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Color Stability of Multi-Layer Fast Fired ZirconiaSaad Joulakh, Yuwei Fan and Russell Giordano Department of Restorative Sciences & BiomaterialsObjectives: This project seeks to examine the effects of rapid firing on shade as well as additional firing cycles needed for staining and glazing. Materials and Methods: Rectangular tiles 2.0±0.2 mm thick were sectioned from blocks of multicolor zirconia manufactured by Kuraray (Katana zirconia, A2 and NW shade) and Denstply–Sirona (inCoris Zirconia, F1 and F0.5 shade). The titles (n=10 for each shade type) were fired using a fast fire cycle (1540°C for 30 minutes, total 1 hour) in inFire HTC furnace and a regular cycle in Zyrcomat furnace serving as a control, (1530°C for 2 hours, total 6 hours). Two additional glazing cycles (850°C for 1 min, total 26 mins) were performed in a Programat CS furnace. Color and shade were measured after each firing cycle. Color was measured using a Ci7600 Spectrophotometer (X-Rite) and a VTIA Easyshade V clinical digital shade-taking device. Comparison of color differences by ANOVA and Tukey-Kramer HSD test (a = 0.05) was done using JMP Pro 13.2. Results: The color changes after one glaze (?E*1) and two glaze (?E*2) cycles for each group are shown in Figure 1. Pooled data showed a color change after first glaze firing ?E* was 0.661 ± 0.584 for inCoris Zirconia, and 0.931± 0.573 for Katana Multilayered Zirconia, while 0.875 ± 0.682 for regular firing, and 0.717 ± 0.418 for fast firing. Linear regression showed firing method, glaze time, and zirconia type shade had no significant effect on the color change. Conclusion: Color change after glazing firing of zirconia sintered using fast firing showed no significant difference compared to that using regular firing. Glazing firing of multilayer zirconia had no significant color change compared to monochromatic zirconia. ?E*1 ?E*2 Material Shade Firing N Mean Std Dev N Mean Std Dev Incoris Mono F0.5 Fast 5 0.484 0.259 5 0.286 0.113 Regular 5 1.254 0.943 5 1.190 1.067 F1 Fast 5 0.478 0.220 5 0.310 0.081 Regular 5 0.427 0.151 5 0.346 0.175 Kanata ML A2 Fast 5 0.997 0.602 5 1.038 0.789 Regular 5 0.964 0.672 5 0.956 0.589 NW Fast 5 0.908 0.570 5 0.770 0.703 Regular 5 0.854 0.635 5 0.747 0.177Automation of Cephalometric Landmarking Using Deep Learning – Preliminary ResultsSvitlana Koval, Caglayan Dicle, Arel Cordero, Lakshmi Garladinne, Melih Motro Departments of General Dentistry and Orthodontics and Dentofacial OrthopedicsIntroduction: Cephalometric tracing is one of the fundamental stages of proper orthodontic diagnosis and treatment planning. However, the process is exhaustive and time consuming. Automation of such a process not only saves time but also may reduce the human errors which may lead to misplanning. The main problem in the task of automation is reaching to the accuracy at human level or surpassing it. Aim: To compare the accuracy of automated landmarking versus manual tracing. Method: 2000 cephalometric images were acquired from Boston University, Department of Orthodontics and Dentofacial Orthopedics Image Database. All of them were traced using 73 landmarks for each image. These images were split to serve as training and testing datasets. State of the art machine learning techniques such as deep learning and convolutional neural networks were used for training and automation of cephalometric landmarking. Results: Our preliminary results show that the mean error in accuracy was within 0.019 x image size which corresponds to mean error of ~20 pixels. Conclusion: Current results are preliminary and resulted from short period of training. With longer training times and using deeper networks, it will be possible to increase the accuracy significantly.Dentigerous Cyst vs Hyperplastic Follicle: Radiographic and Histopathology IdentificationMei-Hsuan Li, Lakshmi Garladinne, Vikki Noonan , Manish Bhagania and Devaki Sundararajan, Departments of General Dentistry and Oral & Maxillofacial SurgeryIntroduction: Dentigerous cysts are one of the most commonly seen developmental odontogenic cysts of the jaw, which are frequently associated with impacted teeth. The histopathologic features of dentigerous cysts vary, depending on whether the cyst is inflamed or not inflamed. They are characterized by squamous stratified epithelium without formation of rete ridges. Hyperplastic dental follicles are described as odontogenic hamartomatous lesion that occurs in pericoronal tissues of the unerupted tooth. They appear radiographically as a well circumscribed cystic radiolucency surrounding the crown of the impacted tooth and has often been misdiagnosed clinically as dentigerous cyst. Microscopically, the hyperplastic dental follicle consists of fibrous connective tissue containing odontogenic epithelium, multinucleated giant cells, and calcification foci. However, despite their long history of discovery and numerous track records concerning the disease, no standardized or well-established measurements to date precisely differentiate the clinical diagnosis of dentigerous cysts from that of hyperplastic dental follicles or other indiscernible radiolucent lesions such as odontogenic cysts through diagnostic images, especially for small dentigerous cysts that are hard to be discerned from large hyperplastic follicles based upon radiographic images and clinical presentations. Some studies stated that if a pericoronal radiolucency of greater than 2.5mm on an intraoral radiograph and greater than 3mm on a rotational panoramic radiograph, a cystic lesion instead of a hyperplastic follicle should be suspected. Aim: Retrospective analysis of the histologically identified specimens for hyperplastic follicle and dentigerous cyst and looking at the radiographic appearance with measurements. 1) We would like to assess the panoramic radiograph of each subject who has had histological assessment made as either the specimen as hyperplastic follicle or dentigerous cyst. 2) Compare the measurements between the hyperplastic follicle vs dentigerous cyst on the panoramic radiographs. Methods and Materials: Retrospective analysis of SDM dental records for known oral pathology report for hyperplastic follicle and dentigerous cyst. Obtaining the panoramic radiograph of the subject and making measurements. All the collected data will have patient’s age, sex and the tooth of concern with histopathology reports. The panoramic images obtained will be identified by using a standardized measuring method based on DAMANTE. Each individual required data alone would be stored on the HIPAA drives for the measurements to be made. Expected Results and Conclusion: Even the perifollicular space less than 2.5mm of an impacted tooth mimicking a hyperplastic dental follicle can still potentially develop cystic changes, namely a dentigerous cyst.We are hoping the criteria of a set measurement can help the clinician diagnose better as dentigerous cysts are mostly seen in younger patients. We would like to minimize the complications followed by the invasive procedures required for the management of the pathological changes, and thus it is crucial for us to be able to identify which lesion simply by assessing the radiographic images.Are You Gritty & Resilient Enough to Succeed? Grit and Resilience May be Related to Dental Students’ Academic Success during School: A National AnalysisMackenson Montas, Hamza Al Atassi, Michael C. Shapiro, Sowmya R. Rao and Andrew R. Salama Department of Oral & Maxillofacial SurgeryPurpose: There has been perennial interest in personal qualities other than cognitive abilities that determine success, including self-control, growth mind-set, and especially grit, and resilience. Grit is defined as trait-level perseverance and passion for long-term goals, while resilience is defined as the ability to bounce back or recover from stress. This study assesses the levels of grit and resilience reported by dental students in the U.S, determines the possible correlation between grit, resilience, and academic achievements, while taking gender, dental school year, race, geographic origin, and socioeconomic status (SES) into consideration. Method: An anonymous twenty-seven (27) questions cross-sectional online survey was sent via e-mail to all dental students in the U.S. The survey consisted of four sections: demographic, grit, resilience, and academic achievement. The demographic section included age, gender, dental school year, race, geographic origin, and SES. The Short Grit and Brief Resilience scales were used to determine grit and resilience respectively. Scores for both scales ranged from (1-5), and were grouped into low, moderate, and high tertiles based on distribution. Dental school GPA, and class rank were used to measure academic achievement, and were similarly grouped into low, moderate, and high tertiles based on distribution. Summary statistics were obtained for the variables, and bivariate analysis were conducted to test for group differences using two-sided chi-square tests (categorical variables) and ANOVA (continuous variables). Odds ratios (OR) and 95% confidence intervals (CI) were obtained from multinomial logistic regression models assessing the relationship of grit and resilience to the outcomes adjusting for other covariates. A two-sided p-value < 0.05 was considered statistically significant. Results: Three thousand two hundred and ninety-two (3292) responses were recorded (18% response rate). 58% of respondents were females. 53% of students had high SES. Male students reported significantly higher resilience scores than female students (mean: 3.8 vs. 3.5; P<0.0001). Hispanics reported significantly higher grit and resilience than any other race, while Asian students reported the lowest grit and resilience scores. 