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To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.

Systemic sclerosis (SSc) is a relatively rare condition characterized by the excessive production and deposition of collagen within tissue. This condition is thought to be immunologically mediated and, in addition to its notorious cutaneous manifestations, often involves multiple organs. A case is presented of systemic sclerosis associated with extensive tumoural calcinosis in the temporomandibular joint. There has been no evidence of recurrence or complications during approximately 2 years of follow up, but long-term follow up is essential.

Therapeutic angiogenesis is a promising approach to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) to the recipient bed of a rat dorsal skin flap by a drug delivery system with acidic gelatin hydrogel microspheres (AGHMs), and assessed augmentation of neovascularization and flap viability. An axial skin flap was elevated on the back of male Sprague-Dawley rats, and bFGF solution or bFGF-impregnated AGHMs were injected into the recipient bed. The dose of bFGF in the bFGF solution was set to 15 (Sol-15 group), 50 (Sol-50 group), or 150 mug (Sol-150 group). Correspondingly, 2 mg AGHMs were impregnated with 15 (AGHM-15 group), 50 (AGHM-50 group), or 150 mug (AGHM-150 group) bFGF. Other groups of animals received phosphate-buffered saline (Sol-Cont group) or phosphate-buffered saline-impregnated AGHMs (AGHM-Cont group) as controls. Seven days later, analyses of the area of necrosis, microangiographic findings, and histological findings in the flap were carried out. The area of necrosis in the AGHM-150 group was significantly smaller than that in the other groups. Microangiographic and histological analyses showed that neovascularization of the ischemic skin flap significantly increased in the AGHM-150 group as compared with the Sol-150 group and the AGHM-Cont group. These findings suggest that continuous delivery of bFGF to the recipient bed by bFGF-impregnated AGHMs enhances the viability of an ischemic skin flap.

小舌症と小下顎症を伴う症例 (23歳, 女性) に対して, 術前および術後の歯科矯正治療と下顎歯列弓狭窄に対する拡大手術による外科的矯正治療を行い, 治療過程の構音の変化と咀嚼機能について検討した.1.構音検査では, 治療前と同様に/t, d, n, r/は舌と口蓋後方でつくられる口蓋化構音に近い歪み音, /s, dz, ts/は上顎前歯と下口唇で音がつくられる/f, v, pf/に近い歪み音であった.構音動態としては, 治療前と比べ構音位置や構音方法に明らかな差は見られなかった.2.語音発語明瞭度は, 術後6ヵ月で低下したものの, 術後1年3ヵ月で治療前とほぼ同程度にまで改善していた.治療過程で口唇音や母音の異聴が一時的に増加したが改善し, 異聴傾向としては治療前とほぼ同様であった.3.文章了解度は, 術後6ヵ月で低下したものの, 術後9ヵ月で治療前とほぼ同程度にまで改善していた.4.咀嚼機能にも改善が見られた.

Many differential diagnoses must be considered in the diagnosis of cervical lymphadenopathy. Lymph nodes, which are responsible for the production of lymphocytes, are crucial to the body's defense mechanism and are responsible for the immune response to bacteria, viruses, toxins, and other materials that invade the lymph vessels. They are also susceptible to metastasis of malignant tumors. Therefore, cervical lymphadenopathy may indicate a variety of disorders. In addition to interviews, visual inspection, and palpation, blood tests and diagnostic imaging such as X-ray, CT, MRI, and ultrasound are also essential. In cases where a diagnosis can not be made based on these tests, biopsy of the swollen lymph node must be performed. Cervical lymph node biopsy requires the utmost care in addition to the patient's informed consent. Many nerves and blood vessels are present in the neck region, and accidental injury to these nerves and blood vessels may result in serious sequelae, which in many cases incur medical malpractice lawsuits. Therefore, cervical lymph node biopsy should not be performed unassisted by inexperienced surgeons. Herein, we present the lymph node disorders and diagnostic methods that are important for differential diagnosis, and describe the points of caution regarding lymph node biopsy.

Background/Aims: CpG island (CGI) methylator phenotype (CIMP) is strongly associated with poor prognosis in neuroblastomas (NBLs; hazard ratios 7–22). Methylation of nonpromoter CGIs is useful to detect the presence of the CIMP, while the poor prognosis is considered to be caused by gene silencing due to promoter methylation. Here, promoter CGIs targeted by the CIMP were searched for. Methods: A genome-wide screening was performed by methylation-sensitive representational difference analysis of CIMP(+) and CIMP(–) NBLs. Results: Promoter CGIs of 9 genes were methylated in CIMP(+) NBL cell lines and caused silencing of their downstream genes. On analysis of 90 clinical specimens, CYP26C1,FERD3L (N-TWIST), CRYBA2 and PCDHGC4 were methylated at significantly higher incidences in CIMP(+) NBLs than CIMP(–) NBLs, while the difference was unclear for NPY, SPAG6, DDIT4L, CHR3SYT and C6Orf141. Methylation of CYP26C1 and FERD3L was significantly associated with poor prognosis, but weaker than the presence of the CIMP. Treatment of an NBL cell line with a demethylating agent caused demethylation of multiple promoter CGIs, and enhanced 13-cis-retinoic acid-induced neuronal differentiation. Conclusion: Our results indicate that the CIMP causes poor prognosis of NBLs by inducing methylation of multiple promoter CGIs with various incidences.

