Relationships among Cortisol, Perceived Stress, and Dental Caries Experience in Adolescents and Young Adults.

INTRODUCTION: Stress can impact mental and physical health, especially during adolescence and young adulthood, but the extent of its contribution to dental caries is poorly understood. The present study assessed the association between perceived stress, cortisol levels (in hair and saliva), and overall caries experience of adolescents and young adults aged 15-25 years. METHODS: Hair and saliva samples were obtained from 93 participants free of periodontal disease. Cortisol in hair and saliva was determined using a competitive enzyme-linked immunosorbent assay. Participants completed a perceived stress questionnaire and underwent full-mouth oral examination by a calibrated examiner. Dental caries experience was based on the decayed, missing, and filled teeth (DMFT) index. Sociodemographic variables were also recorded. RESULTS: There were significantly higher hair cortisol levels and perceived stress scale (PSS) scores in individuals with dental caries experience (DMFT≥1) than in those without (DMFT = 0). However, there was no significant difference in salivary cortisol concentration. A binary logistic regression revealed that higher hair cortisol levels and greater scores on the perceived stress scale were associated with increased odds of having experienced dental caries. In contrast, no significant association was found between salivary cortisol concentration and dental caries. Using multivariable regression models, caries experience was found to be significantly associated with both hair cortisol levels and PSS scores. These associations remained statistically significant even after adjusting for sociodemographic variables. CONCLUSION: Hair cortisol levels and perceived stress have a significant association with dental caries experience, whereas salivary cortisol concentrations do not.

Luciferase-based GloSensor™ cAMP assay: Temperature optimization and application to cell-based kinetic studies.

G protein-coupled receptors (GPCRs) are an important receptor superfamily and common therapeutic targets. The second messenger cyclic adenosine monophosphate (cAMP) is a key mediator in many GPCR signaling pathways. Monitoring intracellular cAMP levels can help identify orthosteric agonists and antagonists, as well as allosteric modulators. In this regard, luminescence-based biosensors have revolutionized our ability to monitor GPCR signaling kinetics. The GloSensor™ cAMP assay enables real-time monitoring of signaling downstream of many GPCRs. However, it is crucial to optimize assay conditions such as temperature. As well, it has not been reported whether the effects of temperature on biosensor activity are reversible. Here, we describe the temperature sensitivity and reversibility of the GloSensor™ cAMP assay, and which GloSensor™ version is optimal for measuring cytosolic cAMP. We also present a detailed protocol for monitoring cAMP levels in live cells expressing endogenous or exogenous GPCRs. Temperature optimization studies were carried out using HEK293H cells transiently transfected with the adenosine receptor A2a and the GloSensor™ plasmid (pGloSensor-20F or -22F). We found that preincubation and luminescence reading at room temperature were optimal as compared to higher temperatures. As well, the GloSensor-22F biosensor had a superior signal-to-background ratio and the effect of temperature on biosensor activity was reversible. However, thermal instability of the biosensor may pose a problem for in vivo studies. Nevertheless, the GloSensor™ cAMP assay can be applied to analyze signaling by a wide range of GPCRs for drug discovery purposes.

Vibration of osteoblastic cells using a novel motion-control platform does not acutely alter cytosolic calcium, but desensitizes subsequent responses to extracellular ATP.

Low-magnitude high-frequency mechanical vibration induces biological responses in many tissues. Like many cell types, osteoblasts respond rapidly to certain forms of mechanostimulation, such as fluid shear, with transient elevation in the concentration of cytosolic free calcium ([Ca2+ ]i ). However, it is not known whether vibration of osteoblastic cells also induces acute elevation in [Ca2+ ]i . To address this question, we built a platform for vibrating live cells that is compatible with microscopy and microspectrofluorometry, enabling us to observe immediate responses of cells to low-magnitude high-frequency vibrations. The horizontal vibration system was mounted on an inverted microscope, and its mechanical performance was evaluated using optical tracking and accelerometry. The platform was driven by a sinusoidal signal at 20-500 Hz, producing peak accelerations from 0.1 to 1 g. Accelerometer-derived displacements matched those observed optically within 10%. We then used this system to investigate the effect of acceleration on [Ca2+ ]i in rodent osteoblastic cells. Cells were loaded with fura-2, and [Ca2+ ]i was monitored using microspectrofluorometry and fluorescence ratio imaging. No acute changes in [Ca2+ ]i or cell morphology were detected in response to vibration over the range of frequencies and accelerations studied. However, vibration did attenuate Ca2+ transients generated subsequently by extracellular ATP, which activates P2 purinoceptors and has been implicated in mechanical signaling in bone. In summary, we developed and validated a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Vibration did not elicit acute elevation of [Ca2+ ]i , but did desensitize responses to later stimulation with ATP.

