Osteocytes Induce Myeloid Proliferation and Inhibit Osteoclast Differentiation

Osteocytes Induce Myeloid Proliferation and Inhibit Osteoclast Differentiation Introduction: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone marrow cells through direct contact and via secreted factors that can reach cells in the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCR) and mice lacking Gsα expression in osteocytes have abnormal myelopoiesis. This study aimed to evaluate the effect of osteocytes on myeloid proliferation and differentiation. To investigate cross talk between osteocytes and the BMM, we established an osteocytic cell line lacking Gsα expression to investigate molecular mechanisms by which osteocytes control macrophages proliferation and differentiation. Methods: CRISPR/Cas9 was used to knockout Gsα in the osteocytic cells line Ocy454. Conditioned medium (CM) from differentiated Ocy-GsαControl and Ocy-GsαKO cells was used to treat myeloid cells and bone marrow mononuclear cells (BMNCs) isolated from long bones of 6 weeks wild type mice. BMNCs were cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) for osteoclast differentiation. Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring and mRNA expression were used to evaluate cell proliferation, differentiation and function. Proteomics analysis of CM was performed to identify osteocyte secreted factors capable of controlling myelopoiesis and osteoclastogenesis. Results: Myeloid cell treated with CM from Ocy-Gsαcontrol or Ocy-GsαKO showed a significant increase in cell proliferation compared to Ocy-GsαControl CM and non- treated control. BMNCs treated with CM from Ocy-Gsαcontrol or Ocy-GsαKO showed a significant increase in cell proliferation as compared to non- treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-Gsαcontrol and further suppressed by CM from Ocy-GsαKO compared to non-treated control. Osteoclasts showed a significant defect in activity and function as compared to non-treated cells. Further experiments are ongoing to identify osteocyte-secreted factors. Conclusions: Osteocytes secrete factor capable of inducing myeloid proliferation and inhibiting osteoclastogenesis and this factor(s) is Gsα-dependent. Dental Care Utilization and Disparities in Access to Dental Care Among Cancer Survivors in U.S. Objectives: This study aims to evaluate the association between cancer history and access to dental care. Methods: We analyzed data of 10,109 subjects, aged 20-80 years from The National Health and Nutrition Examination Survey (NHANES) 2011-2014. The main predictor was self-reported past history of any type of cancer (yes/no). The outcome was having a dental visit in the last year (yes/no) with reasons for the visit and barriers if no visit. Covariates included gender, race, income, education, and marital status. Chi-square and multivariable logistic regression were conducted to evaluate the association of cancer history with dental care utilization. We used SAS survey procedures to account for complex sampling design. Results: Cancer survivors were not more likely to have dental visits within the past year compared to controls (OR=0.95; 95% CI: 0.67-1.19). Both survivors and controls were more likely to have preventive than treatment visits (64% and 65% respectively p=0.68). The most common barrier to accessing dental care was cost (79% for both groups). Cancer survivors with at least a high school degree were significantly more likely to have any dental visit within a year compared to less educated survivors (OR=2.4; 95%CI:1.5-3.9). Survivors with higher SES were significantly more likely to have any dental visit within a year compared to those with lower SES (OR=6.3; 95%CI:3.5-11.6). Conclusion: Cancer survivorship was not significantly associated with any dental visits (yearly, preventive/treatment). However, financial and educational disparities exist among cancer survivors. Further research is recommended to more comprehensively understand oral health disparities among cancer survivors. Pharmaceutical Modification of a Gluten-degrading Enzyme Derived from Oral Bacteria Introduction: Detoxification of gluten immunogenic epitopes is a strategy for the treatment of Celiac disease. Previous studies have demonstrated that the epitopes can be degraded by subtilisin-derived from Rothia mucilaginosa, a natural colonizer of the oral cavity. But the enzyme loses its activity under acidic conditions, as encountered in the stomach. Objective: To protect and maintain enzyme activity under acidic conditions by exploring pharmaceutical microencapsulation techniques. Methods: Subtilisin-A purchased from Sigma was coated with a polymer that commonly used in pharmaceutical industrious. The activity of coated enzyme was assessed by incubation of coated Sub-A, non-coated Sub-A and coating materials only in diluted HCl buffer at pH 3.0 at 37°C for 0, 1, 3, 5 and 24 hours. Aliquots at each time point were collected and their activity were determined using Suc-AAPF-pNA substrate. To assess the abolishment of immunogenic gluten epitopes in vivo. Balb/c mice were fed with mouse chow pellets with and without coated subtilisin-A. Mice were sacrificed and the stomach content were collected. The survival of immunogenic gluten epitopes of the samples were analyzed by ELISA-R5 kit. Results: Subtilisin-A was modified by microencapsulation, achieved ~43% yield of coated Sub-A containing ~8% (w/w) Sub-A. The coated Sub-A showed significantly increased protection against exposure to acid conditions (pH 3.0) in vitro. An in vivo experiment (n=30) showed that gluten immunogenic epitopes decreased by ~60% in the stomach of the mice fed with chow containing coated Sub-A (0.2mg Sub-A/ g chow) compared to that of the mice fed with chow only. Conclusion: Pharmaceutical modification of subtilisins can protect enzyme activity under acidic conditions and such enzymes can be considered a promising modality for future applications to treat celiac disease. Lysyl Oxidase Like-2 Mediates Tumor to Stromal Cell Communication in Oral Cancer Introduction: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study is to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma. Methods: In vitro: To investigate functions of LOXL2 in oral cancer, the effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays, gene knock down and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effect of PXS-S1C on cancer cells expression of LOXL2 and proliferation was determined. To find potential LOXL2 substrates in gingival fibroblasts treated with CM +/- PXS-S1C, carbonyl-containing proteins were purified by affinity chromatography and identified by MS and western blot. In vivo: The effect of PXS-S1C on cancer growth and metastasis in vivo was investigated using an orthotopic oral tongue cancer mouse model in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/ kg and 30 mg/kg was injected immediately following tumor cell injections. Tumors were monitored by caliper measurements, and by in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemistry staining for proliferation markers. Results: PXS-S1C significantly inhibited gingival fibroblast proliferation triggered by tumor cell CM and attenuated phosphorylation of PDGFRβ Y771 and P-PDGFRβ Y857, but not PDGFRβ Y751 in response to CM. PXS-S1C inhibited ERK1/2-signaling in fibroblasts and not AKT in response to CM. PDGFR activation by oral tumor cells was mimicked by PDGF-AB, but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 by 5-fold in HSC3 oral cancer cells in vitro, suggesting a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified both PDGFR and integrin αV. Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 significantly decreases oral cancer progression and metastasis in vivo. Mice without PXS-S1C treatment developed larger tongue volumes (p<0.05), and more mice developed abnormal sized lymph nodes (9 out 12) compared to the PXS-S1C-treated mice (4 out 12) (p<0.05). IVIS imaging revealed inhibition of metastasis by PXS-S1C treatment. The expression of proliferation marker (Ki-67) and LOXL2 was lower in tongue tumors treated with PXS-S1C (p<0.05). Conclusions: LOXL2 secreted by cancer cells stimulates fibroblasts by enhancing PDGFR signaling and proliferation, and by stimulating αV integrin function and cell adhesion. Inhibition of LOXL2 could provide therapeutic strategies to address oral cancer. Scriptaid Induces Osteocyte Respiration through a HDAC Independent Mechanism Background: It has been known that bone has strong capability of self-repairing and it undergoes continuous remodeling to maintain its structural integrity. During development, bones (and in particular osteoblasts) rely on glucose uptake for proper skeletal development whereas in adult bone, the relative role of glucose metabolism, is currently unknown. Osteoblasts take up one fifth of the glucose consumed by muscles and they sense and use glucose through a Glut1 and AMPK-dependent pathway independent of insulin signaling. Glucose uptake in cells of the osteoblast lineage is necessary for osteoblast differentiation, bone formation and glucose homeostasis. Whereas the role of glucose in osteoblasts is well established, its effect on osteocytes is still unknown Osteocytes are terminally differentiated osteoblasts deeply embedded into the mineralized matrix of bone. In vitro studies have shown that PTH promotes bone anabolism, in part, by stimulating, in osteoblasts, anaerobic glycolysis while suppressing glucose oxidation through the TCA cycle. In osteocytes, PTH suppresses Sost expression (a potent inhibitor of bone formation) by inducing HDAC4/5 nuclear translocation and Mef2C suppression. Recently, scriptaid, a HDAC complex co-repressor, has been shown to induce Mef2c expression and exerciselike adaptation in mice. In muscles, scriptaid disrupts the co-repressor complex comprising HDAC3, SMRT/N-cor and induces nuclear export of HDAC4/5 with increase in Mef2c, Glut4 and Pdk4 expression. This suggested that scriptaid might upregulate Mef2C and Sost expression in osteocytes. Methods: To investigate scriptaid influence on osteocyte, we used an osteocytic cell line, Ocy454 12H established in our laboratory. Results: Unexpectedly, the compound potently suppressed Sost, whereas induced Glut4 and Pdk4 expression. Scriptaid also stimulates osteocyte respiration and glucose consumption rate. However, this metabolic regulation is through a HDAC independent mechanism. Moreover, additional genes such as MEPE and RANKL that participate in bone modeling and remodeling are also significantly regulated by scriptaid treatment. Conclusion: Taken together these studies demonstrated that scriptaid induces osteocyte metabolism through a HDAC independent mechanism and regulates key genes related to bone remodeling, which may have profound effect on general glucose level and bone formation. A Trypsinogen Activation Peptide Mutation Worsens Cerulein-induced Pancreatitis in the Mouse Background: Mutations in the activation peptide of human cationic trypsinogen (PRSS1) have been found in association with hereditary pancreatitis. Biochemical