The reconstruction of the soft palate following resection of a tumour remains a challenge to this day. We recently reconstructed the soft palate by a procedure using the palatal mucoperiosteal flap, with blood supply from the greater palatine vessels. This operative procedure is simple, easy to perform and minimally invasive, and has been used on 2 patients with satisfactory results and good postoperative function.

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.

To effectively treat serious bone defects using bone regenerative medicine, there is a need for the development of a small chemical compound that potently induces bone formation. We now report a novel osteogenic helioxanthin-derivative, TH. TH induced osteogenic differentiation in MC3T3-E1 cells, mouse primary osteoblasts, and mouse embryonic stem cells. The combination of TH and bone morphogenetic protein (BMP) 2 induced the mRNA expression of osteoblast marker genes and calcification in primary fibroblasts. The TH induced the mRNA of the inhibitor of DNA-binding 1 (Id-1), and its osteogenic effect was inhibited by Smad6 or Noggin. Furthermore, TH induced the mRNA expression of Bmp4 and Bmp6. These data suggest that TH exerts its potent osteogenic effect in a BMP-dependent manner by enhancing the effects of the existing BMPs and/or increasing the expression of Bmp4 and Bmp6. TH may help establish a more efficient bone regeneration system.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone response after the placement of implants in the femurs of mice. titanium implants 1.0mm in diameter were placed into the middle of the femurs of 9-week-old male COX-2 wild-type ( COX-2 +/+ ) and knockout ( COX-2 −/− ) mice. For RNA analysis, the mice were killed 0, 1, 2, 4, 7 and 56 days after implantation. RNA was extracted from the bone surrounding the implants. For histological analysis, the mice were killed 4 and 8 weeks after treatment, and undecalcified sections were prepared. Contact microradiography was performed, and the sections were stained with 1% toluidine blue for histological examination. Histomorphometric measurements were obtained with a computer-based image analyser to quantify bone newly formed around the implant and the rate of implant–bone contact. Expression of COX-2 and osteocalcin mRNA was induced in bone surrounding implants in COX-2 +/+ mice, but not in COX-2 −/− mice. In cortical bone, the implant surface was in direct contact with newly formed bone lamellae in COX-2 +/+ mice; new bone formation was minimal in COX-2 −/− mice. These results suggest that COX-2 plays an essential role in osseointegration and provide evidence that COX-2-selective non-steroidal anti-inflammatory drugs may interfere with osseointegration clinically.

The CpG island methylator phenotype (CIMP) was closely associated with poor overall survival (OS) in Japanese neuroblastoma (NBL) cases in our previous study. Here, in German NBL cases, CIMP(+) cases (n=95) showed markedly poorer OS (hazard ratio (HR)=9.5; P

We identified original tissue-committed precursors with limited self-renewal capacity from human corneal stromal (HCS) cells and human corneal endothelial (HCE) cells, then tried to determine the distribution and proliferative capacity of the precursors.Experimental study.Eighteen human corneas from donors 56 to 68 years old.Human corneal stromal cells were divided into groups based on distance from the center of the cornea:6 mm (central), 6 to 8 mm (paracentral), and 8 to 10 mm (peripheral). Human corneal endothelial cells were separated into 2 groups:7.5 mm (central) and 7.5 to 10 mm (peripheral) from the center. Each group was subjected to the sphere-forming assay using serum-free medium containing growth factors in floating culture. Sphere numbers and the proliferative capacity of spheres in adherent culture were compared among the groups.Density and proliferative capacity of precursors from each area of HCS and HCE cells.Primary spheres were isolated from all groups of HCS and HCE cells. The rate of primary sphere formation from peripheral HCS cells was higher than those of the other 2 groups, being 1.5-fold greater than in the paracentral cornea and 4-fold greater than in the central cornea. The rate of primary sphere formation by peripheral HCE cells was significantly higher than that by central HCE cells, being 4-fold greater than in the central cornea. There were no differences in the proliferative capacity of HCS and HCE cell spheres from the different areas after adherent culture.All HCS and HCE cells contain a significant number of precursors, but the peripheral cells have a density of precursors higher than that of the central cells. Precursors from each area do not show differences of proliferative capacity. Our findings may in part explain changes after excimer laser treatment and may have implications for corneal transplantation procedures.

To identify potent bioactive factors for in vivo tissue regeneration by comprehensive screening remains a challenge for regenerative medicine. Here we report the development of an ES cell-based monitoring system for osteogenic differentiation, the identification of a potent combination of osteogenic genes using such a system, and an evaluation of its therapeutic potentials. ES cells were isolated from mice carrying a transgene expressing GFP driven by the 2.3 kb fragment of rat type I collagen alpha1 promoter. Using these cells engineered to fluoresce on osteogenic differentiation, we screened cDNA libraries and combinations of major osteogenesis-related genes. Among them, the combination of constitutively active activin receptor-like kinase 6 (caALK6) and runt-related transcription factor 2 (Runx2) was the minimal unit that induced fluorescence. The combination efficiently induced osteogenic differentiation in various cell types, including terminally differentiated nonosteogenic cells. The cooperative action of the combination occurred through protein stabilization of core binding factor beta (Cbfb), induction of Runx2-Cbfb complex formation, and its DNA binding. Furthermore, transplantation of a monolayer sheet of fibroblasts transduced with the combination achieved bone regeneration within 4 wk in mouse calvarial bone defects. Thus, we successfully identified the potent combination of genes for bone regeneration, which helped broaden cell sources.

