Intercellular signaling between ameloblastoma and osteoblasts.

Ameloblastoma is an odontogenic tumor located in the bone jaw with clinical characteristics of extensive bone resorption. It is a locally invasive tumor with a high recurrence rate despite adequate surgical removal. In bone disease, tumors and other cells including osteoblasts, osteoclasts, and osteocytes in the bone microenvironment contribute to the pathogenesis of tumor growth. However, the effect of osteoblasts on ameloblastoma cells is not well-understood, and there has been limited research on interactions between them. This study investigated interactions between ameloblastoma cells and osteoblasts using a human ameloblastoma cell line (AM-3 ameloblastoma cells) and a murine pre-osteoblast cell line (MC3T3-E1 cells). We treated each cell type with the conditioned medium by the other cell type. We analyzed the effect on cytokine production by MC3T3-E1 cells and the production of MMPs by AM-3 cells. Treatment with AM-3-conditioned medium induced inflammatory cytokine production of IL-6, MCP-1, and RANTES from MC3T3-E1 cells. The use of an IL-1 receptor antagonist suppressed the production of these inflammatory cytokines by MC3T3-E1 cells stimulated with AM-3-conditioned medium. The MC3T3-E1-conditioned medium triggered the expression of MMP-2 from AM-3 cells. Furthermore, we have shown that the proliferation and migration activity of AM-3 cells were accelerated by MC3T3-E1 conditioned media. In conclusion, these intercellular signalings between ameloblastoma cells and osteoblasts may play multiple roles in the pathogenesis of ameloblastoma.

Comparison of recurrence rate and speech outcome between two different techniques for cleft palatal fistula closure: A retrospective cohort study.

The aim of this retrospective cohort study was to compare the recurrence rate and speech outcomes between two techniques for palatal fistula closure of cleft palate (CP). Patients with CP who underwent secondary palatal fistula closure using the single hinge-flap method with double-breasted mattress suture (hinge-flap group) and those who were treated with the conventional sliding palatal flap method (sliding-flap group) were retrospectively evaluated for demographic and perioperative variables. Recurrence rate of palatal fistula, perceptual speech outcomes, and nasalance scores were further reviewed in patients who met the inclusion criteria. A total of 31 patients, 21 in the hinge-flap group and 10 in the sliding-flap group, were included in this study. The fistula recurrence rate in the hinge-flap group (0%) was significantly lower than that in the sliding-flap group (30.0%) (P = 0.027). In the speech assessment, hypernasality and nasalance scores decreased post-operatively in both groups and significance was observed in the hinge-flap group (P = 0.013, P < 0.001, respectively). Articulation disorders were significantly improved in the hinge-flap group (P = 0.001). Within the limitations of the study it seems that the single hinge-flap method with double-breasted mattress suture should be preferred whenever appropriate.

Molecular biological findings of ameloblastoma.

Ameloblastoma is benign odontogenic tumours that mainly occur in the jawbone. This tumour induces aggressive invasion into the surrounding bone and has a high recurrence rate after surgery. Therefore, mandibular resection is performed in many patients with this tumour, causing aesthetic and functional problems. It is necessary to develop a novel treatment strategy for ameloblastoma, but there are currently no innovative treatments. Although our understanding of the molecular biological mechanisms of ameloblastoma is still insufficient, there have been many recent reports of new molecular biological findings on ameloblastoma. Therefore, bioactive factors that have potential for novel therapeutic methods, such as molecular targeted therapy, have been discovered in ameloblastoma. In this review, we summarize the molecular biological findings of ameloblastoma reported over several decades, focusing on factors involved in invasion into surrounding tissues and disease-specific gene mutations. We also mention the effect of the interaction between tumour cells and stromal components in ameloblastoma on tumour development. Scientific field of dental Science: Oral surgery, Odontogenic tumor, Ameloblastoma.

Ameloblastoma cell lines derived from different subtypes demonstrate distinct developmental patterns in a novel animal experimental model.

