Introduction – Chemotherapy is an important treatment modality for patients with spinal metastases, with Doxorubicin (Dox) being one of the most effective options. The high systemic doses required for the drug to be effective prevent its prolonged use due to the significant heart failure it causes with cumulative doses. Our lab has previously shown that 3D-printed porous polymer scaffolds are as effective as direct treatment for local delivery of Dox to inhibit bone metastasis cancer cell growth in a 2D culture. However, the effectiveness of this drug delivery system remains to be tested in a model that resembles the in-vivo environment. Bioprinting is an effective tool for creating tissue-like 3-dimensional tumor models for therapeutic screening. Using this technology, our lab is designing a bone-like 3D model for testing the effectiveness of 3D-printed porous polymer scaffolds for drug delivery to cancer cell lines and patient derived cancer cells of breast, lung and prostate origin.
Methods –MDAMB-231 (breast), HCC-827 (lung) and LAPC4 (prostate) cancer cell lines were obtained commercially and 3 different patient derived spine metastasis cell samples per tumor type were obtained from patients participating in this study that underwent tumor resection at the Montreal General Hospital. Primary osteoblasts were isolated from vertebral bodies of organs donors through collaboration with Transplant Quebec. PORO-LAY polymers were 3D printed into porous scaffolds and loaded with 1-3 μg of Dox, for which we have previously determined release kinetics. Tumor or osteoblast cells were fluorescence stained and 3-dimensional co-culture tumor models comprising of an inner tumor core and an outer osteoblast cell perimeter were printed using the CELLINK BIO X bioprinter with a 1% alginate / 7% gelatin bioink in 24 well plates and crosslinked with calcium chloride. 3D printed scaffolds loaded with or without drug were inserted inside the tumor core and plates were incubated for 3 weeks in 10% serum media. Images were captured with an inverted fluorescence microscope every 3 days to assess for migration between the 2 cell compartments. Cell viability and metabolic activity were conducted using the LIVE/DEAD and alamarBlue assay.
Results – To date, we have shown that bioprinted 3D cultures allow for sustained cell viability over a 4-week period. We expect the Dox loaded 3D printed scaffolds will decrease the migration of tumor cells into the surrounding stroma, decrease metabolic activity of 3D printed tumor cells, and decrease the viability of tumor cells more than direct Dox treatment or control.
Conclusion – Use of 3D printed porous scaffolds for targeted drug delivery may allow for effective inhibition of tumor recurrence after resection in patients with metastatic spine disease by targeting the full effect of the drug at the site of the tumor and sparing the rest of the body from side effects that normally arise with systemic treatment. The use of bioprinting in these experiments showcased the potential for this technology for creating physiological screening models with greater resemblance to tissue microenvironments. Future use of these models can identify appropriate therapeutic dosage and facilitate rapid and more effective animal studies.
Aim: The aim of this study is to determine the contribution of acute post-operative pain towards the development of neuropathic pain (NP) 3 months after breast cancer surgery (BCS).
Methodology: In a 3-month prospective cohort study, female breast cancer patients (>18 years) who underwent first BCS were recruited from Segal Cancer Center, Montreal. The study outcome was NP occurrence at 3 months post-surgery, as well as the Douleur Neuropathique-4 (DN-4) score. Collected data was acute post-operative pain, anxiety, depression, type of surgery, axillary status, and NP. Presence of NP was assessed at 3 months post-surgery by telephone using the DN-4 instrument. Linear and logistic regression analyses were used to assess the contribution of acute pain in the incidence of NP.
Results: At 3 months post-surgery, 45 patients (24%) reported NP. The most frequent DN4 terms describing NP were: burning (31%), electric shock (21%), itching (31%), numbness (24%), pin and needles (24%). DN-score at 3 months was positively associated with current pain intensity (β= 0.11, P=0.003), and pain during movement (β=0.59, P=0.006), both assessed at 7 days post- surgery. These associations were not confounded by depression (β=0.15, P=0.01). Furthermore, acute pain increased the likelihood of higher DN-4 score (OR = 1.63), even if this association was not statistically significant. Furthermore, numbness was correlated with current pain intensity(r= 0.13, P=0.02), and tingling and pins and needles were correlated with pain during movement at 7 days (β= 0.17, P < 0.03).
Conclusion: NP at three months is associated with acute postoperative pain and its intensity. Pins and needles and numbness are both associated with acute pain.
BACKGROUND: Osteogenesis Imperfecta (OI) is a rare connective tissue genetic disorder causing bone fragility and growth deficiency. The clinical findings of OI vary widely in severity and presentation, thus leading to circumstances requiring access to a large range of occupational, medical, and supportive services. Whereas families and individuals with rare diseases are known to often bear the financial burden of such services, there is a paucity of research on the topic from the OI perspective. The purpose of this scoping review (Arksey and O’Malley, 2005) was to systematically review and synthesize the nature and magnitude of the direct and indirect costs incurred by individuals living with OI and their families.
METHODS: A scoping review was conducted. Three electronic databases (OVID: EMBASE, MEDLINE and PsychINFO) were searched to identify and select eligible articles in consultation with a librarian scientist. Published quantitative, qualitative or mixed-methods studies assessing costs were screened and reviewed for inclusion. Items for data extraction included author and year,country of origin, study design, data collection method, sample size, employment status, direct costs, indirect costs, types of care mentioned, and allowances and financial assistance provided. The cost-of-illness framework guided the descriptive analysis and synthesis of extracted data.
RESULTS: From the 355 titles identified, 14 records were selected, including 10 journal articles, three poster presentations, and one conference abstract published between 1976 and 2017. Of the 14 studies, four were quantitative, three qualitative, and seven of unreported study designs. These studies were conducted in the USA, Canada, Sweden, the UK, Indonesia, Poland, and Turkey. Sample sizes were small, ranging from n = 3 to n = 47, with an average of n = 27. There was no systemic approach to assess and measure direct or indirect costs. Direct costs included housing modifications, medical and hospital costs, transportation and travel, ambulatory equipment, special camps, in-home caregivers, respite care, support workers, home help, and drugs. Indirect costs included missing work hours for parents, with mothers appearing to be disproportionately affected, and missed school hours for children. Collectively, these costs contributed to families experiencing ‘financial difficulties’ and ‘economic changes’.
CONCLUSION: A paucity of research has been conducted to understand the costs associated with living with OI. Future research should be directed towards better understanding these costs with the ultimate goal of optimally assisting individuals and families in planning for and receiving better financial support.
Background. The mechanical environment plays a major role in bone remodeling and exercise is an effective way of improving and maintaining bone health. However, the skeleton’s ability to adapt to mechanical loading declines with age. The mechanism(s) responsible for this age-related loss in mechano-responsiveness are unknown. ATP and ADP are among the earliest biochemical signals released by mechanically-stimulated bone cells that act via the purinergic (P2) receptor family to evoke downstream intracellular calcium [Ca2+]ielevations. Objectives. The aim of this study was to determine whether mechanotransductive purinergic signaling alters with age. We evaluated ATP release and downstream signalling in osteoblasts isolated from young and old mice. Methods and Results. Compact bone-derived osteoblasts (CB-OBs) were isolated from 10- and 70-week old C57Bl/6J mice by enzymatic digestion and cultured in osteogenic media for 3 days prior to experiments. We observed no age-related differences in the amount of ATP released following mechanical stimulation of CB-OB cells by turbulent fluid shear applied by displacing 50% media 10 times over cell layer. However, when total extracellular ATP and ADP content in tibial and femoral explants was measured, we found 10-fold more ATP and ADP in bones from young mice, compared to older mice. When individual Fura2-loaded CB-OB cells were mechanically-stimulated by a glass-micropipette, the oscillatory fraction of calcium responses in neighbouring non-stimulated cells was significantly higher in CB-OBs isolated from older mice. Consistently, there was a significant age-related difference in the dose-dependence of Ca2+responses to [ATP]. Finally, we demonstrated that mRNA expression of the P2RY12 receptor was significantly elevated in 70-week old CB-OB, compared to younger mice. Conclusion. These data suggest that increasing age is associated with (i) decrease in basal extracellular [ATP] and [ADP], (ii) increase in P2Y12 expression, and (iii) alterations in mechanotransductive purinergic signalling, which together may contribute to lower mechano-adaptive response in the aging skeleton.
Background: The Strategic Regional Plan of the Cree Board of Health and Social Services of James Bay (CBHSSJB), serving Cree communities in Quebec, mandates an objective of integrating oral health within primary care. Emerging evidence suggests that the integration of oral health into primary health care can decrease oral health disparities. This research study aimed to answer the question: How and to what extent does the integration of oral health into primary care address the oral health needs of Cree communities in Quebec?
Methods: This study used a multiple case study design within a qualitative approach and developmental evaluation methodology. Appreciative Inquiry was selected as a study framework among existing frameworks of the developmental evaluation. Maximum variation and snowball sampling were used to identify and recruit participants. Data collection included focus group discussions and individual in-depth interviews. Data analysis included transcription, codification, thematic analysis, and triangulation.
Results: A total of 36 interviews and 6 focus group discussions were conducted with health care providers, administrators, and patients. Preliminary results identified 9 themes within three Appreciative Inquiry phases (Discovery, Dream, and Design). The Discovery phase identified the strengths of this organization in facilitating enablers of integration including strategic planning, organizational structure, cultural integration, coordinated networks, and co-location. In the Dream phase, participants’ oral health care stories expressed various dimensions of integration and they wish for strengthening the integration via extending public oral health care programs, increasing resources and organizational management. In the Design phase, recommendations were formulated for the future action plan within the CBHSSJB.
Conclusion: This study results suggested that the CBHSSJB has been successful in implementing oral health integration into primary care following its strategic planning. At this stage, the organization could extend the level of the integration into full integration by following study recommendations derived from the perspective of local stakeholders.
About 10% of temporomandibular disorder (TMD) patients are associated with chronic pain and disability. The relationship between fatigue, insomnia, or obstructive sleep apnea (OSA), and painful TMD remains unclear and needs further investigation. This aim of this case-control study is to evaluate the association between fatigue, insomnia or OSA on acute and chronic painful TMD groups.
Painful TMD participants were recruited from three dental clinics in Montreal, QC and one in Ottawa, ON. Recruitment involved a clinical examination according to the Research Diagnostic Criteria/TMD and the completion of questionnaires assessing demographics, TMD symptoms and duration, fatigue, insomnia, and OSA. Multivariable logistic analyses were used to assess our primary aim.
80 (20%) subjects with acute and 312 (80%) chronic painful TMD were recruited for this study. The majority in both groups were females (76% and 79%, P = 0.57), and no significant difference on the mean age (P= 0.68) was noted between these groups. Relative to the acute painful TMD participants, chronic cases had a greater likelihood to present fatigue (OR = 1.89, 95%CI: 1.05-3.40), and insomnia (OR = 2.56, 95%CI: 1.09-6.00). A borderline association was noted with STOP-BANG (OR = 1.77, 95%CI: 0.97-3.22), while no association was noted using Epworth Scale (OR = 1.15, 95%CI: 0.57-2.34), both are screening questionnaires for OSA. All these previous logistic regression models were adjusted age, gender, depression and anxiety scores.
Patients with chronic painful TMD pain at three months were more likely to present fatigue and insomnia than acute cases.
Aging is at the core of many degenerative diseases such as osteoporosis. In the past, aging was thought to be immutable; however, recent discoveries have shown that systemic manipulations, such as parabiosis or administration of young plasma, can counteract many important symptoms of aging. Aged bone marrow may regulate at least some of the general hallmark indicators of aging. Therefore, we hypothesized that transplantation of young bone marrow (BM) cells in old mice could reverse aging in bone, brain and metabolism. Our objective is to develop a mouse model of old mouse transplanted with young BM and then examine bone remodeling, cognitive functions and energy metabolism.
C57BL/6-Tg (CAG-EGFP)1Osb/J mice of two different age groups: (6-weeks and 18-months) were used as donors of enhanced-GFP positive BM cells. Recipient C57BL/6 mice (18-month old) were total-body irradiated to eliminate their endogenous BM, and then were transplanted (tail vein injection) with donor EGFP BM cells. Recipient mice were randomized into three groups and transplanted with: a) BM cells from young donor, b) BM cells from old donor, c) Reference group: no irradiation and no BM transplantation.
We successfully created a model of “old mice transplanted with young EGFP BM cells”. The lethal dose required to ensure complete myeloablation of BM in recipient mice was between 10-12 Gray. Also, at least 5 Χ106 BM donor cells were required to ensure complete engraftment in recipient mice. Moreover, our preliminary results showed that the survival rate was not significantly different between the three tested groups.
It was possible to create a mouse model of old mice transplanted with young bone marrow. The findings of this study could radically alter our understanding of aging in bone, and direct further examination of the cell signaling process involved in age-related diseases such as osteoporosis.
Introduction: Mature articular cartilage displays poor intrinsic healing and its degradation is the major hallmark of osteoarthritis (OA). TGF-β is a multifunctional cytokine that plays a critical role in cartilage repair and maintenance. Aberrant TGF- β signaling in chondrocytes has been strongly implicated in the pathogenesis of OA. Our group has previously reported CD109 as a novel TGF-β co-receptor and shown that CD109 is a potent negative regulator of TGF-β signaling in the skin.
Objective: The proposed study is aimed at identifying whether manipulation of CD109 expression levels modulates the balance between TGF-β signaling via ALK1 versus ALK5 signaling pathways and regulates ECM protein expression in vivo in articular chondrocytes.
Methods: Articular cartilage tissue was collected and primary chondrocytes were isolated from CD109 KO and wild-type mice. TGF-β signaling components were analyzed in isolated chondrocytes or cartilage tissue by determining ALK5 versus ALK1 levels and Smad2/3 versus Smad1/5 levels using Western blot. Chondrocyte function was determined by evaluating the expression of, collagen type II, aggrecan, collagenase (MMP-13) and aggrecanase (ADAMTS-5), at the protein and mRNA levels by Western blot, real time PCR or immunocytochemistry (ICC). Histological features and proteoglycan content of cartilage from CD109 KO and wild-type mice were assessed by Safranin O/fast green staining.
Results: Articular chondrocytes isolated from CD109 KO mice display markedly enhanced levels of ALK5, and collagen type II, and increased expression of aggrecan, in comparison with chondrocytes from wild-type mice. On the other hand, loss of CD109 expression in mice articular chondrocytes results in decreased levels of ALK1, MMP13 and ADAMTS5. Moreover, histological results indicate that collagen content in articular cartilage from CD109 KO mice is increased significantly, as compared to cartilage from wild-type mice.
Conclusion: Our findings suggest that CD109 differentially regulates TGF-β signaling pathways and inhibits ECM protein production while promoting proteases expression, in articular chondrocytes in vivo. We conclude that CD109 may play an important role in maintaining cartilage function and integrity.
Introduction: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer with over 500,000 new cases diagnosed yearly, and 4.6% of cancer cases. Despite the improvements in treatment modalities, the five-year survival rate for HNSCC has remained unchanged at ~50% over the past 30 years. One reason for HNSCC treatment failure is related to a subpopulation of cells in the tumor called cancer stem cells (CSCs) which are suggested to have tumor initiating potential, combined with the self-renewal ability and multilineage differentiation. According to many studies, CD44 surface marker can be used to identify CSCs. The purified CD44+ cells from the primary tumors can give rise to tumors faster and by injecting less cell number in animal models compared to CD44- cells, and these xenograft tumors subsequently reproduce the original tumor heterogeneity observed in the primary tumor. Recently, CD271 was identified as a marker of CSCs in many tumors, such as human melanoma and hypopharyngeal carcinoma. We investigated if to whether CD271 has any significant contribution in the context of CD44 population towards defining CSC marker
Methods: SCC12 and SCC38 were used as models for HNSCC and CD44+/CD27+ and CD44+/Cd271- cells were isolated with FACS sorting. The isolated cells with the parental cells were tested for cellular growth using MTT assay, clonogenicity, tumorsphere formation, treatment resistance, and difference in gene expressions using qRT-PCR.
Results: Our results revealed that CD271+ cells are a subpopulation from the CD44+ cells. The CD44+/CD271+ cells have a faster dividing rate, higher proliferation rate, higher self-renewal ability, and chemo/radio-resistance when compared to CD44+/CD271- and the total population. CD44+/CD271+ cells showed higher expression of stemness-related genes such as SOX2 and OCT4, and self-renewal related genes as BMI1 and GLI1 when compared to CD44+/CD271-.
Conclusion: Our results suggest that CD271 is a more accurate marker to purify the CSCs from HNSCC compared to the widely used CD44.
Introduction : Bone adapts to changing mechanical loading conditions via (re)modeling, a process involving bone-forming osteoblasts, bone-resorbing osteoclasts, and mechanosensing osteocytes. There is evidence that circadian rhythm influences bone formation and resorption, but little is known concerning the molecular mechanism(s) regulating this process. In this study, we investigated if time of loading influences gene expression to provide insights into whether load-induced bone formation in mice is regulated by circadian oscillator mechanisms functioning in bone tissue.