Background Collagenous connective tissue membranes (biosheets) are useful for engineering cardiovascular tissue in tissue engineering. The aim was to evaluate the use of biosheets as a potential tracheal substitute material in vivo in a rabbit model. Methods Group 1: Rectangular-shaped Gore-Tex (4×7mm) was implanted into a 3×6mm defect created in the midventral portion of the cervical trachea. Group 2: Rectangular-shaped dermis was implanted into a tracheotomy of similar size. Group 3: Biosheets were prepared by embedding silicone moulds in dorsal subcutaneous pouches in rabbits for 1month. Rectangular-shaped biosheets were implanted into a tracheotomy of similar size in an autologous fashion. All groups (each containing 10 animals) were sacrificed 4weeks after implantation. Main Results All materials maintained airway structure for up to 4weeks after implantation. Regenerative cartilage in implanted Biosheets in group 3 was confirmed by histological analysis. Tracheal epithelial regeneration occurred in the internal lumen of group 3. There were significant differences in the amounts of collagen type II and glycosaminoglycan between group 3 and group 1 or 2. Conclusion We confirm that cartilage can self-regenerate onto an airway patch using Biosheets.

Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, β-tricalcium phosphate (β-TCP) granules, or β-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60 % those of native bones, respectively). All tests showed poor bone regeneration in the β-TCP and β-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

Background Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common human myelopathy that leads to spinal cord compression. No disease-modifying drug for OPLL has been identified, whereas surgery and conservative management have been established. Objectives To evaluate the therapeutic potential of the H2 blocker famotidine for ectopic ossification in the cervical spine in an OPLL mouse model. Methods The H2 blocker famotidine was orally administered to Enpp1 ttw/ttw mice, a model of OPLL, at either 4 or 15 weeks of age. Radiological and survival rate analyses were performed to assess the effects of famotidine on OPLL-like lesions and mortality in Enpp1 ttw/ttw mice. Results Oral administration of famotidine suppressed the progression of OPLL-like ectopic ossification and reduced mortality in Enpp1 ttw/ttw mice when administration began at 4 weeks of age, early in the development of ossification. Conclusions This study points to the use of famotidine as a disease-modifying drug for ectopic ossification of spinal soft tissue, including OPLL.

A 2-stage procedure combining maxillary advancement by distraction technique with mandibular setback surgery was used to correct jaw deformities in 5 patients with severe maxillary retrusion secondary to cleft lip and palate. First, a Le Fort I maxillary osteotomy was performed. Immediately after maxillary distraction, the distraction device was removed. The advanced maxilla was fixed with miniplates after adjusting the length and direction of advancement, and mandibular setback surgery was performed simultaneously to obtain a normal occlusal relationship. This 2-stage procedure resulted in stable occlusion and a markedly improved facial profile.

Objective: To assess the transcriptional regulation of cyclooxygenase-2 by bone morphogenetic protein2 in isolated bone marrow stromal cells obtained from cyclooxygenase-2 wild-type and cyclooxygenase-2defrcient mice. Materials and Methods: Bone marrow stromal cells were cultured for 14 days in the presence of osteogenic medium with or without bone morphogenetic protein-2. Real-time polymerase chain reaction analysis of the relative expression of cyclooxygenase-2 and osteocalcin, a late osteogenic differentiation marker mRNA, were examined. Prostaglandin EZ accumulation in the culture media were measured by radioimmunoassay. Activity and staining of alkaline phosphatase, an early osteogenic differentiation marker, were examined. Results: Bone morphogenetic protein-2 induced cyclooxygenase-2 mRNA and prostaglandin production in cultured bone marrow stromal cells derived from cyclooxygenase-2 wild-type mice. Alkaline phosphatase activity was lower in cyclooxygenase-2-deficient control cultures than in cyclooxygenase-2 wild-type cultures, and treatment with bone morphogenetic protein-2 stimulated alkaline phosphatase activity only in cyclooxygenase-2 wild-type cultures. A similar reduction was seen in cyclooxygenase-2 wild-type cells treated with NS-398, a selective inhibitor of cyclooxygenase-2. Alkaline phosphatase staining revealed a 30% decrease in the cyclooxygenase-2-deficient control cultures, and the addition of bone morphogenetic protein-2 increased alkaline phosphatase staining only in cyclooxygenase-2 wild-type cultures. Bone morphogenetic protein-2 stimulated osteocalcin mRNA expression in bone marrow stromal cells derivedfrom cyclooxygenase2 wild-type mice but not in cyclooxygenase-2-deficient cells. Conclusions: Bone morphogenetic protein-2 induces cyclooxygenase-2 in bone marrow stromal cells transcriptionally and the induction of cyclooxygenase-2 may play a role in the effects of bone morphogenetic protein-2 on bone metabolism in vitro.

