Kenjiro Iwanaga

Local delivery system of cytotoxic agents to tumors by focused sonoporation

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as ‘sonoporation’ has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechsts staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

OBJECTIVES Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. MATERIALS AND METHODS Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. RESULTS BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA. CONCLUSIONS These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation.

Ultrasonography for Intraoperative Determination of Tumor Thickness and Resection Margin in Tongue Carcinomas

PURPOSE Exact estimation of tumor thickness and the status of the resection margin in tongue carcinoma are important prognostic factors for local recurrence, subclinical nodal metastasis, and survival. This study aims to evaluate the accuracy of intraoral ultrasonography-guided measurement of tumor thickness and define an adequate intraoperative resection margin in squamous cell carcinomas of the tongue. MATERIALS AND METHODS In this prospective study, 13 patients with presurgical, biopsy-proven, clinical T1N0 or T2N0 tongue squamous cell carcinomas who underwent a partial glossectomy were examined preoperatively with ultrasonography to assess tumor thickness under general anesthesia. Nine cases underwent resection by a conventional method, whereas we introduced elastic needles with a metal core to mark a deep surgical margin of 10 mm from the deepest tumor invasion front under ultrasonographic monitoring as a new technique in the remaining 4 cases. Each resected specimen was immediately immersed in gelatin solution while maintaining its original shape and orientation and was placed under refrigeration to solidify. Ultrasonographic observations of the gelatin-embedded specimens were performed from the superior surface of the gelatin block. RESULTS Very fine ultrasonographic images of the resected specimen could be easily obtained without any special skills, and surgical clearance could be verified intraoperatively. The ultrasonographic tumor thickness measurements corresponded well with those of histologic sections, with a consistency ratio of 91.4% to 98.2% (Pearson correlation coefficient = 0.981, P < .05). CONCLUSION Intraoperative ultrasonography is a reliable method to objectively evaluate tumor thickness and surgical margin clearance.

Targeted drug delivery system for oral cancer therapy using sonoporation

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.

Role of heme oxygenase-1 in inflammatory response induced by mechanical stretch in synovial cells

ObjectiveThe purpose of this study was to investigate the mechanism by which heme oxygenase-1 (HO-1) regulates inflammatory responses induced by mechanical stretch in human fibroblast-like synoviocyte (HFLS) cells.Materials and methodsHFLS cells were cultured in the presence of hemin and seeded into fibronectin-coated silicon chambers. The chambers were attached to a stretching apparatus which applied a uniaxial sinusoidal stretching force. The genetic expressions of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β) and HO-1 were analyzed using real-time RT-PCR. The expression and localization of HO-1 protein were detected by immunofluorescence staining. The amounts of prostaglandin E2 (PGE2) released into the culture medium were determined using ELISA.ResultsMechanical stretch enhanced the expressions of COX-2 and IL-1β, and the amount of PGE2 synthesis in HFLS cells, whereas that of HO-1 was slightly increased. In contrast, treatment with hemin enhanced HO-1 gene expression and mechanical stretch enhanced this expression in hemin-pretreated cells. In addition, hemin pretreatment suppressed PGE2 synthesis induced by mechanical stretch.ConclusionWe found that constitutive HO-1 expression in hemin-pretreated HFLS cells suppressed mechanical stretch-induced inflammatory responses, suggesting that HO-1 may play a role as a regulation factor in synovial tissue inflammation.

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

OBJECTIVES Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. MATERIALS AND METHODS Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechsts staining was used to detect apoptotic cells. RESULTS Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. CONCLUSION These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.

A novel modification of a bone repositioning device and a new technique for reestablishing facial contours after mandibular resection surgery

A novel modification of a bone repositioning device previously published by the same authors is introduced. A flexible tube to define the intersegmental bony relationship is filled with light-cured resin. It solidifies following exposure to strong visible light for about 1 min. This technique can be used for bone positioning after mandibular resection surgery and during positioning of the proximal segment after sagittal split ramus osteotomy. The authors also propose a simple method for determining the contour of the reconstructed mandible to regain the original shape and form. The advantage of this technique is its simplicity and flexibility compared with other methods of bone positioning during mandibular segmental surgery.

Predictability and accuracy of maxillary repositioning during bimaxillary surgery using a three-dimensional positioning technique

OBJECTIVE The purpose of this retrospective study was to evaluate the predictability and accuracy of maxillary repositioning during bimaxillary surgery using a three-dimensional positioning technique. STUDY DESIGN Twenty-six adult patients who underwent bimaxillary surgery requiring high superior maxillary impactions were divided into 2 groups. In group A, a three-dimensional positioning technique during maxillary repositioning was used along with an intermediate occlusal splint. In group B, only an intermediate occlusal splint with internal reference points was used. Both groups had measurements from predictive tracings compared to postoperative cephalograms to assess the accuracy of horizontal and vertical movements of the maxilla. RESULTS Group A showed excellent correlation between the planned and actual maxillary positions in vertical and horizontal dimensions. In group B, the maxilla tended to move anteriorly than planned. CONCLUSIONS Use of the three-dimensional positioning technique offered a predictive and accurate method for maxillary repositioning.

Inhibition of Adjuvant Arthritis in Rats by Electroporation with Interleukin-1 Receptor Antagonist

To assess the protective effects of the cytokine inhibitor interleukin-1 receptor antagonist (IL-1ra) on gene induction, an electroporation technique to treat adjuvant-induced arthritis (AIA) in rats was established, and its advantage was estimated in the present study. Electroporation with human IL-1ra was performed in Lewis rats before and after induction of AIA. Local inflammation was evaluated by monitoring hind paw swelling, whereas histological evaluations were performed using paraffin embedded sections of hind paw specimens stained with hematoxylin and eosin. In addition, serum IL-1? levels were analyzed using an enzyme-linked immunosorbent assay. Induction of IL-1ra by our electroporation method inhibited systematic body weight loss and enhancement of local inflammation after intradermal injection of heat-killed Mycobacterium tuberculosis. Notably, IL-1ra electroporation reduced paw swelling, inflammation, and bone erosion scores in embedded sections and serum IL-1? levels induced in AIA rats. The IL-1ra gene induction using the present electroporation technique inhibited local and systematic inflammation in AIA rats. These results indicate that this method may represent a novel pharmacotherapy strategy for arthritis.

Maxillary single-jaw surgery combining Le Fort I and modified horseshoe osteotomies for the correction of maxillary excess

A modified technique of horseshoe osteotomy combined with Le Fort I osteotomy for superior and posterior repositioning of the maxilla is presented. Eight patients with maxillary excess associated with retrogenia or microgenia were treated with this technique in combination with genioplasty. The maxillary segment was repositioned a maximum of 5.0mm posteriorly and 7.0mm superiorly at point A. The mandible autorotated anterosuperiorly to achieve sound occlusion. Point B moved 2.0-10.0mm anteriorly and 5.0-10.0mm superiorly. The pogonion moved 7.0-17.0mm anteriorly in combination with genioplasty. All patients obtained sound occlusion and a good profile after the operation. Almost no skeletal relapse was observed during 1 year of postoperative follow-up. Patients with long faces with maxillary excess and retrogenia often have small, unstable condyles. In these cases, because surgical intervention to the ramus can result in postoperative progressive condylar resorption, maxillary single-jaw surgery with a horseshoe osteotomy, thereby avoiding ramus intervention, is a less invasive option.