Methods : The left tibiae of 10 week-old, skeletally-immature female C57Bl/6 mice underwent a single session of non-invasive dynamic compressive loading (1200 µε at midshaft determined by strain gauging, 216 cycles at 4 Hz) at ZT2 (day time) or ZT14 (night time). Right tibiae served as an internal control. Mice were euthanized and dissected 8h or 24h later, corresponding to ZT2, ZT10, ZT14 or ZT22. Expression of bone and clock genes in both tibiae was examined by qPCR.
Results : We found that circadian rhythm genes Clock, Per1 and Per2 were expressed in bone. Per2 displayed a 24h oscillatory expression profile in non-loaded, right tibiae. Bone-specific genes Runx2, Sp7 and Sost (a mechanically-responsive gene that encodes for the secreted factor sclerostin) also showed 24h cyclic expression profiles in control limbs. Expression levels of Sost peaked at ZT22 in non-loaded tibiae. Interestingly, in loaded limbs, loading at ZT2 (day time) led to a significant increase in Sost expression after 8h while loading at night (ZT14) led to a significant decrease after 24h. Osteoclast marker Ctsk levels were also affected differently by mechanical loading, depending on the time of day it was applied.
Conclusions : These data suggest the time of loading influences the expression of mechanically-responsive and bone metabolism genes. These findings could help determine at what time of day patients should undergo physical exercise to enhance load-induced bone formation.
Introduction: Fusobacterium nucleatum has been associated with various forms of periodontitis and is one of the main bacterial species involved in physical interactions between symbiotic and dysbiotic microbiota. It is also a resident of the human gastrointestinal tract and has been involved with inflammatory bowel disease and contributed to the initiation and progression of colorectal cancer.
Objectives: The aim of this study, was to investigate the effects of cranberry extract on virulence properties of F. nucleatum. In addition, the ability of cranberry polyphenols to modulate the gingival and intestinal epithelial tight junction integrity was evaluated. Lastly, the effect of cranberry on the activation of the nuclear factor-κB (NF-κB) and TREM-1 a pro-inflammatory signaling pathway in monocytes were assessed. The antibacterial and anti-biofilm activities were determined with a microplate dilution assay. A fluorescence assay was used to determine the effect of cranberry extract on the adherence of F. nucleatum to a basement membrane matrix model (MatrigelÔ) and to oral and intestinal epithelial cells. The protective effect of cranberry polyphenols on the gingival keratinocyte barrier was evaluated by determination of the transepithelial electrical resistance (TER). The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to monitor NF-κB activation. Finally, TREM-1 gene expression and sTERM-1 secretion were assessed respectively by qPCR and ELISA.
Results: Cranberry extract dose-dependently inhibited the growth of F. nucleatum as well as the biofilm formation. This antibacterial activity appeared to involve iron-chelating activity. This extract reduced the adherence of F. nucleatum to the basement membrane matrix model and oral and intestinal epithelial cell. Moreover, a protective effect of cranberry extract on the keratinocyte tight junction integrity breakdown mediated by F. nucleatum was demonstrated. Lastly, cranberry polyphenols inhibited the F. nucleatum mediated activation of the NF-κB signaling pathway and attenuated the TREM-1 gene expression and in consequence reduced the secretion of sTREM-1.
Conclusions: To be considered as an etiologic agent of periodontitis, F. nucleatum can also cause a variety of oral and extra-oral infections, including colorectal cancer. This dual anti-bacterial and anti-inflammatory action of cranberry polyphenols suggest that they may be promising candidates for novel therapeutic agents.
INTRODUCTION: The bone is a dynamic tissue that is constantly broken down and repaired through a balance between osteoclasts and osteoblast activity. However, when various cancers invade the bone, they can disrupt the physiological balance leading to various deleterious changes in bone structure and function. At the moment, radiotherapy, chemotherapy and surgical resection are the main clinical approaches to bone metastases. Studying cancer cells molecularly in vitro relies on 2D monolayer cultures which in no way represents the physiological tissue microenvironment in vivo. Therefore, preclinical and translational cancer research trends are moving toward organoid studies and 3D biomimetic models to provide more clinically relevance in all aspects. In this case, 3D bioprinting has become an important tool in developing 3D biomimetic in vitro models. Here, we set out to generate a bioink for bioprinting, consisting of alginate, gelatin, nano-crystal hydroxyapatite loaded with primary human osteoblasts to produce a robust 3D bone-like microenvironment in order to study human bone metastasis.
METHODS: A previously reported 3D hydrogel (3% alginate; 7% gelatin) model for cancer cell-migration was modified to incorporate nano-crystal hydroxyapatite, primary human osteoblasts and primary human bone marrow derived stromal cells (2 x 106 cells/mL). Primary osteoblasts were isolated from cadaveric vertebral bodies of organ donors, and bone marrow MSCs are purchased from Rooster Bio. The constructs were firsthand-cast and cultured for 28 days in either normal growth medium (DMEM) or osteogenic medium (OM) with and without 0.5 mg/mL hydroxyapatite (HA). Live/Dead assays will be performed to quantify viability at 28 days of culture. Fixed samples will be stained with Alizarin red for calcified matrix deposition, and hematoxylin and eosin to observe cells within the matrix. A second set was bioprinted using a tissue scribe and western blot will elucidate presence of bone matrix and osteocytes by probing for osteopontin and sclerostin. Finally, GFP labled MDA-MB-231 cells were co plotted with the human osteoblasts and cultured over 7 days
RESULTS: Live/Dead analysis revealed strong primary human osteoblast viability in all conditions after 28 days of culture: 91.3 ± 3.18 % for DMEM/HA+, 92.5 ± 2.5 % for DMEM/HA-, 88.6 ± 0.38 % for OM/HA+ and 85.9 ± 6.2 % for OM/HA-. Primary human MSCs showed approximately 85.1, 69.4, 87.8 and 90% viability in the same four conditions. Alizarin red staining showed that cells grown in DMEM without HA had the least amount of bone mineralized matrix. The combination found to have the most amount of bone mineralized matrix was OM/HA+. Western blot analysis shows presence of osteopontin and sclerostin across MSCs samples. Preliminary results of tumor cells co-cultures, shows cell growth and migration to periphery potentially providing a model for drug screening.
CONCLUSION: This work will allow better understanding of effect of the drugs on non-tumor cells and tumor cells in a more clinically relevant and physiological manner. The model will have broad implications for screening other pathological tissue and cell types as well as establish novel bone tissue engineering avenues.
Introduction: Low back pain is one of the most prevalent medical conditions, affecting up to 84% of all people. Despite its widespread implications, the molecular cause of it has yet to be determined. Low back pain is caused by the degradation of the intervertebral discs—small, compressible discs that aid in the dispersion of weight between your vertebrae. Currently, the disease is thought to occur from a buildup of inflammatory molecules, either from trauma or over time with age. It is known that as the disc degenerates, the pH becomes more acidic, as low as 6.0 and in extreme cases, even lower. Over longer culture periods, more acidic pHs have significantly decreased cell viability and increased inflammatory cytokine production. The role of toll-like receptors in the disc is becoming well studied; TLRs are known to be a major player in the progression of disc degeneration because of the upregulation of inflammatory cytokines resulting from TLR signaling, and their ability to recognize endogenous ligands, including extracellular matrix (ECM) fragments. It is not known if TLR signaling, and the resulting down-stream effects on gene expression, is altered by the pH alone.
Aim: Determine the effect of pH on gene expression of factors associated with disc degeneration and the effect of pH on TLR receptor expression and signaling.
Hypothesis:We expect that acidic pH (<6.8) will increase inflammatory cytokine production.
Methods:We are fortunate to receive human lumbar spines through a partnership with Transplant Quebec, from which we isolate cells and store in our cell bank. It has been shown that acidic pH can decrease cell viability drastically after four days of culture, so a 48-hour culture was chosen. Human nucleus pulposus cells were cultured under three different conditions: pH 7.4, 6.8, and 6.2 with 250,000 cells/well in a 6-well plate. Pam2CSK4, a TLR 2/6 agonist, was used to treat cells for the 48 hours at a concentration of 1ug/mL. RNA was then extracted and PCR was performed to asses relative gene expression of IL-6, MMP3, TLR2, and NGF.
Results:We did not see an effect of pH on gene expression of IL-6, MMP3, TLR2, and NGF after two days of culture. We saw a small increase in IL-6 gene expression at pH 6.8 and 6.2 (between 2- and 3-fold increase for each), a slight increase in MMP3 expression, and a slight down-regulation of TLR2 and NGF gene expression. There was a strong induction of IL-6 and MMP3 after treatment with Pam. However, there was only a slight induction of TLR2 and a decrease in NGF expression across all pHs.
Significance:Since the pH in intervertebral discs is known to decrease as they become more degenerate, it is important to see if it alone can cause changes to catabolic factors. It is also important to see if pH affects cellular signaling after stimulation with an agonist, because there could be the potential for a synergistic effect. We did not see any significant induction of gene expression due to pH alone in either scenario.
Objectif : Évaluer l’effet de la vitamine D (VitD) sur la vitesse et la stabilité du mouvement dentaire orthodontique (MDO) et comparer l’efficacité de deux formes d’administration de la Vit D, systémique et locale.
Méthode : 25 rats Sprague Dawley mâles (250 g) ont été assignés aléatoirement dans 4 groupes : expérimental gavage (EG), contrôle gavage (CG), expérimental injection (EI), contrôle injection (CI). Le groupe EG (administration systémique) a reçu une solution VitD 50 ng/mL quotidiennement et la dose variait en fonction du poids (100 ng/kg). Le groupe EI (administration locale) a reçu une solution VitD 1x10-10 M chaque 2 jours en injection sous-muqueuse dans la région palatine antérieure et linguale à la première molaire maxillaire droite. Les groupes contrôles ont reçu une solution saline 0.1 M en dose identique à celle de la solution VitD pour le groupe expérimental correspondant. Le MDO a été réalisé avec un ressort NiTi de 50 cN fixé entre la première molaire maxillaire droite et les incisives supérieures durant 7 jours. Le côté gauche était utilisé comme contrôle intra-rat. Des scans Micro-CT ont été effectués à 5 temps de mesure : T0 (début d’administration de VitD), T1 (début du MDO), T2 (fin du MDO), T3 (7 jours post-MDO), T4 (30 jours post-MDO). Les distances interdentaires ont été mesurées sur le logiciel CTAn en vues occlusale et sagittale pour chaque rat à chaque temps de mesure. Le MDO était calculé à partir de la plus courte distance entre les première et deuxième molaires.
Résultats : À chaque temps de mesure et pour chaque groupe, une différence statistiquement significative a été trouvée entre le côté du ressort (droit) et celui sans (gauche) (p<0.05), sauf à T4-T3 (7-30 jours) pour EG occlusal, CI occlusal, EI occlusal et sagittal. Le MDO du groupe CG était significativement plus élevé que celui du groupe EG (p=0.040), ainsi que sa récidive globale (p=0.029). Aucune autre différence significative n’a été obtenue pour différencier les groupes. La récidive à court terme était plus marquée que celle à long terme pour les quatre groupes (p<0.001) en vues occlusale et sagittale, sans différence entre les groupes.
Conclusion : Les faibles doses de VitD administrées systémiquement ou localement n’ont pas affecté la vitesse et la stabilité du MDO chez les rats. La récidive du MDO était plus significative à court terme en comparaison à celle à long terme (7 vs 7-30 jours). Un MDO plus grand a montré une récidive plus importante (groupe CG).
INTRODUCTION: Bone repair starts with a localized inflammatory response during which immune and vascular precursor cells are attracted to the defect. Our previous work has demonstrated that the immune system through the action of macrophages and mast cells regulate angiogenesis, osteoclast activity, and the inflammatory environment during bone healing. The goal of this project is to use a mouse model of bone repair to determine if priming immune cells to recognize broken bone in a first fracture accelerate bone repair in a second fracture sustained shortly after.
METHODS: Bone repair in skeletally mature Bl6 mice was quantified in 2mm cortical window defects drilled in the femur. Mice were randomly assigned to one of two treatment groups: 1) Double fracture where the first defect was drilled on the left leg, and 14 days later, a second defect was drilled on the right one; or 2) control where both defects were drilled at the same time. The mice were allowed free ambulation and access to food and water for 14 or 56 days before euthanasia and femur harvest. The amount and quality of bone and revascularization of the defect were quantified using micro CT. Mast cell, osteoblast, osteoclast, vascular endothelial cell, and macrophage distribution pattern and activity were assessed by histology. Comparisons were made by T-tests at the 95% confidence level.
RESULTS: In a standardized region spanning the defect, at 14 days of healing we found no differences in bone quantity between groups. Significantly more and larger mast cells were found in the periosteum of the double fracture mice, along with more osteoclast activity and more macrophages, compared to controls. Blood vessels in the double fracture group also seemed to be more organized. We found no difference in osteoblast activity between groups. At 56 days of healing, all mice exhibited bridging of the defects, with significantly more bone tissue with less difference in the mineral content between the repair tissue and the healthy bone in the double fracture group. This finding was matched by significantly higher number of osteocyte lacunae in the repair tissue in the double fracture group. Interestingly, the differences at 56 days post-operative were similar between mast cell competent WT and mast cell deficient Cpa3Cre/+ mice, suggesting a mechanism independent of mast cell activity.
CONCLUSIONS: Taken together, the results suggest a more intense inflammatory reaction during the early stages of repair in the double fracture group which leads to enhanced bone repair after 8 weeks of healing. Ongoing histological analysis will help identify the spatial pattern of distribution of immune, vascular and skeletal cells at this time point. Our results may have implications for the development of immune cell-based therapies to promote bone healing.
Introduction: Elastogenesis presents a cell surface located hierarchical process that requires the recruitment of several proteins, including fibulin-4 (FBLN4) and latent transforming growth factor beta binding protein-4 (LTBP4). Mutations in FBLN4 and LTBP4 cause autosomal recessive cutis laxa type B and C, respectively. Knockout mouse models of FBLN4 and LTBP4 emphasized the roles of these two proteins in elastogenesis. Previously, we showed that FBLN4 interacts with fibroblasts. Cell interaction with LTBP4 has also been demonstrated. However, the cell receptors for FBLN4 and LTBP4, and the respective molecular mechanisms in elastogenesis remain unknown. In this study, we aimed to identify the FBLN4 and LTBP4 cell receptors, and to determine the functional consequence of their cell interactions in elastogenesis.
Results: Here, we show that skin fibroblasts and vascular smooth muscle cells bind strongly to FBLN4 and LTBP4 using real-time and end-point cell binding assays. Both, synthetic and contractile aortic smooth muscle cells interacted similarly with FBLN4 and LTBP4. We also demonstrate the functionality of FBLN4 multimerization in cell binding. FBLN4 multimers exclusively interact with cells, but not monomers. With a set of FBLN4 deletion mutants, we identified two cell interaction epitopes on FBLN4, one located in cbEGF2-3 and a second one in the C-terminal domain. FBLN4 glycosylation in cbEGF3 and the C-terminal domain does not affect cell binding. We also have investigated FBLN4 and LTBP4 cell receptor(s). Both FBLN4 and LTBP4 have high affinity for heparin. This suggests that heparan sulfate proteoglycans mediate FBLN4 and LTBP4 cell interaction. In the presence of heparin (an experimental model of heparan sulfate), cell binding to FBLN4 was entirely abolished, and reduced to LTBP4. Likewise, cellular interaction with FBLN4 and LTBP4 was reduced in the presence of heparan sulfate. Treating cells with heparinases significantly reduced cell attachment to both proteins. Heparan sulfate deficient cells did not bind to FBLN4. Information about the expressed heparan sulfate proteoglycan cell receptors in skin fibroblasts and vascular smooth muscle cells are required for the downstream identification of the FBLN4 and LTBP4 receptors. Therefore, real-time quantitative PCR assays were conducted to profile the mRNA expression levels for a number of cell surface receptor candidates in primary elastogenic cells including fibroblasts, smooth muscle cells. The expressions of syndecan-1 and glypican-1 were absent in some of the examined elastogenic cells while these cells interact strongly with FBLN4 and LTBP4. That excluded syndecan-1 and glypican-1 from the candidate list as cell receptors for FBLN4 and LTBP4. siRNA knockdowns were used to identify the FBLN4 and LTBP4 cell receptors. Both syndecan-2 and -3 knockdowns in fibroblasts abolished interaction with FBLN4, whereas only syndecan-3 knockdown abolished interaction with LTBP4. Syndecan-2 and -3 knockdowns in cultured skin fibroblast resulted in compromised elastic fiber assembly examined by immunofluorescence staining.
Conclusions: FBLN4 contains two cell interaction sites mapped to cbEGF2-3 and the C-terminal domain. FBLN4 interacts with syndecan-2 and -3, whereas LTBP4 interacts with syndecan-3.The data suggest a new cell-interaction role for FBLN4 and LTBP4 which is essential for proper elastogenesis.
