Cleft palate formation after palatal fusion occurs due to the rupture of epithelial basement membranes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and hydronephrosis in the mouse embryo. Cleft palate occurs due to failure in palatal grow, but the underlying mechanisms are unclear. We investigated the mechanisms of cleft palate development in TCDD-exposed mouse embryos. We administered olive oil (control group) or TCDD diluted in olive oil (40 µg/kg) via gastric tubes to pregnant mice on gestational day (GD) 12. Embryos of control and TCDD-exposed groups were removed from pregnant mice on GD 14 and GD 15, respectively. One mouse embryo from the control group had anteroposterior palatal fusion. Palatal fusion was observed in three TCDD-exposed mouse embryos. Palates of TCDD-exposed mice fused from the interior to the middle of the palates, while the palates were separated in the posterior region. The middle of the embryonic palatal shelves in TCDD-exposed animals was narrow and split at the fusional position. At this position, palatal and blood cells were dispersed from the palatal tissue and the epithelium was split, with a discontinuous basement membrane. The results suggest that decreased intercellular adhesion or insufficient tissue strength of the palatal shelves may be involved in the development of cleft palate following palatal fusion.

Homeobox family Hoxc localization during murine palate formation.

Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words) © 2016 Japanese Teratology Society.

Heparanase localization during palatogenesis in mice.

Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES.

Heparanase and cyclooxygenase-2 gene and protein expressions during progression of oral epithelial dysplasia to carcinoma.

Heparanase and cyclooxygenase-2 (COX-2) are 2 key enzymes that modulate diverse physiological processes during embryonic development and in adult life. Their deregulations have been implicated in the growth and progression of many cancer types. To date, comparatively little is known about the roles of these molecules during oral carcinogenesis. The aim of this study was to investigate the expression patterns of heparanase and COX-2 during progression of oral epithelial dysplasia (OED) to carcinoma. In situ hybridization and immunohistochemistry were performed on 5 cases of normal mucosa, 15 cases of OED, 5 cases of carcinoma in situ and/or microinvasive carcinoma, and 40 cases of oral squamous cell carcinoma (OSCC). Results demonstrated that heparanase and COX-2 messenger RNA and protein were absent in normal oral mucosa but were coexpressed in increasing intensity as OED progressed to OSCC. Concomitant heparanase- and COX-2-positive staining in the stromal cells suggests that OED/OSCC progression may be modulated by stromal-cancer cell interactions. Diffuse intense staining of poorly differentiated OSCC compared with staining localized to tumor nest periphery in well- and moderately differentiated OSCC suggests that heparanase and COX-2 overexpressions correlated with tumor grade. Strong expression of these enzymes in tumor cells at the advancing front suggests a role in local tumor spread. These results, taken together, suggest that heparanase and COX-2 might play complementary roles in the stepwise progression of OED to carcinoma.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin suggests abnormal palate development after palatal fusion.

Mouse embryos exposed to 2,3,7,8-tetrachloridedibenzo-p-dioxin (TCDD) develop cleft palates and hydronephrosis. Cleft palates occur after TCDD exposure due to contact and/or fusion failure. We investigated whether cleft palate can be induced by dissociation of the palatine process after fusion. Pregnant mice on gestational day (GD) 12 were randomly divided into two groups: one group was administered through gastric tubes one dose of olive oil (control group) and the other group was administered one dose of TCDD diluted with olive oil, both at a dose of 40 microg/kg body weight. Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13-18) and the palatal form was observed using a stereoscopic microscope. In TCDD-exposed embryos, palatal fusion was observed on GD 14, 15 and 16 and the incidence of cleft palate was 100% on GD 18. Fusion rates were 17.5 +/- 15.2% and 12.4 +/- 11.8% on GD 15 and 16, respectively. Some palates from the TCDD-exposed mouse embryos showed clearly developed cleft palate after fusion of the lateral palatine processes during palatal formation. A mass of cells, which were chiefly epithelial in the fused palates was observed in the TCDD-exposed mouse embryos. A decrease in E-cadherin expression was observed in this mass of cells, indicating its involvement in the development of cleft palate.

Osteomyelitis of the mandible secondary to infantile osteopetrosis: a case report.