18% of Hispanics were ranked in the top 20 percentile vs. 37% of whites. Neither geographic origin nor SES were associated with grit, resilience, or GPA. Our logistic regression models suggest that dental students with high grit are two times more likely to have high GPA compared to students with low grit (OR= 2.06; 95% CI= 1.57-2.72). Students with high resilience are 1.3 times more likely to have high GPA compared to students with low resilience (OR= 1.36; 95% CI=1.04-1.76). Additionally, students with low grit are nearly two times more likely to be ranked in the bottom third of their classes than students with high grit (OR= 1.86; 95%CI= 1.25- 2.77) and students with low resilience are 1.2 times more likely to be ranked in the bottom third of their classes than students with high resilience (OR= 1.24 95% CI= 0.85- 1.80). Conclusion: It is clear that gritty and resilient dental students achieved higher GPA scores and better class ranks. Therefore, it is crucial for students who would like to achieve academic success to consider the value that these non-cognitive characteristics might have on their overall progress. It is also critical to further discuss and potentially address the relationship between race and academic achievement, by trying to understand the core reason behind this association.Implementation and Utilization of Digital Dentistry (CAD/CAM) in US Dental Schools - An Integration Path from Preclinical to Clinical EducationMohammad Mourad, Ana Keohane, Alexander Bendayan, and Vasiliki Maseli Department of General DentistryObjectives: The objectives of this survey study are: a) to assess the degree to which digital dental technologies (CAD/CAM, Computer-Aided Design/Computer-Aided Manufacturing) are implemented and utilized in the preclinical and clinical educational activities of the 66 U.S. dental schools at the predoctoral level; b) to identify when and what dental technology techniques or systems are integrated into the dental school curriculum; c) to identify reasons why schools are not currently implementing digital technology and d) to assess how dental faculty are trained and calibrated. Methods: After IRB approval (IRB Number: H-37610, Exempt), a survey link (REDCap software) through email was sent to the Associate Deans of Academic and Clinical Affairs to each of the 66 dental schools in the U.S. The email addresses were collected from the websites of each U.S. dental school. Quantitative response data will be analyzed using JMP Pro 13 (SAS, Cary, NC) statistical analysis software. Results: The response rate so far is 40.74%. All the responses should be received by the end of September 2018. The data collected will include the number and percentage of U.S. dental schools implementing and using CAD/CAM technology. In addition, the frequency of dental technology techniques or systems being used in the dental curriculum, the frequency of what CAD/CAM techniques/features are taught, and the reasons why schools are not currently implementing digital technology will be gathered as well. Finally, a summary of how dental faculty can be effectively trained and calibrated will be compiled. Conclusion: The information obtained will be shared, keeping the responses completely anonymous, in order to serve as a guide on how CAD/CAM technology can be better implemented throughout the predoctoral U.S. dental school curriculum.In Vitro Comparison of Tensile Bond Strength of Zirconia to Titanium Substrate with Various Types of Luting CementsAsif Rangoonwala, Yuwei Fan and Dan Nathanson, Department of Restorative Sciences & BiomaterialsIntroduction: In today’s market with the advent of multiple self-adhesive resin cements there remains some unanswered phenomena regarding the bond strengths of these novel self- adhesive cements as compared to the traditional adhesive cements and resin modified glass ionomer cements (RMGIC). This study was done to compare tensile bond strengths of nine commercially available luting cements. Material and Methods: Laboratory pressed 10 mm diameter zirconia disks and 7.9 mm diameter titanium cylinders were prepared (N=108) and cemented using the tested cements. Samples were divided into one of the 9 groups (n=12): CemEZ (Zest Dental), Multilink Automix SG (Ivoclar), NX3(Kerr), Panavia V5 (Kuraray), Maxcem Elite (Kerr), RelyX Unicem 2 (3M), TheraCem (Bisco), Ceramir (Doxa), ACTIVA Bioactive-cement (Pulpdent). After applying cements, the specimens were placed under a constant load of 1390 grams for thirty minutes. After the cement completely set, the specimens were stored in 100% humidity at 37°C for 72 hours. The samples were then subjected to tensile testing until the failure of bounded interface between the zirconia disk and titanium. Data were analysed by one-way ANOVA using JMP Pro 13. Results: The mean tensile bond strengths of tested cements are shown in the table. There is significant difference among the tensile bond strengths of the tested materials. Conclusion: In general, the adhesive luting agents exhibited significantly higher tensile bond strength than the self-adhesive luting agents that exhibited significantly higher tensile bond strength than RMGIC. Bonding strengths of cements in the same category may show significant difference.Salivary Alkaline Phosphatase Activity and Total Protein Levels in Periodontal Disease and DiabetesJessica Wang, Erdjan Salih, Yihong Zhao and Bing LiuDepartment of PeriodontologyObjectives: Periodontal disease affects 47.2% of adults over 30. Early diagnosis is critical to improve quality of life. It is a host inflammatory reaction to bacterial assault which may increase protein concentration in saliva. Alkaline phosphatase (ALP) is a biomarker of osteoblastic activity. We hypothesize that salivary ALP (sALP) level may be associated with periodontal destruction and remodeling. People with diabetes are susceptible to developing periodontal disease, and those with uncontrolled diabetes are especially at risk. This study aims to 1) Compare salivary total protein levels and sALP levels in subjects with and without periodontal disease. 2) Compare sALP levels in subjects with and without diabetes to assess its diagnostic potential. Materials and Methods: Parotid and whole saliva were collected from 15 periodontally diseased and 15 healthy individuals from Boston University Medical Center. Whole saliva was collected from visitors of the Museum of Science Boston. Total protein was assessed using a BCA Protein Assay. sALP activity was determined using StemTAG. Both assays were performed on microtiter plates in duplicates to control for technical variation. Controlling for age and gender, mixed effect model with a random intercept was used to assess group differences in sALP and total protein levels. Results: Individual variability in sALP and total protein was large. Total protein in whole saliva was significantly higher in periodontal disease (p=0.05) whereas sALP was not. sALP and total protein in parotid saliva did not demonstrate statistical significance. There is no statistically significant difference in sALP activity between diabetes and health. Conclusion: From our study, total protein is a potential biomarker of periodontal disease, albeit the mechanism remains to be determined. The lack of significance in sALP activity is likely due to the limited sample size. Although sALP has proven to be a valuable biomarker, others are needed to further elucidate the effect of diabetes on oral health.Early Determinants of Ductal Patterning in the Developing Submandibular GlandWeihao Wang, Edith Lin, Alison Romisher, Janice Walker, Sue Menko, and Maria A. Kukuruzinska, Departments of Pathology, Anatomy & Cell Biology, University of Pensylvania and Department of Molecular & Cell BiologyObjectives: Previous studies have shown that during branching morphogenesis of the submandibular gland (SMG), proximal regions of the terminal buds are sites of presumptive duct formation. The goal of the study was to examine how ductal progenitor cells acquire polarity early in branching morphogenesis and how they specify the pattern of ductal extensions to form branched ductal structures. Methods: We used embryonic SMG explant cultures ex vivo to delineate the establishment of single cell asymmetries from the single bud stage through cellular reorganizations into ducts. We followed the distribution of F-actin and ZO-1 along with cell polarity effectors, aPKCz, ROCK and myosin IIB, by immunofluorescence analyses and in response to functional and pharmacological perturbations. Quantification of F-actin distribution in response to inhibitors was carried out by NIS Elements Analysis software and corresponding changes in bud geometries were quantified using Adobe Illustrator. Analyses of imaging data were performed using Imaris software by Bitplane, and statistical significance was determined by Student’s t-test for comparison of two groups and analyses of variance with Bonferroni’s post-tests were carried out for statistical analyses with Prism 5 software. Results: The F-actin cytoskeleton and the cell adhesion protein ZO-1 are early determinants of cellular asymmetries and duct specification in the embryonic SMG. Apical polarity protein aPKC is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and define the path of duct specification. Next, myosin IIB, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells, and generates apical constriction forces during the formation of ductal lumens to drive duct maturation. Conclusion: Our studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG.Deletion of LSD1 During Dysplasia Epigenetically Attenuates HNSCC PreneoplasiaThabet Alhousami, Faiza Ali, MustafaTashkandi, Andrew Salama, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti, and Manish V. Bais, Departments of Biochemistry and Section of Computational Biomedicine, Boston University School of Medicine, Departments of Endodontics, Oral & Maxillofacial Surgery and Molecular & Cell BiologyIntroduction: We recently showed that the epigenetic regulator lysine-specific demethylase 1 (LSD1) exhibits increased expression in dysplasias, in more advanced tumor grades and stages, and in mouse models of HNSCC (Alsaqer et al. 2017; Bais et al. 2015). LSD1 is a lysine-specific demethylase and flavin adenine dinucleotide (FAD)-dependent amine oxidase. We hypothesize that conditional epithelial cell-specific deletion of LSD1 in dysplasia attenuates HNSCC preneoplasia by regulating aberrant oncogenic signaling pathway and expression of immune check points. Methods: The approvals were obtained from Institutional Review Board and IACUC, Boston University. K14Cre-Lsd1 mice are generated by crossing conditional floxed mice [loxP-Lsd1-loxP) (Kerenyi et al. 2013)] with keratin 14 promoter-driven tamoxifen-inducible Cre mice (K14CreERT, Jackson Laboratory, stock no 005107). To induce OSCC, 4-Nitroquinoline 1-oxide (4NQO) which is carcinogen and tobacco component applied to tongue epithelium of Lsd1 floxed mice and wild type littermates. Results: C57BL/6 4NQO immunocompetent mouse model, LSD1 expression was visible between 9-18 weeks (dysplasia) and was significantly higher at 18-25 weeks (HNSCC). Next, in order to evaluate if conditional deletion of LSD1 in dysplasia inhibits preneoplasia, tamoxifen or vehicle injection were started after 9 week (n=7/ condition) and the effects were evaluted at 19 weeks. The data showed that LSD1 floxed mice showed inhibition of tongue preneoplasia, downregulation of mRNA expression of PD-L1, NF-kB1, Ki-67, CTGF and other oncogenic genes. Immunostaining analsysis confirmed the downregulation of PD-L1, CTGF and other oncogenic factors in LSD1 floxed mice. LSD1 inhibition by lentivirus shRNA or GSK-LSD1 attenuates proliferation and NF-kB-luciferase reporter activity in HSC3 cells. Further, proteomics, genomics and molecular signaling mechanisms are in progress to evaluate LSD1 induced epigenetic mechanisms in HNSCC preneoplasia. Conclusion: Keratin14-LSD1 floxed mice offer a novel model to study epigenetic attenuation of HNSCC preneoplasia for immunotherapy and combination therapeutic applications. Our study showed that LSD1 has a potential target for HNSCC preneoplasia. Support: NIH/NIDCR grants R03DE025274 and R21DE026892 to Manish V. Bais. Conflict of interest: All authors declare that they have no conflicts of interest regarding the contents of this manuscript. Financial Interests: All authors declare that they have no financial interest regarding the contents of this manuscript. Alsaqer SF, Tashkandi MM, Kartha VK, Yang YT, Alkheriji Y, Salama A, Varelas X, Kukuruzinska M, Monti S, Bais MV. 2017. Inhibition of lsd1 epigenetically attenuates oral cancer growth and metastasis. Oncotarget. 