An in vitro three-dimensional co-culture system for ameloblastoma modelling

Ameloblastoma, the most clinically significant odontogenic epithelial tumor, is a locally-invasive and destructive lesion in the jawbones. However, the nature of this infiltrativeness and destructive behavior remains ill-understood. To address this, we established an in vitro three-dimensional (3D) co-culture system to simulate an amelobastoma disease model aimed at investigating the interactions between tumor cells and osteoblasts. Osteoblastic cell lines (KUSA/A1 and MC3T3-E1) and one stromal cell line (ST2) were separately co-seeded with ameloblastoma-derived cell line (AM-1) in a collagen scaffold (representing the extracellular bone matrix) and incubated with mineralization medium. Immunohistochemistry, double immunofluorescence and mineralization assay were performed. Only AM-1/KUSA-A1 co-culture showed a significant increase in AM-1 cell count, suggesting that heterotypic cell-cell interaction promotes tumoral cell growth, while formation of visible AM-1 epithelial nest-like structures resembling ameloblastoma cells in their native state, suggest morphodifferentiation. A RANK-high, RANKL-low and osteoprotegerin-low immunoprofile in co-culture AM-1 cells implies deregulated osteoclastogenesis. Mineralization assays showed diminished calcification in AM-1/KUSA-A1 co-culture extracellular matrix suggesting an altered local bone metabolism. In contrast, KUSA/A1 monocultures showed abundant extracellular matrix calcification. Taken together, these results suggest that a 3D co-culture system as an amelobastoma disease model provides insights that bidirectional ameloblastoma-osteoblastic interactions might play a role in modulating tumor growth and osteoclastogenesis

Technical issues in craniomaxillofacial distraction osteogenesis: a case series

Distraction osteogenesis allows superior skeletal advancement compared to conventional surgical osteotomy. It can be considered as a reliable and predictable surgical procedure and is widely used to correct the craniomaxillofacial bone discrepancy. Nevertheless, the outcome is technically dependent and requires comprehensive peri-operative assessment, preparation, and precision in application.The objective of this study is to highlight some important technical issues in distraction osteogenesis when the technique is indicated in various craniomaxillofacial regions and at the same time to discuss the options of preventing and overcoming these technical complications based on our experience and relevant literature.Important technical issues on the application of distraction osteogenesis in 5 different craniomaxillofacial regions were selectively highlighted based on the completed cases in one centre. Potential complications and its prevention methods were documented and discussed.The 5 highlighted regions of craniomaxillofacial distraction osteogenesis were alveolar, mandibular, cleft maxilla, craniofacial and facial cleft. Technical issues and complications were mostly device related and associated with anatomical limitations and surgical technique. Nevertheless, these complications are preventable and can be appropriately managed. From the literature and our experience, the technical aspects vary according to its application in different craniomaxillofacial regions. Preventing the potential complications contribute to the success of its application. This article also discussed the concept of Ihsan application in the medical field, to achieve the best of treatment in terms of delivery and technical preparation for the patients

Overexpression of MMP13

Matrix metalloproteinase 13 (MMP13) plays a central role in the MMP activation cascade that enables degradation of the extracellular matrix and basement membranes, and it is identified as a potential driver in oral carcinogenesis. Therefore, this study aims to determine the copy number, mRNA, and protein expression of MMP13 in oral squamous cell carcinoma (OSCC) and to associate these expressions with clinicopathological parameters. Copy number, mRNA, and protein expression analysis of MMP13 were determined using real-time quantitative PCR and immunohistochemistry methods in OSCC samples. The correlations between MMP13 expressions and clinicopathological parameters were evaluated, and the significance of MMP13 as a prognostic factor was determined. Despite discrepancies between gene amplification and mRNA and protein overexpression rates, OSCC cases showed high amplification of MMP13 and overexpression of MMP13 at both mRNA and protein levels. High level of MMP13 protein expression showed a significant correlation with lymph node metastasis (P=0.011) and tumor staging (P=0.002). Multivariate Cox regression model analysis revealed that high level of mRNA and protein expression of MMP13 were significantly associated with poor prognosis (P<0.050). Taken together, these observations indicate that the MMP13 protein overexpression could be considered as a prognostic marker of OSCC