A case of infantile malignant osteopetrosis with refractory mandibular osteomyelitis is reported. A female child was diagnosed with osteopetrosis at 2 years 8 months of age, and was scheduled to receive a bone marrow transplantation (BMT) in a pediatric department at 3 years 11 months of age. Her lower incisors were extracted at a dental clinic, after which she had recurring abscesses with progressive severity. She was referred to our department to control local infections before the BMT and was diagnosed with chronic mandibular osteomyelitis caused by osteopetrosis. Sequestrotomy and curettage were performed under general anesthesia. After surgery, daily saline irrigation was continued and osteomyelitis was well controlled. An uneventful BMT was performed, although it was refused and failed. A second BMT was planned, but during chemotherapy, cellulitis occurred after a recurrence of osteomyelitis caused by a newly erupted tooth. The local infection was controlled, but pneumonia recurred. She ultimately died of respiratory failure at 5 years of age.

Bimaxillary protrusion with masseter muscle hypertrophy treated with titanium screw anchorage and masseter surgical reduction.

This case report describes the treatment of a patient with bimaxillary protrusion and masseter muscle hypertrophy. At age 21 years 7 months, this woman had temporomandibular disorder (TMD) symptoms, severe bimaxillary protrusion, and a prominent mandibular angle with facial asymmetry. After an attempt to alleviate the TMD symptoms with occlusal splint stabilization, portions of the masseter muscle and the mandible were surgically removed. Titanium screws were placed bilaterally in both arches, and a retraction force was applied. After active treatment for 38 months, the convexity of the facial profile with lip protrusion was improved remarkably, and good occlusion was achieved. The prominent mandibular angle with facial asymmetry was improved as a result of the surgical reduction of the masseter muscle and the modeling ostectomy near the masseteric tuberosity. The TMD symptoms disappeared, and the jaw movement pattern became normal. Therefore, our results suggest that this combination treatment would be useful for masseter muscle hypertrophy for morphologic and functional problems.

Localization of runx2, osterix, and osteopontin in tooth root formation in rat molars.

Cementogenesis starts with the differentiation of cementoblasts. Mature cementoblasts secrete cementum matrix. Cementum components are similar to bone; moreover, cementoblasts possess many characteristics similar to those of osteoblasts. Runx2 and osterix, the transcriptional factors for osteoblast differentiation, participate in tooth formation. However, the characteristics of Runx2 and osterix during the differentiation process of cementoblasts remain unclear. In this study, we examined the immunolocalization patterns of Runx2, osterix, and osteopontin during rat molar tooth formation. Periodontal ligament cells and osteoblasts located on the alveolar bone surface showed immunoreactivity for Runx2. Colocalization of Runx2 and osterix was detected in cementoblasts, which penetrated the ruptured Hertwig's epithelial root sheath and attached to root dentin. Moreover, osteopontin was observed in Runx2-positive cementoblasts facing the root surface. However, the cells adjacent to cementoblasts showed only Runx2 reactivity. Neither Runx2 nor osterix was seen in cementocytes. These results suggest that both Runx2 and osterix are important for differentiation into cementoblasts. Additionally, osterix may be indispensable for transcription of osteopontin expression.

Postoperative course after SSRO in mandibular asymmetries with or without MMF.

OBJECTIVE: The aim of this study was to assess the necessity of maxillo-mandibular fixation (MMF) from a viewpoint of postoperative stability to treat mandibular asymmetries. MATERIALS AND METHODS: Twenty-four patients who underwent surgical correction of mandibular asymmetry were analyzed. The surgical procedure in all patients consisted of bilateral sagittal splitting ramus osteotomy (SSRO). The segments were then fixed rigidly with titanium screws. A postoperative MMF was performed in 12 patients within 1 day of the SSRO. The MMF lasted for 1 week. The other 12 patients were free to move their jaw on the day of the surgery and received occlusal guidance with elastics starting from the third postoperative day. Posterior-anterior cephalograms were taken preoperatively, 1 day postoperatively, and at 1, 3, 6, 12, and 24 months after surgery. Skeletal and occlusal stabilities along with postoperative complications were then assessed. RESULTS: Nausea and pharyngeal discomforts were observed very often in both groups. Even without MMF, occlusions were guided to the objective positions by an average of 3.5 days after surgery. Occlusal and skeletal stability was satisfactory in both groups, and there was no correlation between the surgical results and the use of postoperative MMF. CONCLUSION: MMF is not necessary after rigid fixation SSRO for mandibular asymmetry, considering the risks of airway distress.