8(43):73372-73386. Bais MV, Kukuruzinska M, Trackman PC. 2015. Orthotopic non-metastatic and metastatic oral cancer mouse models. Oral Oncol. 51(5):476-482. Kerenyi MA, Shao Z, Hsu YJ, Guo G, Luc S, O'Brien K, Fujiwara Y, Peng C, Nguyen M, Orkin SH. 2013. Histone demethylase lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation. Elife. 2:e00633.Key Words: LSD1, CTGF, HNSCC,preneoplasia Tensile Bond Strength of Enamic and Mark II Effected by Etching time of 5% HF Etch GelChayanit Angkananuwat, Gail Yang, Yuwei Fan and Russell Giordano Department of Restorative Sciences & BiomaterialsBackground: Enamic, a polymer-ceramic biphasic interpenetrated network material, was developed to imitate natural teeth and overcome excessive wear of antagonistic teeth. One of factors to determine the success of restoration is the ability to bond with tooth structure. Objective: To investigate the tensile bond strength of Enamic blocks using 5%HF etchant and resin cement with different etching time. Materials and Methods: CAD/CAM VITA Enamic and VITA Mark II blocks were sectioned into 4 mm thickness tiles and polished. Ceramic specimens were randomized into three subgroups (n=7) according to etching time of 30, 60 and 120 seconds. Specimens were etched using a 5% HF gel and cleaned. Then the ceramic tiles were bonded to 3/16-inch diameter Grade 2 titanium rod using Panavia SA cement (Kuraray America) under 1390g load. The tensile bond strength between the tile and the titanium rod were tested using an Instron 4455A. The data were analyzed by one-way ANOVA and linear regression model using JMP Pro 13. Results: The tensile bond strengths of the tested groups are shown in the Table 1. The Vita Enamic has significant higher bond strength than Mark II to the titanium rod. For Enamic, there was no statistically significant different tensile bond strength for 30, 60 and 120 seconds etching. The maximized desirability from linear regression shows the highest tensile bond strength to be Enamic with 60s etching. Table 1 Tensile bond strength of t Enamic and Mark II to Titanium Tensile Bond Strength, MPaMaterialEtching time, sMeanStd DevCVMark II303.4032.63277.36606.8365.64182.521203.7432.10756.30Enamic307.5813.49546.10607.3443.95953.911207.1686.44789.95Conclusion: Dental porcelain types affected bond strength (P?< .05). Vita Enamic has a significantly higher bond strength than Mark II. The surface etching time did not significantly affect the bonding ability of Vita Enamic. Both Enamic and Mark II should be etched at 60s as the manufacturer recommends. Keywords:?Enamic, Tensile bond strength, Etching time. In Vivo Analysis of Lysyl Oxidase Deletion in Mouse Osteoblasts, a Pilot Study Tyler J Guinn, Vrinda Dumbal, Philip Trackman Departments of Endodontics and Molecular & Cell BiologyBackground: Lysyl Oxidase (LOX) is a key copper dependent amine oxidase enzyme present in the final steps of collagen and elastin biosynthesis. Extracellularly, LOX catalyzes the reaction of lysine and hydroxylysine to allysine and hydroxyallysine, respectively. These highly reactive aldehyde residues self-coalesce into normal immature and mature collagen cross-links. Additionally, it is known that the hormone osteocalcin is solely produced by osteoblasts. Osteocalcin is present in the later stages of bone development and mineralization. Aims: This study investigated LOX gene deletion in osteoblasts and its effect on mouse bone development over time. Materials and Methods: Our group employed the Cre-LoxP recombinase system to knockout LOX in mouse osteoblasts using the osteocalcin promoter to drive Cre recombinase. C57BL/6J male and female mice were selected and bred to achieve the breeding pair genotypes OCN-Cre+ heterozygous flox lox and homozygous flox lox. The progeny received were male and female, OCN-Cre+ homozygous flox lox, OCN-Cre- homozygous flox lox, OCN-Cre+ heterozygous flox lox, and OCN-Cre- heterozygous flox lox. Of these, we studied the differences between OCN-Cre+ homozygous lox flox, OCN-Cre- homozygous lox flox using micro CT analysis of femurs and mRNA qPCR from tibia of the same mice. Genotyping was confirmed for each mouse through DNA isolation from tail samples; and subsequent PCR, restriction enzyme digestion, and gel electrophoresis was conducted. The primer sets of interest were the OCN-Cre primer set, Lox primer set 2 that amplified the loxP insertion site downstream of exon 2; and Lox primer set 5 that amplified the loxP insertion site upstream of exon 1. The loxP sites were including new EcoR1 restriction sites were inserted by means of CRISPR/Cas9 technology, and were confirmed by cloning and DNA sequencing. Products of amplifications from both primer sets were digested for 2 hours prior to gel electrophoresis using the restriction enzyme EcoR1 to distinguish the floxed allele from wild type, while the OCN-Cre primer set needed no such digestion prior to the gel electrophoresis. 50uL of each sample was loaded into separate wells of a 1.5% agarose gel with 5uL of ethidium bromide and 1x Tris/Acetate/EDTA (TAE) buffer. 110 Volts of current was then applied for approximately 1 hour and 15 minutes. After which, the gel was placed over an ultraviolet light and analyzed for the DNA of interest. MicroCT analyses of femures were performed at the Center for Skeletal Research at Massachusetts General Hospital. Tibia RNA isolati ons and qPCR analyses of were performed according to established protocols and employed specific Taqman probes for LOX mRNA, and 18S rRNA as an internal control. Results: Micro CT data showed that male OCN-Cre+ mice had lower trabecular bone volume (-41%), trabecular bone mineral density (-31%) as well as trabecular number (-11%). Furthermore, these OCN-Cre+ male mice showed greater trabecular spacing, in comparison to OCN-Cre- male mice. Mid-diaphysis cortex of OCN-Cre+ male mice also yielded a 20% reduction in cortical area, 13% reduction in cortical thickness, and 25% reduction in polar moment of inertia. Results of the female mice showed normal bone morphology without any significant differences or changes of the OCN-Cre+ samples versus OCN-Cre- samples. In addition to the micro CT analysis, qPCR was conducted to measure LOX levels between OCN-Cre+ samples and OCN-Cre- samples. The qPCR showed the amount of LOX expression in OCN-Cre+ samples was 90% lower than OCN-Cre- mice in males and 55% lower in females. The relative quantification fold change of Cre+ males was 0.0969; while the Cre+ female samples were .4460. Conclusion: LOX deletion in mouse osteoblasts significantly reduced bone development in male 9-week old mice. So far there appears to be a sex difference in the effect of LOX deletion in osteocalcin-expressing osteoblasts in 9 week old mice. Additional mice and additional aged mice are being generated and analyzed to further investigate age- and sex-dependent differences as a function of osteoblast-specific LOX deletion.Chronic Disease Associations with Tooth LossPreeti Singh and Elizabeth Kaye Department of Health Policy and Health Services ResearchObjective: To analyze BRFSS data for associations between chronic diseases and tooth loss. Methods: The BRFSS surveys the non-institutionalized household population. Adults, 18 years and older, were analyzed for BRFSS 2012, 2014 and 2016. Self-reported tooth loss was the dependent variable categorized as no tooth loss vs any tooth loss. Self-reported history of chronic disease diagnosis were the primary independent variables (physical health, mental health, weight, diabetes, myocardial infarction, coronary heart disease, stroke, asthma, cancer, respiratory diseases, arthritis and chronic kidney disease). Bi-variate analysis included Proc Survey Freq and Proc Survey Means. Multivariate logistic regression (Proc Survey Logistic) analysis was performed and Odds Ratios computed using SAS V9.4. Results: All the examined chronic diseases demonstrated significant associations with having tooth loss in both bi-variate and multivariate analyses(Table 1,Table 2). The relationship was strongest for respiratory disease (OR=1.8), severe physical health problems (OR=1.7) and myocardial infarction (OR= 1.7). The three weakest relationships, and were for underweight individuals(OR=1.1), mild mental health problem (OR=1.1) and cancer patients (OR=1.2). Conclusion: Individuals with chronic disease are more prone to tooth loss. As the number of chronic diseases accumulates, the odds for having lost at least one tooth increases. These results will help policy makers and dentists target the populations at greatest risk. This may include adding dental benefits into Medicare for retirees and offering other dental insurance plans for younger populations. Focusing preventive programs on those with chronic diseases.Disability, Food Security, Socioeconomic Status and Untreated Dental Caries among US Adults 20-60 years: NHANES 2013-2014 Safaa Ahmed and Elizabeth Kaye, Department of Health Policy and Health Services ResearchObjectives: We examined the association between disability status and untreated dental caries in permanent teeth while exploring the influence of food security, demographic factors (age, race, gender, marital status, place of birth), and socioeconomic (poverty income ratio, education, health insurance status and last visit to the dentist) characteristics on this association. Methods: Data were drawn from the National Health and Nutrition Survey (2013-2014). We focused on adults aged 20-60 years old (N= 3914). The main independent risk factor was disability status. Weighted logistic regressions were used to estimate the odds of having at least one permanent tooth with untreated dental caries among study participants with different household food insecurity levels while controlling for demographic, and socioeconomic model covariates. Results: Having at least one permanent tooth with dental caries was significantly associated with disability status (OR=1.3, 95%CI 1.07,1.55) adjusted for demographic and socioeconomic status indicators, and household food security status. Furthermore, household food security was significantly associated with untreated dental caries. Individuals from households ranked in the lowest category of food security had 1.8 times the odds of having at least one permanent tooth with dental caries compared with those from full food security households (OR=1.76, 95% CI 1.29 to 2.39). Conclusion: The results indicate that untreated dental decay is highly associated with disability and is heavily influenced by socioeconomic inequalities. Disabled adults are more likely to struggle with poverty and access to nutritional foods, forcing them to make economic choices that can lead to a higher risk for dental caries. Our findings have implications on future oral health policies that should consider the delicate balance between equitable access to socioeconomic resources and dental public health interventions. Future research should describe the mechanisms that cause food insecurity to influence dental decay.Association between Vitamin D Deficiency and Permanent Caries among US PopulationAli Al Aqeeli, Safaa Ahmed, Thayer Scott and Elizabeth Kaye Department of Health Policy and Health Services ResearchObjective: To examine the association between vitamin D serum level deficiency and dental caries in the US population, and to evaluate the mediation effects of sunscreen on vitamin D and dental caries. Methods: 2 cycles National Health and Nutrition Examination Survey (NHANES 2011-2014). Vitamin D status: defined as Total 25-Hydroxyvitamin (25OHD2 + 25OHD3) level: sufficient (?? 