Objective Ameloblastoma is a representative odontogenic tumor comprising several characteristic invasive forms, and its pathophysiology has not been sufficiently elucidated. A stable animal experimental model using immortalized cell lines is crucial to explain the factors causing differences among the subtypes of ameloblastoma, but this model has not yet been disclosed. In this study, a novel animal experimental model has been established, using immortalized human ameloblastoma-derived cell lines. Methodology Ameloblastoma cells suspended in Matrigel were subcutaneously transplanted into the heads of immunodeficient mice. Two immortalized human ameloblastoma cell lines were used: AM-1 cells derived from the plexiform type and AM-3 cells derived from the follicular type. The tissues were evaluated histologically 30, 60, and 90 days after transplantation. Results Tumor masses formed in all transplanted mice. In addition, the tumors formed in each group transplanted with different ameloblastoma cells were histologically distinct: the tumors in the group transplanted with AM-1 cells were similar to the plexiform type, and those in the group transplanted with AM-3-cells were similar to the follicular type. Conclusions A novel, stable animal experimental model of ameloblastoma was established using two cell lines derived from different subtypes of the tumor. This model can help clarify its pathophysiology and hasten the development of new ameloblastoma treatment strategies.

Ameloblastoma cell lines derived from different subtypes demonstrate distinct developmental patterns in a novel animal experimental model

Abstract Objective Ameloblastoma is a representative odontogenic tumor comprising several characteristic invasive forms, and its pathophysiology has not been sufficiently elucidated. A stable animal experimental model using immortalized cell lines is crucial to explain the factors causing differences among the subtypes of ameloblastoma, but this model has not yet been disclosed. In this study, a novel animal experimental model has been established, using immortalized human ameloblastoma-derived cell lines. Methodology Ameloblastoma cells suspended in Matrigel were subcutaneously transplanted into the heads of immunodeficient mice. Two immortalized human ameloblastoma cell lines were used: AM-1 cells derived from the plexiform type and AM-3 cells derived from the follicular type. The tissues were evaluated histologically 30, 60, and 90 days after transplantation. Results Tumor masses formed in all transplanted mice. In addition, the tumors formed in each group transplanted with different ameloblastoma cells were histologically distinct: the tumors in the group transplanted with AM-1 cells were similar to the plexiform type, and those in the group transplanted with AM-3-cells were similar to the follicular type. Conclusions A novel, stable animal experimental model of ameloblastoma was established using two cell lines derived from different subtypes of the tumor. This model can help clarify its pathophysiology and hasten the development of new ameloblastoma treatment strategies.

Reliability of Americleft Yardstick Nasolabial Appearance Assessment With/Without Basal View for Japanese Children With Unilateral Complete Cleft Lip and Palate.

OBJECTIVE: There is debate on the reliability of the Americleft Yardstick (AY) global nasolabial appearance assessment method. The objective was to analyze the effect of the additional basal view (BV) feature on the reliability of the AY method for Japanese children with complete cleft lip and palate (CUCLP). DESIGN: Blind retrospective analysis of clinical records on 43 patients (5- to 7-year-old) with nonsyndromic CUCLP who underwent primary lip repair from 2005 to 2011. MAIN OUTCOME MEASURE: Color pictures published in AY and Rubin's studies were used as reference pictures. Patients' photographs were cropped and rated on a 5-point scale for the vermilion border (VB), nasolabial frontal (NLF), and nasolabial profile (NLP) according to AY with/without BV assessment by Rubin's methods. Rating was performed twice by 3 oral surgeons. Intra- and inter-rater reliabilities were analyzed using weighted κ, and correlations between BV and other features were analyzed. RESULTS: Overall average assessment scores were 2.742 (0.573) with AY and 2.702 (0.489) with AY+BV methods (P = .728). Average intra-rater reliabilities were 0.605 and 0.611 and average inter-rater reliabilities were 0.525 and 0.48 with AY and AY+BV, respectively. Inter-rater reliability was the lowest for NLP. ρ scores between BV versus VB, NLF, and NLP were 0.025, 0.659, and 0.092, respectively. CONCLUSIONS: Present study demonstrates moderate intra- and inter-rater reliabilities obtained with the AY assessment method for Japanese children with CUCLP. Nasolabial profile standard ambiguity may lead to the poor reliability of AY assessment. Addition of the BV feature does not improve overall reliability.

Three-dimensional analyses of nasolabial forms and upper lip surface symmetry after primary lip repair in patients with complete unilateral cleft lip and palate.