Objective : To report a simplified technique for nasoalveolar moulding for treatment of infants with unilateral and bilateral cleft lip and palate. Patients and Methods : One or 2 stents made of plastic cobalt-chrome orthodontic wires 0.8-mm thick were used with a conventional palatal plate (Hotz plate) to treat 7 patients with unilateral cleft lip and palate and 3 with bilateral cleft lip and palate. Results : This modified technique for nasoalveolar moulding permitted the nose of patients with unilateral and bilateral cleft lip and palate to be easily and precisely moulded. This technique also reduced stress for the patients and their parents because it did not require extraoral appliances, except for surgical tape over the cleft lip. Time-consuming adjustments of the nasal stent were also unnecessary. Conclusion : The wire nasal stent facilitated nasoalveolar moulding before cheiloplasty for unilateral and bilateral cleft lip and palate.

We report a case of van der Woude syndrome associated with Hirschsprung disease. The patient was delivered vaginally. Our department was consulted because of cleft lip and palate (CLCP). After cheiloplasty, the department of pediatric surgery was consulted because of continuous vomiting. Hirschsprung disease was diagnosed.Hirschsprung disease occurs in 1/5, 000 live births and is caused by defective development of embryonic neural crest cells at the vagal level, leading to intestinal obstruction. The patients show severe vomiting and constipation, but owing to improved surgical techniques, the number of curable patients is increasing. Our patient received radical treatment and has no difficulty in eating or excretion at present.Several reports have described patients with CLCP and Hirschsprung disease, and all have had mental retardation. Moreover, there are several syndromes associated with both CLCP and Hirschsprung disease, such as Goldberg-Shprintzen syndrome, Waardenburg syndrome, and Smith-Lemli-Opitz syndrome. Each syndrome has their own differential diagnosis, but our patient did not meet the diagnostic criteria. We gave our patient a diagnosis of van der Woude syndrome combined with Hirschsprung disease for the present.However, mental retardation cannot be ruled our in this patient, and he might have other congenital malformations that have not been identified. If so, he may have a subtype of a syndromic disease.

Objective Evaluation of the dental arch relationships of Japanese patients with unilateral cleft lip and palate (UCLP) from the orthodontic clinic of the University of Tokyo Hospital (UTH) compared with patients treated by the Oslo Cleft Team, Norway. Design Retrospective study and comparison with previous reports. Materials Dental models of 24 patients with UCLP in UTH taken before orthodontic treatment and before alveolar bone grafting were included. Surgeons in many hospitals performed primary surgeries. These models were matched for age and gender with 24 models from a consecutive series of patients treated by the Oslo Cleft Team as part of the Eurocran Good Practice Archive. A total of 48 models were evaluated. Main Outcome Measure Dental arch relationship was rated with the Goslon Yardstick. The strength of agreement of rating was assessed with weighted kappa statistics. Results Intra- and interexaminer agreements evaluated by weighted kappa statistics were high, indicating good reproducibility. Almost 60% of the patients in UTH were classified into poor or very poor categories, and the mean Goslon score was 3.50. These results show a contrast to those in Oslo and were the poorest in comparison with previous reports. Conclusion Dental arch relationships in patients with UCLP in UTH were poor. This seemed to be attributable to surgical procedures, but a factor of racial difference in the craniofacial morphology was also considered. Further intercenter research is required to clarify this point.

Background: Ocular surface reconstruction with autologous oral mucosal epithelium has attracted attention as a novel treatment strategy that avoids allograft rejection. Objectives: To evaluate the absorption of ultraviolet (UV) A or B irradiation by human oral mucosal epithelium cultured on human amniotic membrane. Methods: Human oral mucosal and limbal epithelial cells were co-cultured on amniotic membrane with inactivated 3T3 fibroblasts. The cell sheets were also subjected to UV-A (365 nm) or UV-B (302 nm) irradiation at energy levels ranging from 50 to 800 µW/cm2, and the UV absorption rate was measured with a UV irradiation meter. Results: Cultured oral mucosal epithelium had a structure with 3–5 layers of cells, consistent with the histological features of cultured corneal limbal epithelium after 4 weeks. The decrease in UV-A absorption of cultivated oral mucosal epithelium ranged from 25 to 36% of that for cultured corneal epithelium. The increase in UV-B absorption by cultured oral mucosal epithelium between 200 and 800 µW/cm2 was approximately 145% of that for cultured corneal limbal epithelium. Conclusion: Our data demonstrated that cultured oral mucosal epithelium has low UV-A and high UV-B absorption capacity as compared with those of cultured corneal epithelium, suggesting that oral mucosal epithelium can compensate for UV absorption of corneal epithelium.

Highlights • Mammary analog secretory carcinoma (MASC) is a rare malignancy of the salivary glands characterized by ETV6–NTRK3 fusion gene. • We diagnosed a case of MASC by molecular approaches (FISH, RT-PCR and sequencing). • Furthermore, we searched novel genetic and epigenetic abnormalities in this MASC., Introduction Mammary analog secretory carcinoma (MASC) is a newly described rare malignancy of the salivary glands characterized by an ETS variant 6 (ETV6)–neurotrophic tyrosine kinase receptor type 3 (NTRK3) fusion gene (EN fusion gene). Presentation of case We present a case of MASC derived from the left upper lip in a 61-year-old woman. ETV6 rearrangement was detected by fluorescence in situ hybridization (FISH). The presence of EN fusion transcripts was verified by reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing of the PCR products. Accordingly, this tumor was diagnosed as MASC. Moreover, we performed mutation analysis of the 50 known cancer-related genes using next-generation sequencing. No mutation of cancer-related genes was identified here. Subsequently, the methylation status in promoter region of tumor-suppressor genes, RASSF1A and RARB2, was examined. Both genes have been reported to be methylated in malignant salivary gland tumors, but they were found to be unmethylated. Discussion Recent studies have demonstrated that distinct types of malignant salivary gland carcinomas are driven by specific, highly recurrent genetic alterations. Detection of molecular abnormalities could be powerful diagnostic tools in the field of salivary gland tumors in near future. Conclusion We experienced a rare malignant salivary gland carcinoma, MASC. We diagnosed this tumor by molecular approach and subsequently tried to identify novel molecular abnormalities. Although no novel molecular alteration except for EN fusion gene was identified, this result might represent the favorable prognosis of patients with MASC.

