P2X7 Nucleotide Receptor Signaling in Osteoblasts

Nucleotides are released from cells of the osteoblast lineage in response to mechanical stimulation, and signal through two families of P2 nucleotide receptors – G protein-coupled P2Y receptors and ligand-gated P2X cation channels. Nearly every cell-type expresses multiple P2 receptor subtypes. However, the significance of these networks of receptors in any system is unclear. In this thesis, we demonstrate that the endogenous network of P2 receptors expressed by osteoblasts permits graded increases in Ca2+ signaling over a million-fold range of ATP concentrations. P2Y receptors mediate transient activation of the Ca2+/NFATc1 pathway, whereas stimulation of P2X7 causes sustained Ca2+/NFATc1 signaling. The P2X7 receptor has been shown previously to promote osteoblast differentiation, though the signaling mechanisms remain poorly defined. In addition to activation of NFATc1, we demonstrate here that P2X7 activation elicits a dramatic increase in proton efflux, which is triggered by elevation of cytosolic free Ca2+ concentration and sustained through PI3K-activated glucose metabolism. Finally, we show that stimulation of P2X7 receptors potentiates β-catenin nuclear localization and transcriptional activation elicited by canonical Wnt signaling. In summary, the findings of this thesis demonstrate for the first time that P2Y-P2X receptor networks allow cells to sense a wide range of ATP concentrations, and transduce this input into distinct cellular responses. Additionally, we have found that P2X7 couples through multiple anabolic pathways in osteoblasts, including Ca2+/NFAT, PI3K/AKT and Wnt/β-catenin signaling. One or more of these pathways in turn may mediate the effects of P2X7 on osteoblast differentiation and mechanotransduction in bone

Optimizing RNA Isolation and Histology Protocols for Characterization of Preclinical Models of Tendon Disease

Tendons join muscle to bone and are essential for posture and movement. Tenocytes are the resident tendon cells. Isolating RNA from small animal models such as mice can be difficult, especially from tendon, as much of the time, they are extremely small samples. Downstream analysis such as qPCR and especially RNA sequencing require greater quantity and quality of RNA than is often achievable. RNA degradation is extremely rapid following tendon isolation, with RNA quality decreasing as time increases between the collection of tendons and RNA isolation. Even tendons whose RNA was isolated immediately following tendon isolation had imperfect quality. The goals for this project were to first, find and optimize a reproducible method for isolating RNA from mouse tendons for downstream qPCR and next generation sequencing that is replicable for people with varying degrees of experience, and second, optimize a method of sectioning mouse joints for the examination of tendon tissue structure

Diet-induced obesity leads to behavioral indicators of pain preceding structural joint damage in wild-type mice

Introduction: Obesity is one of the largest modifiable risk factors for the development of musculoskeletal diseases, including intervertebral disc (IVD) degeneration and back pain. Despite the clinical association, no studies have directly assessed whether diet-induced obesity accelerates IVD degeneration, back pain, or investigated the biological mediators underlying this association. In this study, we examine the effects of chronic consumption of a high-fat or high-fat/high-sugar (western) diet on the IVD, knee joint, and pain-associated outcomes. Methods: Male C57BL/6N mice were randomized into one of three diet groups (chow control; high-fat; high-fat, high-sugar western diet) at 10 weeks of age and remained on the diet for 12, 24, or 40 weeks. At endpoint, animals were assessed for behavioral indicators of pain, joint tissues were collected for histological and molecular analysis, serum was collected to assess for markers of systemic inflammation, and IBA-1, GFAP, and CGRP were measured in spinal cords by immunohistochemistry. Results: Animals fed obesogenic (high-fat or western) diets showed behavioral indicators of pain beginning at 12 weeks and persisting up to 40 weeks of diet consumption. Histological indicators of moderate joint degeneration were detected in the IVD and knee following 40 weeks on the experimental diets. Mice fed the obesogenic diets showed synovitis, increased intradiscal expression of inflammatory cytokines and circulating levels of MCP-1 compared to control. Linear regression modeling demonstrated that age and diet were both significant predictors of most pain-related behavioral outcomes, but not histopathological joint degeneration. Synovitis was associated with alterations in spontaneous activity. Conclusion: Diet-induced obesity accelerates IVD degeneration and knee OA in mice; however, pain-related behaviors precede and are independent of histopathological structural damage. These findings contribute to understanding the source of obesity-related back pain and the contribution of structural IVD degeneration

