Manabu Habu

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.

Relationship Between Inferior Alveolar Nerve Canal Position at Mandibular Second Molar in Patients With Prognathism and Possible Occurrence of Neurosensory Disturbance After Sagittal Split Ramus Osteotomy

PURPOSE To elucidate the relationship between the anatomic position of the inferior alveolar nerve (IAN) at the mandibular second molar and the occurrence of neurosensory disturbances of the IAN after sagittal split ramus osteotomy (SSRO) in patients with mandibular prognathism. Also, the present study evaluated the difference in anatomic position of the IAN between patients with and without mandibular prognathism. PATIENTS AND METHODS Computed tomography images were taken of 28 patients with mandibular prognathism and 30 without prognathism. On these scans, the IANs from the mandibular second molar region to the mandibular foramen in the mandibular ramus were identified. The present study was designed as a cross-sectional study. The distance from the buccal aspect of the IAN canal to the outer buccal cortical margin of the mandible in the mandibular second molar regions was measured on the computed tomography images. Also, the linear distance between the superior aspect of the IAN canal and the alveolar crest in these regions was calculated. In addition, we investigated the presence or absence of contact between the IAN canal and the inner buccal cortical margin of the mandible from the mandibular second molar to the mandibular foramen in the mandibular ramus. Next, we examined whether neurosensory disturbances occurring after SSRO were related to the position of the IAN at the mandibular second molar. RESULTS A significant difference was found in the occurrence of neurosensory disturbances of the IAN after SSRO between men and women (χ(2) test, P < .05). For the distance from the buccal aspect of the IAN canal to the outer buccal cortical margin of the mandible in the mandibular second molar region, a significant difference was found between groups with and without neurosensory disturbances (Students t test, P < .01). The shorter the distance from the buccal aspect of the IAN canal to the outer buccal cortical margin, the more frequent the occurrence of neurosensory disturbances of the IAN. CONCLUSIONS The present results have demonstrated that gender and the anatomic position of the IAN canal at the mandibular second molar are significantly related to the occurrence of neurosensory disturbances of the IAN after SSRO. Therefore, surgeons should clearly inform patients of the increased possibility of neurosensory disturbances after SSRO when the patients are female and are found to have a shorter distance from the buccal aspect of the IAN canal to the outer buccal cortical margin.

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.

Advanced Clinical Usefulness of Ultrasonography for Diseases in Oral and Maxillofacial Regions

Various kinds of diseases may be found in the oral and maxillofacial regions and various modalities may be applied for their diagnosis, including intra-oral radiography, panoramic radiography, ultrasonography, computed tomography, magnetic resonance imaging, and nuclear medicine methods such as positron emission tomography. Of these modalities, ultrasound imaging is easy to use for the detection of noninvasive and soft tissue-related diseases. Doppler ultrasound images taken in the B-mode can provide vascular information associated with the morphology of soft tissues. Thus, ultrasound imaging plays an important role in confirming the diagnosis of many kinds of diseases in such oral and maxillofacial regions as the tongue, lymph nodes, salivary glands, and masticatory muscles. In the present article, we introduce three new applications of ultrasonography: guided fine-needle aspiration, measurement of tongue cancer thickness, and diagnosis of metastasis to cervical lymph nodes.

Correlation of mandibular bone quality with neurosensory disturbance after sagittal split ramus osteotomy

Our aim was to find out whether the quality of bone around the inferior alveolar nerve is correlated with neurosensory disturbance to the nerve after sagittal split ramus osteotomy (SSRO) in patients with mandibular prognathism. Computed tomograms (CT) were taken of 35 patients with mandibular prognathism and 35 without. To assess the density of bone around the inferior alveolar nerve, the width of the buccal cortical bone in the mandibular second molar regions was measured on CT. The Hounsfield units (HU) in the same regions were also measured. The number of HU in the mandible around the second molar regions was significantly higher (p<0.01) in those with neurosensory disturbance (p<0.01). The quality of bone measured by HU is associated with an increased risk of neurosensory disturbance, but the width of buccal bone is not.