75nmol/L), deficiency (<74nmol/L). Bivariate comparisons: We assessed the association between permanent caries and vitamin D deficiency, using sunscreen, race/ethnicity, age, gender, BMI, and poverty level in bivariate survey logistic regression models. Survey logistic procedures were performed to predict dental caries in permanent dentition by vitamin D deficiency adjusting for covariates. Covariates included age, gender, race/ethnicity, BMI, poverty, and sunscreen use. All analyses were weighted and adjusted for the complex design of the NHANES survey, using SAS 9.4 survey procedures. Results: The prevalence of permanent caries was higher among those with vitamin D deficiency (33.3%) compared with vitamin D sufficiency (19%). In multivariate models vitamin D status was associated with permanent caries (vitamin deficient vs sufficient OR 1.5 95% CI 1.3, 1.8; p<.0001) adjusting for demographic variables (age, gender, and race, BMI, poverty level), and sunscreen use. Surprisingly, sunscreen use has a protective effect against vitamin D deficiency (OR 0.43 95% CI 0.34, 0.51; p<0.0001) and permanent caries (OR 0.25 95% CI 0.21, 0.30; p<.0001) even after adjusting for the poverty (OR 0.35 95% CI 0.29, 0.43; p<.0001). Conclusions: The study provided evidence for the association between vitamin D deficiency and the prevalence of caries in permanent dentition. Further research is needed to study the effects of treating the vitamin D deficiency in relation to dental caries prevalence, and to further examine the surprising protective effects of sunscreen.Effect of Silicon and Calcium on Human Dental Pulp Cell Cultures Jomana Alsenan and Laisheng Chou Departments of Endodontics and Restorative Sciences & BiomaterialsAim: This study was designed to investigate the odontogenic effects of Silicon, Calcium and Phosphorous on human dental pulp cells. Methods: Human dental pulp cells derived from extracted pristine teeth were cultured in growth media with supplements of Si 25ppm, Si 25ppm+Ca 8.3ppm, Si 25ppm+Ca 8.3ppm+P 4.16ppm, Si 50ppm, Si 50ppm+Ca 16.7ppm, Si 50ppm+Ca 16.7ppm+P 8.3ppm and media without additional supplement as control, for the time intervals of 16 hours, 7, 12, and 21 days. Cell proliferation rates were measured by the optical density of crystal violet dye stained cells. ALP activity was measured by fluorometric assay. Expression of Dentin Sialoprotein (DSP) was measured by ELISA. Mineralization of cultures was measured by Alizarin Red staining. The data were presented as the mean of triplicates and normalized on a per million cell basis. Statistical analysis was conducted using ANOVA and Tukey HSD post-hoc tests. Results: Culture with 50ppm supplemental Si at day 21 yield significantly higher levels of ALP activity, DSP expression and mineralization (P<0.05) compared to the control group and other supplemented groups. Cultures with Si 25ppm+Ca 8.3ppm supplemental and Si 50ppm+Ca 16.7ppm supplemental displayed significantly higher cell proliferation rates compared to the control group at day 12 (P<0.05) and at day 21 (P<0.05). Supplemental silicon in concentration of 50 ppm could significantly induce differentiation and mineralization of normal human dental pulp cells. Calcium has a synergetic effect in up-regulating the proliferation rates. Conclusion: This is the first report to demonstrate the silicon- and calcium-induced mineralized tissue formation of human dental pulp cell cultures, leading to the potential development and clinical application of a future novel dental pulp capping material.D-Galactose Induced Senescence in Periodontal Ligaments Fibroblast CellsNada Alshehri, Leslie Will, Melih Motro and Alpdogan Kantarci Department of Orthodontics and Forsyth InstituteObjectives:? Aging is one of the main known risk factors for gingival and periodontal diseases. D-Galactose has been used to induce senescence in multiple types of cells including neurocytes and endothelial cells. The effect of D-Galactose on human periodontal ligament fibroblasts (hPDLF) has not been demonstrated. In this study, we aimed to measure the induced senescence effect of D-Galactose on hPDLF. Materials and Methods:? Human Periodontal Ligament Fibroblasts were cultured and passaged. At p3, cells were plated in 24-well plates (SD 3.5x103/cm2) and assigned to control and test groups. The test group received D-Galactose-supplemented media in different concentrations: 50 ?M, 100 ?M, 1 mM, 10 mM, 50 mM. Cells were incubated for 48 hrs. Staining forxb Associated Beta-Galactocidase (SA-?-Gal) enzyme was done. Images were taken by microscope and counted for total cell number and cells that stained positive for SA-?-GAL in both test and control groups. Results: After staining cells from different passages between P1-P9, no apparent change in ([SA-?-Gal)] activity was found. The earliest available passage (P3) was used for further experiments. Expression of (SA-?-GAL) was demonstrated to be higher in test cells compared to control cells. The difference was significant (p<0.05) for cells incubated in 10mM and 50mM D-Ggalactose concentrations, with a 39.32% and 64.83% increase in (SA-?-GAL)-positive +ive cells, respectively compared to the control group. Conclusion:? D-Galactose induced HPDLF to express senescence marker (SA-?-Gal). D-Galactose effect on hPDLF cells was dose-dependent.The Ring: Three-Dimensional Vertical AugmentationHillary Sawyer, Sherif Zareif Tawfik, Taisuke Ohira and Serge Dibart, Department of PeriodontologyBackground: In case of the 3-dimensional bone defects, two-stage surgical procedure is preferred for the implant treatment. However, the bone reconstructive surgery requires at least 6 months for the healing process prior the secondary surgery for implants placement. In 2004, Dr. Giesenhagen introduced the surgical procedure utilized an autogenous bone ring through which an implant could be placed simultaneously. Nevertheless, there were potential risks and complications of donor sites. Recently, a new prefabricated ring material derived from allogenic donor bone (AlloGraft Ring?) has been developed by Straumann collaborated with him. We believe that this material has the ideal graft characteristics of osteoconductive and osteoinductive potential intimate bone to graft contact, and superior space maintenance with the primary stability of implant. Here, we introduce two clinical cases regarding simultaneous implant placement utilized the AlloGraft Ring? for three-dimensional ridge augmentation. Case Presentation #1: A 45 years old Hispanic female patient presented for #7 implant supported restoration due to the tooth fracture. She was classified as ASA class I with no contraindications for surgical procedure. Clinical and radiographic evaluation revealed 6-7 mm labial plate dehiscence defects with adequate apical bone for implant stability. Non-surgical Phase 1 therapy was performed prior minimally traumatic extraction and implant placement with the Ring. Case Presentation #2: A 70 years old Caucasian female patient presented for implant supported restoration of missing tooth #30. She was classified as ASA class I with no contraindications for surgical procedure. Clinical and radiographic evaluation revealed vertical and horizontal defect due to the previous tooth extraction without alveolar ridge preservation. Following the phase 1 therapy, an implant was placed with the Ring and additional bone particulate grafts covered with the type I cross-linked collagen membrane. Conclusion: The Ring technique is a less invasive alternative to bone graft materials with a non-resorbable membrane allowing for expediting treatment time, predictable vertical bone augmentation with simultaneous implant placement, and reduced morbidity. Further studies and cases are needed to evaluate the long term prognosis.Engineering Novel Models for Craniofacial Bone Regeneration Abdelrouf Suwid, Serge Dibart and Erdjan Salih Department of PeriodontologyObjectives: Engineering of bone tissue is a very promising approach for bone regeneration and is believed to be as potential substitute to the existing clinical treatments. Tissue engineering can be used to build bone blocks to be transplanted as grafts, study tissue healing abnormalities to evaluate them at molecular level. So, establishing in-vitro and in-vivo models to study bone regeneration is a crucial step in evaluating the bone regeneration and study the impact of biomaterials used to enhance bone healing. In our previous studies, we created a 3D ex-vivo model to investigate bone injury healing and study the effect of different allografts in bone regeneration. In this in-vivo study model, we transplanted the live mouse calvarial bone defects grafted with different biomaterials onto the developing chicken embryo chorioallantoic membrane to evaluate the efficacy of using the chicken embryo as an in-vivo model bone tissue engineering. Materials and Method: Mouse neonatal calvarial bone were surgically isolated. Fertilized chicken eggs were incubated for 7 days, then we light candled them to locate the veins. A widow was made on the shell and the embryonic membrane dropped. Calvarial bone with different treatments transplanted to the membrane and incubated for 8 days. At the end of 8 days, the chicken embryo dissected and calvarial bone extracted to be examined histologically. Results: This study showed that all the transplanted calvarial bones were vital and integrated to the embryonic membrane. Histological analysis showed that complete healing of bone defects in control group with new bone formation and signs of blood vessels integrated in all