experiments using recombinant proteins demonstrated that the activation peptide mutations strongly stimulate autoactivation of cationic trypsinogen. These observations supported the hypothesis that increased intra-pancreatic trypsinogen activation causes hereditary pancreatitis. Aims: The aim of the present study was to generate a mouse model carrying a hereditary-pancreatitis associated activation peptide mutation and characterize the impact of the mutation on experimentally induced pancreatitis. Methods: Our genetically modified mouse strain carries the p.K24R mutation in the activation peptide of the mouse cationic trypsinogen (isoform T7). This corresponds to the p.K23R mutation in PRSS1. Acute pancreatitis was induced by 12 hourly injections of cerulein. Pancreatitis severity was determined by histology scoring, measurement of edema, MPO and serum amylase. Pancreatic trypsin activity was measured at 30 min after a single dose of cerulein. Results. Intra-pancreatic trypsin activity and all parameters of acute pancreatitis severity were markedly elevated in mice carrying the p.K24R mutation relative to the C57BL/6N controls. Conclusions: A trypsinogen activation peptide mutation associated with hereditary pancreatitis in humans increases intra-pancreatic trypsin activity and worsens pancreatitis responses in this novel mouse model. Pancreas Pathology in a Mouse Model of Rapid Trypsinogen Activation Introduction: Hereditary pancreatitis is a genetic disorder which is caused by mutations in human cationic trypsinogen. Some of these mutations affect the activation peptide of cationic trypsinogen causing intrapancreatic trypsin activation. Earlier in vitro studies showed that such mutant variants autoactivated much faster than wild type and had propensity for trypsinogen activation as in case with well-known human mutation D22G. To investigate this process in more detail the analogous mutation D23A was introduced into mouse T7 trypsinogen. Considering the fact, that this mutant variant had an incredibly high activation rate, 50-fold faster than wild type was generated mouse model with lower expression of T7 trypsinogen and delay the disease progression. Aim: The aim was to generate trypsin-dependent mouse model and to characterize pathological changes which develop and arise spontaneously from a certain age. Materials and methods: Mouse model harboring point mutation D23A with preservation of neomycin cassette was generated by knock-in method on C57BL/6 background. Mice were sacrificed in different ages and animal tissue and blood were taken for analyses. Histological evaluation was made for the presence of characteristic features of the disease such as acinar cell loss, inflammatory cell infiltrate, dilated ducts, acinar-to-ductal metaplasia. Results: Mouse model D23ANeo carrying neomycin cassette exhibit the first signs of disease at the age of 2,5 months. This model develops spontaneous pancreatitis and has features of chronic pancreatitis with loss of acinar cells and preservation of islets. There is a noticeable decrease in pancreas weight with age, which is also consistent with acinar cell loss and indicates pancreatic atrophy. Although there is a small increase in amylase level in mutant mice compared with C57BL/6N. Conclusions: This model supports the theory that increased and uncontrol trypsinogen autoactivation is one the main pathological mechanisms which leads to the development of hereditary pancreatitis. 3D Implantology: Revolutionizing Guided Implant Placement Using 3D Printed Guides Background: The use of surgical guides in dental implant surgery is well-documented and is increasingly being used during surgical placement. It is estimated that 10% of all implant surgeries utilize a surgical guide (iData research, 2015). During semi-guided implanted placement, the surgical guide helps in accurately positioning the initial osteotomy, ultimately affecting the final implant location. In such cases, the implant is placed without a guide into the osteotomy site. However, the use of the guide in semi-guided systems dramatically improves the predictability of the final placement, thus improving the overall outcome. Advances in CAD/CAM dentistry have led to increased use of milled surgical guides; however, recent development of FDA approved dental resins has led to the ability to 3D print guides, using relatively low cost equipment. Aims: The aim of this pilot study is to determine if there is a significant difference in the relative accuracy of location and of the initial osteotomy when using a milled versus a 3D printed surgical guide. Methods: The study involved five CEREC milled guides, and five FormLab 3D printed guides all produced using the same CAD scanned image file of a dentoform. A dentoform mounted on a surveyor table was used to compare the placement and angulation of the osteotomy between the milled and 3D printed guides. Following a semi-guided implant placement protocol, the initial osteotomy was created in two separate dentoforms, each using a guide key, and each made with a milled guide and 3D guide respectively. Static images of the mounted dentoform in a jig were taken, with guides firmly seated to the teeth and secured tightly. The negative control group was a static image of the respective dentoforms without any surgical guide seated. Two of the four positive control groups were static images of the 3D printed guides seated on the dentoform with the osteotomy made with the 3D printed guide and milled guide respectively. The final two of four positive