Exploring the role of competing demands and routines during the implementation of a self-management tool for type 2 diabetes: A theory-based qualitative interview study

Background The implementation of new medical interventions into routine care involves healthcare professionals adopting new clinical behaviours and changing existing ones. Whilst theory-based approaches can help understand healthcare professionals’ behaviours, such approaches often focus on a single behaviour and conceptualise its performance in terms of an underlying reflective process. Such approaches fail to consider the impact of non-reflective influences (e.g. habit and automaticity) and how the myriad of competing demands for their time may influence uptake. The current study aimed to apply a dual process theoretical approach to account for reflective and automatic determinants of healthcare professional behaviour while integrating a multiple behaviour approach to understanding the implementation and use of a new self-management tool by healthcare professionals in the context of diabetes care. Methods Following Diabetes UK’s national release of the ‘Information Prescription’ (DUK IP; a self-management tool targeting the management of cholesterol, blood pressure and HbA1c) in January 2015, we conducted semi-structured interviews with 13 healthcare professionals (general practitioners and nurses) who had started to use the DUK IP during consultations to provide self-management advice to people with type 2 diabetes. A theory-based topic guide included pre-specified constructs from a previously developed logic model. We elicited healthcare professionals’ views on reflective processes (outcome expectations, self-efficacy, intention, action and coping planning), automatic processes (habit), and multiple behaviour processes (goal priority, goal conflict and goal facilitation). All interviews were audio recorded and transcribed verbatim and all transcripts were independently double coded and analysed using content analysis. Results The majority of healthcare professionals interviewed reported strong intentions to use the DUK IP and having formed a habit of using them after a minimum of one month continuous use. Pop-up cues in the electronic patient records were perceived to facilitate the use of the tool. Factors that conflicted with the use of the DUK IP included existing pathways of providing self-management advice. Conclusion Data suggests that constructs from dual process and multiple behaviour approaches are useful to provide supplemental understanding of the implementation of new self-management tools such as the DUK IP and may help to advance behavioural approaches to implementation science

P2X₇-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells.

The P2X₇ receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X₇ function exhibit altered bone formation. Moreover, activation of P2X₇ in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X₇ agonist 2\u27,3\u27-O-(4-benzoylbenzoyl)ATP (BzATP; 300 μM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X₇ receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 μM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (∼2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 μM), which activate P2 receptors other than P2X₇, failed to elicit a sustained increase in proton efflux. Specific P2X₇ receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca²⁺ was required during P2X₇ receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X₇ receptors on osteoblast-like cells triggers a dramatic, Ca²⁺-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

EExtracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1/2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP

Reinforcement of flowable dental composites with titanium dioxide nanotubes

© 2016 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. Objectives Flowable dental composites are used as restorative materials due to their excellent esthetics and rheology. However, they suffer from inferior mechanical properties compared to conventional composites. The aim of this study was to reinforce a flowable dental composite with TiO2 nanotubes (n-TiO2) and to assess the effect of n-TiO2 surface modifications on the mechanical properties of the reinforced composite. Methods n-TiO2 were synthesized using an alkaline hydrothermal process and then functionalized with silane or methacrylic acid (MA). Nanotubes were characterized by scanning and transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. Commercially available flowable composite (Filtek™ Supreme Ultra Flowable Restorative, 3M ESPE) was reinforced with varying amounts of nanotubes (0-5 wt%). Flowability of the resulting composites was evaluated using a Gillmore needle method. Dynamic Young\u27s modulus (E) was measured using an ultrasonic technique. Fracture toughness (KIc) was assessed using a notchless triangular prism and radiopacity was quantified. Viability of NIH/3T3 fibroblasts was evaluated following incubation on composite specimens for 24 h. Results Electron microscopy revealed a tubular morphology of n-TiO2. All reinforced composites exhibited significantly greater values of E than unreinforced composite. Composites reinforced with 3 wt% n-TiO2 functionalized with MA exhibited the greatest values of E and KIc. Cytotoxicity assays revealed that reinforced composites were biocompatible. Taken together, flowable composites reinforced with n-TiO2 exhibited mechanical properties superior to those of unreinforced composite, with minimal effects on flowability and radiopacity. Significance n-TiO2-reinforced flowable composites are promising materials for use in dental restorations