Durant les dernières années, plusieurs études se sont intéressées au rôle du microbiome dans le développement du cancer colorectal (CCR). Dans le cadre de ces investigations, certaines études avaient trouvé de fortes concentrations de la bactérie Fusobacterium nucleatum (F. nucleatum), dans des échantillons intestinaux des patients diagnostiqués avec cancer colorectal (CCR), comparés à des contrôles sains. Ce qui paraissant intrigant, vu que F.nucleatum est une bactérie commensale de la cavité orale et un pathogène essentiel de la maladie parodontale. Plusieurs études ont ensuite suivi et ont rapporté des résultats pour la majorité similaires aux premières. Devant cette croissance notable du nombre de manuscrits publiés sur le sujet, nous avons décidé de mener notre revue systématique afin de répondre à la question de recherche: la bactérie F. nucleatum, est-elle associée au développement du CCR? Les résultats de notre revue sont d’un grand intérêt scientifique pour les chercheurs en épidémiologie du cancer colorectal et en santé buccodentaire.
Objectifs: 1) identifier, réviser et évaluer systématiquement la qualité de la littérature disponible concernant l’association entre F.nucleatum et le CCR; 2) éventuellement, estimer les mesures d'association regroupées entre F.nucleatum et CRC.
Une stratégie de recherche prédéfinie est utilisée dans MEDLINE, EMBASE, Web of Science, CDSR et CINAHL, pour la période de 1946 jusqu'en décembre 2018. Pour être incluses dans cette revue, les études doivent répondre aux critères suivants : 1) étude originale de devis observationnel (les études de cohortes, les études cas-témoins et les études transversales); 2) la quantification ou la détection de la bactérie F.nucleatum doit concerner des spécimens biologiques humains du côlon (tissus et/ou scelles) ; 3) la comparaison concerne un groupe de cas de CCR et un groupe de contrôles sains. Sont alors exclues les études comparant des tumeurs colorectales et des tissus sains adjacents chez les mêmes sujets. Aucune restriction de langue n’est retenue.
Stratégie d’analyse des données: Une analyse descriptive des publications retenues dans le cadre de la revue systématique avec des tableaux qui décrivent les études retenues. Si les données disponibles permettront de mener une méta-analyse, l’effet global de l’association entre F.nucleatum et le CCR sera calculé, avec un test de sensibilité et test d’hétérogénéité (Cochran’s Q test et statistique I2). Des analyses de sous-groupes ou de méta-régression seront éventuellement menées.
La recherche initiale a identifié 563 manuscrits, dont 244 étaient des duplicata. Après une première sélection basée sur les titres et les résumés, 57 manuscrits ont été examinés pour vérifier les critères d’éligibilité. La recherche manuelle a identifié 22 études additionnelles. Suite à la révision du texte intégral, seules 38 études répondaient aux critères d’éligibilité. Toutes les étapes de sélection de manuscrits, de révision du texte intégral et d’extraction des données sont faites par deux réviseurs indépendants (AIJ et CL) et les conflits sont réglés au fur et à mesure. Les données des études retenues sont extraites dans un formulaire conçu et adapté au sujet et à la question de recherche (auteurs, pays, année de publication, but de l'étude, devis de l'étude, taille de l'échantillon, caractéristiques des participants à l'étude, description de l'exposition (infection au F.nucleatum, technique utilisée pour quantifier l'exposition, nombre de F.nucleaum positifs, ainsi que les principaux résultats). L’extraction des données est presque terminée et les résultats seront présentés lors du présent congrès.
Discussion : nous nous attendons à ce que la revue systématique proposée ici ait une grande valeur scientifique et pragmatique. L’étude vise à comprendre l’association entre F.nucleatum et le CCR. Les résultats de cette étude pourraient aider à ouvrir la voie à la mise au point de nouvelles méthodes de prévention, de diagnostic et de traitement du CCR.
Aims: Adolescent Idiopathic Scoliosis (AIS) is a complex disease with unknown etiology characterized by phenotypic heterogeneity. AIS has an important genetic contribution and more than fifteen gene seem to be associated to AIS. Two different approaches were used to identify new candidate genes for AIS including a population-based approach called GWAS and a family-based approach, linkage analysis and whole exome sequencing (WES). In this work, we used family approach to identify new causative genes.
Method: We performed WES of at least two affected people from 37 French-Canadian families. In parallel, the linkage analysis that was performed in 25 British families (Ocaka 2007), identified candidate loci and potential variants (using exome sequencing). Gene variants identified by exome sequencing were analysed by bio-informatics tools and validated by co-segregation analysis (Sanger sequencing).
Results: Exome sequencing of 37 AIS families, after bio-informatics analysis, identified ciliary candidate genes. With linkage analysis, the locus 9q31.2-q34.2 was identified as significantly linked to the disease in a 5-generation family. Exome sequencing of this region identified a new candidate gene for AIS, a gene coding a protein responsible for ciliary integrity.
Conclusion: Exome sequencing of 37 AIS families and linkage analysis identified ciliary genes as possibly involved in spine development. Molecular consequences of these genes will be validated in vitro in cells derived from AIS patients and in vivo in a zebrafish animal model. Almost all identified AIS genes are susceptibility genes. Identifying causative genes is crucial for elucidating AIS pathogenesis.
Background & objective
Ligament and cartilage damage often occurs as a result of sports-related injuries. Poor alignment of the joint, excessive weight, excessive activity, overuse or injury can cause further damage to these tissues. As avascular tissues, self-repair of cartilage and ligament is intrinsically limited, so intervention is required to recover the tissue structure. Mechanically, cartilage resists compressive forces, due to the high osmotic pressure that results from water interacting with negatively charged proteoglycans. Contrarily, ligament tissue exhibits strong properties when in tension; ligaments are subjected to a variety of load conditions that affect their mechanics. With proper scaffold design, the viscoelastic nature of these tissues may be replicated with LAY-FOMM; a thermoplastic polyurethane (TPU) co-polymer with polyvinyl alcohol (PVA). LAY-FOMM scaffolds are produced using additive manufacturing, or 3D printing. These scaffolds themselves are nanoporous, with each scaffold incorporating three micro/macro-pores to allow for the deposition of ligament and chondrocyte cells and the formation of collagen-based matrices. By supplementing these scaffolds using 10% FBS, chondrogenic control and chondrogenic media, their ability to promote deposition and regeneration of collagen-based ligament/chondrocyte matrices can be investigated.
Using fused-deposition modeling, these scaffolds are produced. All scaffolds were printed using a Flashforge Creator Pro, with a 0.3mm nozzle at a print temperature of 220°C, 50°C bed temperature, at 18mm.s-1. After printing, they were washed with distilled water to remove excess PVA, leaving a highly porous, flexible structure. The scaffolds were then washed with 70% ethanol for sterilization.
Ligament and chondrocyte cells were held in culture for 21 days (high glucose DMEM, supplemented with 10% FBS, and 1% penicillin streptomycin), whilst changing media every 2-3 days. Following culture, the cells were counted, and seeded in the scaffolds in a 24-well plate, with 400,000 cells on each scaffold. Samples were prepared for each cell type (ligament and chondrocyte) and supplemented with: 1) 10% FBS media, 2) chondrogenic control media (serum-free), 3) chondrogenic media (serum-free, with TGF-β1). Following 21 days of culture, cell proliferation, viability (live/dead assay), western blot, and qPCR were performed.
The addition of serum (10% FBS) showed increased proliferation of chondrocyte and ligament cells. Deposition of ligament/chondrocyte extracellular matrix was visible in the pores of these scaffolds. Preliminary results show the expression of genes specific to chondrocytes and ligament-derived fibrocytes. Specifically, using qPCR, it was shown that cells from ligaments expressed higher levels of scleraxis and collagen type VI. Live/dead assay will further demonstrate the viability of ligament and chondrocyte cells, along with the viability of cells within the collagen-meshes. The proteins of interest for western blot of chondrocyte tissue are collagen II and aggrecan, while the protein of interest for ligament tissue is collagen I.
The implantation of these LAYFOMM scaffolds can lead to the deposition of collagen-rich matrices. The anticipated results include the repair and regeneration of ligament and chondrocyte tissue, which can in-turn generate a novel treatment for tissue damage caused by sports-related injuries. This application can be easily translated to a clinical setting as the cell types involved are human-derived.
Fibronectin is a multimodular glycoprotein existing in two isoforms. The soluble form is known as the plasma fibronectin, produced and secreted by the liver. The insoluble cellular fibronectin forms fibers in the extracellular matrix (ECM). Cartilage development involves aggregation of undifferentiated mesenchyme into a condensed mass of cells. The cells then proceed to differentiate into chondrocytes, which facilitate cartilage development through ECM protein production and secretion. During mesenchymal condensation, fibronectin is abundantly expressed, promoting migration of mesenchymal cells as well as their differentiation. Thus, impaired production or secretion of fibronectin may affect chondrocyte differentiation and development of cartilage. Spondylometaphyseal dysplasias (SMD) are a heterogeneous group of conditions affecting the spine and growth plates. One of these conditions include the corner-fracture type SMD, a rare skeletal dysplasia with short stature and numerous other skeletal phenotypes. We previously discovered several heterozygous mutations in the fibronectin gene (FN1), upon whole exome sequencing of 12 suspected corner fracture SMD patients. These mutations are dispersed across the N-terminal type -1-module domains, and include p.Cys87Phe, p.Tyr240Asp and P.Cys260Gly, among others.
To determine the consequences of these missense mutations in fibronectin production and secretion, we produced recombinant, secreted 70kDa N-terminal fragment (rF70K) as well as a full-length recombinant fibronectin (rFN). We also developed mutant constructs by introducing the SMD mutations p.Cys87Phe, p.Tyr240Asp, and p.Cys260Gly into rF70K and rFN. The full-length wild-type rFN as well as the mutants were transfected into the chondrogenic cell line ATDC5. Wild type and mutant rFNs showed similar levels of expression on the mRNA level when analysed by RT-PCR. Protein expression analysis using Western blotting and immunofluorescence staining revealed synthesis of both transfected wild type and mutant rFN proteins. ATDC5 cells secreted wild type rFN into the culture medium. However, the transfected cells secreted very little or no mutant rFN into the culture medium. Immunofluorescence analysis revealed intracellular retention of these mutant proteins. We are now analysing the consequences of these fibronectin mutations on chondrocyte differentiation, using the transfected ATDC5 cells.
In summary, mutations in the fibronectin gene, found in corner - fracture type SMD patients do not alter fibronectin production by cells, but impair secretion of the mutant proteins. The results indicate the involvement of these fibronectin mutations in dysregulating chondrocyte differentiation and impairing proper cartilage development, leading to spondylometaphyseal dysplasia.
OBJECTIVE: Paget's disease of bone (PDB) is characterized by an increased bone remodeling. Genetic and environmental factors are involved in its pathogenesis. The mutation p.Pro392Leu in the SQSTM1 gene is the most frequently reported mutation in 46% of familial forms. A whole exome sequencing in two families in which PDB patients with or without the p. Pro392Leu mutation coexisted, allowed us to identify the rare p. Val45Ile variant in the DOCK6 gene. This study aims to determine the impact on cellular phenotype of this new variant.
MATERIALS AND METHODS: The study of the impact of the rare variant in the DOCK6 gene on osteoclastic phenotype (osteoclastogenesis, number of nuclei per osteoclast and bone resorption) was performed by in vitro differentiation of monocytes from peripheral blood to mature osteoclasts with RANKL and hMCSF for 21 days. The statistical analysis of the data, comparing pagtic patients with or without variants against healthy controls was performed using Graphpad prism.
RESULTS: We observed an increase in osteoclastogenesis in patients with each of the variants vs. non-mutated patients (70% and 67% vs. 40%, NS). The mean number of nuclei was significantly higher in patients with each of the variants compared to controls (10.7 and 11.3 vs. 5.9; P=0.02 and P<0.0001). Bone resorption was increased in patients with variants vs. controls (81% and 78% vs. 42%, NS).
CONCLUSION: The impact of the rare variant of DOCK6 on the osteoclastic phenotype is similar to that observed with the mutation in the SQSTM1 gene. The modifying effect of the rare variant of DOCK6 on the mutation in SQSTM1 is under study.
Background: Preclinical studies revealed that bone remodeling regulates insulin sensitivity and secretion via the decarboxylation of osteocalcin. It is unknown whether this mechanism contributes to the improvement in glucose homeostasis after biliopancreatic diversion (BPD).
Objectives: To determine if uncarboxylated osteocalcin (unOCN) increases after BPD and whether this increase correlates with changes in glucose homeostasis markers.
Methods: Ancillary study using fasting frozen plasma from 16 individuals (11F/5M, 69% with type 2 diabetes, mean BMI 49.4 kg/m2) assessed before, 3 days, 3 months and 12 months after BPD. unOCN, insulin sensitivity indices (HOMA-IR; adipose tissue insulin resistance index (ADIPO-IR) and insulin sensitivity index (Si) derived from the euglycemic-hyperinsulinemic clamp), insulin secretion rate (ISR) derived from the hyperglycemic clamp, insulin disposition index (DI= Si x ISR) and HbA1cwere analyzed at each visit. Changes in unOCN were correlated with changes in glucose homeostasis indices using Spearman correlations adjusted for weight loss.
Results: There was a non-significant decrease in unOCN at 3 days after BPD (-11%, p=0.12) but a dramatic rise at 3 months (+207%, p<0.001) and 12 months (+392%, p<0.001). The change in unOCN correlated negatively with the change in HOMA-IR (-0.54, p=0.03) and positively with the change in Si(r=0.52, p=0.04) at 3 days after BPD. At 3 months, there was an inverse correlation between change in unOCN and change in ADIPO-IR (r=-0.67, p<0.01) and HbA1c(r=-0.68, p<0.01). No correlations were seen with ISR and DI.
Conclusions: The increase in unOCN may contribute to the amelioration of glucose homeostasis observed after BPD independently of weight loss.
Introduction: Giant cell tumour of bone (GCTB) is a benign (non-cancerous) tumour that affects people between the ages of 20 and 40 years. Over-expression of receptor activator of nuclear kappa-B ligand (RANKL), upsets the RANKL/OPG (osteoprotegerin) synergy that regulates bone remodelling. As a result, there are increased numbers of osteoclasts, which can fuse to form giant cells, causing increased bone resorption. This process can remain clinically silent for extended periods of time and can lead to large voids in the trabecular bone, often at the distal femur. Treatment strategies are limited to either bone grafts, structural support or joint replacement surgeries. In vitro disease models are being developed for a number of cancers as they can recapitulate the tumour microenvironment. This reduces the need for animal models in early stages of drug development, as well as their use in investigating tumour-healthy tissue interactions. The use of hydrogels for 3D cultures better mimics the in vitro environment so allows for better models and bioprinting enables precise spatial control over the deposition of cell-seeded hydrogels.
Aim: The cause of GCTB is relatively unknown and treatments are limited. This study aims to develop an in vitro model of the healthy bone and tumour environment. To achieve this, constructs containing both healthy and tumour-like regions were bioprinted to enable spatial control over the deposition of cells.
Methods: To model both the healthy and tumour environments, co-cultures were bioprinted. The healthy environment consisted of a patient derived osteoblast population while the tumour environment encompassed both osteoclasts and stromal cells (derived from patient tumour samples). These constructs were produced using a BioX printer (Cell Ink, Sweden) with a methacrylated gelatin (GelMA) bioink (Cell Ink, Sweden). Semi-circular regions that interfaced on the flat edge were co-printed for fusion at the boundary so that the direct interactions between cells could be investigated. Cell viability, proliferation and migration assays were performed to determine the effect of tumour cell populations on the healthy cells.
Results: Following printing and UV crosslinking, osteoblasts and patient-derived stromal cells showed very good viability (over 90%) and were able to proliferate in the GelMA bioinks. Longer-term investigations of healthy cell survival with the secretion of factors from the stromal cells are currently in progress. Further, migration assays are expected to show that the cell populations can mix by migrating into opposing regions.
Preschool aged children have been reported to be the most common age group to visit the emergency department (ED) for caries-related dental problems. Although children’s oral health is heavily reliant on their parents’ practices, there is currently little evidence about the influence of parents’ oral health knowledge and beliefs on their child’s utilization of ED. This study aims to assess the association of parents’ oral health knowledge and beliefs, family sociodemographic characteristics and children’s utilization of ED for treatment of early childhood caries.
Data was from a cross sectional study carried out at the Montreal Children’s Hospital (MCH) investigating the utilization of ED for caries-related dental problems. The study involved children aged 1 to 17 years who sought care at the MCH ED. The current study focuses on data collected relating to preschool aged children. Measures included clinical examination and a semi-structured questionnaire. The child’s diagnosis (early childhood caries, pulpitis and/or odontogenic infection) and the number of teeth affected by caries (Dt) was assessed by the treating ED dentist. A questionnaire answered by parents assessed the child’s oral health care utilization and behaviours, familial sociodemographic characteristics and parent’s oral health knowledge and beliefs. Descriptive analysis and multivariate logistic regression analyses were conducted to examine predictor variables of use of ED for ECC.