PURPOSE: To analyze three-dimensional (3D) nasolabial forms and upper lip surface symmetry after primary lip repair in children with unilateral cleft lip and palate (UCLP). METHODS: Subjects were 22 Japanese children with complete UCLP who underwent primary lip repair and were followed-up for 4-6 years. The 3D coordinates of facial landmarks and the angle and radius of the approximate nasal alar circle were calculated. Upper lip surface symmetry was analyzed using histogram intersection. RESULTS: The nasal tip and columella base were slightly dislocated to the cleft side, and the midpoint of Cupid's bow shifted to the non-cleft side. The nasal alar and the top of Cupid's bow were reconstructed at the same height, while the approximate nasal alar circle was smaller on the cleft side. The mean value of similarity for upper lip surface symmetry was 0.82; a subject with a higher value had more symmetrical contour lines in the visualized surface image. CONCLUSIONS: Postoperative nasolabial forms were almost restored to symmetrical levels, while retaining a small nasal alar. Histogram intersection is applicable as a method for the quantitative evaluation of upper lip surface symmetry in UCLP.

Pilot Study of Visual and Quantitative Image Analysis of Facial Surface Asymmetry in Unilateral Complete Cleft Lip and Palate.

OBJECTIVE: To visualize and quantitatively analyze facial surface asymmetry following primary cleft lip repair in patients with unilateral cleft lip and palate (UCLP) and to compare this with noncleft controls. DESIGN: Retrospective comparative study. PATIENTS: Twenty-two patients with complete UCLP who underwent primary lip repair from 2009 to 2013 were enrolled in this study. The preserved 3-dimensional (3D) data of 23 healthy Japanese participants with the same age were used as controls. INTERVENTIONS: All patients had received primary labioplasty in accordance with Cronin triangular flap method with orbicular oris muscle reconstruction. MAIN OUTCOME MEASURES: Shadow and zebra images established from moiré images, which were reconstructed from 3D facial data using stereophotogrammetry, were bisected and reversed by the symmetry axes (the middle line of the face). The discrepancies of the gravity and density between cleft and noncleft sides in 2 regions of interest, facial and lip areas, were then calculated and compared with those of healthy participants. RESULTS: In the UCLP group, the mean discrepancies of gravity on shadow and zebra images were 1.76 ± 0.70 and 2.63 ± 1.72 pixels, respectively, in the facial area and 1.31 ± 0.36 and 3.83 ± 2.08 pixels, respectively, in the lip area. There was a significant difference in the mean discrepancies of gravity and density on zebra images in the lip area between the UCLP and control groups. CONCLUSIONS: Our image analysis of digital facial surface asymmetry in patients with UCLP provides visual and quantitative information, and it may contribute to improvements in muscle reconstruction on cleft lip repair.

Fibroblasts promote the collective invasion of ameloblastoma tumor cells in a 3D coculture model.

Ameloblastoma is a benign tumor of the odontogenic epithelium with several histological subtypes. All subtypes of ameloblastoma contain abundant stroma; the tumor cells invade collectively into the surrounding tissues without losing intratumor cell attachments. However, the molecular mechanisms mediating ameloblastoma invasion remain unclear. Here, we evaluated the functional significance of the interactions between ameloblastoma tumor cells and stromal fibroblasts on collective cellular invasion using a three-dimensional cultivation method, double-layered collagen gel hemisphere (DL-CGH) culture. The AM-1 plexiform and AM-3 follicular human ameloblastoma cell lines and HFF-2 human fibroblasts were labeled with GFP and DsRed, respectively. Collective cellular invasion of ameloblastoma cells was assessed in the presence or absence of fibroblasts. Notably, without fibroblasts, AM-1 cells formed sharp, plexiform-like invasive processes, whereas AM-3 cells formed a series of blunt processes often observed during collective migration. In comparison, under the cocultures with HFF-2 fibroblasts, AM-3 cells formed tuft-like invasive processes and collectively invaded into outer layer more than that observed with AM-1 cells. Moreover, HFF-2 fibroblasts localized to the tips of the invasive tumor processes. These findings suggest that tumor-associated cells assist tumor cell invasion. Microscopic analysis of sectioned three-dimensional cultures revealed that AM-3/HFF-2 hemispheres were histologically similar to follicular ameloblastoma tumor samples. Therefore, our findings suggest that ameloblastoma subtypes exhibit distinct invasion patterns and that fibroblasts promote collective tumor invasion in follicular ameloblastoma.

Evaluation of maxillary central incisors on the noncleft and cleft sides in patients with unilateral cleft lip and palate-Part 2: Relationship between root resorption, horizontal tooth movement, and quantity of grafted autogenous bone.