Loss of ENT1 increases cell proliferation in the annulus fibrosus of the intervertebral disc.

Mice lacking equilibrative nucleoside transporter 1 (ENT1 -/- ) demonstrate progressive calcification of spinal tissues including the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously established ENT1 as the primary nucleoside transporter in the AF and demonstrated dysregulation of biomineralization pathways. To identify cellular pathways altered by loss of ENT1, we conducted microarray analysis of AF tissue from wild-type (WT) and ENT1 -/- mice before calcification (2 months of age) and associated with calcification (6 months of age). Bioinformatic analyses identified cell cycle dysregulation in ENT1 -/- AF tissues and implicated the E2f family of transcription factors as potential effectors. Quantitative polymerase chain reaction analysis confirmed increased expression of multiple E2f transcription factors and E2f interacting proteins ( Rb1 and Cdk2) in ENT1 -/- AF cells compared with WT at 6 months of age. At this time point, ENT1 -/- AF tissues showed increased JNK MAPK pathway activation, CDK1, minichromosome maintenance complex component 5 (Mcm5), and proliferating cell nuclear antigen (PCNA) protein expression, and PCNA-positive proliferating cells compared with WT controls. The current study demonstrates that loss of ENT1-mediated adenosine transport leads to increased cell proliferation in the AF of the IVD.

Whole-body vibration of mice induces progressive degeneration of intervertebral discs associated with increased expression of Il-1ß and multiple matrix degrading enzymes.

OBJECTIVE: Whole-body vibration (WBV) is a popular fitness trend based on claims of increased muscle mass, weight loss and reduced joint pain. Following its original implementation as a treatment to increase bone mass in patients with osteoporosis, WBV has been incorporated into clinical practice for musculoskeletal disorders, including back pain. However, our recent studies revealed damaging effects of WBV on joint health in a murine model. In this report, we examined potential mechanisms underlying disc degeneration following exposure of mice to WBV. METHODS: Ten-week-old male mice were exposed to WBV (45 Hz, 0.3 g peak acceleration, 30 min/day, 5 days/week) for 4 weeks, 8 weeks, or 4 weeks WBV followed by 4 weeks recovery. Micro-computed tomography (micro-CT), histological, and gene expression analyses were used to assess the effects of WBV on spinal tissues. RESULTS: Exposure of mice to 4 or 8 weeks of WBV did not alter total body composition or induce significant changes in vertebral bone density. On the other hand, WBV-induced intervertebral disc (IVD) degeneration, associated with decreased disc height and degenerative changes in the annulus fibrosus (AF) that did not recover within 4 weeks after cessation of WBV. Gene expression analysis showed that WBV for 8 weeks induced expression of Mmp3, Mmp13, and Adamts5 in IVD tissues, changes preceded by increased expression of Il-1ß. CONCLUSIONS: Progressive IVD degeneration induced by WBV was associated with increased expression of Il-1ß within the IVD that preceded Mmp and Adamts gene induction. Moreover, WBV-induced IVD degeneration is not reversed following cessation of vibration.

Interaction of primary human trabecular meshwork cells with metal alloy candidates for microinvasive glaucoma surgery.