A total of 109 children participated in this study. Fifty-seven percent were male and the mean age was 3 years. The majority of children had never visited a dentist before and was mainly from families of low socioeconomic status and recently immigrated backgrounds. Parents demonstrated generally good oral health knowledge; however, parental perception of their children’s oral health was low. After adjusting for child’s age and gender, children from lower income families were more likely (OR 3.02, 95% Cl: 1.01-9.07) to utilize the ED instead of seeking regular dental care services. Parents’ knowledge and beliefs were not associated with children’s utilization of ED.
Overall, this study shows that low socioeconomic status was associated with preschool children utilizing the ED for ECC. Parents demonstrated adequate knowledge with regards to fundamental oral hygiene practices, as well the importance of maintaining the primary dentition. However, parents revealed an inaccurate perception of their child’s oral health care needs. As parental knowledge alone is not effective in preventing the use of ED, implementation of community-based oral health promotion programs should be explored.
Introduction: Mutation of fubrillin-1 can cause Marfan syndrome (MFS), which is characterized with abnormalities in various tissues, including bone, aorta, lung and skeletal muscle etc. Among them, thoracic aortic aneurysm (TAA) is the leading cause of mortality, which is characterized by elastic laminae fragmentation, localized inflammatory cell infiltration, loss of smooth muscle contractility, elevated canonical and non-canonical TGFβ signaling. Several microRNAs (miRNAs) are reported to participate in various vascular pathologies, including aneurysm, atherosclerosis, and vascular calcification. Among them, miR-29b inhibition was shown to ameliorate TAA in a MFS mouse model, by regulating ECM synthesis/deposition and rescuing smooth muscle cell (SMC) apoptosis. However, the role of miRNA networks during the TAA formation in MFS is largely unknown. This study investigates the global miRNA expression profiles during the course of TAA formation, and explores the relationship between relevant miRNAs and mRNAs.
Results: To investigate potentially dysregulated miRNAs during TAA formation, microarrays were performed with RNA isolated from the ascending aortae of 4- and 10-week old Fbn1mgR/mgR mice, a fibrillin-1 haploinsufficient mouse model for MFS. These mice are characterized by TAA starting at about 8-week of age. At 4 weeks, 14 miRNAs were upregulated and 3 miRNAs were downregulated more than 2-fold, comparing the MFS mice with wild-type mice. At 10 weeks after the TAA formed, there were 124 miRNAs upregulated and 5 downregulated, 8 of which were upregulated at both time points. Bioinformatic prediction of the 8 shared miRNAs revealed MAPK, ECM receptor interaction, focal adhesion, mTOR and inflammatory receptor signaling pathways to be dysregulated, all closely related to TAA formation. Comparative mRNA microarrays at 10-week showed 471 upregulated and 253 downregulated mRNAs, 109 (15%) of which reversely correlated with 89 (69%) miRNAs predicted to target them. Gene ontology analysis of the 109 mRNAs revealed the inflammatory response, cell adhesion, MAPK cascade, protein metabolism to be the top hits. miR-122, the most downregulated miRNA at 10-week (-5.87 fold), is predicted to target MCP1 (4.05 fold), CXCL13 (8.74 fold), IL6 (3.03 fold) and IL1b (2.92 fold), which are known to promote the inflammatory response in tissue. Immunohistochemical staining of the mutant aorta displayed infiltration of T-cells and macrophages into the media from the adventitia. miR-221/222, upregulated at both 4-week (2.26/7.3 fold) and 10-week (4.64/3.54 fold), are reported to be necessary for SMC proliferation. This correlates with the downregulation of smooth muscle contractility related genes in the 10-week Fbn1mgR/mgR aorta, indicating a SMC switch to the synthetic phenotype. Additionally, functional analysis showed that the mimics of an upregulated miRNA, let-7g (2.37 fold) can significantly down-regulate total ERK1/2 in SMCs isolated from mouse ascending aortae.
Conclusion: 17 miRNAs and 129miRNAs were dysregulated in the ascending aortae of Fbn1mgR/mgR mice at 4-week and 10-week respectively, 8 of which were upregulated at both time points. These miRNAs might contribute to TAA formation by regulating MAPK, ECM receptor interaction, focal adhesion, mTOR and inflammatory receptor signaling. Furthermore, our results suggest the involvement of miR-122 in the inflammatory cell infiltration at the aneurysm site, and miR-221/222’s role in the SMC phenotype switch in the course of TAA formation. In addition, the regulatory role of let-7g in ERK1/2 signaling is also highlighted.
Is maternal oral health status a risk factor for dental caries in children?
Radhika Chhibber1, Belinda Nicolau1
1McGill University, Montreal, Quebec, Canada
Introduction: Several studies have shown that the health and health behaviours of parents affect the health and behaviour of their offspring. However, only a few studies have investigated the association between mothers’ and their children’s oral health status. In addition, the majority of them are cross-sectional in nature.
Objective: to investigate the extent to which maternal oral health status at baseline is associated with the 7-year caries increment of their children among the participants of the Quebec Adipose and Lifestyle Investigation in Youth (QUALITY) cohort.
Methodology: We use data from the first 3 visits from the QUALITY cohort, an ongoing prospective study aimed at understanding the natural course of obesity. Caucasian children aged 8–10 years and their biological parents, at least one of them being obese, were recruited in Montreal, Canada between 2005 and 2008. Data collection included a dental clinical examination and questionnaires administered to children and parents. Dental caries was measured using the Decayed, Missing, Filled Surfaces (DMFS) index. The exposure of interest is mothers’ self-reported oral health status. Descriptive statistics and negative binomial regression will be used.
Results: I am currently performing the data analysis and in this presentation, I will discuss the preliminary results.
Significance: while it is well known that families share common risk factors, oral health literature lacks evidence on the subject. The results of this study will contribute to this evidence and may provide support for preventive strategies targeting families.
The risk of oral squamous cell carcinoma (OSCC) is 2.5-fold higher among males compared to females. Greater prevalence of risk-taking behaviors (e.g., tobacco and alcohol consumption, betel quid chewing) among males is a possible explanation. However, reason behind this behavioral difference between sex remains unclear. Ratio of index and ring digits lengths (2D:4D ratio), a sex dimorphic proxy of intra-utero levels of testosterone and estrogen exposure may affect a series of behavioral characteristics forming prenatally and collectively referred to as “masculinity” or “femininity”, which in turn may influence OSCC risk.
Objective: To estimate the extent to which 2D:4D ratio as an indicator of intra-utero hormone level is associated with an increase in OSCC risk in a population from Southern India.
Methods: We used data from a hospital-based case-control study conducted in Kozhikode, Kerala, India, between 2008 and 2012. 348 cases (153 females, 195 males) with newly histologically confirmed OSCC and 371 controls (167 females, 204 males) frequency matched by age and sex from two main referral hospitals were recruited. In-person interviews collected information on an array of life course exposures (e.g., sociodemographic, anthropometric, behavioral factors). We used logistic regression to estimate the odds ratios (OR) and 95% confidence intervals (95% CI) for the association between 2D:4D ratio and OSCC.
Results: Overall, comparing to people with 2D:4D ratio lower than 1, risk of developing OSCC for people with ratio equaled to or higher than 1 decreased by 25% (OR=0.75, 95% CI: 0.53-1.07) before adjustment and 31% (OR=0.69, 95% CI: 0.45-1.04) after adjustment. When stratified by sex, this negative association was found both among males (OR=0.68, 95% CI: 0.401.17) and females (OR=0.76, 95% CI: 0.381.51) after adjustment. Among people who did not consume tobacco, alcohol or betel quid, with 2D:4D ratio equaled to or higher than 1 was still associated with lower risk of OSCC than those with ratio lower than 1 in both sexes (male: OR=0.68, 95% CI: 0.11-4.16; female: OR=0.87, 95% CI: 0.29-2.58) after adjustment.
Conclusion: Higher 2D:4D ratio (equaled to or higher than 1) is associated with decreased OSCC risk compared to lower 2D:4D ratio (lower than 1).
Objectif: Bien que les services intégrés continuent d'attirer l'attention des décideurs des politiques de santé, l’intégration des soins buccodentaires aux soins primaires demeure un défi considérable. Par conséquent, l'objectif de cette étude est d'explorer les perceptions des intervenants de première ligne du Québec à l'égard de l'intégration des soins buccodentaires dans leur pratique.
Méthode: Pour cette étude de cas multiples, nous avons utilisé une approche qualitative :interpretive description. Les sources de données proviennent de 74 entretiens semi-structurés et 5 groupes de discussion, avec les intervenants de première ligne et les gestionnaires des deux établissements de santé, urbain et rural. Le guide d’entrevue et le cadre conceptuel de l’étude ont été basés sur le modèle de soins primaires intégrés de Valentijin et al. L’échantillonnage à variation maximale et la technique boule de neige sont les méthodes utilisées pour le recrutement des participants. La collecte de données s’est poursuivie jusqu'à la saturation. La démarche d’analyse de chacun des cas, réalisée en quasi-simultanéité avec la collecte des données, s’est déroulée selon les étapes suivantes : la décontextualisation, la récontextualisation, la catégorisation et la compilation des données et nous avons utilisé différentes formes de triangulation (des méthodes, des sources de données et des chercheurs). Les données ont été analysées manuellement et en utilisant le logiciel Atlas-ti.
Résultats: Quatre thèmes ont été identifiés, couvrant tous les domaines du cadre conceptuel de l’étude: 1) Moteurs d’intégration, avec les sous-thèmes : Le manque de services buccodentaires publics et Les besoins de santé buccodentaire; 2) Importance de l’intégration; 3) Rôle des professionnels dans les soins intégrés; et 4) Barrières et les facilitateurs de l’intégration. Les intervenants s’identifiaient des rôles de liaison et/ou coordination et ont souligné l’importance du personnel dentaire dans la prestation de ces services. La plupart des obstacles exprimés par les participants à l’étude étaient liés aux domaines organisationnel et systémique de l’intégration. La mise en œuvre des politiques de soins buccodentaires intégrés, la priorisation de la formation du personnel et la collaboration interprofessionnelle pourraient faciliter cette intégration.
Conclusion : Ces résultats suggèrent que des préoccupations concernant les services buccodentaires primaires sont présentes chez les intervenants de première ligne. Le développement futur de modelés de services intégrés nécessite des approches spécifiques aux rôles professionnels, une analyse du contexte et des rapports interprofessionnels au niveau organisationnel.
Introduction: Studies have already demonstrated that oral parafunctional behaviours (OPB) increase the risk of temporomandibular disorders (TMD). This case-control study aimed to investigate whether sleep OPB (SOPB) is more common among chronic painful TMD (PTMD) in comparison the acute PTMD. Methods: Patients with acute or chronic PTMD were recruited from four dental clinics. TMD diagnoses were established by dental specialists using Diagnostic Criteria (DC/TMD). Acute PTMD was defined as PTMD lasting less than three months, whereas chronic PTMD should have occurred for at least three months. The validated Oral Behaviour Checklist, General Anxiety Disorder (GAD-7), and Patient Health Questionnaire (PHQ-9) were used to assess SOPBs, anxiety, and depression respectively. SOPBs include clenching and grinding during sleep and sleep position that puts pressure on the jaw. Results: A total of 45 acute PTMD and 212 chronic PTMD were recruited for this study. Mean scores were calculated for the SOPBs frequencies; “None of the time” “, <1 Night/month”, “13nights/month”, “1-3 nights/week”, “4-7 nights/week”. A multivariable logistic regression analysis adjusted for anxiety, depression, age, and gender revealed the following results: low frequency (mean score of 0 to < 6) OR= 3.67, 95% CI= 1.43-9.42; moderate (mean score of > 6 to < 8), OR= 3.16, 95% CI= 1.20-8.33; or high frequency (mean score of > 8), OR= 3.44, 95% CI= 1.34-8.83). Conclusion: The odds of SOPBs are higher among chronic PTMD relative to acute patients, independent of the SOPBs frequency, demographics and psychological disorders. This result suggests that SOPBs may contribute to the transition from acute to chronic painful TMD
Bone adapts to mechanical loading by forming and resorbing bone to meet functional demands. It has previously been shown that bone formation markers oscillate based on time of day. The aim of this project is to determine how the time of day affects the bone formation and resorption response to mechanical loading. In vivo cyclic compressive loading was applied to left tibiae of 10-week-old C57BL/6 female mice for 2 weeks (+1200 με at midshaft determined by strain gauging, 216 cycles/day at 4 Hz) and right tibiae served as internal controls. Loading was performed daily (Mon-Fri) at either Zeitgeber (ZT)2 or ZT14. The mice were housed in metabolic cages and in-vivo microcomputed tomography was performed. Results indicate that loading led to significant increases in cortical and trabecular microstructural parameters (e.g. cortical area, cortical thickness, bone volume fraction, trabecular thickness) in the loaded compared to the control limb. No significant differences in bone mass or microstructure were observed after loading between the ZT2 or ZT14 time points. Further studies will analyze longer loading periods and other loading time points to determine if time of loading affects molecular or material level properties. These data may enhance load-included bone formation in patients undergoing exercise.
Introduction: Misclassification is an important, but often overlooked, source of bias in medical and dental research. It is when a diseased case is classified as non-diseased or vice versa. This occurs because our tools of measurement and testing are not perfect and are often prone to error. Quantitative Bias Analysis (QBA) estimates the magnitude and direction of systematic errors to enhance validity using external information. However, strong assumptions are needed to achieve this numerically. We will use an example of human papillomavirus (HPV) infection in a case control study to discuss the analytical challenges and assumptions involved in accounting for HPV misclassification, and the effect that might have on our research conclusions.
Methods: we use data from a case-control study conducted in 4 Montreal hospitals investigating the etiology of cancer of the upper aerodigestive tract (UADTC). Cases, incident squamous cell carcinoma of UADT (460), were frequency matched by age and sex to non-cancer controls (458) selected in the same hospitals as the cases. In-person interviews collected information on socio-demographic and behavioral factors. We collected oral rinse and oral brush biopsy samples and detected HPV-DNA with consensus PCR. We conducted QBA exploring three deterministic non-differential misclassification scenarios: (i) pessimistic (assuming high level of error), (ii) informed point estimates (assuming moderate level of error), and (iii) optimistic (assuming low level of error). Under each scenario, the impact of potential error is assessed on a multiplicative scale (odds ratio(OR)).
Results: 818 individuals were tested for HPV, 79.1% of cases and 20.9% of controls were oncogenic HPV positive. The uncorrected and unadjusted odds ratio (OR) was 6.15 (CI95% 4.11-9.20).Observed data are not compatible with low sensitivity under the first two scenarios, highlighting the analytical challenges of this technique. The optimistic scenario resulted in a corrected unadjusted OR of 11.14 and a 45% bias toward the null.
Conclusion: Our findings suggest that HPV association with UADTC us often underestimated in the literature due to imperfect measurement of HPV among studied individuals. This can have a great public health impact on screening and HPV vaccination efforts. We therefore believe that robustness checks of results through QBA is advisable when policy decisions can be made using evidence as in this case for HPV prevention programs.
Nanoscale physical modifications of medically-relevant metals are a compelling determinant of cell behavior. Cell-substrate interactions and related signalling pathways determine the response of the host tissue and therefore the success of implants. Here, we demonstrate the versatility of a simple chemical oxidative treatment with H2SO4/H2O2 to nanocavitate titanium surfaces and achieve a unique mesoporous surface network. Our previous work has revealed that such surfaces significantly influence osteogenic activity, both in vivo and in vitro. They also exhibit antibacterial properties. Here, the objective of our work was to determine the effect of this nanocavitated surface on the cell adhesion apparatus. Osteogenic cells were cultured on polished (control) and nanotextured surfaces for periods of 6, 24, and 72 h. Results from immunofluorescence analysis revealed an increase in the number of focal adhesions per cell area, and in their length and maturity on the mesoporous surface as compared to the control. Gene expression for various focal adhesion markers, including paxillin and talin, and different integrins (e.g. α1, β1, and α5) was also significantly increased. Scanning electron microscopy results revealed that the mesoporous surface promoted the presence of more filopodia on cells. Initial analysis using atomic force microscopy suggest that filopodia on the nanocavitated surface require more lateral force to detach. These cell extensions displayed abundant and distinctive nanoscale lateral protrusions of around 10-15 nm in diameter that intimately molded the nanopore walls. The increase in number of focal adhesions, as well as the abundance of filopodia with nanoprotrusions, that exhibit an apparent ‘stronger’ adhesive strength, altogether likely positively contribute to increasing cell adhesion, and thereby alter the nanoscale biomechanical relationships that regulate cell behavior.
Keywords: nanotopography, filopodia, focal adhesions, gene expression.
Poly-methyl methacrylate (PMMA) bone cement is one of the most commonly used bone substitutes in orthopedic surgery. In clinical practice it can be loaded with various drugs, such as antibiotics or anti-cancer therapeutics, as a means of local drug delivery. However, studies have shown that drugs loaded into PMMA cement tend to release in small bursts in the first 48-72 hours, and the remaining drug is trapped without any significant release over time. The objective of this study is to develop a nanoparticle-functionalized PMMA cement for use as a sustained doxorubicin delivery device. We hypothesize that PMMA cement containing mesoporous silica nanoparticles will release more doxorubicin than regular PMMA.