control groups were static images of the milled guides seated on the dentoform with the osteotomy made with the milled guide and 3D printed guide respectively. Results: Using image analysis software, the distance was measured from a point on the osteotomy to a pre-determined point on the dentoforms. Measurements were taken for the negative control group, and each of the four positive control groups. No significant difference in location and angulation of the initial osteotomy was noted between the five milled guides. Similarly, no significant difference in location and angulation of the initial osteotomy was noted between the five 3D printed guides. When the milled guides were compared to the 3D printed guides, no significant difference between the placement and angulation of the initial osteotomy was noted. Conclusions: Guides that were 3D printed demonstrated an equal level of accuracy in site location and angulation of initial dental implant osteotomy during semi-guided implant placement. Our results show evidence that 3D printed guides serve as a viable alternative to milled surgical guides when used for semi-guided implant placement. Further comparative studies between milled and 3D printed surgical guides are necessary to demonstrate the accuracy and efficacy in the use of 3D printed surgical guides for dental implant placement. MMP-20 Expression in Human Cancer Cells Interacting with Forming/Resorbing Bone Background: Matrix Metalloprotease (MMP)-20 is a proteolytic enzyme previously thought to be restricted to enamel formation and development. Recent studies have found that MMP20 is expressed in different body tissues and interestingly, found to be increased in cancer pathology. Aggressive cancer can metastasize to bone, creating bone-cancer biology to be an area of interesting study. Objectives: The objective of this study is to investigate MMP20 protein expression in tumor cell lines interacting with bone in a ex vivo bone/cancer co-culture system. Materials and methods: Using a calvaria bone/cancer cell 3D co-culture roller tube model, bone remodeling was separated into a formation and resorption model to identify the novel effects of bone-cancer interaction on MMP20 expression in osteoblastic and osteoclastic differentiated states. Oral Squamous Carcinoma cell line (OSCC) and two breast cancer cell lines were investigated; MDA-MB-231 and a more aggressive/bone seeking MDA-BO tumor cell line. Results: Using ELISA, our study found an increase in MMP20 expression secreted from all tumor cell types interacting with calvaria in an osteoclastic differentiated state compared to calvaria in an osteoblastic differentiated state. MDA-BO is found to have an increased MMP20 expression compared to the less aggressive tumor cell lines. The cell lines MDA-MB-231 and MBA-DO have MMP20 protein expressed which were previously not investigated or found to contain MMP20. The last novel finding of our study is that bone calvaria from an ex vivo model have an inherently high expression of MMP20 protein regardless of formation or resorption status. Conclusions: In conclusion, our study offers support to previous studies that suggest MMP20 effects cancer prognosis and aggressiveness with our studies novel finding that the more aggressive breast cancer cell line had increased MMP20 expression. Further studies are warranted to investigate MMP20’s role in more aggressive cell lines in a bone/cancer co-culture system. Analysis of Nasal Airway Symmetry and Pharyngeal Airway Following Rapid Maxillary Expansion (RME) Objectives: This retrospective cohort study tested the effect of Rapid Maxillary Expansion (RME) on symmetrical volumetric changes in the nasal cavity. Volumetric changes were also analyzed for nasopharyngeal and oropharyngeal cavities as well as linear minimum cross-sectional measurements for left and right overall nasal cavities. Methods: CBCT scans of before and after RME treatment for 28 subjects (17 females, 11 males, average age 9.85 ± 2.42 years) were collected from a previously de-identified database. All subjects were treated for maxillary constriction using banded hyrax expanders. Mimics software was utilized to segment nasal and pharyngeal airways and a 3-D model was created. Anatomical points and subsequent planes using these points were established to divide the 3-D model into anterior nasal cavity right and left (ANC-R, ANC-L), posterior nasal cavity right and left (PNC-R, PNC-L), nasopharyngeal cavity, and oropharyngeal cavity. Volumetric changes for each of these compartments were then collected. For minimum cross-section analysis, initial scans were inspected visually until the thinnest portion of the left and right nasal airway was confirmed in the coronal plane. The number of slices from pronasale was recorded and the thinnest portion of the left and right nasal cavity was measured linearly. The analogous slices in the post-expansion scans were identified from pronasale and the same measurements were recorded. Paired T-test was used to quantify the pre- and post-expansion volumetric/ cross-sectional changes. Statistical significance was set at the 0.05 level. Results: Posterior expansion as measured between right and left greater palatine foramen (GPF) averaged 2.41 mm (SD = 1.03 mm). There were statistically significant differences in overall nasal cavity (3240.7 ± 2726 mm3, 32.63 ± 26.47%), ANC-R (325.4 ± 237.0 mm3, 28.71 ± 21.35%), ANC-L (420.1 ± 328.6 mm3, 40.03 ± 30.52%), PNC-R (643.4 ± 923.6 mm3, 27.01 ± 36.82%), PNC-L (767.0 ± 1343.8 mm3, 40.55 ± 68.89%), nasopharyngeal (1000.6 ± 917.7 mm3, 43.92 ± 42.36%), and oropharyngeal (2349.2 ± 2520.8 mm3, 33.76 ± 39.16%) cavity volumes. For cross-sectional analysis, the right nasal cavity (0.13 ± 0.07 mm, 41.13 ± 24.72%) and left nasal cavity (0.11 ± 0.06 mm, 38.53 ± 24.33%) showed similar increases in thinnest cross-sectional linear measurements. Conclusions: Results suggest that RME has significant benefits to increasing nasal and pharyngeal airway cavity volumes in all segments of the airway. Taking a closer look at the segments of the nasal cavity shows that the left side, both anterior and posterior, has more improvement than the right side counterparts by approximately 10-13%. Furthermore, in analyzing the left or right side of the nasal cavity alone, results show that the anterior and posterior segments have similar percent increases. Additionally, linear minimum cross-section of the left and right nasal cavities showed highly symmetrical improvements. 3D Evaluation of Condylar Changes After Rapid Maxillary Expansion Objectives: The aim of this retrospective cohort study was to determine the effect of Rapid Maxillary Expansion (RME) on condylar shape, volume and orientation as well as on mandibular rotation. Method: Pre- and post-expansion cone beam computerized tomography (CBCT) images of forty-four healthy subjects (mean=13.65, SD=1.93) who previously had undergone banded RME were selected from a CBCT repository. Linear, angular and volumetric changes as well as condylar surface area differences and changes in mandibular rotation were assed using Mimics software. The condyles were segmented by a plane passing through the sigmoid notch (Snp) and parallel to a Frankfort Horizontal derivative plane (FHD). All the linear, angular, volumetric and surface area assessments were performed on the isolated 3D condyles. Changes in mandibular rotation were evaluated by analyzing the alterations in the angle between mandibular plane and FHD plane before and after RME. Paired T test was performed to compare pre- and post- expansion for all variables stated above. Statistical significance was set at <0.05. Results: The right and left condylar widths significantly increased by 0.56mm ±0.88mm (p=0.01) and 0.61mm ±0.82mm (p=0.002), respectively. The right and left condylar volumes significantly increased by 48.06mm3 ±110.1mm3(p=0.05) and 73.62mm3 ±104.2mm3 (p=0.003), respectively. Finally, the right and left condylar surfaces significantly increased by 24.06mm2 ±36.46mm2 (p=0.006) and 37.37mm2 ±44.67mm2 (p=0.001), respectively. No significant changes were found in condylar height, condylar orientation and mandibular plane angle. Conclusion: Condylar width, volume and surface were significantly increased after Rapid Maxillary Expansion. However, condylar growth still remains to be a cofounding factor that affects the results. No statistically significant effect on condylar orientation and mandibular plane was found. Future studies will be required to assess the three dimensional changes of the condyles with growth. 3D Cephalometric Analysis Using Novel Landmarks Objectives: The aim of this project is to use new landmarks to develop a novel 3D cephalometric analysis using anatomical landmarks that cannot be easily viewed on a planar film but on a CBCT image and study the reliability of these measurements between different examiners. Our implication for completing this study is to attempt to create new cephalometric norms on CBCT scans which have not been defined yet in current literature. Methods: Fifty CBCT images of patients with normodivergent, Class I skeletal patterns and without any noticeable craniofacial deformities were screened and included in the study based on the lateral cephalograms generated from CBCT data using the Roth/Jarabak Analysis. Ffifteen predetermined landmarks that found to be reliable in another associated study were used to define six planes. Eleven different cephalometric measurements were performed using landmarks and planes in 3D. Descriptive statistics (mean and standard deviation), overall interclass correlation coefficient, and individual interclass correlation coefficient of all of the measurements was calculated among two operators. Results: The measurements showed that the means and standard deviations of all three measurers are: for Mandibular A/P: 14.49mm ±2.26; Mandibular deviation: avg: 1.63mm, ±1.19; Maxilla/ Mandibular divergence: 24.51°, ±3.85; Maxillary deviation: 0.94mm ±0.5; Pitch (Mandibular Plane), 50.38° ±2.8; Pitch (Maxillary Plane): 74.66° ±3.53; Roll (Mandibular Plane): 87.66° ±2.38, 0.05; Roll (Maxillary Plane): 87.70° ±1.85; Yaw (Maxillary Plane): 4.41°± 2.11; Yaw (Mandibular): 3.61°± 2.43; Maxillary A/P: 2.96mm ±2.26. An interclass correlation was calculated at a range from 0.53 to 0.95 with an average of 0.76±0.12. Conclusions: Our conclusions for this pilot study indicate that we have our first results for creating Cephalometric norms on a 3D scan. Amongst the operators, we indicate between moderate and excellent reliability. The measurements and points need to be plotted again two more times to confirm intra and inter examiner reliability. Determining the Reproducibility of Locating Anatomical Land Marks on Human Skulls on CBCT for Orthodontic Measurements Introduction: The objective of the study was to evaluate the reproducibility of identifying various landmarks solely from a CBCT. Landmarks include points from traditional cephalometry and points which are uniquely found on CBCT. The landmarks were strategically selected in order to be markers of different