transplanted grafted calvarial bones. Conclusion: We have developed novel 3D in-vivo live bone organ model which can be used to study bone healing biology. The results of our studies provided the first in-vivo bone model system that can be used for variety of biological studies to further understand the behavior of bone, bone repair and regeneration using clinically utilized biomaterials in dentistry. The model is promising and has a great potential to bring a practical strategies and innovations for bone tissue engineering.CBCT Evaluation of the Root and Canal Morphology of Mandibular First and Second Permanent Molars in Patient Population Visiting BUSDM – A Retrospective Study.Sowjanya Tadakapalli, Daniel Messer, Sami Chogle, Hong- Sheng Liu and Lakshmi Garladinne, Departments of Endodontics and General DentistryAim: Determining root and canal morphology of mandibular first and second molars. Evaluate the level and nature of merging of two separate root canals from the root apex in the mesial and distal roots presenting with Vertucci type II canal configuration. Methods: CBCT volumes of 100 subjects were analyzed for variations in the number of roots and canal configuration based on Vertucci classification. The level and nature of merging of two separate root canals were measured from the root apex in the mesial and distal roots with type II canal configuration. Results: Mandibular first molars had 2 roots in 95.5%, 3 roots 4%, and 4 roots in 0.4 %. Mesial roots had type IV canal 85.5%, type II 19.5%. Distal roots had type I canal 74 %, type IV 21.5%, type II 3.5%, type III 0.5% and type V 0.5%. Mandibular second molars had 2 roots 95.5%, 3 roots 4. 5%. Mesial roots had type II canal 74%, type IV 14%, type III 5 % and C - shaped 7 %. Distal roots showed type I canal 94.7%, type III 1% and type IV 4.3%. The results of nature and level of merging of the two canals for type II configuration are being determined in the ongoing analysis. Conclusion: In our study mandibular first molars with mesial and distal roots have type IV and type I canals respectively. Mandibular second molars with mesial and distal roots have type IV and type I respectively.The Effect of Different Zirconia Surface Treatments on the Bond Strength to Resin CementAbeer Tashkandi, Yuwei Fan, Dan Nathanson, Department of Restorative Sciences & BiomaterialsObjective: The aim of this study is evaluate the effect of different zirconia surface pre-treatment on its bond strength cemented to titanium abutments with resin cement. Methods: Forty Y-TZP zirconia rings with tapered hole were uniaxial dry pressed from powder (TZ-3YSB-E Tosoh zirconia) with a steel die set, and divided to 4 groups (10/group). Forty titanium tapered pin-shaped abutments were machined with 4.8 mm diameter and 7.3° tapering angle and sandblasted with 125 ?m alumina particle at 2.8 bar for 20s. After full sintering at 1530°C, all zirconia rings had 6.8 mm height, 9.8 mm outer diameter, 3.7 mm inner diameter at bottom, and 7.3° tapered hole. Group A: fully-sintered zirconia rings without surface treatment were cemented to titanium pin abutments (Control). Group B: inner surfaces of fully-sintered zirconia rings was APA treated with 50 ?m alumina at 2.8 bar for 10s (APA-FS). In Group C: the inner surfaces of 1100°C partially-sintered zirconia rings were APA treated with 50 ?m alumina particle, then fully-sintered at 1530°C (APA-PS). Group D: fully-sintered zirconia rings treated with Nano-structured alumina coating (diluted aquas suspention 3 wt.% AlN powder) cemented to titanium pin abutments (AlN-FS). TheraCem resin cement (Bisco) was used to cement all the zirconia rings to titanium pins following manufacturer’s instruction under 14 N load for 5 mins. All cemented specimens were kept in 37°C water for 24 hours before testing. Instron machine (Model 5566A) with a pull-out fixture was used to test the ultimate retention of the zirconia ring on titanium abutments under tensile. One-way ANOVA post hoc Tukey HSD (JMP Pro 13.0.) was used for data analysis (α=0.05). Results: The comparison of cement retention of tested groups are shown in Figure 1. Significant different retention loads were found among tested groups. Conclusion: APA surface treatment of zirconia in partially sintered stage and Nano-structured alumina coating surface treatemnt can significant increase the cement retention level.Optical Properties of Alumina Matrix Interpenetrating Phase CeramicsSupontep Teerakanok, Yuwei Fan and Russell Giordano, Department of Restorative Sciences & BiomaterialsObjectives: 1. To determine the effects of the use of a porogen and sintering temperature on the optical properties of infiltrated alumina ceramics. 2. To determine the effects of various polymers used for infusion of the alumina matrix. Methods: Alumina powder: (A16SG, Almatis Inc., Leetsdale, PA) was mixed a dispersant or with a porogen (10% wt) and dispersant. Alumina discs were fabricated using a slip cast technique in which the suspension is poured into a gypsum mold and dried. After partially sintering at 1050 C for 2 hours, each 2 mm-thick disc was randomly assigned according to groups with various final sintering temperatures (1250 C, 1350 C, and 1550 C): 15 groups (n=10). After silane infiltration, each disc was dried in a vacuum oven at 90 °C for 24 hours. Two different resin solutions were prepared by mixing Urethane dimethacrylate (UDMA) with Triethylene glycol dimethacrylate (TEGDMA) or Poly(propylene glycol) (400) dimethacrylate (PPGDMA 400). Polymer infused alumina discs were cured with heat under pressure. The density was measured. The transparency parameter and visible light transmission were measured using a spectrophotometer (Color i7, X-rite Inc., Grand Rapid, MI). Analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (p < 0.05) was used to analyze the density and optical properties using JMP 14.0 statistical program. Results: The average and standard deviation of % density and optical properties of different groups are shown in Table 1. Table 1. Density and optical properties of different groups of porous alumina discs. Conclusion: While increasing the porogen content during alumina disc preparation and increasing sintering temperature significantly improved optical properties, infiltration with different polymers has no significant effect on optical properties. KEYWORDS: Infused alumina, Porogen, Optical properties, PPGDMA, Sintering temperature.Role of Caregivers’ Oral beliefs and Social Influences on their Child’s Utilization of Preventive Dental care ServicesJhansi Tulasi, Thayer Scott, Elizabeth kaye Department of Health Policy & Health Services ResearchObjective: The objective of the study is to determine the effect of caregiver’s oral beliefs and social influence on their child’s utilization of preventive dental care services. Methods: Data from Detroit Dental Health project encompassed 1021 low income African American caregivers of children 0-5 years of age, living in Detroit, MI. The associations between caregivers’ oral belief scores, influence scores and preventive dental care utilization in their children was analyzed after controlling for gender, age, education, and dental insurance coverage. Bivariate and multivariate logistic regression models were performed using SAS V.9.4 survey procedures. All analyses were weighted and adjusted for complex sampling. Results: Higher scores reflected strong influence and worse oral beliefs. In bivariate analysis neither influence scale nor oral belief scale were significantly related to preventive dental visits. After controlling for confounders in multivariate regression analysis, caregivers with higher scores on the influence scale (OR:0.81, pvalue:0.02) were less likely to use preventive dental care services for their children. Though worse oral belief scores (OR:0.72, p value: 0.15) reduce the utilization of preventive dental visits, the results were not statistically significant. Conclusion: The findings suggest that social influence and worse oral beliefs of caregivers can reduce the utilization of preventive dental care services in their children. The findings underscore the need to focus on caregivers’ social networks to improve the oral health status of children.The Mechanism of Osteoblast Response to Resolvin E1Wael Yaghmoor, Amin Marghalani, Nada Tashkandi, Hatice Hasturk, Thomas Van Dyke and Alpdogan Kantarci Department of Periodontology - Forsyth InstituteBackground: Inflammation and its resolution are key mechanisms involved in several diseases, including periodontitis. Resolvin E1 (RvE1) is an endogenous pro-resolving lipid mediator. Animal studies revealed that topical treatment of periodontitis with RvE1 significantly decreased osteoclast counts, prevented alveolar bone loss, and regenerated lost periodontal tissues including bone. RvE1 inhibits osteoclast differentiation, proliferation, and reduces resorption pit formation in vitro. Our knowledge of the direct actions of RvE1 on osteoblasts in periodontal bone regeneration is limited. Methods: Primary osteoblasts were isolated from neonatal mouse calvaria. Cells were differentiated in ascorbic acid and β-glycerophosphate for 10 days and then incubated with 10ng/ml interleukin-6 (IL-6) and interleukin-6 receptor (IL-6R) to simulate pro-inflammatory conditions, with or without 10 nM RvE1. The production of OPG and RANKL was measured by ELISA. The proliferation of osteoblasts was assessed with BrdU. Signaling pathways were examined by Western blot using phosphospecific antibodies to phosphorylated substrates of AKT and MAP Kinases. Results: IL-6/IL-6R treatment resulted in a significant reduction of OPG production by osteoblasts after 2 days of culture compared to untreated cells (P<0.05). RvE1 treatment rescued OPG production compared to the IL-6/IL-6R treatment (P<0.01) and decreased RANKL. Overall, RvE1 increased the OPG/RANKL ratio to favor bone formation. RvE1 significantly increased the proliferation of osteoblasts compared to IL-6/IL-6R treatment (P<0.01). RvE1 treatment induced a significant increase in the phosphorylation of S6 ribosomal protein (P<0.05), Akt (P<0.01), and ERK1/2 pathways (P<0.01) in osteoblasts. Conclusion: RvE1 directly acts on osteoblasts by increasing their proliferation as well as the OPG/RANKL production ratio favoring bone formation through a pathway that includes phosphorylation of S6 ribosomal protein via Akt, and ERK1/2 pathways. The data suggest that RvE1 resolves inflammation and stimulates bone formation by directly modulating osteoblast functions.Novel Transcriptome Subpopulations from Sj?gren’s and Non-Sj?gren’s Salivary Gland EpitheliaAriana Dela Cruz1, Vinay Kartha3, Rongjuan Mi1,2, Stefano Monti3, Janicke Jensen4, Kathrine Skarstein5, *Xaralabos Varelas2 and *Maria Kukuruzinska11Department of Molecular and Cell Biology, Boston University School of Dental Medicine; 2Department of Biochemistry, Boston University School of Medicine; 3Department of Medicine, Boston University School of Medicine; 4Faculty of Dentistry, University of Oslo, Norway; 5Department of Clinical Medicine, University of Bergen, Norway.