High viscosity SmartSet ™ PMMA cement by DePuy Synthes was used in this study. The experimental group consisted of 3 replicates each containing 0.24 g of mesoporous silica nanoparticles, 1.76 g of cement powder, 1ml of liquid cement monomer and 1 mg of doxorubicin. The control group consisted 3 replicates each containing 2.0 g of cement powder, 1ml of liquid cement monomer and 1 mg of doxorubicin. Each replicate was casted into a cylindrical block, and then incubated in a phosphate-buffered saline solution which was changed at predetermined intervals for 24 days. The concentration of eluted doxorubicin in each solution was then measured using a florescent plate reader against a doxorubicin standard curve.
Each cement block weighed 2.43 ± 0.08 g representing 82.70 ± 2.72% of the initial mixture weight. The experimental group contained an average of 8.18 ± 0.008 % (W/W) mesoporous silica nanoparticles. After 24 days the experimental group released 2.04 ± 0.33 % of the initially loaded doxorubicin which was more than the 1.18 ± 0.11% released by the control group (p 0.03).
This Study has shown that nanoparticle-functionalized PMMA cement can release up to 1.7 times more doxorubicin than the standard PMMA. The use of mesoporous silica nanoparticles to improve drug release from PMMA cement shows promise. In the future, long term doxorubicin elution data from PMMA cement should be analyzed to assess whether or not nanoparticles result in sustained release from PMMA Cement. Furthermore, in vitro and in vivo experiments are required to test the efficacy of released doxorubicin on tumor cell growth.
Decreased tissue oxygenation is related to microvascular disease, critical limb ischemia, compartment syndrome, sepsis, traumatic brain injury, wound healing, and other disease states and tissue malfunctions. Currently, continuously monitoring of tissue oxygen levels involves Clark probes, microdialysis, MRI, and EPR. These techniques are time intensive, require costly large equipment, lack accuracy, and/or typically require anesthesia or restraint, thereby excluding the possibility for real-time monitoring during daily life. Electrochemistry can offer rapid, sensitive, selective, and low-cost quantitative or semi-quantitative analytical information and is only beginning to be applied to medical decision making. Because no expensive infrastructure is required to run an electrochemical test and the equipment is simple and could be as small as a mobile phone; electrochemical analysis could be easily implemented in the operating room, near the patient bedside, and even at home. Through engineering a simple metallic injection needle, we developed a novel, low-cost, tissue-integrating sensors that enable real-time continuous measurement of tissue oxygen. Laccase is a copper-containing oxidase whose catalysis reactions are tightly coupled to the four-electron reduction of oxygen to water; the enzyme uses only oxygen as a final electron acceptor. We hypothesise that by monitoring the electron exchange rate between the enzyme and the electrode we are able to continuously track tissue hypoxia. To limit invasiveness of such procedures, SS316L acupuncture needle (0.3 mm diameter) have been applied as the working electrode. To increase the surface/volume ratio, we successfully developed a controlled corrosion environment that produces reproducible and robustly pitted needles. To provide specificity and selectivity in eliciting a response to oxygen without the need separation membrane or sacrificial layer, laccase biocatalyst was immobilised through an innovative spinning process at the SS electrode. One of the problems with the use of enzymes as catalysts in sensing device is to achieve good and direct contact between the enzyme and the electrode surface. To overcome this problem, we combined the use of carbon nanostructures with electropolymerized conductive coating for the construction of electrode.
Minimally invasive, low-cost stainless steel acupuncture needles (AN) were modified via a two-step process: 1) controlled corrosion microfabrication, and 2) carbon nanoparticle (CNP) spinning deposition, 3) electropolymerization laccase coupled polypyrrole. Following physical, electrical and chemical characterization of the probe (AFM, CV, SEM, microCT, mechanical strength testing via Instron), we validated this electrochemical detection needle in vitro with cyclic voltammetry, long term stability in N2 and O2 environment. AFM and microCT data show that microporous structures cover ~75% of the electrode surface, with an average diameter of 0.166 µm ±0.09. Mechanical testing of pitted versus unpitted needles showed no significant compromise to the integrity of the needles when simulating tissue penetration. Further, upon successful CNP+laccase co-immobilisation, the needle showed a direct electron transfer without the need of mediator and an ample sensitivity in vitro. O2 needle was stable over time and showed a great selectivity towards oxygen as no noticeable changes were observed upon GABA and catechol injection. Future works include the development of a rodent model of ACS with ischemic injury induced by tourniquet application followed by a reperfusion injury in upper hind limb of a rat as proof of principle.
Introduction: Transcranial magnetic stimulation (TMS) is a non-invasive brain stimulation technique that is being explored as a therapeutic alternative for the management of various chronic pain conditions. Chronic orofacial pain (OFP) disorders are subserved by the trigeminal nerve, which has unique somatosensory and pain processing properties, making these pain disorders different and less comparable to extra-trigeminal ones. While the efficacy of TMS in reducing clinical pain intensity has been assessed systematically in spinally-mediated pain disorders, to our knowledge, there has been no systematic review on the analgesic effect of TMS in OFP.
Aim of the investigation: The primary objective of this systematic review is to assess the efficacy of TMS in reducing pain intensity. The secondary objectives are to describe adverse effects, duration of relief, and TMS methodologies used in chronic OFP disorders
Methods: A search was performed in Medline, Embase, Web of Science, Scopus, and Google Scholar. Inclusion criteria were: 1) Population: Adults diagnosed with chronic OFP including neuropathic and non-neuropathic disorders. 2) Intervention: active TMS stimulation regardless of the used protocol. 3) Comparison: sham TMS stimulation. 4) Outcome: primary outcome was clinical pain intensity. Secondary outcomes were duration of pain relief, adverse effects, and methodological parameters. Risk of bias and quality of study reporting were also assessed.
Results: A total of 556 individual citations were identified by the search strategy, with 11 articles meeting selection criteria. The specific disorders included in these 11 articles, which totaled 156 patients, were: trigeminal neuralgia (TN), trigeminal neuropathy, burning mouth syndrome (BMS), and atypical facial pain. Qualitative heterogeneity was present among samples, methods, and outcomes. All studies used repetitive TMS (rTMS) protocols except for one that used intermittent theta burst stimulation (iTBS). rTMS frequencies ranged from 10Hz to 20Hz and intensities varied between 80% and 110% of the resting motor threshold. Stimulated brain areas were left prefrontal cortex (LPFC), M1 representation of face and hand, and M1/S1 area. Stimulations over the LPFC (k=2) and M1/S1 (k=1) were associated with significant pain reduction. Pain reduction was also found following stimulation of the hand representation over M1. However, stimulation of the face representation over M1 face showed conflicting results. Studies that included more stimulation pulses per session and more sessions obtained more durable analgesic effects (from 14 to 60 days). Adverse effects were minimal, with only two studies reporting mild transient headaches.
Conclusions: TMS appears to be a safe and promising alternative to reduce pain intensity in different chronic OFP disorders. Additional research effort is needed to reduce bias, improve quality, and characterize optimal brain stimulation parameters to promote its efficacy.
Acknowledgments/Disclosures: Authors would like to acknowledge the contribution of Natalie Clairoux from University of Montreal for her guidance and Dr. Estephan Moana-Filho for his contribution. Authors report no conflict of interest or financial disclosure.
Refugees are migrants who have experienced harsh conditions, such as environmental disasters, violence, and war, and are forced to leave their home country. Children represent almost half of the refugee population. The complex migration trajectory and underdeveloped healthcare systems in source countries often lead to poor oral health in the refugee population. Literature suggests that improving the oral health of refugees is a global priority. It is important to explore the oral health experiences of refugee children to improve their oral health status. Thus, this study aims to explore how refugee children in Canada experience oral health and access to oral health care. Furthermore, we want to better understand how refugee parents in Montreal experience accessing oral health care for their children.
This study will use a focused ethnographic methodology. The participants will be children aged 6-12 years and their parents. Participants will be recruited from the Student Dental Clinic for Pediatric Dentistry at the Division of Dentistry of the Montreal Children’s Hospital. Snowball technique will also be used to recruit study participants, if required. Face to face, semi-structured interviews will be conducted. Data will be analyzed using a thematic approach including interview debriefing, transcript coding, data display and interpretation.
This study will contribute to the oral health of refugee populations in Canada by addressing the gap in knowledge related to the experience of refugee children in Montreal regarding accessing oral health care and their experience of care. This study will also contribute towards improving the oral health-related quality of life of refugee children.
Our aim is to work together with refugee families to improve the overall health status of refugee children. With this study, we will endeavor to understand the perspective of these children. The study results will contribute to oral health of refugee children in Canada by exploring their experiences in accessing and receiving oral health care.
Objectives: Previous studies have shown that osteoporosis could be a risk factor for the progression of periodontitis although the underlying mechanisms are poorly understood. Objective: To determine whether upregulation of the transcription factor PITX1 contributes to periodontitis by inducing agerelated osteoporosis.
Methods: Transgenic mice were engineered with the 2.3 kb mColα1 promoter to overexpress murine Pitx1 cDNA in osteoblast cell lineages and odontoblasts. Twentyfour mice were analyzed at 3months (6 males and 6 females for each genotype). Highresolution radiographs were taken of the skulls, mandibles, and teeth and complemented by a dualenergy Xray absorptiometric (DXA scan). Threepoint bending tests were performed on a Mach1 TM micromechanical testing system to assess changes in bone strength. Hematoxylin/eosin staining was performed on bone sections and complemented by immunohistochemistry assays and expression analyzes by qPCR.
Results: Pitx1 overexpression decreased BMD by 18.4% and 15.3% in the maxilla and the mandible respectively, while BMC was reduced by 30.5% and 26.2% in the maxilla and the mandible respectively. Cortical bone thickness in transgenic mice was decreased by 66.7% and 62% at the maxilla and mandible level respectively compared to wild type mice. Bone stiffness and the work to ultimate breakpoint were decreased by 37.7% and 56.5% respectively in the mandibles of transgenic mice compared with the wild type ones. Alveolar crest height and mobility testing revealed early onset periodontal breakdown of the periodontium of the transgenic mice. Histological analysis showed combined periodontalendodontic lesions in the transgenic mice with a decrease in cementoblast and odontoblast numbers, hypomineralization of dental hard tissues. At the molecular level, MMP8, a known prominent collagenase involved in periodontal disease, was overexpressed by ~20fold in the mandible of transgenic mice.
Conclusions: Collectively, our findings further define a novel role of PITX1 in agerelated osteoporosis and periodontitis and could represent the missing link between both diseases.
INTRODUCTION: Mutations in human matrix Gla protein gene (MGP) lead to Keutel syndrome (KS), a rare genetic disorder hallmarked by ectopic calcification of cartilage and vascular tissues, midface hypoplasia and skeletal abnormalities. Although MGP is a potent inhibitor of ectopic calcification, its mode of action is poorly understood. It has been suggested that two sets of conserved residues undergoing post-translational modifications are essential for MGP’s function. These are i) 4 glutamic acid residues which are g-carboxylated (Gla) by the enzyme gamma-glutamyl carboxylase (GGCX); and ii) 3 serine residues which are phosphorylated (pSer) by an unknown kinase. Until now, the requirement of these residues in MGP’s function in vascular and cartilaginous tissues has not been investigated in vivo.
PURPOSE: To investigate the role of the conserved glutamic acid and serine residues in MGP’s function in blood vessels and cartilaginous tissues.
METHODS: MGP-deficient (Mgp-/-) mice recapitulate the phenotypic features of KS. Considering this, we decided to use murine models for our study. Using a combination of gene modification techniques, we mutated Mgp or Ggcx in vascular and cartilaginous tissues. Ectopic calcification phenotypes of these mice have been analyzed by micro-CT-, histology- and molecular biology-based techniques.
RESULTS: First, we generated a transgenic line (SM22-GlamutMgp) expressing a mutant form of MGP, in which the four conserved glutamic acid residues were mutated to alanine. The transgene was introduced to Mgp-/- mice to generate a compound mutant, which did not produce the native MGP, but the mutated MGP only in the vascular smooth muscle cells (VSMCs). Surprisingly, blood vessels in these mice were not calcified. These data were further supported by another model lacking GGCX specifically in the VSMCs, as vascular calcification was also absent in this model. Interestingly, when we ablated Ggcx specifically in chondrocytes, only cartilaginous growth plates were calcified; there was no calcification of the nasal septum cartilage, a hallmark feature of KS patients and Mgp-/- mice. We next used a CRISPR-Cas9-based approach to mutate the codons for the conserved serine residues to that for alanine in vivo. These newly generated mice recapitulated both vascular and cartilage calcification traits of Mgp-/- mice, although with a lesser severity.
CONCLUSIONS: For the first time, using a genetic approach, we show here that MGP’s Gla and pSer residues are critical for its anti-mineralization functions in cartilage and blood vessels. However, the functional roles of these residues may vary in these tissues ̶ Gla residues are required for MGP’s functions in the growth plates only, while its pSer residues are required to prevent calcification at all vascular and cartilaginous sites. Our data raises the possibility that the Gla and pSer residues may work in a cooperative manner to confer MGP’s anti-mineralization function.
Background: Bone and bone marrow do not exist in isolation and disruption of one system affects bone-blood system as a whole. In a recent systematic review, we examined bone health in patients with hematopoietic disorders and demonstrated that an increased hematopoietic cell proliferation, such as observed in patients with hemolytic anemias, was associated with bone loss, while bone marrow hypocellularity, such as in patients with chronic myelofibrosis (CMF), was associated with bone density increase. The mechanism of bone gain in CMF is unclear, but it contributes to patients’ morbidity as it is associated with bone pain, and mortality as it may lead to bone marrow failure. Recently, a mouse model with a global knock-out of the G6b-B receptor was shown to develop myelofibrosis secondary to megakaryocyte/platelet differentiation defect. Moreover, a group of patients with primary myelofibrosis was identified to have a loss-of-function mutation in the G6b-B gene. Objective: The objective of this study was to characterize temporal changes in the skeleton of the G6b-B knock out mice. Results: Age- and sex-related changes were examined in 4-, 8-, 16-, and 32-week-old G6b-B+/+, G6b-B+/-, G6b-B-/- female and male mice. G6b-B+/- mice of both sexes were not different from G6b-B+/+ mice. Spleen, a site of extramedullary hematopoiesis, progressively increased in female, but not male, G6b-B-/- mice compared to corresponding G6b-B+/+ mice, reaching 3.2-fold increase at 32 weeks (p < 0.01). Liver, another potential site of extramedullary hematopoiesis, was not affected by the mutation. Microcomputed tomography of femur metaphysis of 32-week-old female G6b-B-/- mice demonstrated a 3.8-fold increase in BV/TV and trabecular number and a 2.8-fold decrease in trabecular separation (p < 0.001) compared to G6b-B+/+, while in 32-week-old male G6b-B-/- mice trabecular thickness increased 1.3-fold (p < 0.01). Femur diaphysis showed the presence of trabecula in female G6b-B-/- mice, with trabecular BV/TV increasing by 24-fold and trabecular number by 20-fold (p < 0.001) compared to G6b-B+/+. Mid-shaft cortical bone area was increased 1.2-fold in both female (p < 0.01) and male (p < 0.1) G6b-B-/- mice compared to corresponding G6b-B+/+. Gene expression analysis of osteoblast, osteoclast, and osteocyte-specific genes as well as TGF-β1, a protein implicated into the development of fibrosis, in bone-derived samples demonstrated no difference between G6b-B-/- and G6b-B+/+ 32-week-old female and male mice. Conclusion: G6b-B-/- mice demonstrate an age and sex-related bone gain, which was especially strong in female G6b-B-/- mice that also demonstrated severe splenomegaly. These data suggest that the observed bone gain is consequent to a hematopoietic disturbance reported in G6b-B-/- mice, rather than alterations in bone cells. This mouse model will be helpful in understanding the pathophysiology and finding new treatments for myelofibrosis.
Sedation is an important adjunct in providing oral health care for pediatric patients with uncooperative age-appropriate behavior, anxiety, disabilities, or medical conditions. However, little is known about how sedation services are offered in Canadian pediatric hospitals and the profile of children receiving them. Thus this study aims to analyze the differences between Canadian pediatric hospitals in matter of availability, recommendation and waiting time for dental treatments with sedation.
We conducted a cross-sectional web-based survey of sedation services provided at Canadian pediatric hospitals. The survey instrument was sent to the chiefs of Dentistry of all Canadian pediatric hospitals with a dental division. The questionnaire included 42 questions and assessed: the sedation services offered; the eligibility criteria for their use, the waiting time for access and the characteristics of patients receiving them during the year 2017. Descriptive analyses of the data were undertaken to summarize the results. Chi-square tests and 1-way ANOVA were used to assess differences in number of cases performed and waiting time between provinces.