In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

We created sugar-based crosslinkers for precise chemical crosslinking of atelocollagen to form a stable hydrogel microenvironment for three-dimensional (3D) cell culture. Crosslinkers were synthesized by partially oxidizing glucose, fructose, maltose, sucrose, or raffinose with periodate. The partial oxidization of sugar generated multiple aldehydes in the molecule, which acted as multifunctional crosslinkers, allowing atelocollagen to form chemical hydrogels. The crosslink reaction of atelocollagen was competitively suppressed by the addition of amino acid-rich medium, enabling flexible control of 3D molecular density in the acquired hydrogel. To evaluate structural stability, ultrasonic stress was loaded onto the acquired hydrogel and sequential measurement of water content showed that the crosslinking considerably stabilized the hydrogel structure. The effectiveness of the atelocollagen hydrogel as a 3D culture scaffold was analyzed by embedding and culturing endothelial cells or cardiomyocytes in it. Endothelial cells formed 3D capillary-like structures at 12 h, and cardiomyocytes formed a beating 3D netlike structure by 7 days. These findings suggest that crosslinked atelocollagen hydrogel effectively functions as a stable scaffold in 3D culture.

In vitro assays revealed that COX-2 inhibitors with CA II inhibitory potency suppressed both differentiation and activity of osteoclasts, whereas that without the potency reduced only osteoclast differentiation. However, all COX-2 inhibitors similarly suppressed bone destruction in adjuvant-induced arthritic rats, indicating that suppression of osteoclast differentiation is more effective than that of osteoclast activity for the treatment. Introduction: Cyclooxygenase (COX)-2 and carbonic anhydrase II (CA II) are known to play important roles in the differentiation of osteoclasts and the activity of mature osteoclasts, respectively. Because several COX-2 selective agents were recently found to possess an inhibitory potency against CA II, this study compared the bone sparing effects of COX-2 selective agents with and without the CA II inhibitory potency. Materials and Methods: Osteoclast differentiation was determined by the mouse co-culture system of osteoblasts and bone marrow cells, and mature osteoclast activity was measured by the pit area on a dentine slice resorbed by osteoclasts generated and isolated from bone marrow cells. In vivo effects on arthritic bone destruction were determined by radiological and histological analyses of hind-paws of adjuvant-induced arthritic (AIA) rats. Results: CA II was expressed predominantly in mature osteoclasts, but not in the precursors. CA II activity was inhibited by sulfonamide-type COX-2 selective agents celecoxib and JTE-522 similarly to a CA II inhibitor acetazolamide, but not by a methylsulfone-type COX-2 inhibitor rofecoxib. In vitro assays clearly revealed that celecoxib and JTE-522 suppressed both differentiation and activity of osteoclasts, whereas rofecoxib and acetazolamide suppressed only osteoclast differentiation and activation, respectively. However, bone destruction in AIA rats was potently and similarly suppressed by all COX-2 selective agents whether with or without CA II inhibitory potency, although only moderately by acetazolamide. Conclusions: Suppression of osteoclast differentiation by COX-2 inhibition is more effective than suppression of mature osteoclast activity by CA II inhibition for the treatment of arthritic bone destruction.

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor(FGF)-2, insulin-like growth factor(IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1α25-dihydroxy vitamin D3, L-3,3′,5′-triodothyronine, interleukine-1 receptor antagonist, 17β-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10–12-fold increase with 5 μg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 μg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.

Objectives: To assess the effects of platelet-rich plasma on the proliferation of cultured mouse bone marrow stromal cells and on the production of cyclooxygenase-2 and prostaglandin E2 by these cells, and to determine whether the mitogenic activity of platelet-rich plasma is enhanced by combination with a selective cyclooxygenase-2 inhibitor Materials and Methods: A dish with 2 chambers, separated by a porous membrane, and platelet-rich plasma gel were used to assess the effects of platelet-rich plasma on cultured mouse bone marrow stromal cells in the absence of serum. The cells were cultured for 7 days with low (0.05 mL), medium (0.25 mL), or high (0.5 mL) volumes of platelet-rich plasma gel, and the effects on bone marrow stromal cell proliferation were examined. Concentrations of cyclooxygenase-2 and prostaglandin E2 in the culture media were assayed. The effect of celecoxib, a cyclooxygenase-2 inhibitor, on the mitogenic activity of platelet-rich plasma was examined. The mitogenic activity of platelet-rich plasma was compared between bone marrow stromal cells from cyclooxygenase-2-deficient mice and bone marrow stromal cells from wild-type littermates. Results: Platelet-rich plasma stimulated bone marrow stromal cell proliferation over a 7-day period. Platelet-rich plasma potently increased the cyclooxygenase-2 mRNA level and prostaglandin E2 production, and endogenous prostaglandin E2 treatment inhibited the proliferation of bone marrow stromal cells. The cyclooxygenase-2 inhibitor celecoxib stimulated the mitogenic activity of platelet-rich plasma. The mitogenic activity of platelet-rich plasma that was added to the culture medium of bone marrow stromal cells derived from cyclooxygenase-2-deficient mice was approximately twice that of platelet-rich plasma added to the culture medium of bone marrow stromal cells derived from wild-type mice. Conclusions: Platelet-rich plasma may directly induce bone formation by stimulating the proliferation of bone marrow stromal cells. Because cyclooxygenase-2 induction by platelet-rich plasma blunts its mitogenic activity, combination with a cyclooxygenase-2 inhibitor may enhance the osteogenic activity of platelet-rich plasma.