parts of the skull in order to be used to measure the skull in three dimensions in further studies. Methods: 50 CBCTs were taken from a database belonging to patients that required orthodontic treatment. With mimics software landmarks were identified from diverse parts of the skull that ranged from bony land marks, foramina and dentition. Three examiners located the land marks three times over a year long period. Three dimensional coordinates were determined in the x, y and z plane. Interpersonal and intrapersonal reliabilities were determined as well as deviations from the average. Results: Bony protrusions such as the lingulae, anterior clinoids and dentition proved to be the most reliable with the highest precision between interexaminer readings. Conclusion: Choosing different landmarks purely from CBCTs are found to be precise and reliable for landmarks that have clear bony protrusions. These points may demonstrate a new type of standard when determining land marks for 3-d evaluation of the skull. Heritability of Facial Growth of Children using Lateral Cephalograms Objective: The purpose of this study was to test the association of parental mid-face and nasal soft and hard tissue morphology with their offspring. Methods: This retrospective pilot study was comprised of twenty western European families from the Forysth/Moorrees Twin Study. The lateral cephalograms of each parent and post-pubertal child, who is at least 2 years past peak growth (from literature age ≥ 16 yrs for females and ≥ 17 yrs for males) were evaluated on thirteen cephalometric variables including ANB, overjet, ratio of the nose to total face height (Nasion (N)-Anterior Nasal Spine (ANS)/N-Menton), angle of nasal bone relative to Sella (S)-N, nasal bone length, distance from rhinion to ANS, angle of ANS to S-N, distance of rhinion to pronasale, distance of ANS to pronasale, nasolabial angle, projection of nose, soft tissue convexity and nasal height. A two-way ANOVA with multiple comparisons (TUKEY) was performed to test for the differences between family members controlling for the effect of the individual family. Statistical significance was set at p< 0.05. Results: Twelve male and 8 female offspring were studied. There was a significant difference between the child and father but not the mother for angle of nasal bone relative to S-N (p = 0.04), rhinion to ANS (mm) (p = 0.048), rhinion to pronasale (mm) (p < 0.0001), ANS to pronasale (mm) (p = 0.005), projection of nose (p = 0.0006) and nose height (p = 0.03). There was no difference between males and females in the extent of their similarity to their mothers or fathers except for nasal height, where females were similar to their mother but different from their fathers (p = 0.03). Conclusion: Children are morphologically more similar to their mothers than their fathers when comparing mid-face and nasal bone soft and hard tissue parameters. Student Learning of Digital vs Plaster Orthodontic Model Analysis Introduction: Cast analysis plays an essential role in orthodontic diagnosis. Intraoral scanning producing digital models is a relatively new but increasingly common practice in graduate orthodontic programs. It is unknown how incorporating digital models in post-graduate orthodontic programs will influence student learning of model analysis. The purpose of this pilot study was to investigate the possible differences in learning speed and accuracy of basic model analysis using digital models or traditional plaster casts. Materials and methods: Two groups of senior dental students participated, one for plaster casts and one for digital. A study moderator provided a 15-minute tutorial instructing participants on how to analyze the casts. A standardized scoring sheet was used for data collection. Each group was given five sets of orthodontic models to analyze the following measurements: Right molar occlusion, overbite, overjet, arch length, required arch space, crowding, and incisor irregularity. The accuracy of the measurements as well as the time taken to complete all measurements on each model were recorded. Learning as measured by increasing accuracy or decreased time over the group of five casts was determined. Five orthodontic faculty served as the control group. Results: Sixteen students analyzed plaster casts; 15 students analyzed digital casts. Molar occlusion was judged as either correct or incorrect; means of the millimetric measurements of the other parameters were compared between groups using GLM. The digital learning group had 15 measurements that were significantly different from the faculty mean; the plaster learning group had only 2(p<0.05). Regarding molar occlusion, the plaster group was always more accurate. The time required for the measurements decreased in each group to a similar extent, with the greatest decrease between casts 1 and 2. Conclusions: Senior dental students learned how to analyze plaster orthodontic models more accurately. The time required for analysis decreased over 5 trials. The Effect of the Modified Kavain Compound on Inhibition of P.Gingivalis /LPSinduced TNF-Alpha Production in Human THP-1 Cells Background: TNF-α is an important cytokine mediator of inflammation which suggests that inhibition of TNF activity may provide potential for clinical application. Recent data indicated that treatment of both human and mouse cells with Kavain significantly modulates P. gingivalis- and LPS-induced TNF-α expression. Materials and methods: In order to obtain a selective analog with optimized biological activity and structural