Effect of Sustained Joint Loading on TMJ Disc Nutrient Environment.

The temporomandibular joint (TMJ) disc nutrient environment profoundly affects cell energy metabolism, proliferation, and biosynthesis. Due to technical challenges of in vivo measurements, the human TMJ disc extracellular nutrient environment under load, which depends on metabolic rates, solute diffusion, and disc morphometry, remains unknown. Therefore, the study objective was to predict the TMJ disc nutrient environment under loading conditions using combined experimental and computational modeling approaches. Specifically, glucose consumption and lactate production rates of porcine TMJ discs were measured under varying tissue culture conditions (n = 40 discs), and mechanical strain-dependent glucose and lactate diffusivities were measured using a custom diffusion chamber (n = 6 discs). TMJ anatomy and loading area were obtained from magnetic resonance imaging of healthy human volunteers (n = 11, male, 30 ± 9 y). Using experimentally determined nutrient metabolic rates, solute diffusivities, TMJ anatomy, and loading areas, subject-specific finite element (FE) models were developed to predict the 3-dimensional nutrient profiles in unloaded and loaded TMJ discs (unloaded, 0% strain, 20% strain). From the FE models, glucose, lactate, and oxygen concentration ranges for unloaded healthy human TMJ discs were 0.6 to 4.0 mM, 0.9 to 5.0 mM, and 0% to 6%, respectively, with steep gradients in the anterior and posterior bands. Sustained mechanical loading significantly reduced nutrient levels (P < 0.001), with a critical zone in which cells may die representing approximately 13.5% of the total disc volume. In conclusion, this study experimentally determined TMJ disc metabolic rates, solute diffusivities, and disc morphometry, and through subject-specific FE modeling, revealed critical interactions between mechanical loading and nutrient supply and metabolism for the in vivo human TMJ disc. The results suggest that TMJ disc homeostasis may be vulnerable to pathological loading (e.g., clenching, bruxism), which impedes nutrient supply. Given difficulties associated with direct in vivo measurements, this study provides a new approach to systematically investigate homeostatic and degenerative mechanisms associated with the TMJ disc.

Overexpression of miR-489 derails mammary hierarchy structure and inhibits HER2/neu-induced tumorigenesis.

Although it has been demonstrated that transformed progenitor cell population can contribute to tumor initiation, factors contributing to this malignant transformation are poorly known. Using in vitro and xenograft-based models, previous studies demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic pathways. It has been demonstrated that miR-489 directly targets HER2 and inhibits the HER2 signaling pathway; however, its role in mammary gland development and HER2-induced tumor initiation hasn't been studied. To dissect the role of miR-489, we sorted different populations of mammary epithelial cells and determined that miR-489 was highly expressed in mammary stem cells. MMTV-miR-489 mice that overexpressed miR-489 in mammary epithelial cells were developed and these mice exhibited an inhibition of mammary gland development in early ages with a specific impact on highly proliferative cells. Double transgenic MMTV-Her2-miR489 mice were then generated to observe how miR-489 overexpression affects HER2-induced tumorigenesis. miR-489 overexpression delayed HER2-induced tumor initiation significantly. Moreover, miR-489 overexpression inhibited tumor growth and lung metastasis. miR-489 overexpression reduced mammary progenitor cell population significantly in preneoplastic mammary glands of MMTV-Her2 mice which showed a putative transformed population in HER2-induced tumorigenesis. The miR-489 overexpression reduced CD49fhiCD61hi populations in tumors that have stem-like properties, and miR-489 overexpression altered the HER2 signaling pathway in mammary tumors. Altogether, these data indicate that the inhibition of HER2-induced tumorigenesis by miR-489 overexpression was due to altering progenitor cell populations while decreasing tumor growth and metastasis via influencing tumor promoting genes DEK and SHP2.

Correction: Overexpression of miR-489 derails mammary hierarchy structure and inhibits HER2/neu-induced tumorigenesis.

In the published version of this paper the author A. Awgulewitsch's surname was incorrectly given as Awagulerwitsch instead of Awgulewitsch. This has now been corrected in the HTML version of the paper, the PDF was correct at the time of publication.