*co-senior authorsBackground: Sjogren’s syndrome (SS) is a complex autoimmune disease associated with lymphocytic infiltration and secretory dysfunction of salivary glands frequently accompanied by pro-fibrotic changes in the stroma. Although the current model postulates that primary SS is a secondary effect of lymphocytic infiltrates, increasing evidence suggests that structural defects in salivary gland epithelia precede and contribute to pathological responses in SS. To gain insights into the underlying molecular changes in SS epithelia, we evaluated global gene expression of salivary gland epithelia from SS and non-SS patients using laser capture microdissection (LCM) followed by computational genomic analyses. Methods: Labial salivary glands were obtained from biopsies of 16 patients with sicca symptoms. Based on the American-European Consensus Criteria, patients were sub-divided into 8 SS (focus score (FS) 1-2) and 8 non-SS controls (FS<1). RNA was isolated from epithelia by LCM and analyzed by RNAseq for computational genome-wide differential expression testing (DEseq2). Differential gene expression was further evaluated by RNAscope.? Results: Analyses of RNAseq-based gene expression profiles from SS and non-SS samples revealed three distinct subclusters: a SS (n=3), a non-SS (n=3) and a subcluster of both SS and non-SS samples (n=10). Differential expression analysis between subclusters revealed signals that may be associated with stages of SS epithelial pathology. Notable signals included the enrichment of IFN-gamma and JAK/STAT-regulated genes and genes implicated in immune responses. Increased lactoferrin mRNA expression in the SS subcluster detected by RNAseq was confirmed with RNAscope.Conclusion: We identified potential molecular subtypes of SS pathology, defined by gene expression signatures of salivary epithelia, associated with mixed clinical and histopathological characteristics. Our observations suggest that gene expression alterations arising in the epithelia contribute to the etiology of SS. We postulate that these sub-groups reflect varying stages of disease predisposition and may offer novel insight into the signals contributing to SS progression.Mechanical Strengths of Translucent Zirconia MaterialsTaylor Paek, Yuwei Fan and Russell Giordano Department of Restorative Sciences & BiomaterialsObjectives: To evaluate the biaxial flexural strengths of three different types of commercially available zirconia. Methods: Forty-five sectioned disks of ADML A-Dark Multilayered zirconia (conventional, n=15), STML Super Translucent Multilayered zirconia (intermediate translucency, n=15), and UTML Ultra Translucent Multilayered zirconia (high translucency, n=15) zirconia were prepared by core-drill cylinders from disc puck (Kitana from Kuraray). The disks were sintered according to the manufacturers recommendations. The sintering was completed in a furnace (Zircar Hot Spot 110) with a slow firing cycle ramping to 1500C in 2.25 hours, soaked for 4.5 hours, then cooled to 90C in 2.25 hours. The sectioned specimens were prepared to compensate for 20% shrinkage in order to get the desired fully-sintered dimensions of 1.5mm thickness and 16.3 mm in diameter. All the specimens were tested in biaxial flexural, with failure recorded at max load (MPa) using a universal testing machine (Instron 5566A) until complete fracture. Data was analyzed with ANOVA and Tukey HSD using JMP Pro 13.2. Results: The mean biaxial flexural strengths for ADML, STML, and UTML were as follows: 931.76 ± 108.94 MPa, 758.01 ± 80.00 MPa, 654.92 ± 40.78 MPa. ADML had significantly higher biaxial flexural strength than others. UTML had the significantly lower biaxial flexural strength than others. Conclusion: The increase in translucency decreased the biaxial flexural strength of the zirconia material.Effect of Different Firing Cycles, Etchant Concentration and Etching Time on Tensile Bond Strength of E.max CADMohammed Ahmed, Yuwei Fan and Dan Nathanson, Department of Restorative Sciences & BiomaterialsObjective: To evaluate if the firing cycles of e.max CAD, duration of etching, and etchant concentration have effect on the tensile bonding strength. Methods: Lithium-disilicate (IPS e.max CAD, Ivoclar) ceramic blocks were sectioned into rectangular tiles of 1.8mm thickness. The tiles were heat treated (1, 5, or 9 firing cycles) in Programat CS furnace, etched for different times (20,160,300 seconds) with hydrofluoric-acid gel (5% or 9.6%). Titanium pins (Grade V Ti-alloy, 6.4 mm in diameter, 1 inch in length) were sand blasted on the flat base end, cleaned and dried for use. A randomized experimental table was made by design of experiment (DOE) method using JMP Pro 13. 2 with 40 specimens and 5 blocks design. Forty ceramic tiles and forty titanium pins were prepared as described above. Self-adhesive resin cement (Theracem) was used for cementing the titanium-pins on the etched lithium disilicate tiles. A vertical load of 10 N was placed for 40 minutes. All the 40 cemented specimens were stored in 100% moisture in a 37 °C incubator for 48 hours. A tensile test was performed on all specimens using a universal mechanical testing machine (Instron 5566A). The load at failure was recorded for each specimen and the tensile bonding strength was calculated. To test the etching time effect only, the same procedure was followed on another set of 18 rectangular ceramic tiles (fired for 5 firing cycles) and titanium pins. The ceramic tile were etched with 5%HF for 10, 20, 60, 160 and 300 seconds. The same cementation procedure was followed and tensile bonding strengths were tested. Linear regression model was used to analyze the acquired the tensile bond strength data using JMP Pro 13.2. Results: The tensile bond strength was significantly affected by etching duration and firing cycles (p<0.001), but not significantly affected by etchant concentration (p=0.31). The test with concentrated interval of etching time indicated that the highest load to failure was observed around 60-100 s etching. Conclusion: The etching time and firing cycle directly are dominate effects to the tensile bond strength. The etchant concentration does not significantly affect the tensile bonding strength.3D Treatment Changes in Alveolar Bone Thickness Around the Mandibular IncisorsKelsey George, Ahmed Alsulaiman, Emilia Smith and David Briss Department of Orthodontics and Dentofacial OrthopedicsThe limit of mandibular incisor correction is dependent on the amount of crowding and the incisor position within the mandibular alveolar bone. ?Moving teeth outside of the alveolar bone can have detrimental effects on the periodontium.? Aim: The purpose of this study was to evaluate in 3D incisor angulation, B point, root apex position changes, facial and lingual cortical bone thickness at four levels on each tooth between the CEJ and apex, and 2D lower incisor angulation to the true vertical plane and Intercanine width changes.?Pre- and post-treatment CBCT images of 69 orthodontic patients were included from the BU repository (IRB number: H32515); 276 mandibular incisors and 138 canines were evaluated.? ?A mandibular plane generated through menton and left and right lingula was used as a horizontal reference plane. 3D measurements of angular changes in apex to constructed menton-lingula plane for all four lower incisors increased by a statistically significant amount. (p<0.0001) ?Intercanine width (p=0.004), arch length discrepancy (p<0.0001) incisor angulation for each incisor also showed statistically significant differences (mean change 2.407°, mean p=0.0151).? Changes in L1-NB were also found to be statistically significant between pre-and post-treatment records (p < 0.05).? Incisor bone thickness changes were statistically significant.? Lingual bone change in LL1 at point A, was –0.17 mm, (SD=0.54), p=0.010: at point B, -0.35 (SD=0.76), p=0.0002: at point C, -0.54 (SD=0.94), p<0.0001: at apex point -0.64 mm (SD=1.17), p<0.0001.? Similar lingual bone thickness changes were noted for all incisors.? Buccal bone change was not significant (p=0.06-0.88). Results: The results show that lingual bone loss increased from superior reference point (A) to apex, suggesting that roots are tipped around the center of resistance.? Conclusion: This finding contradicts other studies suggesting labial bone loss is greater than lingual bone loss.Effect of Simvastatin on the Biocompatibility of Human Dental Pulp CellsKanwal Maheshwari, Chang Zhang and Laisheng Chou Departments of Endodontics and Restorative Sciences & BiomaterialsHydroxymethylglutaryl-coenzyme-A reductase inhibitors (HMG-Co-A), statins, are known to reduce plasma cholesterol levels and cardiovascular morbidity and mortality. Moreover, administration of Simvastatin has exhibited positive effect on proliferation and osteoblastic differentiation of human periodontal ligament cells. Aim: The purpose of this study was to compare the effectiveness of different concentrations of Simvastatin on the attachment and proliferation of human dental pulp cells (HDPCs). Methods: HDPCs were cultured with Simvastatin at various concentrations of: 1, 10, 50, 100 umol/L, and 0 umol/L was used as a control. The cell attachment was investigated at 4, 8, 12, 16, and 20 hours. The proliferation rate was investigated at 7, 14 and 21 days. Crystal violet staining was used to determine attachment efficiency and proliferation. The optical density of cell-bound crystal violet was measured using a spectrophotometer at a wavelength of 590 nm. Student’s T-test and ANOVA were used to perform the statistical analysis. P-values ≤0.05 were considered statistically significant. Results: The results showed that all tested concentrations of Simvastatin reduced cell attachment at 16 hours by 10% at 1 umol/L, 30% at 10 umol/L, 15% at 50 umol/L and 35% at 100 umol/L (P<0.001). The proliferation rates at day 21 were also decreased by 50% for all concentrations (P<0.001). Conclusion: our results showed that Simvastatin downregulated cell attachment and proliferation rates of human dental pulp cells. Cephalometric Variability among SiblingsKatharine Marcks, Yihong Zhao, Melih Motro and Leslie Will Department of Orthodontics and Dentofacial OrthopedicsObjective: To determine whether multiple siblings in a family resemble one another in terms of their craniofacial characteristics as measured on cephalometric radiographs. Methods: This study was conducted retrospectively using data from the Forsyth Twin sample. 