Hypernasality in singing among children with cleft palate: a preliminary study

The aim of this study was to document differences in hypernasality during speaking and singing among children with cleft palate and to compare nasality score ratings of trained and untrained listeners. Twenty subjects with cleft palate aged between 7 and 12 years participated in this study. Audio recordings were made of the children reading a passage and singing a common local song, both in the Malay language. The degree of hypernasality was judged through perceptual assessment. Three trained listeners (a speech therapist, a classical singer, and a linguistic expert – all academicians) and two untrained listeners (a cleft volunteer worker and a national high school teacher) assessed the recordings using a visual analogue scale (VAS). Inter-rater and intra-rater reliability for hypernasality in both speaking and singing were verified using the intra-class correlation coefficient (ICC). A significant reduction in hypernasality was observed during singing as compared to speaking, indicating that hypernasality reduces when a child with cleft palate sings. The act of singing significantly reduces hypernasality. The outcome of this study suggests that children with cleft palate would benefit from singing exercises to ultimately reduce hypernasality. However, future research is needed to objectively measure nasality in singing compared to speaking. © 2019 International Association of Oral and Maxillofacial Surgeon

Oblique facial cleft

Introduction: Oblique facial clefts are an extremely rare disorder, accounting for only 0.24% of facial clefts.The condition causes pronounced facial deformity which can lead to major cosmetic and functional problems. Due to its nature of complexity, it becomes one of the reconstructive challenges which require careful planning and multiple surgeries throughout life.In this case report, we would like to share our experience with orbital reconstruction by means of distraction osteogenesis in a 1 and a half-year-old girl

Determining the Accuracy of the Mandibular Canal Region in 3D Biomodels Fabricated from CBCT Scanned Data: A Cadaveric Study

Objective: To validate the accuracy of the mandibular canal region in 3D biomodel produced by using data obtained from Cone-Beam Computed Tomography (CBCT) of cadaveric mandibles. Methods: Six hemi-mandible samples were scanned using the i-CAT CBCT system. The scanned data was transferred to the OsiriX software for measurement protocol and subsequently into Mimics software to fabricate customized cutting jigs and 3D biomodels based on rapid prototyping technology. The hemi-mandibles were segmented into 5 dentoalveolar blocks using the customized jigs. Digital calliper was used to measure six distances surrounding the mandibular canal on each section. The same distances were measured on the corresponding cross-sectional OsiriX images and the 3D biomodels of each dentoalveolar block. Results: Statistically no significant difference was found when measurements from OsiriX images and 3D biomodels were compared to the “gold standard” -direct digital calliper measurement of the cadaveric dentoalveolar blocks. Moreover, the mean value difference of the various measurements between the different study components was also minimal. Conclusion: Various distances surrounding the mandibular canal from 3D biomodels produced from the CBCT scanned data was similar to that of direct digital calliper measurements of the cadaveric specimens. © 2019 Bentham Science Publishers

Metal organic chemical vapor deposition of m-plane GaN epi-layer using a three-step approach towards enhanced surface morphology

Specular m-plane (101¯0) gallium nitride (m-GaN) epi-layer are grown on m-plane (101¯0) sapphire substrates by metal organic chemical vapor deposition using a three-step approach. A two-step approach was used to grow m-GaN buffer layer (BL), while a three-step approach was applied to improve the surface morphology of the top m-GaN epi-layer at high temperature. The three-step approach started with growing m-aluminum nitride nucleation layer with an optimized ammonia flux during the growth of aluminum nitride. Then the temperature was ramped up during the recrystallization step before the m-GaN BL deposition at low-temperature and the growth of m-GaN layer at high-temperature for the final step. Unexpectedly, when ammonia flow was intentionally halted during the recrystallization step, the surface morphology of the BL drastically changed from three- to two- dimensional with an abrupt cross-sectional structure. This in turn facilitated the complete coalescence of the m-GaN layer as revealed by field emission scanning electron microscopy. The three-step technique was found to affect the quality of m-GaN epi-layer as the samples exhibit improved crystallinity with X-ray diffraction rocking curves widths of 4680 and 1980 arcsec along the azimuth, perpendicular and parallel to [101¯0] directions, respectively

Zerumbone targets the CXCR4-RhoA and PI3K-mTOR signaling axis to reduce motility and proliferation of oral cancer cells

