEL-positive cells., Conclusions: The results of the present study suggest that TNF-alpha may be involved in apoptosis and/or apoptotic necrosis of chondrocytes as TMJ arthritis progresses from the acute to chronic stage.

Background: The molecular weight (MW) of hyaluronic acid (HA) in joints generally declines in inflammatory arthritis. As inflammation exists in certain temporomandibular disorders (TMD), we measured the MW of HA in synovial fluid (SF) recovered from patients with TMD and compared it with that from normal controls., Methods: Synovial fluid was obtained from patients with TMD (21 TMJs) and normal controls (5 TMJs). The MW of HA was measured using high-performance liquid chromatography (HPLC)., Results: In the controls, the MW of HA in SF exceeded the detection limit, 3000 kDa. In contrast, 19 (90.5%) of 21 SF samples from patients showed a decreased MW of HA. The median MW of HA (1570 kDa) in TMD was significantly lower than that in the controls (P < 0.01)., Conclusion: The MW of HA in SF from patients with TMD is decreased.

Background: In temporomandibular joint (TMJ) arthritis, knowledge is limited about the source of the inflammatory mediators. The aim of this study was to investigate the immunohistochemical role of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) in the development of the antigen-induced arthritis of the rabbit TMJ., Methods: Unilateral TMJ arthritis was induced in 28 adult rabbits. From 6 h to 6 weeks after induction of arthritis, the topology of TNF-alpha and IL-8 was observed., Results: Positive reaction for TNF-alpha of synovial cells was observed within 3 days after induction and at 3 weeks after induction. TNF-alpha positive vascular endothelial cells and chondrocytes were identified throughout the observation period. IL-8 was detected only during the acute stage., Conclusions: The cytokines TNF-alpha and IL-8 were observed in specific cells depending on the stage. TNF-alpha was particularly related with angiogenesis and cartilage destruction and IL-8 was involved in the acute stage of inflammation.

Background: In temporomandibular joint (TMJ) arthritis, there is limited knowledge of the relationship between interleukin-1beta (IL-1beta) and interleukin-1 receptor antagonist (IL-1ra), as well as the source of these cytokines. We investigated the development of an antigen-induced arthritis in the rabbit TMJ immunohistochemically., Methods: Unilateral TMJ arthritis was induced in 32 adult New Zealand White rabbits. From 6 h to 12 weeks after induction of arthritis, topology of IL-1beta and IL-1ra were observed., Result: The acute stage of induced arthritis lasted for one week after induction, thereafter it became chronic. In the early phase of the acute stage, infiltrating inflammatory cells, as well as synovial cells, produced IL-1beta and IL-1ra. In the late phase of the acute stage, the main source of these cytokines was subsynovial fibroblasts. In this phase of arthritis, IL-1beta and IL-1ra did not appear to be produced by synovial cells. From the early to intermediate phase of the chronic stage, proliferating synovial cells produced IL-1beta and IL-1ra. In this phase of the arthritis, these cytokines were also observed in a cluster formation in chondrocytes., Conclusion: This arthritis model shows a staging of the joint inflammation process with time. IL-1beta and IL-1ra are produced by a certain kind of cells depending on the stage of inflammation.

Fas antigen is a cell surface protein that mediates apoptosis via signal transduction from the plasma membrane. Using reverse transcriptase-polymerase chain reaction (RT-PCR), messenger RNA for Fas antigen was detected in the human oral squamous cell carcinoma cell line, SCC-25. In serum-free medium, a monoclonal anti-Fas antibody (CH-11) induced Fas antigen expression in SCC-25 cells, as determined by immunocytochemistry and Western blotting, using an anti-Fas polyclonal antibody (Fas D) as primary antibody. Fas antigen was localized to the cytoplasm and the cell membrane. The molecular weight of the protein recognized by Western blot analysis was 35,000, consistent with the value reported for the Fas antigen. The CH-11 antibody did not induce Fas antigen expression in serum-containing medium. To determine whether CH-11 could induce apoptosis in oral squamous cell carcinoma, we examined its effects on the survival of cultured SCC-25 cells. Anti-Fas monoclonal antibody in serum-free medium induced cytotoxicity in SCC-25 cells in a time-dependent manner up to 8 h, as determined by phase-contrast microscopy and WST-1 assay. Marked nuclear condensation and fragmentation of chromatin were observed in the CH-11-treated cells using Hoechst 33342 staining. This anti-Fas monoclonal antibody also induced DNA ladder formation in SCC-25 cells in a time-dependent manner. The present results indicate that the anti-Fas monoclonal antibody (CH-11) may mediate apoptosis by binding to the Fas antigen expressed in SCC-25 cells.

A monospecific and high titer polyclonal antibody, designated as Fas D, raised against synthetic polypeptides selected from a part of the human Fas antigen (aa 104-114), was used to identify the Fas antigen in human oral epithelia in normal and pathological states. The human gingival proteins had been extracted and analysed by an ABC (avidin-biotin complex) immunoblotting technique. The antibody interacted with a single band in gingival proteins with an estimated molecular weight of 35,000, which is in good agreement with that calculated from amino acid sequences of the human Fas antigen. Using an indirect immunohistochemical method, the antibody localized on the stratum spinosum and the basal part of the stratum corneum of normal human gingiva. Specimens obtained from patients with odontogenic keratocysts, leukoplakia, lichen planus, and squamous cell carcinoma were also stained with the antibody. The pattern of the Fas antigen distribution in oral stratified squamous epithelia was, with some overlapping, characteristic for each disease.