32 families were included, each with at least 4 siblings who had lateral cephalometric radiographs taken after skeletal maturity had been documented, for a total of 142 subjects. Only one of a pair of monozygotic twins was included per family. Headfilms were digitized and skeletal landmarks located to allow the measurement of 6 parameters that indicated sagittal jaw relationships and vertical status. Statistical analysis was carried out. Dixon’s Q test was applied to see if there were any outliers in a family for a given parameter. Manhattan distance was then calculated for each sibling per parameter to demonstrate individual variability. Linear cluster plots were examined relative to the mean and standard deviation of each parameter to assess clinical relevance of the differences. Results: 21 families (65.6%) had at least one outlier among the 6 measurements, with a total of 29 individual outliers (of 142; 20.4%). Only 2 individuals (1.4%) were significantly different from their siblings for more than 1 measurement. However, the remaining 11 families (34.4%) had no outliers, and 13 families (40.6%) had only 1 measurement with an outlier. Although the majority of the families had measurements that were statistically clustered, the range of the measurements were clinically relevant as they might lead to differing orthodontic treatment plans. For example, the mean range of SNB angles was 7.23 degrees, and the mean range of mandibular plane angles was 9.42 degrees. Conclusion: Although families are generally similar in their craniofacial characteristics as measured on cephalometric radiographs, measurements from siblings cannot be used to predict the measurements of another sibling.Soft Tissue Profile Preference of Layperson Among Different CulturesAhmed Mohammed, Ahmed Alsumaiman, Monika Hersberger-Zurfluh, Joseph Bouserhal, Nayla Bassil-Nassif, Rachel Satherkge, Kazuhito Arai, Misa Ikoma, Ghassan Al-Turki, Abdulrahman Idrees, Beyza Tagrikulu and Melih Motro, Zurich, Saint-Joseph, Nippon Dental, King Abdul Aziz and Istanbul universities, Military Hospital of Pretoria and Department of Orthodontics & Dentofacial OrthopedicsObjectives: The aim of this study was to assess the profile preferences of laypeople in 7 demographic regions (USA, Switzerland, Lebanon, South Africa, Japan, Saudi Arabia and Turkey), and to assess if background such as, gender, age, race, education and income had any influence on decision. Methods: 535 laypersons were asked to rate fifty profile silhouettes of a female after altering the lip in five 1 mm increments in the sagittal direction and nose in three sagittal and vertical directions, creating 45 combinations. The study was conducted in 7 cities (Boston, Zurich, Beirut, Pretoria, Tokyo, Jeddah and Istanbul). Demographics were collected. Once identified in each location, the soft tissue values were incorporated to the most preferred profile. (Nasolabial angle/lip position to the E and S plane). ANOVA with post hocTukey test was used to compare difference in mean preference in each location. Multivariate regression model was used to assess the effect of demographics on preference. Results: The mean for profile preference was significantly different across locations at p-value of <.0001. In the United States and Lebanon, the most preferred profile was Original Lip and Original Nose. In Switzerland and South Africa, the most preferred profile was Lip -2, Nose A -1, V -1. In Japan and Saudi Arabia, the most preferred profile was Original Lip, Nose A +1, V-1. And in Turkey, the most preferred profile was Lip +2, Nose A -1, V +1. Profile change, location, gender and race were significant confounders at p-value of <.0001, <.0001, <.0001 and 0.02 respectively, on the other hand, age, education and income were not. Conclusion: Layperson’s perception of lip and nose position is different among locations. Gender and race are significant confounders unlike age, education and income.3D Soft Tissue Effects of Rapid Palatal ExpansionDiana Torres, Ahmed Alsulaiman and Melih Motro Department of Orthodontics and Dentofacial OrthopedicsObjective: The aim of this study was to investigate the 3D effects of rapid maxillary expansion on the soft tissues of the face using CBCT. Materials and Methods: Images were acquired from the CBCT repository of Boston University Department of Orthodontics. The experimental group consisted of 30 patients (10 males, 20 females. Mean age, 10.93 ± 2.20) who were treated with a hyrax expander. The control group comprised of 30 patients (11 males, 19 females. Mean age 11.43 ± 2.8). Initial and progress CBCT scans were analyzed using MIMICS 20.0 software. Soft tissue measurements were made using soft tissue landmarks. A paired t-test was used to compare the changes after expansion, and a student t-test was used to compare the experimental and the control group. Intraclass correlation coefficient was used to evaluate intra-examiner reliability. Results: Statistically significant differences were noted when comparing the experimental to the control group in transverse and anterior posterior dimensions. There was more widening or increase in the experimental group when looking at the bialar distance P=0.0363, Angle of pronasale to nostril base right and left P<.0001, Nostril medium left to midsagittal plane P=0.0423, Nostril medium right to nostril medium left P=0.0272, Nostril base left to midsagittal plane P=0.0207, Chelion right to endocanthus right P=0.0086, Chelion left to endocanthus left P=0.0015. In the anteroposterior plane, the tip of the nose moved forward (Pronasale to coronal plane P=0.0018, Pronasale to Nostril Base left P <.0001, Pronasale to Nostril Base Right P<.0001. Finally, the increase from Subnasale to coronal plane was marginally significant (P= 0.0561) Suggesting that subnasale moved forward as well. ICC was >0.99, indicating excellent reliability. Conclusion: Rapid palatal expansion influences soft tissue changes of the face, specifically in the area of the nose.Skeletal Maturity: Past vs. PresentAshley Ta, Ahmed Alsulaiman, Leslie Will Department of Orthodontics and Dentofacial OrthopedicsObjectives: To compare skeletal maturation of female and male subjects from historic samples to present day subjects by assessing Fishman’s skeletal maturity index. Present day eating habits and lifestyle have been suggested as factors in accelerating pubertal maturation seen within the last century. Consequently, body mass index was evaluated to determine whether it is a factor in skeletal maturation. Methods: This pilot study included hand-wrist films from 92 subjects from the Burlington and Forsyth longitudinal growth studies taken from 1959-1970 and 90 patients currently enrolled in the Orthodontic department. Each group was divided by gender and included at least four subjects from each SMI stage. The age of the subjects ranged between 7 and 16 years. Skeletal maturation was evaluated using Fishman’s skeletal maturity index. BMI was calculated using the subjects’ heights and weights. Results: Females in the present sample reached SMI stages 7 and 8 significantly earlier: 11.5 versus 13.2 years for SMI 7 (P=0.0021) and 11.3 vs 12.7 years for SMI 8 (P = 0.0025). Among males, only stage 5 showed statistically significant differences: 12.4 vs 13.8 years (P=0.0033). It was also seen that present day females at SMI stage 7 had a significantly higher mean BMI than the females at the same stage in the historic sample. This suggests that BMI is associated with acceleration of maturation among females at SMI stage 7 (P=0.044). Conclusion: In both male and female subjects, there are differences between past and present populations at certain SMI stages. This evidence suggests that patterns of skeletal maturation may have changed and BMI may contribute to such changes. Increasing our sample size may further help elucidate these changes.Cellular Uptake of Chitosan Nanoparticles Induced Genotoxicity in Human Dental Pulp CellsRami Alhomrany, Chang Zhang and Laisheng Chou Departments of Endodontics and Restorative Sciences & BiomaterialsObjective: Recent studies have shown that chitosan nanoparticles could enhance the antimicrobial activity of several dental materials. The aim of this study was to evaluate cellular uptake and genotoxicity of various sizes and concentrations of chitosan nanoparticles cultured with human dental pulp cells. Methods: Human dental pulp cells were derived from human dental pulp tissues and cultured for 24 hours with 50 nm and 350 nm FITC tagged chitosan nanoparticles in concentrations: 0.1 mg/mL, 0.5 mg/mL, and 2 mg/mL as study groups, and 0 mg/mL as a control. The fluorescence intensity of the FITC tagged chitosan nanoparticles was measured using spectrophotometer to determine the cellular uptake. Genotoxicity was assessed by Cytokinesis-block micronucleus method and by measuring the fluorescent intensity of the phosphorylated H2AX nuclear foci. Statistical analysis was performed using One-Way ANOVA, post-hoc Tukey, and Chi-square tests. Results: Human dental pulp cells exposed to chitosan nanoparticles at a concentration of 0.1 mg/mL showed limited cellular uptake with no genotoxicity. At concentrations of 0.5 mg/mL and 2 mg/mL, chitosan nanoparticles were able to internalize the cells and significantly induced higher proportions of micronuclei (P=0.001), nuclear buds (P=0.009), and pH2AX nuclear foci (P=0.00004) as compared to the control. The genotoxic effect appeared to be in dose-dependent and size-associated manners. Conclusion: Chitosan nanoparticles at a concentration of 0.1 mg/mL showed no genotoxicity, however, higher concentrations, 0.5 mg/mL and 2 mg/mL possessed a DNA damaging effect on human dental pulp cells.Three-Dimensional Stability Following Two-Jaw Class II Surgery: A Pilot StudySarah AlMugairin, Melih Motro, Goli Parsi, Pushkar.Mehra and Leslie Will Departments of Oral & Maxillofacial Surgery and Orthodontics and Dentofacial OrthopedicsThe objective of orthognathic surgery is to establish proper oral function and facial esthetics. Implicit in this goal is obtaining a stable result. Reports studying the stability of surgery in three dimensions typically utilized Voxel Based Registration method on images using the cranial base as a reference. To this date, there is insufficient detailed data for three-dimensional stability of orthognathic surgeries. The aim of this pilot study was to evaluate the stability of different orthognathic surgical procedures. Materials and Methods: CBCT scans of 9 patients who had orthognathic surgery were reviewed. These included pre-operative (T0), post-surgery (T1), and at least one year postoperative (T2) (12-47 months) scans. All patients had maxillary 4-piece LeFort I osteotomies and mandibular advancement surgery. Mimics Innovation Suite? Research Edition software (version 21) (Leuven, Belgium) was used for analysis. Forty-three parameters were chosen that represented the maxillary and mandibular segments. Measurement between points and/or planes on the 3D surface models were used to detect the sagittal, vertical, and transverse movements in each time interval. The Friedman test was used to compare the average change