A total of 10 pediatric hospitals answered the survey (response rate of 83%). General anesthesia (GA) and inhalation sedation with nitrous oxide (N2O/O2) were provided in all surveyed hospitals; whereas sedation with benzodiazepines was offered in only 60% of them. Criteria for the use of GA were fairly homogenous; however, several discrepancies were noted for the use of benzodiazepine sedation and N2O/O2 inhalation sedation. The majority of children receiving dental care under GA aged between 3-5 years; whilst N2O/O2 inhalation sedation was mainly used in children aged 6-9 years. Moreover, the average wait time for access to sedation varied significantly between provinces.
This is the first nationwide study of the provision of sedation for dental care in Canadian pediatric hospitals. Our results show that institutional variations exist on the delivery of sedation services as an adjunct to pediatric dental care. There are some important discrepancies between provinces in the waiting time to access these services, the number of cases performed and accessibility to treat patients under GA. Furthermore, the lack of national guidelines for sedation impairs standardized dental care amongst provinces. The relationship between social determinants and the use of sedation services merits further investigation to establish best dental practice and preventive care within Canadian pediatric hospitals.
Introduction: Upper aerodigestive tract cancer (UADTC) is a group of malignancies arising in the mouth, throat and larynx. Each year there are approximately half a million UADTC cases and 300,000 deaths due to these cancers worldwide. The existence of a gradient between socioeconomic position (SEP) and UADTC is well established, and greater exposure to psychosocial stress in low-SEP environments has been suggested as a major explanatory mechanism underlying this association. The concept of allostatic load (AL) has been proposed as a physiological explanatory mechanism linking stress to health. However, to date only a few studies have investigated these associations.
Aim: To estimate the extent to which low SEP is associated with oral cancer risk. In addition, we will estimate how much of this association is explained by psychosocial stressors.
Methodology: We use data from the India HeNCe Life study, a hospital based case-control study investigating the aetiology of UADTC. Cases (n=350) newly diagnosed with primary squamous cell carcinomas in the oral cavity and non-cancer outpatient controls (n=371) frequency-matched to cases according to age and sex were selected at the Governmental Dental and Medical Colleges in Calicut, Kerala, India. In-person interviews using a structured questionnaire and life grid collected information on several exposures (e.g., behavioural, SEP, family environment factors). We also collected biological specimens for human papillomavirus detection. Data analysis will involve: (i) descriptive statistics; (ii) t-test and χ2 test to explore bivariate associations between key variables; (iii) unconditional logistic regression to investigate associations between indicators of SEP and indicators of cumulative psychosocial stress and oral cancer adjusting for potential confounders (e.g., socio-demographic, age); and (iv) path analysis to identify the potential pathways linking SEP and oral cancer.
Conclusions: Findings from this project will provide new information on the role of stress in the relationship between SEP and oral cancer.
Rates of growth in childhood and dental caries experience in adolescence.
Amarjot Kaur1, Sreenath Madathil1, Belinda Nicolau1
1McGill University, Montreal, Quebec, Canada
INTRODUCTION: Several studies have shown an association between anthropometric measures including birth weight and body mass index and children dental caries. However, there is limited evidence on the role of growth during childhood and dental caries in adolescents.
OBJECTIVE: To estimate the extent to which rate of growth from birth to 2 years is associated with dental caries increment over a 7-year follow up period among children participating in the Quebec Adipose Lifestyle Investigation in Youth (Quality) Cohort Study.
METHODOLOGY: Data from the first 3 visits of the QUALITY cohort, an ongoing prospective study investigating the natural history of obesity among the youth of Quebec. A total of 630 Caucasian children ages 8-10 years at baseline and their family were recruited from schools in Montreal and Quebec City between 2005 and 2008. Data collection involved dental clinical examination along with administration of questionnaires to parents and children. Rate of growth, our main exposure, was measured as change in growth (slope of body mass index and intercept). Dental caries was measured using the Decayed, Missing, and Filled Surfaces (DMFS) index. Descriptive statistics and regression analysis will be used.
RESULTS: The analysis is ongoing and we will present preliminary results in this conference.
SIGNIFICANCE: This study may clarify whether rate of growth may be used as an indicator of future risk of dental caries and may support early prevention of dental caries.
Platelet concentrates (PCs) are biological autologous substances derived from the patient’s whole blood and consist mainly of supraphysiologic concentration of platelets and plasma proteins. There are three main families of PCs: platelet-rich plasma (PRP), platelet-rich fibrin (PRF) and plasma rich in growth factors (PRGF). PC contains valuable concentrations of growth factors (GFs). These molecules have anti-inflammatory and healing enhancing properties and play an important role in the healing of various tissue. Moreover, PC contains small molecules such as serotonin, Adenosine triphosphate (ATP), Adenosine diphosphate (ADP), calcium, glucose, and polyphosphate. PCs seem to have a beneficial role in bone and soft tissue regeneration in different clinical procedures, such as socket preservation, endodontic surgery, sinus augmentation, periodontal surgery, as well as ridge augmentation. Also, studies have reported that PC involved in reducing pain and inflammatory complications after oral surgeries. However, these findings were not consistent in all studies. Different study designs, types of PC, variable platelet and growth factor concentrations, presence of leucocytes, and various application forms and techniques could explain these contradictory results. Recently, our group has emphasized on the possible role of dense granule continents; mainly serotonin in inhibiting bone regeneration and this could be another explanation for this inconsistency. It is essential to analyze different molecular components of PC used in different protocols, to better understand their regenerative role and their beneficial concentration. This study aims to review the molecular components of PC, oral clinical applications, and their regenerative outcomes.
Background: Fibrillins are large extracellular glycoproteins ubiquitously expressed in elastic and non-elastic tissues throughout the body. Marfan syndrome (MFS) is a heritable disorder caused by mutations in fibrillin-1. Lipodystrophy as well as obesity are relatively common in adults with MFS, and both are known to increase the risk of cardiovascular complications. Moreover, fibrillin-1 expression in obese women correlates with obesity and increased adipocyte size.
Aim: The aim of this study is to understand the role of fibrillin-1 in adipocyte differentiation and fat metabolism using MFS mouse models.
Methods: In vitro, mouse bone marrow-derived mesenchymal cells (MSCs) were cultured for 10 days with an adipogenic cocktail (insulin, dexamethasone, isobutylmethylxanthine, and indomethacin) with or without recombinantly produced fibrillin-1 N-terminal (rFBN1-N) and C-terminal (rFBN1-C) halves. Adipocytes were identified with the lipophilic dye Oil Red O, and adipocyte count was evaluated. Surface plasmon resonance spectroscopy (SPR) was used to study the interaction of fibrillin-1 with insulin. For in vivo experiments, we analyzed two MFS mouse models, Fbn1mgR/mgR and Fbn1C1039G/+. Fbn1mgR/mgR are hypomorphic mice that produce reduced amounts (25%) of normal fibrillin-1. Fbn1C1039G/+ carry a missense mutation in fibrillin-1 (C1039G). MSCs from Fbn1mgR/mgR, FbnC1039G/+ and wildtype mice were cultured with adipogenic cocktail and adipocyte count was determined with Oil Red staining. We measured the weight of white adipose tissue (WAT), brown adipose tissue (BAT) and serum insulin concentrations at 16 weeks old Fbn1mgR/mgR and 35 weeks old Fbn1C1039G/+ mice and their respective wildtype littermates controls (LC). Serum insulin concentrations were analyzed by ELISA.
Results: We found that rFBN1-C significantly reduced adipocyte differentiation in vitro while rFBN1-N did not influence the adipocyte differentiation. While maximum inhibition of adipogenesis was achieved when rFBN1-C was present for the entire duration of the experiment, a significant decrease in adipocyte numbers was evident when rFBN1-C was present during the first 3 days of differentiation. SPR analysis showed that insulin can directly interact with fibrillin-1. The dissociation constant (Kd) indicated stronger binding of rFBN1-C (Kd=143 nM) to insulin compared to rFBN1-N (Kd=359 nM). An increased number of adipocytes was observed when MSCs from Fbn1mgR/mgR mice were cultured in adipogenic medium compared to LC, while no significant difference was observed between adipogenic differentiation of MSCs from FbnC1039G/+ mice and their LC. Male Fbn1mgR/mgR mice displayed a significantly higher weight of BAT and WAT compared to the LC. Male FbnC1039G/+ mice showed a trend in increased weight of BAT, but not WAT, compared to LC. The whole-body weight was not significantly different between Fbn1mgR/mgR and Fbn1C1039G/+ mice and their LC. Hyperinsulinemia was observed in Fbn1mgR/mgR male mice compared to LC. Adipose tissue indices in female Fbn1mgR/mgR and Fbn1C1039G/+ mice were similar to their LC.
Conclusion: Fibrillin-1 haploinsufficiency or a fibrillin-1 missense mutation in male mice caused an increase in fat mass. Moreover, C-terminal half of fibrillin-1 interfered with adipogenesis likely by sequestering insulin from the culture medium. Thus, our study indicates a novel mechanism of action by which fibrillin-1 reduction or mutation may affect whole body adipose tissue homeostasis.
Introduction: Adolescents living with Chronic Pain (CP) are vulnerable to negative outcomes such as disability and impaired quality of life; they often miss schools, are unable to maintain social contacts, have sleep disturbances, and suffer from anxiety and depression. The continuation of avoidance coping behaviour beyond normal healing time had also been shown to result in negative consequences such as Disuse Syndrome—a state associated with physical deconditioning, sick role behaviour, psychosocial withdrawal, as well as negative and catastrophic beliefs.
Objective: This study uses an interpretative phenomenological approach to explore individual positive thought processes, adaptation efforts, coping mechanisms, as well as resilience resources (beneficial social situations and solid family ties) that adolescents adopt to minimize the impact of pain and its consequences. The ultimate goal is to liaise with fellow physicians, allied researchers, and policy makers to modify, adapt, and improve current adolescent CP services. This way, we can help patients foster skills that will allow them to adapt positively, regain a balanced social life, and live successfully despite their pain.
Results / Findings: Central to all accounts is a sense of interrupted life—phrases with negative connotations such as “couldn’t do” and “had to stop” are repeatedly used to express feelings of loss of control. To regain control over their situation, participants create within themselves a positive internal dialogue: they reconstruct the meaning of normalcy, practice acceptance, make downward social comparisons, and engage in daily positive affirmations. While chronic pain disrupts their career trajectories, the experience of living with pain has instilled in them the pursuit of significance. This pursuit is propelled by the imagery of a full life and seems to be particular to this age group. Ironically, some participants are seen to be grateful for their pain. Living with pain has gifted them with intuitive empathy for the suffering of others, as well as the emotional credibility to help.
Background: According to the literature, rural communities report lower levels of quality of life and satisfaction with oral health care than urban populations. Furthermore, this rural-urban disparity in oral health and satisfaction with oral health care has been reported worldwide. Identifying spatial variation in patient satisfaction is essential to improve quality of care. To our knowledge, there is no study in which rural-urban disparities in patient satisfaction with oral health care in Canada was specifically examined. Therefore, the objective of this study was to estimate the rural-urban differences in satisfaction with oral health care and to identify predictors of satisfaction amongst rural and urban Quebec residents.
Methods: In this study, we used data from a previous survey entitled “Dent ma region” that was conducted in 8 Quebec regions. Data from 1,788 parents/caregivers of children who participated in a Quebec Ministry of Health clinical study were analysed. Satisfaction with oral health care was measured using the WHO-sponsored International Collaborative Study of Oral Health Outcomes (ICS-II). Other data were collected by means of self-administered structured questionnaires that contain items regarding patient characteristics and enabling resources. The residential postal codes were used to determine the participants geographic locations by using Conversion File Plus. An adapted model (Perneger) of patient satisfaction was used as the conceptual framework for data analysis. Statistical analyses were comprised of descriptive statistics, as well as bivariate and logistic regression.
Preliminary results: Preliminary results demonstrated significant differences between urban and rural residents regarding satisfaction with quality and experience of health care. Rural residents were significantly less satisfied with cost of treatment (P< 0.001) and waiting time to see the dentist (P< 0.005) than were urban residents.
Conclusion: Based on these findings in a Quebec population, there are rural-urban differences in patient satisfaction with oral health care. Additional investigations to determine the reasons for these differences should be carried out so that dental public health and policy makers can implement strategies to address these issues.
Introduction: Intervertebral disc (IVD) degeneration is one of the major causes of back pain, a condition that represents a serious socio-economic burden. Upon degeneration, intervertebral disc tissues become inflamed, and this inflammatory microenvironment is associated with a cascade of degenerative events that may eventually cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including DNA damaging agents, oxidative stress, mitochondrial dysfunctions, adverse load and disruption of epigenetic regulation. The accumulation of senescent IVD cells in the tissue suggests crucial roles of these cells in the initiation and development of painful IVD degeneration through the secretion of an array of diverse cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in intervertebral disc thereby accelerating the process of its degeneration. In this study we evaluated the effects of an FDA approved drug to remove IVD senescent cells while preserving cells health, proliferation and overall matrix production of the remaining cells.
Methods: Human IVDs were obtained from organ donors with no history of back pain through collaboration with Transplant Quebec. Pellet or monolayer cultures were prepared from freshly isolated cells in culture media. They were then cultured in the presence or absence of the FDA approved drug. Monolayer cultures were analyzed after 4-days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed on monolayer cells treated and non-treated using Alamar blue assay. Cells were plated in chamber-slides and treated with this drug at 3 concentrations (0.5, 5, 50) mM and at 3 time points (6, 6+12, 6+24) hr. Following treatment, RNA was extracted with TRIzol reagent and gene expression of senescence and inflammatory markers (p16, IL6 and MMP3) and GAPDH, was evaluated by real-time quantitative-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Five fields of view were analyzed with an in-house developed MatLab script. Immunofluorescence, Alamar Blue Assay and caspase-3/7 Kit were used to confirm the senolytic activity of the tested drug. Pooled conditioned media (days 4 to 21) from treated and untreated pellets was applied to protein arrays that detect 105 human cytokines and chemokines. Cytokines arrays significant changes were confirmed with ELISA. SafraninO to stain for proteoglycan synthesis and DMMB to assess the effect of senolytics on sulfated glycosaminoglycan (sGAG) levels were used to determine the percentage of senescent cells removal and if the remaining cells exhibit an improved matrix producing capacity.
Results: The FDA approved drug was evaluated for an effective senolytic dose and duration that at the same time did not affect viability of non-senescent cells. We determined that 5mM was the optimal senolytic dose in NP and AF cells pellets. This senolytic cleared 49.83% of the NP and 66.97% of the AF senescent cells in pellet cultures. Also, we observed 68.97% and 63.15% increase of the number of proliferating AF and NP cells respectively. Apoptotic NP cells number increased by 18.16% in treated group while AF cells show an increase of 65.86%. Also, we observed p16 co-expression with caspase-3 and separate staining with Ki-67 positive cells. In addition, caspase-3 activity increased by 13.33% following treatment while metabolic activity was unaffected in degenerate compared with non-degenerate cells. All together these findings confirm the senolytic activity of the drug. mRNA expression levels and cytokines arrays quantitation showed a greater decrease in secreted cytokines and proteases in treated group. These findings were confirmed by ELISA and validated by an increase in proteoglycan content (Safranin-O) and release into the culture media (sGAG levels) observed in treated groups.
Discussion & Conclusions: Elucidation of the complex and fine relationship between disc degeneration, tissue inflammation and the molecular mechanism of disc cell senescence appears to be critical to improve the current ineffective therapies. This work identifies a novel senolytic for the treatment of IVD degeneration that could provide therapeutic interventions and ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain.
Osteoarthritis (OA) is a major chronic musculoskeletal disease that affects up to 15% of adults across the planet and results in pain, disability and a reduced quality of life. Pain is the major complaint of osteoarthritis patients, and it can be provoked by innocuous stimuli such as the bending of the knee during walking, a condition called mechanical allodynia. During OA, the nerve terminals of nociceptors (pain-sensing neurons) in the affected joints are sensitized to innocuous stimuli, resulting in mechanical allodynia. Work from our group has demonstrated that selective blockade of these mechanosensitive ion channels (MSICs) produced significant analgesia in OA mice, indicating that these channels may be valuable therapeutic targets in the treatment of OA pain. Our group has recently identified a candidate MSIC, called TACAN, which is expressed in nociceptors. TACAN generates a MSIC when transfected in heterologous systems and its knockdown in sensory neurons in vivo selectively reduces the mice ability to sense mechanical pain, while leaving touch and heat sensing intact. The aim of this project is to determine whether TACAN contributes to mechanical allodynia and cartilage degradation in OA mice.
We used the validated chemical mouse model of OA (intra-articular injection of Sodium Mono-IodoAcetate (MIA), 250mg in 5mL) in which the pathology develops within three weeks. Two different approaches were used to reduce TACAN expression: first, Mrgprd::CreERT2 mice were crossed to TACAN-floxed mice and injected as adults with tamoxifen to delete TACAN expression prior to OA induction. The second approach used MIA-injected wildtype C57Bl/6 mice that had developed mechanical allodynia, which were then injected in their OA knee with an AAV2/6 encoding shRNA against TACAN.