BACKGROUND: Microinvasive glaucoma surgery (MIGS) is a relatively new addition to the glaucoma treatment paradigm. Small metallic stents are inserted into the trabecular meshwork in order to increase aqueous humour drainage. MIGS procedures are rapidly being adopted owing to a more favourable side effect profile when compared with traditional surgery. Remarkably, this rapid rate of utilization has occurred without any published studies on the effect of metal alloys used in these stents on human trabecular meshwork cells (HTMCs). Therefore, this study aimed to determine the effect of candidate metal alloys for MIGS on HTMC morphology, viability and function. METHODS: Human trabecular meshwork cells were cultured on the surfaces of titanium (polished and sandblasted), a titanium-nickel (nitinol) alloy and glass (as control substratum). Fluorescence imaging was used to assess cell morphology and spreading. A lactate dehydrogenase cytotoxicity assay, cell death detection ELISA, MTT cell viability assay, BrdU cell proliferation assay and fibronectin ELISA were also conducted. RESULTS: Cells cultured on sandblasted titanium exhibited significantly greater spreading than cells cultured on other substrata. In comparison, HTMCs cultured on nitinol displayed poor spreading. Significantly more cell death, by both necrosis and apoptosis, occurred on nitinol than on titanium and glass. Also, cell viability and proliferation were suppressed on nitinol compared with titanium or glass. Finally, HTMCs on both titanium and nitinol produced greater amounts of fibronectin than cells grown on glass. CONCLUSIONS: Substratum topography and metal alloy composition were found to impact morphology, viability and function of primary HTMC cultures.

A Comparison of the Mechanical Measures Used for Assessing Orthodontic Mini-Implant Stability.

PURPOSE: Mechanical loosening remains a common complication associated with mini-implant failure. The purpose of this study was to compare common mechanical measures of mini-implant stability to determine their association and reliability. MATERIALS AND METHODS: Ninety self-drilling orthodontic mini-implants from 6 manufacturers were inserted into artificial bone blocks. Insertion torques (ITs) and Periotest values (PVs) were measured. Subsequently, mini-implants underwent pull-out testing for measures of pull-out load (POL) and screw displacement (ScrD). Stability measurements were compared using one-way ANOVA, associations among them were assessed using correlation analyses, and reliability was evaluated using coefficients of variation (COVs). RESULTS: Variations in stability of mini-implants were found, specific to the mechanical measure used for assessment (P < 0.05). The strongest correlations were found between IT and PV (r = -0.68) and between IT and POL (r = 0.66). Overall, PV showed the greatest variability (COV: 11%-100%) compared with IT (≤11%), POL (≤4%), and ScrD (≤19%). CONCLUSIONS: IT, PV, and POLs only agreed moderately in their assessment of mini-implant stability, and Periotest showed the least reliability in predicting mini-implant stability. As such, independent and interchangeable use of these stability measures should be avoided.

Practical fabrication of microfluidic platforms for live-cell microscopy.

We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.

P2X7 nucleotide receptor signaling potentiates the Wnt/ß-catenin pathway in cells of the osteoblast lineage.

The P2X7 and Wnt/ß-catenin signaling pathways regulate osteoblast differentiation and are critical for the anabolic responses of bone to mechanical loading. However, whether these pathways interact to control osteoblast activity is unknown. The purpose of this study was to investigate the effects of P2X7 activation on Wnt/ß-catenin signaling in osteoblasts. Using MC3T3-E1 cells, we found that combined treatment with Wnt3a and the P2X7 agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) elicited more sustained ß-catenin nuclear localization than that induced by Wnt3a alone. Wnt3a-induced increases in ß-catenin transcriptional activity were also potentiated by treatment with BzATP. Consistent with involvement of P2X7, a high ATP concentration (1 mM) potentiated Wnt3a-induced ß-catenin transcriptional activity, whereas a low concentration (10 µM) of ATP, adenosine 5'-diphosphate (ADP), or uridine 5'-triphosphate (UTP) failed to elicit a response. The potentiation of ß-catenin transcriptional activity elicited by BzATP was also inhibited by two distinct P2X7 antagonists: A 438079 and A 740003. Furthermore, responses to Wnt3a in calvarial cells isolated from P2rx7 knockout mice were significantly less than in cells from wild-type controls. In MC3T3-E1 cells, BzATP increased inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß), a process that was blocked by A 438079 and diminished by inhibition of protein kinase C. Thus, P2X7 signaling may potentiate the canonical Wnt pathway through GSK3ß inhibition. Taken together, we show that P2X7 activation prolongs and potentiates Wnt/ß-catenin signaling. Consequently, cross-talk between P2X7 and Wnt/ß-catenin pathways may modulate osteoblast activity in response to mechanical loading.