between the different time points and the Wilcoxon signed rank test was used to detect changes during each time interval. Results: During surgery, the anterior maxilla and anterior mandible both advanced (p= 0.008, p=0.008 respectively) while the posterior maxillary segments differed in their movements. Maxillary expansion was noted (p= 0.002) but no vertical change occurred in any segment. No movement was noted in any landmark on the condyles. In the postoperative interval, the only significant changes were seen in the right and left mental foramen-coronal plane (p=0.015, 0.021) and the left gonion – Frankfort Horizontal plane(p= 0.008). These were thought to be due to sample size. Long term, significant surgical changes were maintained. Conclusion: Surgical changes were stable at least one year postoperatively.Amniotic Growth Factors Induced Bone Formation in a Mouse Ex-vivo ModelAbdullah Bamashmous, Jeremy Kernitsky, Taisuke Ohira and Serge Dibart Department of PeriodontologyBackground: Cells, Growth factors and Scaffold are fundamental 3 factors for tissue regeneration. These components have to be orchestrated precisely for the ideal functional tissue formation. Growth factors act to enhance cellular activities that lead to angiogenesis, cell proliferation and extracellular matrix biosynthesis. However, a single growth factor has limitation for such activities. In order to solve this problem, a new biomaterial made of growth factors derived from human Amniotic Membrane Extracts has been recently approved by the FDA. Aim: In this study, we investigated the potential of bone formation by amniotic extracted growth factors (AGF) under ex-vivo calvaria organ culture conditions. Materials and Methods: 60 Calvarias from 7-9 days old neonatal CD-1 mice were surgically harvested under sterile conditions. A 2mm diameter critical defect was created by biopsy punch on the calvaria, and was filled with same size of non-crosslinked type I collagen membrane as a scaffold with or without growth factors. To evaluate AGF potential of tissue regeneration, we divided into four experimental group: 1) Defect only, 2) Defect with scaffold, 3) Scaffold with single growth factor (rhPDGF-BB), and 4) Scaffold with AGF (N=5). A static organ culture method was used with DMEM medium supplemented Ascorbic Acid (150 ug/ml) and Bovine Serum Albumin (5 mg/ml) for 7 weeks. Culture medium was exchanged every 2 days and used for the following biological analyses: A) Alkaline phosphatase (ALP) as an osteoblastic activity, and B) Tartrate Resistant Acid phosphatase (TRAP) as an osteoclastic activity. Scanning electron microscope (SEM) image analysis was performed to evaluate surface condition. Histological analysis was performed from paraffin sections. Results: ALP activity was significantly higher and persistent until post-operative 2 weeks in AGF group (P<0.05) while there was a dramatic decline in all other groups within a week. TRAP activity was not detectable in all groups. SEM images showed that osteoblasts like cells were dramatically accumulated over the surface of the scaffold with AGF stimulus. Histologic images illustrated an organized distribution of cells along the surface of the scaffold and new bone formation around the periphery of the defect in the AGF Group. Discussion: Our results indicate that growth factors derived from amniotic extract (AGF) have a critical potential to enhance calvaria bone regeneration under ex-vivo culture condition. These findings emphasize that AGF is possibly an important candidate for a new type of therapeutic material for regenerative medicine. The Relationship between Adiposity Indices and Skeletal Maturation in OrthodonticsLaila Bamashmous, Leslie Will, Mohamed Bamashmous, Elizabeth Kaye and Melih Motro Department of Orthodontics and Dentofacial OrthopedicsObjectives: It is well established that BMI percentile is correlated with measuring skeletal maturation in adolescence by means of Skeletal Maturity Index (SMI) and Cervical Vertebral Maturity Index (CVMI). However, BMI does not account for fat content that is found to be correlated with maturation. Therefore, the aim of this study is to assess the association between different adiposity indices and skeletal maturation in children and adolescents. Methods: In this cross-sectional study 110 patients who presented to the orthodontic post graduate clinics at Boston University were examined for their height, weight, skeletal maturation, and 10 different adiposity indices. Simple bivariate regression was used to model the relationship between skeletal maturity and adiposity indices. Results: In the sample (59% female, 41% male) mean age was 14 yoa (SD:2.5), height 160 cm (SD:11.9), weight 61 kg (SD:19.1), and ethnicity was 31% African Americans, 31% Hispanics, 27% Caucasians, and 9% Asians. A bivariate regression analysis showed that the following indices were most to least significantly associated with SMI are Hip Circumference, Fat Free Mass (FFM), Body Adiposity Index (BAI), Total Body Water (TBW), Fat Mass, Waist circumference, Subscapular Skinfold, BMI Percentile, Bio-electrical Impedance Analysis (BIA), and Triceps Skinfold (p<0.01), Waist/Hip ratio and Waist/Height ratio (p<0.05). For CVMI, it was FFM, Hips, TBW, BAI, Fat Mass, Waist, Subscapular skinfold, BIA, and Waist/Hip ratio (p<0.01), BMI, Fat percentage and Triceps Skinfold (p<0.05). However, Waist/Height ratio was not significant. Conclusion: All fat indices associated with CVMI (except waist/height ratio) and SMI were significantly higher than BMI Percentile. This represents the first cross-sectional study demonstrating a different method to assess skeletal maturity other than BMI percentile.Osteocyte MetabolismNingyuan Sun, Yuhei Uda,?Alejandro Kochen,?Roberto Nunes, Campos E?Santos and?Paola?Divieti?Pajevic Department of Molecular & Cell BiologyBone is a highly metabolic organ that undergoes continuous remodeling to maintain its structural integrity. During development, bones, and in particular osteoblasts, rely on glucose uptake. However, the role of glucose metabolism in osteocytes is currently unknown. Osteocytes are terminally differentiated osteoblasts orchestrating bone modeling and remodeling. In these cells, parathyroid hormone (PTH) suppresses Sost/sclerostin expression (a potent inhibitor of bone formation) by promoting nuclear translocation of class IIa histone deacetylase (HDAC) 4 and 5, and repression of myocyte enhancer factor 2 (MEF2) type C. Recently, Scriptaid, an HDAC complex corepressor inhibitor, has been shown to induce MEF2 activation and exercise-like adaptation in mice. In muscles, Scriptaid disrupts the HDAC4/5 co-repressor complex with increase in MEF2C function and cell respiration. We hypothesized that Scriptaid, by affecting HDAC4/5 localization and MEF2C activation might affect osteocyte functions. Treatment of the osteocytic cells Ocy454-12H with Scriptaid increased metabolic genes expression, cell respiration and glucose uptake. Surprisingly, similar effects were also seen upon treatment with PTH, suggesting that both Scriptaid and PTH can promote osteocyte metabolism. Similar to PTH, Scriptaid potently suppressed Sost expression. Silencing of HDAC5 in Ocy454-12H cells abolished Sost suppression but not glucose transporter type 4 (Glut4) up-regulation induced by Scriptaid. These results demonstrate that Scriptaid increases osteocyte respiration and glucose uptake by mechanisms independent of HDACs complex inhibition. In osteocytes, Scriptaid, similar to PTH, increases binding of HDAC5 to Mef2c with suppression of Sost whereas only partially increase Rankl expression, suggestion a potential bone anabolic effect.Lysyl Oxidase Like-2 Role in TMJ and Knee OsteoarthritisMustafa Tashkandi, Saqer Alsaqer, Thabet Alhousami, Faiza Ali, Pushkar Mehra, Larry M. Wolford, Louis C. Gerstenfeld, Mary B. Goldring and Manish V. Bais, Texas A&M University College of Dentistry, Baylor University Medical Center, Dallas, TX; Department of Cell and Developmental Biology, Weill Cornell Medical College; Department of Orthopedic Surgery, Boston University School of Medicine and Hospital for Special Surgery Research Institute and Departments of Oral & Maxillofacial Surgery & Molecular & Cell BiologyIntroduction: In the United States, 33% of adults have at least one symptom of temporomandibular joint (TMJ) disorders including TMJ osteoarthritis (TMJ-OA). However, there is no chondroprotective agent approved for clinical application. We showed that LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses, and attenuates NF-kB signaling pathway (Alshenibr et al. 2017). The goal of the present study was to evaluate if systemic administration of LOXL2 adenovirus by in vivo promotes an anabolic response in TMJ-OA cartilage. Methods: The approval to obtain human tissues was obtained from Institutional Review Board and animal experiments were approved by Institutional Animal Care and Use Committee, Boston University. We employed two different models to assess LOXL2 adenovirus function in TMJ-OA. One model is a genetic model of the chondrodysplasia (Cho/+) OA mouse strain (n=56), which develops progressive TMJ-OA due to a mutation in the Col11a1 gene. The second model assessed anabolic activity in 5 human TMJ-OA cartilage /Matrigel constructs implanted subcutaneously in nude mice (n=10). Results: Bi-monthly intraperitoneal injection of Adv-RFP-LOXL2 in four-month-old Cho/+ male and female mice (n=14/condition) for 16 weeks upregulated Sox9, aggrecan (Acan) other anabolic genes and increased proteoglycan staining in the TMJ compared to Adv-RFP-Empty group. LOXL2 is co-expresses in in human degenerative TMJ-OA along with TGF-β1, TNF-α, and IL-1β as detected by confocal imaging and 3D-reconstruction. Next, human TMJ-OA cartilage/Matrigel implants in nude mice treated with Adv-RFP-LOXL2 showed higher levels of SOX9, ACAN, and other anabolic mRNAs by RT-qPCR and RNA-seq analysis compared to the Adv-RFP-Empty-treated group. Gene set enrichment analysis showed that LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. Next, these studies are extended to Cho/+ mice knee-OA and LOXL2 transgenic mice. The data showed that LOXL2 induce anabolic effects knee joint-OA validated by marker gene experssion and immunostaining. Further, proteomics, genomics and molecular signaling mechanism are in progress to evaluate LOXL2 induced effects by unbiased approaches. Conclusion: LOXL2 has a potential to induce anabolic gene expression in the progressive TMJ-OA mouse model and human cartilage nude mouse implant preclinical models. Our study demonstrates for the first time that LOXL2 adenovirus administered systemically induces anabolic gene expression in vivo in TMJ-OA preclinical models. Thus, manipulation of LOXL2-related functions has potential as a strategy for anabolic gene therapy to treat OA. ................
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