Summary Background The recipient bed is a promising target of angiogenic therapy to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) gene to the recipient bed by a plasmid-based method with electroporation, and assessed the effects on flap viability in a rat dorsal skin flap model. Methods A 25×90mm 2 axial skin flap was elevated on the back of male Sprague–Dawley rats. Two days before flap elevation, an expression plasmid vector containing the bFGF gene with the signal sequence was injected into the dorsal muscles beneath the skin flap, and then electroporation was delivered (FGF-E + group). As control, rats were injected with a plasmid vector containing LacZ gene (LacZ-E + group), instead of bFGF gene. Other groups of animals received plasmid vector containing bFGF (FGF-E − group) or LacZ (LacZ-E − group) gene without electroporation. Seven days later, the area of necrosis and neovascularisation of the skin flap were evaluated. Results The bFGF gene was successfully transferred to the dorsal muscles, and bFGF was expressed in muscle tissue. The area of flap necrosis (%) in the FGF-E + group (21.7±5.3%) was significantly smaller than that in the LacZ-E + (28.3±4.1%), FGF-E − (29.7±3.3%), and LacZ-E − (28.1±2.5%) groups. Postmortem angiograms and histological analyses showed that vascularisation in the distal part of the skin flap was significantly increased in the FGF-E + group compared with the other groups. Conclusion These findings suggested that gene delivery of bFGF to the recipient bed muscles enhanced vascularity and viability of an ischemic skin flap, and that plasmid-based gene delivery with electroporation was a suitable delivery method.

Neuroblastoma, one of the most common pediatric solid tumors, is characterized by two extreme disease courses, spontaneous regression and life-threatening progression. Here, we conducted a genome-wide search for differences in DNA methylation that distinguish between neuroblastomas of the two types. Three CpG islands (CGI) and two groups of CGIs were found to be methylated specifically in neuroblastomas with a poor prognosis. By quantitative analysis of 140 independent cases, methylation of all the five CGI (groups) was shown to be closely associated with each other, conforming to the CpG island methylator phenotype (CIMP) concept. The presence of CIMP was sensitively detected by methylation of the PCDHB CGIs and associated with significantly poor survival (hazard ratio, 22.1; 95% confidence interval, 5.3-93.4; P < 0.0001). Almost all cases with N-myc amplification (37 of 38 cases) exhibited CIMP. Even in 102 cases without N-myc amplification, the presence of CIMP (30 cases) strongly predicted poor survival (hazard ratio, 12.4; 95% confidence interval, 2.6-58.9; P = 0.002). Methylation of PCDHB CGIs, located in their gene bodies, did not suppress gene expression or induce histone modifications. However, CIMP was significantly associated with methylation of promoter CGIs of the RASSF1A and BLU tumor suppressor genes. The results showed that neuroblastomas with CIMP have a poor prognosis and suggested induction of silencing of important genes as an underlying mechanism.

Background— Various growth factors promote collateral vessel development and are regarded as promising for the treatment of vascular occlusive diseases. However, an efficacious delivery system for them has yet to be established. We devised a strategy to augment functional collateral vessels by using acidic gelatin hydrogel microspheres (AGHMs) incorporating basic fibroblast growth factor (bFGF). The aim of the present study was to investigate the hypothesis that by intra-arterial (IA) administration of bFGF-impregnated AGHMs, bFGF could be delivered from AGHMs trapped in distal small-diameter vessels and thereby induce functional collateral vessels with an assured blood supply through the process of arteriogenesis. Methods and Results— Various sizes of AGHMs (3 mg) incorporating 125 I-labeled bFGF were injected into the left internal iliac artery of a rabbit model of hindlimb ischemia. Less than 50% of radioactivity accumulated in the ischemic hindlimb after injection of AGHMs that were 10 μm in diameter, whereas ≈80% of radioactivity was counted in the ischemic limb after administration of 29- or 59-μm-diameter AGHMs. Calf blood pressure ratio and the ratio of regional blood flow of the bilateral hindlimbs immediately before and after IA administration of 29-μm–diameter AGHMs showed no significant change. Then we evaluated the function of the developed collateral vessels 28 days after IA administration of bFGF-impregnated, 29-μm-diameter AGHMs. IA administration of bFGF-impregnated AGHMs induced marked collateral vessel improvement compared with IA administration of phosphate buffered saline–treated AGHMs and intramuscular administration of bFGF-impregnated AGHMs. Conclusions— IA administration of bFGF-impregnated, 29-μm-diameter AGHMs strongly induced functional collateral vessels without worsening ischemia, indicating the possible therapeutic usefulness of this approach.