P2 receptor networks regulate signaling duration over a wide dynamic range of ATP concentrations.

The primordial intercellular signaling molecule ATP acts through two families of cell-surface P2 receptors - the P2Y family of G-protein-coupled receptors and the P2X family of ligand-gated cation channels. Multiple P2 receptors are expressed in a variety of cell types. However, the significance of these networks of receptors in any biological system remains unknown. Using osteoblasts as a model system, we found that a low concentration of ATP (10 µM, ATPlow) induced transient elevation of cytosolic Ca(2+), whereas a high concentration of ATP (1 mM, ATPhigh) elicited more sustained elevation. Moreover, graded increases in the Ca(2+) signal were achieved over a remarkable million-fold range of ATP concentrations (1 nM to 1 mM). Next, we demonstrated that ATPlow caused transient nuclear localization of the Ca(2+)-regulated transcription factor NFATc1; whereas, ATPhigh elicited more sustained localization. When stimulated with ATPhigh, osteoblasts from P2X7 loss-of-function mice showed only transient Ca(2+)-NFATc1 signaling; in contrast, sustained signaling was observed in wild-type cells. Additional experiments revealed a role for P2Y receptors in mediating transient signaling induced by low ATP concentrations. Thus, distinct P2 receptors with varying affinities for ATP account for this wide range of sensitivity to extracellular nucleotides. Finally, ATPhigh, but not ATPlow, was shown to elicit robust expression of the NFAT target gene Ptgs2 (encoding COX-2), consistent with a crucial role for the duration of Ca(2+)-NFAT signaling in regulating target gene expression. Taken together, ensembles of P2 receptors provide a mechanism by which cells sense ATP over a wide concentration range and transduce this input into distinct cellular signals.

Effects of isoform-selective phosphatidylinositol 3-kinase inhibitors on osteoclasts: actions on cytoskeletal organization, survival, and resorption.

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (ß), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15-20 min to 65-75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kß and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.

Lysophosphatidic acid: a potential mediator of osteoblast-osteoclast signaling in bone.

Osteoclasts (bone resorbing cells) and osteoblasts (bone forming cells) play essential roles in skeletal development, mineral homeostasis and bone remodeling. The actions of these two cell types are tightly coordinated, and imbalances in bone formation and resorption can result in disease states, such as osteoporosis. Lysophosphatidic acid (LPA) is a potent bioactive phospholipid that influences a number of cellular processes, including proliferation, survival and migration. LPA is also involved in wound healing and pathological conditions, such as tumor metastasis and autoimmune disorders. During trauma, activated platelets are likely a source of LPA in bone. Physiologically, osteoblasts themselves can also produce LPA, which in turn promotes osteogenesis. The capacity for local production of LPA, coupled with the proximity of osteoblasts and osteoclasts, leads to the intriguing possibility that LPA acts as a paracrine mediator of osteoblast-osteoclast signaling. Here we summarize emerging evidence that LPA enhances the differentiation of osteoclast precursors, and regulates the morphology, resorptive activity and survival of mature osteoclasts. These actions arise through stimulation of multiple LPA receptors and intracellular signaling pathways. Moreover, LPA is a potent mitogen implicated in promoting the metastasis of breast and ovarian tumors to bone. Thus, LPA released from osteoblasts is potentially an important autocrine and paracrine mediator - physiologically regulating skeletal development and remodeling, while contributing pathologically to metastatic bone disease. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Loss of P2X7 nucleotide receptor function leads to abnormal fat distribution in mice.

