Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Direct comparison of the bond strength results of the different test methods: A critical literature review

Objective. The goal of this paper is to undertake a literature search collecting all dentin bond strength data obtained for six adhesives with four tests ( shear, microshear, tensile and microtensile) and to critically analyze the results with respect to average bond strength, coefficient of variation, mode of failure and product ranking. Method. A PubMed search was carried out for the years between 1998 and 2009 identifying publications on bond strength measurements of resin composite to dentin using four tests: shear, tensile, microshear and microtensile. The six adhesive resins were selected covering three step systems ( OptiBond FL, Scotch Bond Multi-Purpose Plus), two-step (Prime & Bond NT, Single Bond, Clear. l SE Bond) and one step (Adper Prompt L Pop). Results. Pooling results from 147 references showed an ongoing high scatter in the bond strength data regardless which adhesive and which bond test was used. Coefficients of variation remained high (20-50%) even with the microbond test. The reported modes of failure for all tests still included high number of cohesive failures. The ranking seemed to be dependant on the test used. Significance. The scatter in dentin bond strength data remains regardless which test is used confirming Finite Element Analysis predicting non-uniform stress distributions due to a number of geometrical, loading, material properties and specimens preparation variables. This reopens the question whether, an interfacial fracture mechanics approach to analyze the dentin - adhesive bond is not more appropriate for obtaining better agreement among dentin bond related papers. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

A critical view on biaxial and short-beam uniaxial flexural strength tests applied to resin composites using Weibull, fractographic and finite element analyses

Objective. To evaluate the biaxial and short-beam uniaxial strength tests applied to resin composites based upon their Weibull parameters, fractographic features and stress distribution. Methods. Disk- (15 mm x 1 mm) and beam-shaped specimens (10 mm x 2 mm x 1 mm) of three commercial composites (Concept/Vigodent, CA; Heliomolar/Ivoclar-Vivadent, HE; Z250/3M ESPE, FZ) were prepared. After 48h dry storage at 37 degrees C, disks and beams were submitted to piston-on-three-balls (BI) and three-point bending (UNI) tests, respectively. Data were analyzed by Weibull statistics. Fractured surfaces were observed under stereomicroscope and scanning electron microscope. Maximum principal stress (sigma(1)) distribution was determined by finite element analysis (FEA). Maximum sigma(1-BI) and sigma(1-UNI) were compared to FZ strengths calculated by applying the average failure loads to the analytical equations (sigma(a-BI) and sigma(a-UNI)). Results. For BI, characteristic strengths were: 169.9a (FZ), 122.4b (CA) and 104.8c (HE), and for UNI were: 160.3a (FZ), 98.2b (CA) and 91.6b (HE). Weibull moduli ( m) were similar within the same test. CA and HE presented statistically higher m for BI. Surface pores ( BI) and edge flaws ( UNI) were the most frequent fracture origins. sigma(1-BI) was 14% lower than sigma(a-BI.) sigma(1-UNI) was 43% higher than sigma(a-UNI). Significance. Compared to the short-beam uniaxial test, the biaxial test detected more differences among composites and displayed less data scattering for two of the tested materials. Also, biaxial strength was closer to the material`s strength estimated by FEA. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Effect of the C-factor and Dentin Preparation Method in the Bond Strength of a Mild Self-etch Adhesive

This study evaluated the effect of the C-factor and dentin preparation method (DPM) in the bond strength (BS) of a mild self-etch adhesive; the study also observed the SEM superficial aspects of the corresponding smear layer. For purposes of this study, 25 molars (n=5) were used in a bond strength test. The molars were divided into two parts (buccal and lingual): one part received a Class V cavity (C-factor=3) and the other received a flat surface (C-factor=0) with the same bur type (coarse diamond or carbide bur and fine diamond or carbide bur), both within the same dentin depth. Five teeth were prepared with wet 60-grit and 600-grit SiC papers. After restoration with Clearfil SE Bond, microtensile beans (0.8 mm(2)) were prepared and tested after 24 hours in a universal testing machine (0.5 mm/minute). An additional two teeth for each DPM were prepared for SEM evaluation of the smear layer superficial aspects. The BS values were submitted to one-way ANOVA, considering only the DPM (flat surfaces) and two-way ANOVA (C-Factor x DPM, considering only burs) with p=0.05. Although the DPM in the flat surfaces was not significant, the standard deviations of carbide bur-prepared specimens were markedly lower. The BS was significantly lower in cavities. The fine carbide bur presented the most favorable smear layer aspect. It was concluded that different dentin preparation methods could not prevent the adverse effect in bond strength of a high C-factor. A coarse cut carbide bur should be avoided prior to a mild self-etch adhesive, because it adversely affected bond strength. In contrast, a fine cut carbide bur provided the best combination: high bond strength with low variability, which suggests a more reliable bond strength performance.