Immobilized recombinant human bone morphogenetic protein-2 enhances the phosphorylation of receptor-activated Smads.

Bone morphogenetic protein (BMP)-2 plays an important role in bone growth and regeneration; however, BMP-2 is easily lost by diffusion through body fluid and has some inhibitory pathways. To address this problem, we previously immobilized recombinant human BMP-2 (rhBMP-2) on succinylated type I atelocollagen. Here, we examined the effect of immobilized rhBMP-2 in vitro and vivo. In ST2, MC3T3-E1, and C2C12 cells, alkaline phosphatase activity, which is a marker of osteoblast differentiation, was enhanced more by immobilized than nonimmobilized rhBMP-2. In addition, the phosphorylation of receptor-activated Smads, part of the signaling pathway activated by BMP-2, was prolonged by immobilized rhBMP-2 in these cells. Furthermore, implantation of immobilized rhBMP-2 into the backs of rats promoted the formation of mature bone-like structure. These results demonstrate that immobilized rhBMP-2 has higher bioactivity than nonimmobilized rhBMP-2, and, therefore, immobilization of rhBMP-2 can prolong BMP signaling.

Mandibular stability after SSRO with or without postoperative maxillo-mandibular fixation for Class III malocclusion.

OBJECTIVE: The objective of this study was to assess the usefulness of maxillo-mandibular fixation (MMF) from a viewpoint of skeletal and occlusal stability and to investigate the complications after surgery with and without MMF. MATERIALS AND METHODS: Twenty-seven patients who underwent surgical correction of class III malocclusion were analyzed. The surgical procedure in all cases consisted of a bilateral sagittal splitting ramus osteotomy (SSRO). The segments were then fixed rigidly with titanium screws. A postoperative MMF was performed in 13 patients within 1 day of the SSRO. The MMF lasted for 1 week. The other 14 patients were free to move their jaw on the day of the surgery and received occlusal guidance with elastics starting from the third postoperative day. Cephalograms were taken preoperatively, at 1 day postoperatively and at 1, 3, 6, 12, and 24 months after surgery. Skeletal and occlusal stabilities along with postoperative complications were then assessed. RESULTS: Discomfort of the pharynx and postoperative pain was more severe in the MMF group. Even without MMF, occlusion was guided to the ideal position by an average of 5.3 days after surgery. Occlusal and skeletal stability was satisfactory in both groups, and there was no correlation between the surgical results and postoperative MMF. CONCLUSION: MMF may not be necessary after a rigid fixation SSRO, considering the risks of airway problems.

Morphological and immunohistochemical studies on cleft palates induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice.

Morphological and immunohistological examinations were performed to reveal the mechanisms of cleft palate induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). ICR strain mice 8-10 weeks of age were used in the study. TCDD was administered in olive oil on gestation day (GD) 12.5 with gastric tubes at 40 microg/kg. From GD 13.5 to 16.5, palates were examined by scanning electron microscopy (SEM), hematoxyline-eosin (HE) staining, and immunohistochemical staining of FGFR1/2, TGF-beta3, MSX1 and LHX8. In the control group, both of the palatal shelves began elevating on GD 14.0 and finished within 6 h. After the elevation, all of the shelves had completely fused with each other on GD 14.5. In the TCDD-treated group, palatal shelves elevated 1 day later than in the control group. However, all palates had elevated by GD 15.0. After the elevation, the shelves contacted each other and fused; however, they were separated on GD16.0. HE staining showed that medial edge epithelium (MEE) was thinner in the TCDD group than in the control group. MEE observed under a high magnification (x2500) exhibited filopodia-like filaments and the cells were bulged in the control group. In contrast, in the TCDD group, no filaments were observed and the cells were flat with unclear boundaries. Immunohistologically, there were no characteristic findings except for FGFR1. FGFR1 was not expressed in the TCDD group after the fusion phase (GD 14.5). TCDD induces many morphological and molecular changes to MEE cells and causes cleft palates.

Dental implant treatment for a patient with bilateral cleft lip and palate.