Background: The CXCR4-RhoA and PI3K-mTOR signaling pathways play crucial roles in the dissemination and tumorigenesis of oral squamous cell carcinoma (OSCC). Activation of these pathways have made them promising molecular targets in the treatment of OSCC. Zerumbone, a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. has displayed promising anticancer properties with the ability to modulate multiple molecular targets involved in carcinogenesis. While the anticancer activities of zerumbone have been well explored across different types of cancer, the molecular mechanism of action of zerumbone in OSCC remains largely unknown. Purpose: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action. Methods: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting. Results: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC50 values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC50 values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins. Conclusion: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment

Effects of palbociclib in oral squamous cell carcinoma and the role of PIK3CA in conferring resistance

Objective: Lack of effective therapies remains a problem in the treatment of oral squamous cell carcinoma (OSCC), especially in patients with advanced tumors. OSCC development is driven by multiple aberrancies within the cell cycle pathway, including amplification of cyclin D1 and loss of p16. Hence, cell cycle inhibitors of the CDK4/6-cyclin D axis are appealing targets for OSCC treatment. Here, we determined the potency of palbociclib and identified genetic features that are associated with the response of palbociclib in OSCC. Methods: The effect of palbociclib was evaluated in a panel of well-characterized OSCC cell lines by cell proliferation assays and further confirmed by in vivo evaluation in xenograft models. PIK3CA-mutant isogenic cell lines were used to investigate the effect of PIK3CA mutation towards palbociclib response. Results: We demonstrated that 80% of OSCC cell lines are sensitive to palbociclib at sub-micromolar concentrations. Consistently, palbociclib was effective in controlling tumor growth in mice. We identified that palbociclib-resistant cells harbored mutations in PIK3CA. Using isogenic cell lines, we showed that PIK3CA mutant cells are less responsive to palbociclib as compared to wild-type cells with concurrent upregulation of CDK2 and cyclin El protein levels. We further demonstrated that the combination of a PI3K/mTOR inhibitor (PF-04691502) and palbociclib completely controlled tumor growth in mice. Conclusions: This study demonstrated the potency of palbociclib in OSCC models and provides a rationale for the inclusion of PIK3CA testing in the clinical evaluation of CDK4/6 inhibitors and suggests combination approaches for further clinical studies

Synergistic Growth Inhibition by Afatinib and Trametinib in Preclinical Oral Squamous Cell Carcinoma Models

Background: Given that aberrant activation of epidermal growth factor receptor family receptors (ErbB) is a common event in oral squamous cell carcinoma, and that high expression of these receptor proteins is often associated with poor prognosis, this rationalizes the approach of targeting ErbB signaling pathways to improve the survival of patients with oral squamous cell carcinoma. However, monotherapy with the ErbB blocker afatinib has shown limited survival benefits. Objectives: This study was performed to identify mechanisms of afatinib resistance and to explore potential afatinib-based combination treatments with other targeted inhibitors in oral squamous cell carcinoma. Methods: We determined the anti-proliferative effects of afatinib on a panel of oral squamous cell carcinoma cell lines using a crystal violet-growth inhibition assay, click-iT 5-ethynyl-2′-deoxyuridine staining, and cell-cycle analysis. Biochemical assays were performed to study the underlying mechanism of drug treatment as a single agent or in combination with the MEK inhibitor trametinib. We further evaluated and compared the anti-tumor effects of single agent and combined treatment by using oral squamous cell carcinoma xenograft models. Results: In this study, we showed that afatinib inhibited oral squamous cell carcinoma cell proliferation via cell-cycle arrest at the G0/G1 phase, and inhibited tumor growth in xenograft mouse models. Interestingly, we demonstrated reactivation of the mitogen-activated protein kinase (ERK1/2) pathway in vitro, which possibly reduced the effects of ErbB inhibition. Concomitant treatment of oral squamous cell carcinoma cells with afatinib and trametinib synergized the anti-tumor effects in oral squamous cell carcinoma-bearing mouse models. Conclusions: Our findings provide insight into the molecular mechanism of resistance to afatinib and support further clinical evaluation into the combination of afatinib and MEK inhibition in the treatment of oral squamous cell carcinoma. © 2019, Springer Nature Switzerland AG