Effect of bone quality and position of the inferior alveolar nerve canal in continuous, long-term, neurosensory disturbance after sagittal split ramus osteotomy

OBJECTIVES To examine the relationship between the anatomical position and the bone quality of the inferior alveolar nerve (IFAN) canal and long-term neurosensory disturbance (NSD) of the IFAN after a sagittal split ramus osteotomy (SSRO). MATERIAL AND METHODS CT images were taken of patients with mandibular prognathism. The location of the IFAN and the bone density around the IFAN were measured on CT images. Whether NSD at 6 months and 1 year after SSRO was related to the position and bone quality of the IFAN canal was analyzed. RESULTS Significant correlations were found between the anatomical position and the bone quality of the IFAN canal and the period to the resolution of NSD of the IFAN from 3 months to 1 year after SSRO. If the HU values around the IFAN were greater than 300 HU and/or the distance from the buccal aspect of the IFAN canal to the outer buccal cortical margin was less than 6 mm, NSD of the IFAN at 1 year after SSRO was significantly increased. CONCLUSION It is important to pay particular attention to the anatomical position of the IFAN and the bone quality around the IFAN to predict long-term NSD of the IFAN after SSRO.

A study of the efficacy of ultrasonic waves in removing biofilms

OBJECTIVE The removal of adherent biofilms was assessed using ultrasonic waves in a non-contact mode. MATERIALS AND METHODS In in vitro experiments, Streptococcus mutans (S. mutans) biofilms were exposed to ultrasonic waves at various frequencies (280 kHz, 1 MHz, or 2 MHz), duty ratios (0-90%), and exposure times (1-3 minutes), and the optimal conditions for biofilm removal were identified. Furthermore, the effect of adding a contrast medium, such as micro bubbles (Sonazoid), was examined. The spatial distribution and architecture of S. mutans biofilms before and after ultrasonic wave exposure were examined via scanning electron microscopy. The biofilm removal effect was also examined in in vivo experiments, using a custom-made oral cleaning device. RESULTS When a 280 kHz probe was used, the biofilm-removing effect increased significantly compared to 1 and 2 MHz probes; more than 80% of the adherent biofilm was removed with a duty cycle of 50-90% and a 3 minutes exposure time. The maximum biofilm-removing effect was observed with a duty cycle of 80%. Furthermore, the addition of micro bubbles enhanced this biofilm-removing effect. In in vivo experiments, moderate biofilm removal was observed when a 280 kHz probe was used for 5 minutes. CONCLUSIONS This study demonstrated that ultrasonic wave exposure in a non-contact mode effectively removed adherent biofilms composed of S. mutans in vitro.

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.

Dynamic magnetic resonance sialography for patients with xerostomia.

OBJECTIVE The aim was to evaluate the utility of dynamic magnetic resonance (MR) sialographic images as a diagnostic tool for patients with xerostomia as chief complaint. METHODS Various parameters of dynamic MR sialographic images from 40 healthy volunteers were compared with those from 28 patients having dry mouth as chief complaint. Dynamic MR sialographic images were acquired using a 1.5-T full-body MR system and 2-dimensional fast asymmetric spin-echo (2D-FASE) sequences. Acquisition of the optimal section using 2D-FASE sequencing with single-section acquisition of thick sections was repeated every 30 s before and after a few drops of 5% citric acid was placed on the tongue. Student t test was used to examine differences between the healthy volunteer and patient groups regarding the maximum area of salivary gland ducts before and after citric acid stimulation. RESULTS The maximum area of the detectable ducts in the patient group was significantly smaller (P < 0.001), whereas the change in detectable duct area was significantly lower (P < 0.001). CONCLUSIONS This study suggests that dynamic MR sialographic images and data can be useful in the diagnosis of patients with dry mouth as chief complaint.