Dental reconstruction in the cleft space is difficult in some patients with cleft lip and palate because of oronasal fistulas. Most of these patients receive a particle cancellous bone marrow (PCBM) graft to close the alveolar cleft, and secondary bone grafting is also required. Treatment options for the alveolar cleft including fixed or removable prostheses require the preparation of healthy teeth and are associated with functional or social difficulties. Recently, the effectiveness of dental implant treatment for cleft lip and palate patients has been reported. However, there have been few reports on the use of this treatment in bilateral cleft lip and palate patients. We report the case of a patient who had bilateral cleft lip and palate and was missing both lateral incisors. She received dental implant treatment after a PCBM graft and ramus bone onlay grafting (RBOG). A 34-month postoperative course was uneventful.

Relationships between nasalance scores and nasopharyngeal shapes in cleft palate patients.

OBJECTIVES: The aim of the present study is to clarify the relationship between nasalance scores and nasopharyngeal shapes obtained by lateral cephalograms. PATIENTS: Eight patients who underwent a Wardill-Kilner push-back palatoplasty were included in this study. Perceptual judgment by a speech pathologist indicated that these patients had no hypernasality and no nasal emission at blowing. As normal controls, 33 non-cleft individuals, 4 boys and 10 girls aged 6 years old and 5 boys and 14 girls aged 7 years old, were investigated. METHODS: Lateral cephalograms at rest were taken for both groups. For the cleft (palate) patients, lateral cephalograms at phonation /a/ and blowing were analyzed and nasometries were also performed using a kitsutsuki passage. RESULTS AND CONCLUSION: There was no significant difference in the velar length, the pharyngeal depth, the ratio of the velar length to the pharyngeal depth and the velar angle between the cleft patients and the non-cleft individuals. Multiple regression analyses indicated that standardized regression coefficients of ratios for the velar length to the pharyngeal depth and the velar ascent at blowing had higher nasalance scores for sentences 1 and 3, which had high coefficients of determination, respectively.

Histological comparison of bone induced from autogenously grafted periosteum with bone induced from autogenously grafted bone marrow in the rat calvarial defect model.

Both periosteum and bone marrow have the potential to induce heterotopic bone when grafted. Whether the process of bone formation is controlled by the recipient environment where the donor graft is placed or by factors from the donor site is not well documented. The purpose of this study was to examine the histology of new bone induced by either autogenously grafted periosteum or autogenously grafted bone marrow using the rat calvarial defect model in Sprague-Dawley rats. Grafts of either bone marrow or periosteum obtained from tibias were placed in calvarial defects with beta-tricalcium phosphate. Ten days after grafting, active cell proliferation was observed in the defects of both types of grafts. After 20 days, cancellous bone formation was observed in the defects with bone marrow grafts, and intramembranous bone formation was observed in the defects with periosteal grafts. After 30 days, bone marrow grafts had developed bone with a bone marrow-like structure, and the periosteal grafts had produced cortical bone structure in the defects. The findings suggest that the type of bone formation is determined by characteristics of the donor site.

Dialectal and gender differences in nasalance scores in a Japanese population.

OBJECTIVES: The aim of the present study was to determine whether there are dialectal and gender-related differences in nasalance scores for normal Japanese speakers. MATERIALS: Sixty-eight volunteers consisting of 31 males (age 23.8+/-2.0) and 37 females (age 23.2+/-2.5) were included in this study. They had no diseases affecting speech, and lived in the same region until high school from birth. According to geography, they were divided into four regional groups: Chugoku region, Kinki region, Shikoku region, and other regions. METHODS: A kitsutsuki passage, which consisted of Japanese non-nasal consonants and vowels, and the Japanese vowels /a/, /i/, /u/, /e/ and /o/, were read three times, and the mean nasalance scores were then obtained with a Nasometer II 6400. The scores of males and females were compared statistically by means of a Student's t-test. The differences among the three regions, Chugoku, Kinki and Shikoku region, were also investigated by means of a one-way analysis of variance (ANOVA). RESULTS AND CONCLUSION: For all sentences and vowels, the nasalance scores were significantly different between males and females. The one-way ANOVA showed that there were no significant differences among the three regions in both males and females.

The evaluation of bone formation of the whole-tissue periosteum transplantation in combination with beta-tricalcium phosphate (TCP).