The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells.

OBJECTIVE: To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. DESIGN: TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively. RESULTS: TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001). CONCLUSIONS: Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply.

Viscoelastic shear properties of porcine temporomandibular joint disc.

OBJECTIVES: To investigate the intrinsic viscoelastic shear properties in porcine TMJ discs. MATERIALS AND METHODS: Twelve fresh porcine TMJ discs from young adult pigs (6-8 months) were used. Cylindrical samples (5 mm diameter) with uniform thickness (~1.2 mm) were prepared from five regions of the TMJ disc. Torsional shear tests were performed under 10% compressive strain. Dynamic shear was applied in two methods: 1) a frequency sweep test over the frequency range of 0.1-10 rad/s with a constant shear strain amplitude of 0.05 rad and 2) a strain sweep test over the range of 0.005-0.15 rad at a constant frequency of 10 rad/s. Transient stress relaxation tests were also performed to determine the equilibrium shear properties. RESULTS: As the frequency increased in the frequency sweep test, the dynamic shear complex modulus increased, with values ranging from 7 to 17 kPa. The phase angle, ranging from 11 to 15 degrees, displayed no pattern of regional variation as the frequency increased. The dynamic shear modulus decreased as the shear strain increased. The equilibrium shear modulus had values ranging from 2.6 to 4 kPa. The posterior region had significantly higher values for dynamic shear modulus than those in the anterior region, while no significant regional difference was found for equilibrium shear modulus. CONCLUSION: Our results suggest that the intrinsic region-dependent viscoelastic shear characteristics of TMJ disc may play a crucial role in determining the local strain of the TMJ disc under mechanical loading.

Regional cell density distribution and oxygen consumption rates in porcine TMJ discs: an explant study.

OBJECTIVE: To determine the regional cell density distribution and basal oxygen consumption rates (based on tissue volume and cell number) of temporomandibular joint (TMJ) discs and further examine the impact of oxygen tension on these rates. DESIGN: TMJ discs from pigs aged 6-8 months were divided into five regions: anterior, intermediate, posterior, lateral and medial. The cell density was determined using confocal laser scanning microscopy. The change in oxygen tension was recorded while TMJ disc explants were cultured in sealed metabolism chambers. The volume based oxygen consumption rate of explants was determined by theoretical curve-fitting of the recorded oxygen tension data with the Michaelis-Menten equation. The rate on a per-cell basis was calculated based on the cell density measurements and volume based rate measured in another group of discs. RESULTS: The overall cell density [mean, 95% confidence interval (CI)] was 51.3 (21.3-81.3) × 10(6) cells/mL wet tissue. Along the anteroposterior axis, the anterior band had 25.5% higher cell density than the intermediate zone (P<0.02) and 29.1% higher than the posterior band (P<0.008). Along the mediolateral axes, the medial region had 26.2% higher cell density than the intermediate zone (P<0.04) and 25.4% higher than the lateral region (P<0.045). The overall volume and cell based maximum oxygen consumption rates were 1.44 (0.44-2.44) µmol/mL wet tissue/h and 28.7 (12.2-45.2)nmol/10(6)cells/h, respectively. The central regions (intermediate, lateral, and medial) had significantly higher volume based (P<0.02) and cell based (P<0.005) oxygen consumption rates than the anterior and posterior bands. At high oxygen tension, the oxygen consumption rate remained constant, but dropped as oxygen tension fell below 5%. CONCLUSIONS: The TMJ disc had higher cell density and oxygen consumption rates than articular cartilage reported in the literature. These results suggest that a steeper oxygen gradient may exist in the TMJ disc and may be vulnerable to pathological events that impede nutrient supply.

Periostin is expressed by cells of the human and sand rat intervertebral discs.

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.

The Prx1 homeobox gene is critical for molar tooth morphogenesis.