physico-chemical properties of Kavain, Kavain analogs were designed and synthesized. Results: one Kavain analogue that is similar to Kavain but soluble and does not induce a significant toxicity. Conclusion: Both studies in vitro and in vivo treatment showed stronger biological function as compared to Kavain. Incretin Dysregulation of Lysyl Oxidase: A Novel Mechanism for Diabetic Bone Disease Background: Incretins are gastric hormones released by intestinal K-cells into the circulation in response to food consumption and stimulate insulin secretion from pancreatic beta cells. One of these hormones, glucose-dependent insulinotropic peptide (GIP) is also anabolic in bone. Individuals with diabetes experience diminished bone quality caused by a low bone formation osteopenia, and impaired cellular response to GIP in the pancreas, however GIP regulation of bone quality in diabetes has not been extensively explored. Objective: The present study seeks to identify a potential mechanism for diabetic osteopenia in which diabetes interferes with GIP-stimulated increases in the collagen cross-linking enzyme lysyl oxidase (LOX) in osteoblasts, leading to decreased collagen integrity and the trabecular abnormalities seen in diabetic bone. Materials and Methods: Micro-CT analysis of long bones and L5 vertebrae from LOX +/- and wild type mice made diabetic by low dose streptozotocin (STZ) induction revealed a profound exacerbation of the decreased bone volume and impaired trabecular structure seen in diabetic wildtype and LOX +/- mutant mice when the mice were both diabetic and haploinsufficient for LOX. Furthermore, qPCR of RNA isolated from diabetic wild type and mutant bones revealed a more than 20 fold decrease in LOX expression in diabetic bone. Studies done in healthy MC3T3 osteoblast cultures treated with GIP show that under normal conditions GIP directly increases LOX transcript and protein levels, mediated by the cAMP/PKA pathway and TCF/LEF response elements in the LOX promoter. The specific molecular mechanisms by which diabetes inhibits GIP-stimulated LOX expression in osteoblasts were also investigated. A novel inhibitory circuit of glucose stimulated insulin secretion in the pancreas has been discovered in which dopamine produced in the foregut can act as an “anti-incretin”, antagonizing and thus regulating the effects of incretins in healthy individuals. Therefore the ability of dopamine to inhibit GIP-stimulated signaling in MC3T3 osteoblasts was examined. Results: Data indicate a strong dose- and time-dependent inhibition of GIP-stimulated cAMP production and LOX expression when MC3T3 osteoblasts are pretreated with dopamine. ELISA assays on serum from our STZ diabetic mouse model also confirmed significantly elevated serum levels of dopamine in diabetic mice. Finally, pretreatment of primary bone explant cultures with the dopamine receptor inhibitor amisulpride restores the impaired GIP stimulated increases in LOX expression in diabetic bone cells. These results define for the first time a potential mechanism for diabetic osteopenia in which elevated serum dopamine levels arising from peripheral tissues under diabetic conditions attenuate GIPstimulated LOX production in osteoblasts, resulting in decreased bone strength, and the increased fracture incidence typical of diabetic bone disease. Conclusion: Interference with dopamine signaling would likely restore bone health in diabetes. Edge Chipping Analysis of CAD/CAM Bur Milled Block Materials Objectives: To evaluate the edge chipping of different CAD/CAM dental materials using a three-axis milling machine. Methods: Five CAD/CAM dental materials were used in this study: 1. Lithium disilicate glass ceramic, IPS e.max CAD, Ivoclar Vivadent. 2. Leucitereinforced glass ceramic, IPS Empress CAD, Ivoclar Vivadent. 3. Interpenetrating Phase Ceramic, Enamic, VITA. 4. Feldspathic porcelain, Vitablocs MarkII, VITA. 5. Nanoceramic filled composite, Lava ultimate, 3M. Rectangular bars with dimensions of 4 mm x 2 mm x 14 mm were designed using the Sirona inLab software system (SW 4.2.5). Ten specimens from each material were milled by CEREC inLab MC XL using a new set of burs for each material. Three bars at the milling sequence number 1, 5 and 10 were selected for the edge chipping analysis. All the edges of the selected bars were imaged using a scanning electron microscope (SU6600, Hitachi High Tech, Ltd, Tokyo, Japan). The length, depth, and area of each edge chip on the specimens were measured using an image processing software Quartz PCI Version 9. The chip measurements were recorded with the corresponding milling surface (cylinder bur surface and step bur surface) and edge locations (top, right and left edge). The chipping factor (ratio of the sum of chip lengths to total edge length) and chipping area of different surface locations and different materials were analyzed by one-way ANOVA and post hoc Tukey test to detect the difference between groups using JMP13.0 with α=0.05. Results and Conclusions: Chipping factor and chipping area were affected by material type. Conventional ceramic materials had significantly higher chipping factor values than composite resin and interpenetrating phase materials. Step bur surfaces had a higher chipping area value than cylinder bur surfaces. Effect of Inorganic Phosphate on the Proliferation and Odontogenic Differentiation of Human Dental Pulp Cells Introduction: This study was designed to investigate the effect of inorganic phosphate (Pi) at different concentrations on odontogenesis of the normal human