We investigated the osteogenic potential of a combination graft of beta-tricalcium phosphate (TCP) and periosteum in the rat calvarial defect model. The combination beta-TCP and periosteum graft was grafted into rat calvarial defects; the newly formed bone in the defect was studied histologically and radiographically and compared with periosteum grafts and TCP grafts. Ten days after combination grafting, the grafted periosteum showed cell proliferation and Runx2 immunoreaction; 20 days after grafting, new bone formation was seen around the beta-TCP; and 30 days after grafting, new bone developed and actively replaced beta-TCP, while radiography showed calcified areas. Total bone formation of the combination periosteum and beta-TCP graft was significantly increased compared with single grafts of beta-TCP or periosteum (P < 0.01). The combination graft of periosteum and beta-TCP showed marked bone formation in rat calvarial defects. This result suggests that combination grafts may be effective for repairing bone defects.

Osteogenic potential of cultured human periosteum-derived cells - a pilot study of human cell transplantation into a rat calvarial defect model.

BACKGROUND: Periosteum shows osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. The osteogenic potential of cultured periosteal cells has also been reported. The findings of bone formation induced by cultured human periosteum-derived cells using a rat model are presented. MATERIAL AND METHODS: Human mandibular periosteum was placed into a culture medium with 10% foetal bovine serum for 14 days. After reaching confluence, periosteal cells were re-suspended with 0.25% trypsin/EDTA and then re-cultured three dimensionally on a collagen sponge. The periosteal cell/collagen complex was grafted into rat calvarial defects and an immunosuppressant (FK506, 1.0 mg/kg/day) was administered intramuscularly. At 2, 3, and 5 weeks postoperatively, grafted tissue was extirpated and compared histologically and radiographically with tissue from a collagen-only grafted group. RESULTS: In the experimental group, periosteal cells had proliferated and differentiated into osteogenic cells by 2 weeks post grafting. At 3 weeks, new bone formation was evident. By 5 weeks, bone growth was observed and new calcification was detected in the defect. CONCLUSION: Cultured human periosteum-derived cells showed osteogenic potential in a xenogeneic graft model using rat calvarial defects.

Comparison of bone formation ingrafted periosteum harvested from tibia and calvaria.

Periosteum covers the bone surface and displays the potential to initiate bone formation, after injury to the bone. Numerous studies have demonstrated that the periosteum plays major roles in the healing process after bone fracture. Some reports have described that in the healing of long bone fractures, the periosteum forms new bone by intramembranous and endochondral ossification. Other researchers insist that healing of defects in membrane bone shows bone formation by intramembranous ossification. However, previous studies have not been able to clarify differences in bone formation patterns. We hypothesized that differences in bone formation pattern are associated with the periosteal potential for cell differentiation. The present study grafted periosteum, harvested from the tibia and calvaria, into the suprahyoid muscle, with the aim of interrupting release of factors from bone matrix. Bone formation, after grafting periosteum, harvested from the tibia and calvaria, was examined histologically and radiographically. Grafted tibial periosteum formed a large area of new bone by intramembranous and endochondral ossification, while grafted calvarial periosteum displayed intramembranous ossification. Grafted tibial periosteum formed a larger area of bone than grafted calvarial periosteum. Patterns of cell differentiation thus differ between grafted periosteum, harvested from the tibia and calvaria.

Cleft lip and palate in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: a morphological in vivo study.

It is well-known that TCDD (2,3,7,8, tetrachloridedibenzo-p-dioxin) induces cleft palates (CPs) in pregnant C57BL mice. However, it is unclear if TCDD is a possible teratogen for cleft lip. We examined maxillofacial malformations including cleft lip in three animal strains: A/J mice, C57BL/6J mice and ICR mice. The A/J mouse develops cleft lip and palate spontaneously at a 5-10% rate. TCDD was administered in olive oil on gestation day (GD) 12.5 with gastric tubes at 10 microg/kg, 20 microg/kg, or 40 microg/kg to examine the dose-response, and on a single day from GD 8.5-14.5 to examine the timing effects of TCDD administration on lip and palate formation. Furthermore, the palatal shelf movements during GD 8.5-14.5 were observed with a stereoscopic microscope. All embryos had cleft palates when the TCDD was administered just before palatogenesis (GD11.5-GD12.5). With respect to the TCDD effects, there were large differences among the strains. In the A/J mice, the difference between a lethal dose and a dose that could induce a cleft palate was close. Cleft lips were not induced, even when the TCDD was given just before labiogenesis. Morphologically, both palatal shelves contacted perfectly along their lengths, but separated and formed cleft palates. In conclusion, TCDD is a strong inducer of cleft palates, and interferes with the fusion phase of the secondary palate, but has no effect on the lip.