Objective To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis. Methods Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes. Results No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time. Conclusion These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage.

Mammalian bones have three distinct origins (paraxial mesoderm, lateral plate mesoderm, and neural crest) and undergo two different modes of formation (intra-membranous and endochondral). Bones derived from the paraxial mesoderm and lateral plate mesoderm mainly form through the endochondral process. During this process, hypertrophic chondrocytes play a vital role in inducing both osteogenesis and angiogenesis. One of the essential osteogenic factors secreted from hypertrophic chondrocytes is Indian hedgehog (Ihh). In contrast, bones derived from the neural crest mainly form through the intramembranous pro-cess and do not require Ihh. Thus, depending on their origin, bones have distinct signaling properties, which need to be considered in the research and application of bone biology.

This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-κB signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-κB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IκB kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-κB pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-κB-mediated downregulation is essential for efficient osteoclast differentiation.

This study attempted to characterize the ability of thermoreversible gelation polymer (TGP) to induce differentiation of human mesenchymal stem cells (hMSC) into osteoblasts. Using a long oligo microarray system consisting of 3760 genes, we compared the expression profiles of the cells in 2-dimensional (2D) culture, 3D culture in collagen gel, and 3D culture in TGP with or without osteogenic induction. Compared to 2D culture, the gene expression profile of hMSC showed almost the same pattern in TGP without osteogenic induction, but 72% of genes (2701/3760) were up-regulated in collagen gel. With osteogenic induction, hMSC showed higher ALP activity and osteocalcin production in TGP as compared to 2D culture. Moreover, up-regulation and down-regulation of osteogenic genes were augmented in 3D culture in TGP as compared to 2D culture. As TGP is chemically synthesized and completely free from pathogen such as prion in bovine spongiform encephalopathy, these results suggest that TGP could be applied clinically to induce osteogenic differentiation of hMSC.

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.

The rotation culture method adopted in our experiments induced numerous aggregates of bone marrow stromal cells (BMSCs); this method augments the frequency of cell–cell contacts and thereby facilitates cell aggregation. Cell condensation plays an important role in differentiation and development. Cell adhesion molecules and the extracellular matrix (ECM) are important regulators of cell adhesion and apoptosis. Therefore, we investigated not only whether aggregates of BMSCs could be formed by rotation culture, but also whether expression of N-cadherin and β1-integrin was involved in the formation of cell aggregates and in the avoidance of apoptosis. The culture medium was supplemented with fibroblast growth factor-2 (FGF-2) or transforming growth factor-β1 (TGF-β1), expected to prompt the formation of cell aggregates. This study enabled the formation of numerous aggregates of BMSCs by rotation culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that FGF-2 and TGF-β1 increased expression of N-cadherin and β1-integrin. Morphological analysis by light microscopy indicated that FGF-2 and TGF-β1 increased cell–cell adhesion and formation of cell aggregates. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay indicated that FGF-2 and TGF-β1 prevented apoptosis. These results suggest that numerous aggregates of BMSCs were formed efficiently by rotation culture since FGF-2 and TGF-β1 increase cell–cell adhesion and rotation increases cell–cell contact.

Regenerative medicine, which takes advantage of the unique capacity of stem cells, is a novel medical strategy to cure irreversible organ failure. There are three important elements in regenerative medicine: cells, scaffolds and signals. Although substantial progress regarding each element has been made in the past few years, it still falls short of clinical applications. In the geriatric field, osteoporosis, osteoarthritis and periodontitis are the major targets of regenerative medicine. They are usually not life-threatening, but often severely affect QOL of elderly patients. Since elderly people have already reduced number of stem cells in bone and cartilage, we need to know the sufficient conditions for osteogenesis and chondrogenesis by comprehensively screening various conditions. We developed a marker gene system expressing green fluorescence protein (GFP) under the control of osteoblast- and chondrocyte-specific promoters. This system helps us monitor osteoblast and chondrocyte differentiation easily, precisely and non-invasively. Using this system, we are now trying to find the sufficient conditions for osteogenesis and chondrogenesis, and to discover osteogenic and chondrogenic small compounds.

Objective : In the present study, we delivered vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene to a rabbit model of hind limb ischemia utilizing an ex vivo method of gene transfer, and evaluated the functional performance of the developed collateral vessels. Method : The left femoral artery of a male Japanese White rabbit was excised to induce limb ischemia, and a section of skin was resected for culture of auto-fibroblasts. Twenty days later, the VEGF gene, bFGF gene or β-galactosidase gene (LacZ) was adenovirally transferred to the cultured auto-fibroblasts (5 × 106 cells), and the next day, a pair of specifically infected fibroblasts (total 1 × 107 cells) was injected via the left internal iliac artery of the same rabbit. Pairs of transferred genes into the fibroblasts were as follows: LacZ/LacZ (control group), VEGF/LacZ (VEGF group), bFGF/LacZ (FGF group) and VEGF/bFGF (combination group). Twenty-eight days after cell administration, collateral development and its function were evaluated. Results : Calf blood pressure ratio, resting blood flow of the left iliac artery and capillary density of ischemic muscle showed similar degrees of angiogenic effects in the VEGF and FGF groups, which were significantly greater than those in the control group. On the contrary, angiographic score, collateral conductance and smooth muscle cell (SMC)-positive vessel density in the FGF group were significantly greater than those in the VEGF group. In the combination group, collateral conductance showed synergistic effects, and in vivo blood flow and smooth muscle cell-positive vessel density revealed additive effects of VEGF and bFGF. Conclusion : These findings suggested that bFGF-induced collateral development exceeded VEGF-induced collateral development in the induction of arteriogenesis, and that combined gene delivery of VEGF and bFGF produced additive or synergistic effects of collateral development as compared with the effects induced by transfer of each gene alone.