The paired-related homeobox genes, Prx1 and Prx2, encode transcription factors critical for orofacial development. Prx1(-/-)/Prx2(-/-) neonates have mandibular hypoplasia and malformed mandibular incisors. Although the mandibular incisor phenotype has been briefly described (ten Berge et al., 1998, 2001; Lu et al., 1999), very little is known about the role of Prx proteins during tooth morphogenesis. Since the posterior mandibular region was relatively normal, we examined molar tooth development in Prx1(-/-)/Prx2(-/-) embryos to determine whether the tooth malformation is primary to the loss of Prx protein or secondary to defects in surrounding tissues. Three-dimensional (3D) morphological reconstructions demonstrated that Prx1(-/-)/Prx2(-/-) embryos had molar malformations, including cuspal changes and ectopic epithelial projections. Although we demonstrate that Prx1 protein is expressed only mesenchymally, 3D reconstructions showed important morphological defects in epithelial tissues at the cap and bell stages. Analysis of these data suggests that the Prx homeoproteins are critical for mesenchymal-epithelial signaling during tooth morphogenesis.

Impact of final coronary flow velocity reserve on late outcome following stent implantation.

Aims To assess whether coronary flow velocity reserve following stent implantation is predictive of the subsequent need of target lesion revascularization. Methods and Results The outcome was examined of 417 patients enrolled in a multicentre prospective randomized study (DESTINI), who received a successful single vessel stent implantation in native coronary arteries and in whom coronary flow velocity reserve was measured. Logistic regression analysis and the receiver operator characteristic curve were used. When compared with 358 patients not requiring target lesion revascularization, 59 patients (14%) who underwent target lesion revascularization had a lower final coronary flow velocity reserve (2.33 +/- 0.87 vs 2.48+/- 0.80, P= 0.20) and smaller final minimal lumen diameter (2.62 +/- 0.66 mm vs 2.73+/- 0.60, P= 0.19); however, those differences were not statistically significant. Patients with a coronary flow velocity reserve of < 2.0 (n=109, 26%) exhibited a significantly higher target lesion revascularization rate than patients with a coronary flow velocity reserve of > or = 2.0 (22% vs 11%, P= 0.010). This difference remained significant (odds ratio=2.01, 95% CI=1.11 to 3.66) after adjustment for other variables that were also correlated with the incidence of target lesion revascularization. Conclusion The presence of a final coronary flow velocity reserve of < 2.0 is an independent predictor of the need for target lesion revascularization after stent implantation in native coronary artery lesions.

Investigation of Prx1 protein expression provides evidence for conservation of cardiac-specific posttranscriptional regulation in vertebrates.

Gene targeting experiments have defined that the homeobox gene Prx1 is essential for normal craniofacial, limb, and vascular development. Although its RNA expression pattern is well established, Prx1 protein expression in the developing embryo has not been examined. A novel Prx1 antibody was produced to define the normal Prx1 protein expression pattern in the developing mouse embryo. In craniofacial and limb mesenchyme, Prx1 protein expression is consistent with previously published data on RNA localization. However, a remarkable discrepancy was found in cardiac tissue. Prx1 protein is undetectable in the murine embryonic and adult heart, despite the presence of Prx1 transcripts. These data demonstrate that Prx1 expression is posttranscriptionally regulated. This discrepancy between the presence of Prx1 transcript and the absence of detectable protein was also observed in embryonic chick heart, suggesting conservation of the regulatory mechanism in vertebrates. This observation provides a new explanation of why the Prx null mice lack cardiac malformations.

Fractional flow reserve compared with intravascular ultrasound guidance for optimizing stent deployment.