OBJECTIVE: To evaluate the relationship between external apical root resorption (EARR) of the maxillary central incisors (U1), horizontal orthodontic tooth movement, and quantity of grafted bone in subjects with unilateral cleft lip and palate (UCLP) over an average duration of 8 years. MATERIALS AND METHODS: Thirty subjects with UCLP were evaluated for EARR of U1 after edgewise treatment (T2). The teeth were classified as having no EARR, moderate EARR (combined into "no/moderate" EARR), or severe EARR. Frontal cephalometric radiographs acquired at eruption of U1 (T0), less than 6 months before secondary alveolar bone grafting (T1), and T2 were evaluated to determine the horizontal inclination (U1-axis angle) and distance of the root apex from the median line (U1-root-VL distance). On the cleft side, the quantities of grafted bone at less than 12 months postsecondary bone grafting and at T2 were evaluated using the alveolar bone graft (ABG) scale. RESULTS: Cleft-adjacent teeth exhibited more severe EARR than did teeth on the noncleft side. The cleft side exhibited greater changes in U1-axis angle and U1-root-VL distance between T0 and T2 than did the noncleft side. On the cleft side, the ABG score at T2 in the severe EARR group was significantly lower than that in the no/moderate EARR group. These measurements were correlated with EARR grade. CONCLUSIONS: Cleft-adjacent U1 exhibited more severe EARR than did the U1 on the noncleft side, which might be associated with orthodontic treatment-induced changes in horizontal inclination and root apex movement. On the cleft side, severity of EARR may be correlated with the success of ABG.

Examination of the early wound healing process under different wound dressing conditions.

OBJECTIVE: Various types of wound-healing dressings have been used to assist in the healing of surgical wounds. We analyzed the wound-healing process in an animal model using different existing wound dressings. STUDY DESIGN: Full-thickness defects were created using a biopsy punch on the backs of 7-week-old rats. The wounded areas were covered with NEOVEIL (polyglycolic acid [PGA]) or TERUDERMIS (collagen sponge [CS]) affixed using a rat jacket. The wound area, neo-epithelium length, and α-smooth muscle actin (α-SMA) expression were evaluated and compared among the control, PGA, and CS groups. RESULTS: The wound areas in the control group on days 4 and 7 were significantly smaller than those in the PGA and CS groups. The expression of α-SMA in granulation tissue peaked on day 4 for all groups. The expression of α-SMA in the control group on days 4 and 7 after injury was greater than in the PGA and CS groups. However, there was no significant difference in the expression of α-SMA between the PGA and CS groups. CONCLUSIONS: In this study, PGA and CS suppressed wound contracture and reduced expression of α-SMA in wound areas. However, PGA and CS did not affect the neo-epithelium length at the wound site.

Clinical and 3-Dimensional Analyses of Nasal Forms After Secondary Correction of Cleft Lip-Nose Deformities Using Extended Spreader Cartilage Graft With a Cross-Lap Joint Technique.

PURPOSE: A surgical strategy for definitive cleft lip-nasal correction that stably provides symmetric and natural nasal forms has not been established to date. The purpose of this study was to describe our surgical techniques and 3-dimensional (3D) assessment results after the definitive correction of cleft lip-nose deformity using an extended spreader cartilage graft with a cross-lap joint technique to achieve a rigid strut for lower lateral cartilage repositioning to obtain a desirable nasal tip projection. PATIENTS AND METHODS: This study enrolled 14 patients with unilateral cleft lip (UCL) with or without cleft palate and 8 patients with bilateral cleft lip (BCL) with or without cleft palate who underwent definitive nose correction and were followed for 1 to 3 years. All patients were treated by open rhinoplasty, repositioning of the lower lateral cartilage, use of an extended spreader cartilage graft with a cross-lap joint technique for nasal tip support, and medial-upward advancement of nasolabial components with vestibular expansion by a free mucosal graft. For the BCL nose, pedicle flaps from rim skin were used for columella lengthening. Preoperative and postoperative nasal forms were 3-dimensionally analyzed by use of 3D images serially obtained in 12 patients. RESULTS: The postoperative nasal forms were satisfactorily improved in all patients, without any serious postoperative complications. Preoperative and postoperative 3D analyses showed a significantly projected nasal tip in the postoperative noses of patients in both the UCL group and the BCL group (P < .01 and P < .05, respectively) and a sharper nasal tip angle in the BCL group (P < .05). Lateral deviation of the nasal tip was significantly improved in the center of the face in patients in the UCL group (P < .01). CONCLUSIONS: The results of this study suggest that the extended spreader cartilage graft using the cross-lap joint graft technique is useful to provide a desirable projection of the nasal tip in the center of the face on definitive correction of both UCL and BCL nose deformities.