Reducing TACAN expression in the inducible conditional knockout mice prior to MIA injection prevented the development of mechanical allodynia. Furthermore, reducing TACAN expression in knee-innervating nociceptors after the development of mechanical allodynia caused a significant reduction in mechanical allodynia.
Removing nociceptors’ capacity to sense painful mechanical stimuli caused a significant reduction in OA pain. Our study indicates that TACAN is an essential ion channel in the development of OA pain, and the development of selective inhibitors of this channel may be valuable tools in the treatment of OA pain.
Background:Pain in intervertebral disc (IVD) degeneration is one of the most common disabilities in young and middle-aged individuals. Although very common, treatment is costly and limited; there are no early treatments available and for patients with advanced stages of the disease invasive surgery is the only option to relieve the pain. A major problem with available treatment options, is that there is limited understanding regarding the early onset and progression of painful IVD degeneration. More specifically, it is unclear how pain and degeneration are initiated and how they can be prevented. Current evidence suggests that changes in the biomechanical properties of degenerating discs are associated with matrix fragmentation, inflammation and pain. Moving away from costly conventional pharmacotherapy which often has significant negative side effects, there is now interest in natural plant products with anti-inflammatory and regenerative properties such as Curcumin (diferuloylmethane) and Vanillin; a metabolite of Curcumin. The overall objectiveof this project is to evaluate the potential of Curcumin and Vanillinto reduce inflammatory mediators and proteases in cells from painful degenerating human IVD.Having both anti-inflammatory and anti-oxidant properties in chondrocytes, we hypothesize that Vanillin will reduce the level of cytokines and proteases present to reduce spinal pain and delay the need for surgical interventions in patients with low back pain. To address this hypothesis, we used painful degenerating IVD cells obtained from patients undergoing surgery for low back painto; 1)quantify the potential of Vanillin to reduce cytokines and proteases.2)examine the expression profile of known target receptors for Vanillin at steady state and following treatment.
Results: Vanillin reduced the levels of cytokines and proteases present at the transcript and protein level in cells from surgical specimens of patients undergoing surgery for low back pain that are treated versus those that are not treated. Also, it is anticipated that the pain degenerating IVD treated cells will have significantly less receptor expression at the transcript level when compared to the to the non-treated cells from pain suffering patients. Our initial results have already shown a significant decrease in pain mediators NGF and IL-8 at the protein level through immunohistochemistry.
Conclusions: Vanillin is known to have anti-inflammatory and anti-oxidant properties in chondrocytes. Therefore, it can be hypothesized that these will have a therapeutic effect on cartilaginous tissues such as those found in IVDs. By measuring the transcript and protein level expression ofcytokines and proteases associated with pain following treatment with vanillin we can begin to identify if these compounds can help reduce pain in IVD degeneration. Moreover, by studying receptors known to be targeted by these compounds, we can begin to determine drug targets. Demonstrating that these compounds can reduce the expression of pain mediators can open novel avenues for early prevention of back pain to avoid/prolong the need for invasive surgery.
Imaging that you need to go to the dental clinic to have a wisdom tooth extraction or filling. Dentists always have to use a long and sharp needle to insert some anesthetic to freeze your feeling. What kind of emotion will you feel if you are receiving an injection?
There are many patients, not only children but also adults, experienced pain and anxiety from conventional dental anesthesia with needles. Thus they may avoid necessary dental treatments.
These problems could be solved by needle-free device.
Our research focuses on understanding the mechanism of needle-free device and developing the optimal technique to apply this device in dental anesthesia. A needle-free device delivers drug solutions by creating a liquid jet that similar to the pressure washer you use to wash your cars, but with much higher pressure and much thinner diameter.
The needle-free device can overcome all the limitations and avoid all the problems related to needles. the most important thing is: the injection is no-pain at all. Image how big impact it could bring to the dental anesthesia field. but why people never seen this needle-free anesthesia in a dental clinic. Because there are still challenges.
Actually, all the anesthesia techniques in dentistry were developed for needles. Dentists do not know how to use the needle-free device correctly.
We solved this problem with 3 steps. Step one, understand the mechanism of needle-free device. We used mimicking materials similar to human tissue to test the needle-free injection. Study the relationship between the effect of injection and setups of the device. Step 2, develop the optimal techniques. On dead human bodied we tried different techniques and validated our technique by dissections to find the reproducible and optimal techniques. The main nerves for dental anesthesia could be successfully blocked with the newly developed needle-free injection techniques. Step 3, we checked our optimal technique by performing needle-free anesthesia on volunteers.
To conclude: This study sets the basis of how to use the needle-free device in the dental clinic in the future. We hope we will bring the pain-free dental clinic dream into reality.
INTRODUCTION: Scaffold microporosity plays an important role in bone tissue engineering as it promotes osteogenesis by improving protein adsorption and cell adhesion1. Current techniques to impart microporosity in scaffolds like thermally induced phase separation generates tubular pores in the size range of 10-50 μm, but have some limitations, like scaffold shrinkage and long solvent sublimation time2. In our study, we propose a simple technique to create microporosity in poly(lactic-co-glycolic acid) (PLGA) – Bioglass (BG) composite scaffolds and we investigate the effect of the micropores on the bioactivity of these scaffolds for bone regeneration.
METHODS: We prepared Bioglass-Poly(lactic-co-glycolic acid) (BG-PLGA) scaffolds with a microporous PLGA phase by solvent casting/particulate leaching, using paraffin microspheres as a porogen and citrisolv as the leaching solvent. Microporosity was absent in scaffolds leached with hexane. We hypothesize that the microporosity in PLGA can enhance the bioactivity of the BG-PLGA composite and promote osteogenesis by enhancing BG dissolution and cell attachment. To test this hypothesis, we did simulated body fluid (SBF) immersion tests and cell assays on both citrisolv leached (CL) and hexane leached (HL) scaffolds.
RESULTS: Both CL and HL scaffolds showed well interconnected spherical pores created by the paraffin microspheres. The main factors determining the polymer microporosity were the solvent used for leaching and the duration of leaching. We found that longer leaching time created a higher microporosity.
We observed larger apatite deposition upon SBF immersion on CL than HL scaffolds at all time points analyzed (from 96 hours to two weeks), showing that microporosity did enhance the bioactivity of the BG-PLGA composites. Both CL and HL scaffolds supported Mesenchymal stem cell adhesion, proliferation, and differentiation.
DISCUSSION & CONCLUSIONS: We are currently doing further tests to understand if the enhanced bioactivity is due to changes in BG reactivity in the BG-PLGA scaffolds and if this could lead to differences in cell activity at early time points.
1 Zhang, Ke, et al. "Effect of microporosity on scaffolds for bone tissue engineering." Regenerative biomaterials 5.2 (2018): 115-124. 2 Rezwan, Kurosh, et al. "Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering." Biomaterials 27.18 (2006): 3413-3431.
The oral soft tissues, including the epithelial and connective tissue elements that envelop the teeth, are also important in maintaining functional dental implants. Tobacco smoking was reported as a risk factor for dental implant failure. To prevent these adverse effects, electronic cigarettes (e-cigarette) was proposed as a safe alternative. Although e-cigarettes were reported safer than tobacco smoke, multiple studies demonstrated potential health concerns.
The Objective of this study was to investigate the effects of e-cigarettes on the primary human gingival fibroblasts interaction with dental implant material.
Methods: Gingival fibroblasts were cultured onto Ti6Al-4V titanium implant disks being layered or not with titanium nitride and were then exposed or not to combustible cigarette smoke (CCS), to nicotine-rich (NR) or to nicotine-free (NF) e-vapor for 15 min. The exposure was two times a day for one, two, or three days. After each period, various analyses were performed.
Results: Gingival fibroblast adhesion/attachment to the dental implant material was dysregulated by CCS, NR and slightly by NF e-vapors as compared to the non-exposed cultures. This was supported by a reduced expression of F-actin, a key focal adhesion protein. We also demonstrated that gingival fibroblast growth on both types of the titanium implant disks was significantly (p < 0.001) reduced following exposures to CCS, NR, and NF e-vapors. The growth inhibition was greater at 2 and 3 days as compared with one-day exposure. Exposures to NR or NF showed a decrease of vinculin but an increase of caveolin-1 suggesting that e-cigarettes inhibit fibroblast interaction with the implant through vinculin but not caveolin-1. The measurement of LDH activity showed that both CCS and NR promoted cell damage as ascertained by increased LDH activity.
Conclusion: E-cigarette exposure had lower effects on fibroblast interaction with dental implant materials. However, the effect of e-cigarette still significant reducing cell adhesion to the implant, and growth. The e-cigarette effect may go through adhesion proteins such as F-actin and vinculin. This study was financed by funds from the AAID Foundation and the "Fonds Émile-Beaulieu, Université Laval."
BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic condition that often causes debilitating bone fragility and other physical limitations. Informal caregivers (such as parents) play a critical role in helping children with OI live well at home. Yet, despite their important role, there is a paucity of knowledge concerning these caregivers’ experiences.
PURPOSE: The purpose of this review was to comprehensively review, appraise, and synthesize the research literature relating to the experiences of informal caregivers of children living with OI, to identify recurring themes and implications for research, policy, and practice.
METHODS: Using an integrative review methodology, studies were accessed through database searching, assessed for eligibility, and appraised. Data from the included papers were thematically analyzed.
RESULTS: Eighteen studies of various methodologies and quality met the inclusion criteria. The experiential themes identified corresponded with temporal caregiving phases. During the time of diagnosis, caregivers experienced suspicions of child abuse, challenges accessing resources, and mostly negative emotions. While providing care in the years following the diagnosis, caregivers’ experiences related to: the energy expenses of caregiving, the family and social effects of OI care, the emotional “ups and downs,” and coping strategies. These experiences depended on several factors, including social support and access to specialized OI care. Caregivers shared their concerns for their children’s futures. Caregivers and authors made recommendations for practice, policy, and research for OI care: they advocate for improved communication by clinicians at the time of diagnosis, increased education regarding OI, and more community supports for families.
CONCLUSIONS: This review provides an enriched understanding of the experiences faced by OI caregivers over time. However, it also reveals that there is limited research of varying quality on this topic. More research is needed on the OI informal caregiving experience to learn how to support these caregivers better.
Activating transcription factor 4 (ATF4), a member of the basic domain/leucine zipper family of transcription factors that can dimerize with other leucine zipper proteins, plays a pivotal role in regulating osteoblast differentiation and function. Our laboratory has identified FIAT (Factor Inhibiting ATF4-mediated Transcription) as a leucine zipper binding partner that inhibits the transcriptional activity of ATF4. Transgenic mice overexpressing Fiat exhibit a low bone mass phenotype while a global Fiat knockout showed reciprocal changes. Through these and other studies FIAT emerged as a valid drug target for bone regeneration. The goal of this project was to use a high throughput screen to identify compounds that disrupt the ATF4/FIAT interaction to find small molecules that could increase bone mass. We have generated a FIATzipper-GAL4-DNA Binding Domain fusion (DBD-FIAT ‘bait’) and an ATF4zipper-VP16 Activation Domain fusion (AD-ATF4 ‘target’) to establish a mammalian two-hybrid assay. In this assay, the DBD-FIAT fusion binds the promoter of a secreted luciferase reporter construct. Interaction of the bait with the AD-ATF4 target through the leucine zipper interface reconstitutes a strong transcriptional activator and results in high levels of luciferase expression. Compounds that disrupted luciferase expression in the two-hybrid assay without influencing transcription from the fused GAL4-VP16 control were considered hits. After screening 135,000 compounds and performing dose-response testing, 8 compounds were selected for additional characterization. Preliminary data has defined the tolerance/survival of MC3T3-E1 osteoblastic cells upon treatment with the drugs, allowing for further investigation of the effects of the compounds on differentiation and mineralization. Two (2) compounds have a positive effect on differentiation and mineralization of MC3T3-E1 cells as measured by alkaline phosphatase and alizarin red staining, respectively. These two compounds also stimulate expression of osteogenic gene markers, such as osteocalcin and COL1A1, as observed using RT-qPCR. These preliminary data support the hypothesis that compound(s) blocking the interaction of FIAT with ATF4 would increase osteoblast activity and provide valuable information for further in-depth in vitro experiments and in vivo pilot studies.
When bone implants are loaded, they are inevitably subjected to displacement relative to bone. Such micromotion generates stress/strain states at the interface that can cause beneficial or detrimental sequels. The objective of this study is to better understand the mechanobiology of bone healing at the tissue-implant interface during repeated loading. Screw shaped Ti implants were placed in a hole slightly bigger than the implant diameter in two anatomical sites in rats, (i) the edentulous ridge in maxillae and (ii) the tibia. In both cases, specially-designed systems were developed to hold the implants, allow initial stabilization, protection from external forces, and controlled axial loading. Three loading regimens were applied, (a) no loading, (b) one daily session of 60 cycles with an axial force of 1.5 N/cycle for 7 days, and (c) two such daily sessions also for 7 days. The implants with surrounding interfacial tissue were harvested and processed for histological and histomorphometric analyses. Two-way ANOVA were performed to determine the differences in histomorphometric analysis between the groups. At both sites, histomorphometric analyses revealed that implants subjected to repeated loading sessions exhibited a significant decrease in bone-implant contact and increase in bone-implant distance, as compared to unloaded implants and those subjected to only one loading session (p< 0.05). The results indicate that increasing the daily cyclic loading of implants induces deleterious changes in the bone healing response, most likely due to the accumulation of tissue damage at the bone-implant interface. They also suggest some commonality in mechanobiological factors influencing bone healing around implants in tibial and maxillary sites.
All animal procedures and experimental protocols were approved by the Comité de déontologie de l’expérimentation sur les animaux of Université de Montréal.
Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. 3D printing with inexpensive desktop printers show promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable and cost-effective FDA approved thermoplastics. This study will demonstrate the appropriate pore size to assess the ability of low-cost 3D printed polylactic acid (PLA) scaffolds in conducting primary human osteoblast adhesion, growth, and osteogenic matrix deposition. It will also test the hypothesis that human mesenchymal stem cells (MSCs) will adhere, proliferate, and adopt osteogenic phenotype when seeded within these scaffolds.
Micro-porous 3D-printed PLA scaffolds with different pore sizes (500 µm, 750 µm, and 1000 µm), were designed and manufactured using Flashforge Creator Pro desktop printer. Scaffold properties, mechanical stiffness, and material characteristics were assessed using µCT, unconfined compression and contact angel testing respectively. Scaffolds were seeded with 5x105 primary osteoblasts for 21 Days. Cells activity and proliferation were assessed using DNA quantification and scanning electron microscopy. calcified matrix production was visualized and quantified by Alizarin red staining and western blotting. Furthermore, MSCs were seeded within the appropriate pore size scaffold and osteogenic differentiation was evidenced by gene expression, calcified matrix formation and bone-like matrix deposition.
PLA constructs demonstrated high accuracy and reproducibility of fabrication evidenced by µCT. Additionally, scaffolds showed compressive properties comparable to trabecular bone when strained between 5% and 10%. Material surface testing determined hydrophilic features suggesting good interaction with cells. Cells were growing and covering the surface and between the additive layers of the scaffolds indicating cell adaptation to the 3D environment. (750 µm) pore size showed significant statistical superiority (p < 0.0001) in cell population expansion, doubling and calcified matrix deposition and over the other sizes. MSCs demonstrated osteogenic phenotypic characteristics evidenced by the significant expression of osteogenic markers (BSP, ALP, RUNX-2, COL-1, and ON), matrix protein (Osteopontin), Calcium deposition and mineralization.
The current study focused on emphasizing the value of inexpensive desktop 3D printers and off-the-shelf materials in keeping pace with the advancement of tissue engineering. Three different pore-sized 3D printed PLA scaffolds have been successfully generated using an inexpensive desktop 3D printer. Out of three pore-sizes, (750 mm) scaffold exhibited highest biocompatibility and bioactivity of primary human osteoblasts. For clinical relevance, human MSCs seeded into (750 mm) size scaffolds showed strong osteogenic differentiation capacity. Therefore, we believe this simple, low-cost approach can be extended to future in vivo studies and potential clinical applications incorporating custom-made, on
Background: The constantly rising numbers of sports-related injuries, cancer resections, congenital diseases, trauma, and spine surgery generate a high demand for bone grafting. The current gold standard for repair is the autologous bone graft. However, there is a limited quantity and associated donor site morbidity. Therefore, there is a need for alternative bone substitutes, which justifies the increased number of tissue engineering studies including the use of strong and inexpensive three-dimensional (3D)-printed scaffolds for bone regeneration. Several off-the-shelf materials are available for low-cost fused deposition 3D printing such as polylactic acid (PLA) and polycaprolactone (PCL). These polymers can easily be made in composites to include other factors such as minerals. In this study, we assessed three materials - PLA, Lactoprene 1 and Lactoprene 2 with and without mineral additives for their osteoinductive properties.