BACKGROUND: Determination of fractional flow reserve (FFR) has been proposed as a means to assess stent deployment. In this prospective, multicenter trial, we evaluate the use of FFR to optimize stenting by comparing it with standard intravascular ultrasound (IVUS) criteria. METHODS AND RESULTS: Eighty-four stable patients with isolated coronary lesions underwent coronary stent deployment starting at 10 atm and increased serially by 2 atm until the FFR was >/=0.94 or 16 atm was achieved. IVUS was then performed. FFR was measured with a coronary pressure wire with intracoronary adenosine to induce hyperemia. The diagnostic characteristics of an FFR <0.94 to predict suboptimal stent expansion by IVUS, defined in both absolute and relative terms, were calculated. Over a range of IVUS criteria, the highest sensitivity, specificity, and predictive accuracy of FFR were 80%, 30%, and 42%, respectively. Receiver operator characteristic analysis defined an optimal FFR cut point at >/=0.96; at this threshold, the sensitivity, specificity, and predictive accuracy of FFR were 75%, 58%, and 62%, respectively (P=0.03 for comparison of predictive accuracy, P=0.01 for concordance between FFR and IVUS). The negative predictive value was 88%. Significantly better diagnostic performance was achieved in a subgroup that received higher doses (>30 microgram) of intracoronary adenosine during pressure measurements, suggesting that FFR might be overestimated in the other group. CONCLUSIONS: A fractional flow reserve <0.96, measured after stent deployment, predicts a suboptimal result based on validated intravascular ultrasound criteria; however, an FFR >/=0.96 does not reliably predict an optimal stent result. Higher doses of intracoronary adenosine than previously used to measure FFR improve these results.

Comparison of relative coronary Doppler flow velocity reserve to stress myocardial perfusion imaging in patients with coronary artery disease.

To compare relative coronary artery vasodilator reserve (rCVR = CVRtarget/CVRreference) to myocardial perfusion stress imaging, 48 patients with coronary artery stenoses (61% +/- 16%; mean, +/- SD; range, 30%-91%) had measurements of target and reference vessel CVR (Doppler-tipped guidewire). rCVR was computed and compared to stress 201thallium or (99m)technetium-sestamibi myocardial tomography. Compared to 24 patients with negative stress imaging studies, 24 patients with positive stress studies had angiographically more severe stenoses (74% +/- 13% vs. 44% +/- 24%; P = 0.0005) with lower CVR(target) (1.68 +/- 0.55 vs. 2.46 +/- 0.74; P = 0.002) and lower rCVR (0.72 +/- 0.22 vs. 1.0 +/- 0.26; P < 0.003). Based on receiver-operator characteristic (ROC) cut points (CVR > 1.9; rCVR > 0.75), compared to CVR, rCVR had similar agreement (Kappa 0.54 vs. 0.50), sensitivity (63% vs. 71%), specificity (88% vs. 83%), and positive predictive value (83% vs. 81%) with myocardial perfusion tomography. A concordant CVRtarget/rCVR only slightly increased sensitivity, specificity, and positive predictive values (77%, 90%, and 87%, respectively). Although rCVR, like CVR, correlates with stress myocardial perfusion imaging results, rCVR did not have significant incremental prognostic value over CVR alone for myocardial perfusion imaging. However, rCVR does provide additional information regarding the status of the microcirculation in patients with coronary artery disease and complements the CVR for lesion assessment.

Randomized comparison of coronary angiography using 4F catheters: 4F manual versus "Acisted" power injection technique.

Compared with 6F catheters, diagnostic coronary angiographic and ventriculographic images with 4F catheters can be obtained with equivalent results using less radiographic contrast volume. Whether 4F coronary angiography would be superior using a power-assisted, operator-controlled technique compared with manual technique is unknown. To determine whether 4F coronary angiography using operator-controlled power injection (Acist, Minneapolis, MN) was equivalent or superior to the 4F manual technique, 96 unselected patients undergoing transfemoral coronary angiography were randomized to 4F catheter using a power injection or manual technique. Procedural characteristics and angiographic quality scores were analyzed. Comparing the 4F manual with the 4F power-injection technique, coronary angiographic quality scores were equivalent (left coronary artery 4.7 +/- 0.5 vs. 4.7 +/- 0.6, P = 0.99; right coronary artery 4.94 +/- 0.2 vs. 4.88 +/- 0.1, P = 0.21). Left ventriculography scores were lower in 4F Acist with similar contrast volumes. The total study contrast volume was significantly less in the 4F Acist group (119 +/- 35 vs. 149 +/- 49 ml, P = 0.001). Compared with the 4F manual contrast injection technique, diagnostic angiography through 4F catheters with power contrast injection resulted in equivalent coronary angiographic image quality with significantly less radiographic contrast volume.