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma.

Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave interactively via these cytokines to create a microenvironment that leads to the extension of ameloblastomas.

Ryk is essential for Wnt-5a-dependent invasiveness in human glioma.

Glioblastoma is characterized by marked invasiveness, but little is known about the mechanism of invasion in glioblastoma cells. Wnts are secreted ligands that regulate cell proliferation, differentiation, motility and fate at various developmental stages. In adults, misregulation of the Wnt pathway is associated with several diseases. Recently, we reported that Wnt-5a was overexpressed and correlated with cell motility and infiltrative activity through the regulation of matrix metalloproteinase (MMP)-2 in glioma-derived cells. Although several receptors for Wnt-5a were identified, the receptors of Wnt-5a that mediate cellular responses of glioma were not clearly identified. Knockdown of receptor-like tyrosine kinase (Ryk) but not that of Ror2 suppressed the activity of MMP-2 and Wnt-5a-dependent invasive activity in glioma cells. These results suggest that Ryk is important for the Wnt-5a-dependent induction of MMP-2 and invasive activity in glioma-derived cells and that Ryk might have a novel patho-physiological function in adult cancer invasion. Furthermore, not only the expression of Wnt-5a but also that of Frizzled (Fz)-2 and Ryk was correlated with the WHO histological grade in 38 human glioma tissues. Taking these findings together, Fz-2 and Ryk could be therapeutic or pharmacological target molecules for the control of Wnt-5a-dependent invasion of human glioma in the near future.

Short-term molding effects on the upper alveolar arch following unilateral cleft lip repair with/without nasal vestibular expansion.

Objective : To elucidate the various effects on maxillary growth following different procedures for vestibular expansion at the time of primary lip repair for unilateral cleft lip and palate (UCLP). Participants : Thirty patients with complete UCLP who underwent primary lip repair using a triangular-flap technique with nasal vestibular expansion (NVE; the NVE group) and 30 patients who underwent the same lip repair with closure of the nasal floor (non-NVE group) were enrolled in this study. Interventions : Serial dental casts on lip and palatal repair were scanned with a laser scanner. The three-dimensional coordinates of seven anatomical landmarks and their growth changes, the curvature radius rate between major/minor segments, and the collapse rates were compared between the two groups. Results : At the time of lip repair, the incisal point was located slightly anteriorly in the non-NVE group. At the time of palatal repair, the cleft edge of the alveolar process in the minor segment was located significantly anteriorly and laterally in the NVE group, showing the significantly forward change of the minor segment. The minor segment collapsed in the non-NVE group. The collapse rate of the NVE group (3.3%) was significantly lower than that of the non-NVE group (40.0%). Conclusions : NVE following simultaneous advancement of nasolabial components on the affected side at the time of primary lip repair for UCLP facilitates the forward molding of the maxilla, resulting in a more symmetrical alveolar arch form.

A novel ameloblastoma cell line (AM-3) secretes MMP-9 in response to Wnt-3a and induces osteoclastogenesis.

OBJECTIVE: Ameloblastoma has a high risk of bone invasion and local recurrence. However, the mechanisms of bone invasion in ameloblastoma remain unclear. In this study, we established an experimental model for matrix metalloproteinase (MMP) induction and osteoclastogenesis using ameloblastoma-derived cells. STUDY DESIGN: We established an ameloblastoma-derived cell line without viral genes and analyzed the expression of all Wnt and Frizzled members and MMPs by real-time reverse transcription-polymerase chain reaction, and analyzed the activity of MMP-2 and MMP-9 by the in-gel-gelatinase assay. RESULTS: AM-3, newly established ameloblastoma-derived cells retained the morphology of primary-cultured ameloblastoma cells. AM-3 cells overexpressed the messenger RNA of Wnt-5a, Frizzled-2, MMP-2, and MMP-9 and showed the potential of osteoclastogenesis. In addition, Wnt-3a-treatment induced expression and activation of MMP-9 in AM-3 cells. CONCLUSIONS: Our study suggests that AM-3 cells retained the characteristics of ameloblastoma, without acquiring typical features of cancer cells. Furthermore, Wnt signaling induced MMP-9 in ameloblastoma cells.