Methods: Porous scaffolds (10x10x4mm, pore size 750μm) of PLA and two novel materials Lactoprene 1 and 2 supplemented with and without mineral additives were 3D-printed using a FlashForge Creator Pro desktop 3D printer. Their stiffness and hydrophilicity were assessed by unconfined compression and contact angle tests. Five hundred thousand human bone-marrow-derived mesenchymal stem cells (hMSCs) were seeded on the scaffolds and cultured for 21 days in standard and osteogenic cell culture medium respectively. Cell proliferation was assessed by Hoechst test for total DNA concentrations. Extracellular matrix (ECM) generation was evaluated by scanning electron microscope (SEM) and the calcified matrix was visualized and quantified by Alizarin red staining. Furthermore, we assessed osteogenic markers by Western blot for the evidence of bone matrix proteins.
Results: 3D-printed scaffolds of all materials were successfully fabricated using a low-cost 3D printer. There was no significant difference in stiffness between PLA and Lactoprene 1/ Lactoprene 1+mineral. However, Lactoprene 2/ Lactoprene 2+mineral are ~69% weaker (P<0.0001) compared to PLA and Lactoprene 1 (+mineral). All scaffolds showed hydrophilic material properties. Lactoprene 1/ Lactoprene 1+mineral are ~20% more hydrophilic (P<0.05) than Lactoprene 2/ Lactoprene 2+mineral, and may be more biocompatible for tissue fusion in future implantation test. However, the number of cells adhering were ~71% and ~77% lower in Lactoprene 1 compared to Lactoprene 2 (P<0.01)/ Lactoprene 2+mineral (P<0.001), and ~53% and ~63% lower in Lactoprene 1+mineral compared to Lactoprene 2 (P<0.05)/ Lactoprene 2+mineral (P<0.001). All materials supported cell proliferation and maintained cell numbers. Lactoprene 1+mineral maintained ~12% more cells (P<0.05) than Lactoprene 2+mineral in osteogenic condition. Though having ~63% less number (P<0.001) of cells at the beginning, Lactoprene 1+mineral promoted ~60% and ~57% more calcified ECM formation than Lactoprene 2+mineral in both standard (P<0.0001) and osteogenic (P<0.0001) conditions.
Conclusion: The current study focused on evaluating two innovative materials for bone tissue engineering and compared them to standard PLA. Our results highlight an improved osteogenesis and mineral deposition on Lactoprene 1 and 2. Our data also demonstrated superior properties of Lactoprene 1, in its mechanical strength, hydrophilicity, and its significant promotion of cell proliferation and calcified extracellular matrix deposition. Further tests will evaluate bone matrix formation and biocompatibility in vivo and carry forward our goal of developing novel materials for bone regeneration.
Introduction: Multiple myeloma (MM) is an incurable plasma cell derived neoplasia. Patients develop devastating osteolytic lesions that lead to non-healing fractures and pain. We aimed to assess the adaptive cortical bone (re)modeling response to mechanical loading in a mouse model of MM. We hypothesized in vivo tibial loading would have an anabolic and anti-catabolic effect in tumor-injected tibiae, rescuing bone loss.
Methods: Ten-week-old female Balb/c mice underwent in vivo strain-matched dynamic compressive loading of the left tibia (5d/wk for 20 days, M-F; right tibia internal nonloaded control). The mice were injected 14 days prior to loading with either MOPC315.BM cells (mimic human MM) or PBS (left limb injected, right limb noninjected) or were not injected (n=7, left limb noninjected, right limb noninjected). Tumor- and PBS-injected mice were randomized into loaded (left limb loaded, right limb nonloaded) or nonloaded (left limb nonloaded, right limb nonloaded) groups (n=6-9/group). In vivo microCT was performed at day 13, 18, 23, 28 and 33 post injection. MicroCT images from day 13 and 33 were geometrically registered to monitor bone formation and resorption at the endosteal and periosteal tibial cortical metaphyseal surface: normalized newly mineralized and eroded bone volume (MV/BVday0-20, EV/BVday0-20), bone surface areas (MS/BSday0-20, ES/BSday0-20), and mineralized thickness and eroded depth (MTh day0-20, ED day0-20). An ANOVA assessed effects of treatment (tumor injected/PBS injected /noninjected) and loading (loaded/nonloaded) (p≤0.05). Fluorochrome-based histomorphometry was also performed. Tumor progression was monitored using bioluminescence imaging and enzyme-linked immunosorbent assay to detect MOPC315.BM specific immunoglobulin A (IgA) levels.
Results: Loading increased periosteal MV/BV and MS/BS and decreased periosteal EV/BV and ES/BS compared to nonloaded limbs in all groups. Loading-induced increases in endosteal MV/BV was only observed in the tumor group. Endosteal MS/BS was diminished, while ES/BS was increased in tumor injected mice compared to PBS injected and noninjected groups. Loading-induced effects on the eroded surface and volume suggests fewer resorption sites at the periosteal surface. Injection, either tumor or PBS, altered periosteal and endosteal bone formation and resorption, suggesting a regional acceleratory phenomenon. Load induced increases in cortical bone mass were accompanied by decreased tumor burden as evidenced by MOPC315.BM specific IgA levels. Our data in young mice with MMBD indicate that mechanical loading not only rescues osteolytic bone loss but also has anti-myeloma effects. These data suggesting a combination of load-bearing exercise with bone resorption inhibitors in future clinical therapeutic strategies.
Adolescent Idiopathic Scoliosis (AIS) is a progressive 3-dimmensional bending of the spine which affects the intervertebral disc (IVD) and the facet joints. Our facet joint characterization study has revealed the presence of degeneration in scoliotic cartilage through decreased proteoglycan content and elevated secretion of inflammatory cytokines and matrix degrading proteases. Recently, a novel degenerative pathway in IVDs and cartilage involving abherrant activation of TLRs was found. When activated by endogenous danger signals (alarmins), TLRs in IVD and chondrocytes initiate the production of inflammatory cytokines, proteases and neurotrophins leading to tissue degradation and pain. In this study, we investigate the potential role of TLRs activation in facet joints from AIS patients as a cause of early degeneration. TLR presence was assessed by rt-qPCR and immunocytochemistry on cells isolated from AIS and cadaveric non-scoliotic cartilage samples with consent. TLR2 receptors were activated in monolayer AIS chondrocytes using a TLR2 agonist (Pam2CSK4, Invivogen). Cartilage explants were isolated from the subchondral bone and cultured in the presence and absence of Pam2CSK4 in chondrocyte media, which was analyzed by ELISA for degenerative marker secretion. After the culture period, the cartilage was cryosectionned and stained with SafraninO – Fast green dyes to reveal proteoglycan content. Gene expression analysis revealed a significant upregulation of TLR2 in AIS chondrocytes compared to controls. Interestingly, TLR mRNA in AIS chondrocytes correlated positively and significantly with degenerative factor (MMP3, MMP13, IL-1b, IL-6 and IL-8) mRNA levels. These correlations were mostly absent in non-scoliotic healthy chondrocytes. In the presence of the TLR2/6 agonist, gene and protein expression analysis showed significantly (p<0.05) elevated levels of TLR2 receptor, proteases, inflammatory cytokines and pain-related factors such as MMP3, MMP13, IL-6 and NGF. Furthermore, histological staining of cultured cartilage explants revealed a significant decrease in proteoglycan content after treatment with Pam2CSK4 in AIS cartilage, but not in the non-scoliotic controls. In conclusion, our data show that TLR2 activation lead to a degenerative cycle with the upregulation of the receptor itself and secretion of proteases, inflammatory cytokines and pain-related factors that ultimately trigger proteoglycan loss in the affected cartilage. Our study reveals TLR2 as a potential therapeutic target to treat degenerating articular cartilage and subsequent pain.
Nanoparticles prepared from polysaccharides, such as chitosan, are extensively investigated due to their biocompatility, biodegradability and their potential as carriers for therapeutic compounds. They represent a promising therapeutic strategy to deliver peptides for the treatment of diseases afflicting poorly vascularized tissues, such as cartilage in osteoarthritis (OA). Our group suggests a new therapeutic strategy, capable of protecting cartilage against inflammation and degradation, by using a combination of ETA and B1BKR receptors antagonists (respectively BQ123 and R-954). The protective effect was observed on nociception in a rat model of OA (Kaufman, Arthritis Research and therapy, 2011). The aim of this work was to assess the possible toxicity of polysaccharidic nanoparticles conjugated to therapeutic peptides BQ123 and R-954, both in vitro and in vivo.
In vitro, human cells (chondrocytes, osteoblasts and synoviocytes) were investigated. Cells were derived from patients undergoing Total Knee Arthroplasty for Knee Osteoarthritis. Cells were expanded in vitro and exposed to nanoparticles conjugated to therapeutic peptides BQ123 and R-954 in vitro. We measured the cell viability of the three cell types when exposed to different doses of polysaccharide nanoparticles conjugated to BQ123 or R-954 (0.03-200 µg/mL) using MTS and LDH assays. Potential embryotoxicity of nanoparticles was also assessed in an in vivo model of WT zebrafish (Danio Rerio). A concentration range of 0.3 to 70 µg/mL was added directly in the fish media at 3 hours post-fecundation (hpf). Mortality, hatching and birth-defect rates were measured every 24 hours over a period of 5 days. All studies were independently replicated 3 times (n=3).
All concentrations (0.03-200 µg/mL) of polysaccharide nanoparticles showed no effect on cell viability and mortality in vitro (two-way ANOVA, P>0.05, n=3/cell type). In vivo studies revealed that 70 µg/mL was the only concentration found to influence zebra fish larvae survival (LogRank test, P<0.05). All other tested doses (0.3 to 18 µg/mL) had no significant effect on mortality incidence (LogRank test, P<0.05). Furthermore, no differences in survival curves were seen between the two nanoparticles formulation (BQ123 or R-954 conjugated), indicating similar toxicity profiles. Birth-defects were observed at low incidence (< 2%) in both control and all treatment groups, suggesting that polysaccharidic nanoparticles conjugated to BQ123 or R-954 did not influence the incidence of the early birth defects and zebrafish development. Premature hatching was detected at concentration of 4.6 µg/mL and above (χ2 tests, P<0.05, n=3). Premature hatching occurring in zebrafish suggested a stress response induced by the nanoparticles (Barton, integrative and comparative biology, 2002). The nature of this response is yet to be determined by additional studies.
Conclusion and significance:
We conclude that the concentration range of polysaccharide nanoparticles conjugated to BQ123 or R-954 (NPs) is not toxic for cartilage derived human primary cells in vitro. The data obtained herein supports the evidence that NPs are promising biocompatible carriers for therapeutic agents.
Supported by FQRNT - Projet de recherche en équipe
Introduction. The implementation of universal dental coverage has been a topic of discussion since Medicare was introduced in Canada in 1947. At that time, most dentists did not support the inclusion of dental care into Medicare. Even today, the debate has been seen re-opening, but we do not know future dentists' perspectives on this issue.
Objectives. The aim of our study is to understand the perspectives of future dentists towards universal dental coverage. Specifically, we want to understand what benefits and inconveniences they perceive, and how much they would be ready to advocate in favor or against if such a measure is to be implemented in Quebec in the near future.
Methods. We will conduct an exploratory research by conducting individual face-to-face interviews with “purposefully selected” participants (information-rich on our themes of interest) who will be undergraduate students recruited from the McGill Faculty of Dentistry. We are planning to recruit between 12 and 15 participants and conduct interviews using open-ended semi-structured questionnaires. The discussions will be audio-recorded and last between 30 minutes and 1 hour. Thematic analysis will follow the 6 phases of Braun and Clark’s method.
Expected Results/Outcome. It is an exploratory research, so we don't know what the future of the dental profession thinks, and what kind of arguments they have in mind. So, based on informal discussions, readings and the media, we expect that along-with their opinions regarding universal dental coverage, they may mention the costs of dental treatment and they may perceive necessary rights to autonomy as a profession.
INTRODUCTION: The daily injection of parathyroid hormone (PTH) increases bone mass and protects against osteoporotic fractures, however a complete picture of its mechanism of action is still missing. We have shown that PTH induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (aNAC), leading to nuclear translocation of aNAC and activation of target genes. PURPOSE: The aim of the study is to identify and characterize novel target genes regulated by PTH-activated aNAC in osteoblasts. METHODS: We performed Chromatin Immunoprecipitation with deep sequencing (ChIP-Seq) against aNAC with parallel RNA-sequencing in PTH-treated osteoblast cells. The biological role of candidate genes was studied in vitro in osteoblastic cells and bone marrow stromal cells (BMSCs) cultures using shRNA-mediated knockdown techniques and in vivo by deleting candidate genes using a CMV-Cre driver. RESULTS: This strategy has identified the Ubiquitin-Specific Peptidase 53 (Usp53) as a candidate gene target of PTH-activated aNAC in osteoblasts. Little is known about the biological function of Usp53 in bone biology. PTH treatment of osteoblast cells increased the mRNA expression of Usp53 by 3-fold as compared to vehicle-treated cells but PTH-driven transcriptional induction of Usp53 was completely blunted following aNAC knockdown. The global deletion of Usp53 in vivo led to poorer biomechanical properties in the mutant bones, assessed by 3-point bending and identation mechanical testing. Usp53(-/-) femurs and tibias showed an increased tendency to yield and break as compared to control samples. Osteogenic cultures of Usp53(-/-) BMSCs revealed elevated levels of RANKL. Besides, Usp53(-/-) tibial RNA showed an increase in osteoclast-related markers as compared to controls. Serum bone turnover markers including CTX-1 and PINP were also elevated. Usp53(-/-) BMSCs cultures showed decreased adipogenesis, mainly due to decreased levels of Pparγ. Proteomics has revealed a novel interaction of USP53 with TAK1-TAB1/2 complex proteins. Our data showed that USP53 is critical for the TAK1-TAB1/2 complex assembly. The absence of USP53 disrupted JNK-MAP kinase signaling downstream of the TAK1-TAB complex. CONCLUSION: This is the first report characterizing the physiological role of Usp53 in bone. Our data suggest that Usp53 affects bone homeostasis and mass in vivo, mainly by regulating bone turnover and osteoclastogenesis. The novel interaction of USP53 with the TAK1-TAB1/2 complex; a TGF-β/BMP- regulated complex orchestrating the crosstalk between osteogenesis and adipogenesis, suggests that Usp53 is a regulator of lineage fate decisions.
We are what we are: Religious discrimination and Oral Health of the Muslim community in Quebec.
Tandale M1, Bedos C1
1 Division of Oral Health and Society, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
The issue of ‘how to accommodate religious minorities in Quebec’ has been boiling over more than 10 years. Since, the Bouchard Taylor commission, the Muslim community has been in the centre of these debates, with controversies on religious signs and symbols, such as the Niqab. In this debate, violence has also occurred, especially with a Quebec city mosque shooting.
All these events have made the situation for Muslim people very uncomfortable in Quebec. It needs to be mentioned that Islam is the second largest religious group in Canada. Muslims represent around 3 % of the population, and it is going to increase in the coming years.
We know that religious or racial discriminations may have an impact on mental and physical health through stress, anxiety and chronic diseases. But we do not know if these discriminations, experienced in daily life, may have an impact on oral health and oral-health-related behaviours.
How does religious discrimination may affect the oral health of Muslim people?
To identify potential pathways between religious discrimination and people's oral health.
Methodology: This qualitative exploratory study focuses on the Muslim community living in Montreal, Quebec. We adopted a purposeful sampling technique with a maximum variation in terms of age, education level, marital, employment status and ethnicity. The inclusion criteria were having experienced religious discrimination as a Muslim in daily life, practising the Muslim religion (women wearing hijab and man head caps), being 18 years old or more, and being able to communicate in English. We recruited participants from various Muslim Student Association (MSA) at McGill and mosques. We conducted face-to-face, semi-structured interviews with them. To date, we have interviewed ten women and one man, and Interviews are audio recorded and transcribed verbatim. We analyse these transcripts using a thematic content analysis strategy.
Participants reported discriminatory experiences in daily life like judgemental stares and bad comments in public places. These events often instigated frustrations, and the participants started to be wary about their safety in public areas. In the health care system, some participants reported experiencing longer waiting time, less appointment time with various health care professionals and, in some cases, disrespectfulness toward Islamic religious beliefs (i.e. enacted stigma). The consequences of these experiences generated a lack of trust in health care professionals in general, a preference to consult Muslim doctors, and in some cases anticipation of discrimination in the dental healthcare system (i.e. felt stigma). Three participants also shared their discriminatory experiences in the dental healthcare system. They thought that they were treated more disrespectfully because of their Islamic identity (Muslim name, hijab): one of them explained receiving a limited explanation from a dentist about her illness; another felt that she was not welcomed appropriately and spent more time in the waiting area than non-Muslim patients. The consequences of these discriminatory experiences in the dental health care system generated participants' lack of trust towards dental healthcare professionals, avoidance of the dental clinic that discriminated them and, ultimately, unresolved dental problems.
The knowledge generated by this research will not only enrich the current literature in dentistry, but we also hope that it will contribute to improving the oral health-related quality of life of discriminated communities. We expect to use our findings in dental education to raise awareness and develop critical consciousness among healthcare professional about minority groups.