The P2X7 receptor is an ATP-gated cation channel expressed by a number of cell types. We have shown previously that disruption of P2X7 receptor function results in downregulation of osteogenic markers and upregulation of adipogenic markers in calvarial cell cultures. In the present study, we assessed whether loss of P2X7 receptor function results in changes to adipocyte distribution and lipid accumulation in vivo. Male P2X7 loss-of-function (KO) mice exhibited significantly greater body weight and epididymal fat pad mass than wild-type (WT) mice at 9 months of age. Fat pad adipocytes did not differ in size, consistent with adipocyte hyperplasia rather than hypertrophy. Histological examination revealed ectopic lipid accumulation in the form of adipocytes and/or lipid droplets in several non-adipose tissues of older male KO mice (9-12 months of age). Ectopic lipid was observed in kidney, extraorbital lacrimal gland and pancreas, but not in liver, heart or skeletal muscle. Specifically, lacrimal gland and pancreas from 12-month-old male KO mice had greater numbers of adipocytes in perivascular, periductal and acinar regions. As well, lipid droplets accumulated in the renal tubular epithelium and lacrimal acinar cells. Blood plasma analyses revealed diminished total cholesterol levels in 9- and 12-month-old male KO mice compared with WT controls. Interestingly, no differences were observed in female mice. Moreover, there were no significant differences in food consumption between male KO and WT mice. Taken together, these data establish novel in vivo roles for the P2X7 receptor in regulating adipogenesis and lipid metabolism in an age- and sex-dependent manner.

Acute vibration induces transient expression of anabolic genes in the murine intervertebral disc.

OBJECTIVE: Low-amplitude whole-body vibration has been adopted for the treatment of back pain and spinal disorders. However, there is limited knowledge of the impact of vibration on the intervertebral disc (IVD). This study was undertaken to examine the effects of acute vibration on anabolic and catabolic pathways in the IVD and to characterize the dependence of these changes on time and frequency. METHODS: Custom-designed platforms were developed to apply acute vibration to ex vivo and in vivo mouse models. Spinal segments (ex vivo) or mice (in vivo) were subjected to vibration (for 30 minutes at 15-90 Hz with peak acceleration at 0.3g), and IVDs were examined at specific time points after vibration. Gene expression was quantified using real-time polymerase chain reaction, and protein levels were examined by quantitative mass spectrometry and immunofluorescence. RESULTS: In the ex vivo model, acute vibration at 15 Hz induced expression of anabolic genes (aggrecan, biglycan, decorin, type I collagen, and Sox9) and suppressed expression of Mmp13, with the most pronounced changes detected 6 hours following vibration. These beneficial effects were frequency dependent and were no longer evident between 45 and 90 Hz. In vivo, the effects on anabolic gene expression were even more robust and were accompanied by decreased expression of Adamts4, Adamts5, and Mmp3. Moreover, significant increases in the protein levels of aggrecan, biglycan, decorin, and type I collagen were detected in vivo. CONCLUSION: These findings demonstrate dramatic anabolic effects of acute vibration on IVD tissue, responses that are dependent on frequency. The similarity of the in vivo and ex vivo responses indicates that at least some effects of vibration are tissue autonomous.

Monocytes from patients with osteoarthritis display increased osteoclastogenesis and bone resorption: the In Vitro Osteoclast Differentiation in Arthritis study.

OBJECTIVE: To compare the osteoclastogenic capacity of peripheral blood mononuclear cells (PBMCs) from patients with osteoarthritis (OA) to that of PBMCs from self-reported normal individuals. METHODS: PBMCs from 140 patients with OA and 45 healthy donors were assayed for CD14+ expression and induced to differentiate into osteoclasts over 3 weeks in vitro. We assessed the number of osteoclasts, their resorptive activity, osteoclast apoptosis, and expression of the following cytokine receptors: RANK, interleukin-1 receptor type I (IL-1RI), and IL-1RII. A ridge logistic regression classifier was developed to discriminate OA patients from controls. RESULTS: PBMCs from OA patients gave rise to more osteoclasts that resorbed more bone surface than did PBMCs from controls. The number of CD14+ precursors was comparable in both groups, but there was less apoptosis in osteoclasts obtained from OA patients. Although no correlation was found between osteoclastogenic capacity and clinical or radiographic scores, levels of IL-1RI were significantly lower in cultures from patients with OA than in cultures from controls. Osteoclast apoptosis and expression levels of IL-1RI and IL-1RII were used to build a multivariate predictive model for OA. CONCLUSION: During 3 weeks of culture under identical conditions, monocytes from patients with OA display enhanced capacity to generate osteoclasts compared to cells from controls. Enhanced osteoclastogenesis is accompanied by increased resorptive activity, reduced osteoclast apoptosis, and diminished IL-1RI expression. These findings support the possibility that generalized changes in bone metabolism affecting osteoclasts participate in the pathophysiology of OA.