dental pulp cells (hDPCs). Method: hDPCs derived from extracted teeth were cultured in growth media with supplements of Pi in 0, 2, 4, 5 and 8 ppm, for the time intervals of 16 hours, 7, 14, 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. The data were presented as the mean of triplicates and normalized on a per million cell basis. Statistical analysis was conducted using JMP Pro 12 (ver. 12.1.0) in one-way ANOVA and Tukey HSD post-hoc tests. Result: Cell attachment efficiency was reduced significantly by additional Pi of 2, 4 and 5 ppm (P<0.05). At 21 days, cultures with 2, 4 and 5 ppm supplemental phosphorous displayed significantly higher cell proliferation rates compared to the control group at day14 (P<0.05) and at day 21 (P<0.05). At day 7, cultures with 2, 4, 5 and 8 ppm supplemental Pi yield significantly higher levels of ALP activity (P<0.05) compared to the control group. At day 7, cultures with 5 ppm Pi supplement showed significantly higher levels of DSP expression (P<0.05) compared to the control group and the rest of the other groups. Conclusion: Supplemental Pi in concentration of 5 ppm could significantly induce proliferation and odontogenesis of hDPCs. This is the first report to demonstrate Pi-induced odontogenesis, leading to potential development and clinical application of future Pi containing dental pulp capping or root canal filling materials. Spontaneous Pancreatitis in a Novel Mouse Model With aTrypsinogen Mutation Background: Premature intrapancreatic activation of trypsinogen has been implicated in pancreatic injury and chronic pancreatitis. Heterozygous mutations in the activation peptide of human cationic trypsinogen have been identified in patients with hereditary pancreatitis. Biochemical analyses of trypsinogen activation peptide mutants revealed that the common phenotypic change is a markedly increased propensity for autoactivation. Earlier we tested whether mutations in the activation peptide of the mouse T7 trypsinogen recapitulate the human phenotype. Our in vitro data showed that T7 mutant D23A exhibited approximately 50-fold increase in trypsinogen autoactivation. Aim: Our aim was to generate and characterize a novel T7 trypsinogen knock-in mouse model of human hereditary pancreatitis, which develops pancreatitis spontaneously. Methods: T7 D23A knock-in animals were generated by homologous recombination. Wild-type and heterozygous animals carrying the D23A mutation (T7D23A) were sacrificed in different ages and were analyzed for spontaneous pancreatitis. Histological features of pancreatitis (acinar cell loss, duct dilations, tubular complexes and inflammatory cell infiltration) were evaluated on hematoxylin-eosin (HE) stained sections. Fibrosis was detected by trichrome staining. Plasma amylase levels and intra-pancreatic trypsin activities were measured using kinetic assays. Results: Animals carrying T7D23A mutation showed significant changes in their pancreas starting from 3-4 weeks of age. HE stained pancreatic sections of 3-4 weeks old mice displayed extensive pseudotubular complexes and inflammatory cells. Elevated amylase levels and increased pancreas weight measured in 5 weeks old T7D23A animals indicate the occurrence of an acute pancreatitis attack. With progressing age we observed complete disorganization of the pancreas with loss of acinar cells, dilatation of ducts, fibrosis and replacement with adipose tissue. We found significantly higher intra-pancreatic trypsin activity in T7D23A mice relative to wild type controls. Conclusions: The T7D23A mouse is the first animal model that exhibits early onset spontaneous pancreatitis due to a trypsinogen mutation offering convincing evidence that increased trypsinogen autoactivation is a pathologically relevant mechanism in hereditary pancreatitis. Chymotrypsin Like Protease 1 Knockout Mouse Background. Chymotrypsin is a digestive enzyme, produced by the pancreas as four isoforms (CTRB1, CTRB2, CTRC, CTRL1). Chymotrypsin C (CTRC) degrades trypsinogen, thereby it protects the pancreas against premature trypsinogen activation. Loss of function mutations in the CTRC gene contribute to development of chronic pancreatitis (CP). Mutations in chymotrypsin B (CTRB) gene may also have an effect on the development of CP while the role of chymotrypsin like protease 1 (CTRL1) in CP is unknown. Aims. Our aim was to investigate the effect of Ctrl1 deletion on pancreatitis responses in a mouse model. Methods. A Ctrl1 knockout (KO) mouse model was created with CRISPR Cas9 technique. Successful deletion of Ctrl1 was verified by reverse transcription (RT) PCR and western blot. Total trypsinogen and chymotrypsinogen content were measured by enzymatic assay. To generate pancreatitis we used intraperitoneal cerulein injection 10 times hourly. We measured pancreas mass, pancreatic water content (edema), plasma amylase activity, pancreas MPO and evaluated histology sections for edema, inflammatory cell infiltration and necrosis. We measured pancreas trypsin and chymotrypsin activity 30 minutes after a single injection of cerulein. Results. RT-PCR and western blot demonstrated no Ctrl1 expression in the KO strain. Total trypsinogen and chymotrypsinogen content was comparable between wild-type and KO mice. In experimental pancreatitis studies, we found no significant difference in pancreas mass, edema, plasma amylase activity and histological evaluation between wild type and Ctrl1 KO strains. Conclusion. Ctrl1 is expressed at low levels in the pancreas and plays no role in the development of acute pancreatitis.