Proinflammatry cytokines, tumor necrosis factor-α combined with interleukin-1β, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite peroxynitrite (ONOO-) via inducible nitric oxide synthase (iNOS) in murine osteoblasts. In this study, to properly estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid that specifically targets the iNOS gene for potential long-lasting inhibition. Transformed antisense cell lines were identified by 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NO synthase activity, and 4) the level of NO production. These cell lines targeting iNOS, which showed decreased production of both NO and ONOO-, prevented the inhibition of osteoblastic differentiation as was assayed by the mRNA expression of type I collagen, alkaline phosphatase, osteocalcin, and Core binding factor in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to reduce the cytokine-induced inhibition of osteoblastic activity.

A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.

Secondary correction of bilateral nasal deformity associated with a cleft lip is common. However, few reports have referred to the correction of the wide nasal root. In this study we describe a technique other than osteotomy for the correction of the wide nasal root used in five Oriental patients with bilateral nasal deformity associated with cleft lip. Satisfactory results were obtained, and two representative cases are described.

The surgical treatment of a patient with hereditary angioneurotic edema (BANE) is described in the present report. HANE is an autosomal dominant hereditary disease that causes acute and local submucocutaneous edema. It is a very rare disease in Japan and has been reported in only 20 femilies.The subject was a 24-year-old woman with right upper pericoronitis who had been given a diagnosis of HANE at the age of 13 years. Patients with HANE often have fatal laryngeal edema during dental treatment such as extraction of teeth. Therefore, our patient received ginigivectomy after being hospitalized. Postoperatively, she had mild respiratory distress, but it was controlled by intravenous drip infusion of C 1 esterase inhibitor. When treating patients with HANE, we should take sufficient measures to avoid the development of edema, even when treatment is minimally invasive. Furthermore, sufficient preparation for urgent tracheotomy is necessary owing to the risk of laryngeal edema.

Hypermethylation of the 5' upstream region (5' region) of the human p16(CDKN2A) (p16) gene is known to cause silencing, which is involved in a wide range of human cancers. For the rat p16 gene, its 5' region has not been cloned, and it is uncertain whether surrogate use of exon 1 alpha is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon 1 alpha gave false positive results in three samples of normal rat mammary epithelia and in two of six primary mammary carcinomas. Therefore, we determined the nucleotide sequence of the 5' region of the rat p16 gene. To confirm that methylation status of the 5' region is correlated with p16 expression, the methylation status was analyzed by bisulfite sequencing and methylation-specific PCR in three samples of normal mammary glands, six samples of mammary carcinomas and four cell lines. The 5' region was demethylated in all of the three normal and six carcinoma samples that fully expressed p16. On the other hand, the 5' region was highly methylated in the 3Y1 cell line, which lacked p16 expression, but without deletion. These results showed that the methylation status of the 5' region was more closely correlated with p16 expression than that of the exon 1 alpha and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.

Inverted papilloma of the nasal and paranasal sinuses is an uncommon, histopathologically benign neoplasm. However, because this tumor has an aggressive nature and a risk of recurrence, complete resection is essential.We report two cases of inverted papilloma of the maxillary sinus. Patient 1 was an 80-year-old man with a tumor of the tongue. Accidentally, computed tomography showed a mass of a different density in the maxillary sinus and nasal cavity. The suspected diagnosis was a tumor of the left maxillary sinus. Inverted papilloma was diagnosed on histopathological examination of a frozen-section biopsy specimen, and the tumor was excised under general anesthesia. The postoperative course was uneventful. However, he died of an other disease 18 months after operation. Patient 2 was a 65-year-old man with left nasal obstruction. Computed tomography showed a mass in the maxillary sinus and nasal cavity. The suspected diagnosis was left maxillary sinusitis. Inverted papilloma was diagnosed on histopathological examination of a frozen-section biopsy specimen, and the tumor was excised under general anesthesia. The postoperative course was uneventful, and there has been no evidence of recurrence.

Congenital maxillomandibular fusion (syngnathia) is a rare anomaly associated with soft tissue (synechia) or bony adhesions (synostosis).We describe a girl with synechia who had two mucosal bands between the margins of the cleft palate and the floor of the mouth. We separated the bands surgically at the age of 2 months. Subsequently, some respiratory problems remained. We performed anterior traction of the tongue with a tongue-lip adhesion procedure. Her respiration improved. Seven months after the tongue-lip adhesion procedure, the adhesion was released. At the age of 18 months, the cleft palate was repaired by a push-back method. We describe our treatment and review the literature concerning the origin of the mucosal bands.