Receptor-independent effects of 2'(3')-O-(4-benzoylbenzoyl)ATP triethylammonium salt on cytosolic pH.

The effect of the relatively potent P2X7 receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP-TEA) on cytosolic pH (pHi) was studied using MC3T3-E1 osteoblast-like cells, which endogenously express P2X7 receptors. pHi was measured fluorimetrically using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. BzATP-TEA (0.3-1.5 mM) elicited fast-onset alkalinization responses. In contrast, adenosine 5'-triphosphate disodium salt (5 mM) failed to reproduce the BzATP-TEA-induced responses, indicating a P2 receptor-independent mechanism. We speculated that triethylamine, which is present in solutions of BzATP-TEA, permeates the plasma membrane, and is protonated intracellularly, leading to an increase in pHi. Consistent with this hypothesis, triethylammonium (TEA) chloride mimicked the effects of BzATP-TEA on pHi. Moreover, measurements using a Cytosensor microphysiometer revealed that TEA chloride transiently suppressed proton efflux from cells, whereas washout of TEA transiently enhanced proton efflux. BzATP-TEA also elicited a sustained increase in proton efflux that was blocked specifically by the P2X7 antagonist A-438079. Taken together, we conclude that BzATP-TEA-induced alkalinization is unrelated to P2X7 activation, but is due to the presence of TEA. This effect may confound assessment of the outcomes of P2X7 activation by BzATP-TEA in other systems. Thus, control experiments using TEA chloride are recommended to distinguish between receptor-mediated and nonspecific effects of this widely used agonist. We performed such a control and confirmed that BzATP-TEA, but not TEA chloride, caused the elevation of cytosolic free Ca(2+) in MC3T3-E1 cells, ruling out the possibility that receptor-independent effects on pHi underlie BzATP-TEA-induced Ca(2+) signaling.

Titanium Alloy Implants with Lattice Structures for Mandibular Reconstruction.

In recent years, the field of mandibular reconstruction has made great strides in terms of hardware innovations and their clinical applications. There has been considerable interest in using computer-aided design, finite element modelling, and additive manufacturing techniques to build patient-specific surgical implants. Moreover, lattice implants can mimic mandibular bone's mechanical and structural properties. This article reviews current approaches for mandibular reconstruction, their applications, and their drawbacks. Then, we discuss the potential of mandibular devices with lattice structures, their development and applications, and the challenges for their use in clinical settings.

Osteopontin signals through calcium and nuclear factor of activated T cells (NFAT) in osteoclasts: a novel RGD-dependent pathway promoting cell survival.

Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca(2+)-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca(2+) (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) suppressed Ca(2+) oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca(2+)-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo.

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',3'-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 Ca²âº 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, Ca²âº-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.

Control of surface topography in biomimetic calcium phosphate coatings.

The behavior of cells responsible for bone formation, osseointegration, and bone bonding in vivo are governed by both the surface chemistry and topography of scaffold matrices. Bone-like apatite coatings represent a promising method to improve the osteoconductivity and bonding of synthetic scaffold materials to mineralized tissues for regenerative procedures in orthopedics and dentistry. Polycaprolactone (PCL) films were coated with calcium phosphates (CaP) by incubation in simulated body fluid (SBF). We investigated the effect of SBF ion concentration and soaking time on the surface properties of the resulting apatite coatings. CaP coatings were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FTIR), and energy dispersive X-ray spectrometry (EDX). Young's modulus (E(s)) was determined by nanoindentation, and surface roughness was assessed by atomic force microscopy (AFM) and mechanical stylus profilometry. CaP such as carbonate-substituted apatite were deposited onto PCL films. SEM and AFM images of the apatite coatings revealed an increase in topographical complexity and surface roughness with increasing ion concentration of SBF solutions. Young's moduli (E(s)) of various CaP coatings were not significantly different, regardless of the CaP phase or surface roughness. Thus, SBF with high ion concentrations may be used to coat synthetic polymers with CaP layers of different surface topography and roughness to improve the osteoconductivity and bone-bonding ability of the scaffold.