Stress intensity factor threshold in dental porcelains

The stress intensity factor threshold (K(IO)) is related to the stress level at which cracks start to grow stably, causing the weakening of porcelain prostheses during their use. The values of K(IO) of seven dental porcelains (with and without reinforcing leucite crystal, KAlSi(2)O(6)) stored in air (22 degrees C, 60% relative humidity) and artificial saliva (37 degrees C) were determined by measuring the crack growth velocity of radial cracks generated at the corner of Vickers indentations. The results of K(IO) were correlated with the leucite content, fracture toughness (K(Ic)), and chemical composition of the porcelains. It was observed that K(IO) increased with the increase of leucite content (only for the leucite-based porcelains) and with the increase of K(Ic). The increase in Al(2)O(3) content or the decrease in the alkali oxide (K(2)O and Na(2)O) content of the material`s glassy matrix tended to increase the K(IO) values. Storage media (air and saliva) did not significantly affect the K(IO) of porcelains tested, indicating that the control parameter of K(IO) value was not the water content of the storage media.

Absence of TLR2 influences survival of neutrophils after infection with Candida albicans

Candida albicans is an opportunistic pathogen, which causes local and/or disseminated diseases in immunosuppressed humans. Phagocytic cells play a critical role in the immune response against C. albicans. Toll like receptors (TLR) are important in the identification of invading microorganisms and in the regulation of neutrophil survival. TLR2 has been shown to participate in the response against pathogenic yeasts and to increase the functional life span of neutrophils. In view of these observations, we studied the involvement of TLR2 in neutrophil function after C. albicans infection. The absence of TLR2 resulted in lower chemotaxis of neutrophils to the site of infection. This in turn was associated with lower levels of chemokines from neutrophils, facilitating the dissemination of the pathogen to the lymph nodes and spleen. A high frequency of apoptotic neutrophils and macrophages in the inflammatory exudates from TLR2(-/-) mice was found. In addition, the phagocytic activity of neutrophils and macrophages, nitric oxide production and myeloperoxidase, activity were diminished in cells from TLR2(-/-) mice. Together, these data demonstrate the importance of TLR2 signals for neutrophils activation and survival after C albicans infection.

Healing of critical-size cranial defects in guinea pigs using a bovine bone-derived resorbable membrane

Purpose: To investigate the healing of critical-size cranial bone defects (9-mm-diameter) in guinea pigs treated with a bovine bone-derived resorbable membrane. Materials and Methods: A sample of 42 guinea pigs was divided into test (n = 20), control (n = 20), and standard (n = 2) groups. A full-thickness trephine defect was made in the fronto-parietal bone of each animal. In the test group, the internal and external openings of the defect were each closed with a separate membrane, and the space between them was filled with blood clot and a central spacer. In the control group, the defect was filled only with the blood clot and spacer. At 1, 3, 6, and 9 months later, the calvarias (5 per period) for both the test and control groups were collected, fixed, radiographed, and histologically processed. The Standard-group animals were sacrificed immediately after surgery and used to determine the initial size of defect radiographically. The areas of defects in the radiographs were measured with image-analysis software and were compared between groups and periods by multiple regression analysis with the Bonferroni correction. Results: At 1 and 3 months, newly formed woven bone was histologically observed in both test and control groups. Radiographically, this new bone occupied an average of 32% of the defect area at 1 month and 60% at 3 months in the test group. In the control group, 21% of the defect was filled at 1 month and 39% at 3 months. However, the differences between treatments were not statistically significant (P > .05). At 6 and 9 months, a significant increase in newly formed lamellar bone was seen histologically in both groups. Radiographically, for the test group, the new bone occupied an average of 82% of the defect area at 6 months and 96% at 9 months. For the control group, new bone composed an average of 45% of the defect area at 6 months and 40% at 9 months. The differences between the test and control groups were statistically significant at 6 and 9 months (P < .05). Complete or almost complete filling of the defect was observed in several cases. Conclusion: It was concluded that the bovine bone-derived membrane is highly biocompatible and is able to promote good healing of critical-size defects in calvaria of guinea pig.

Biological monitoring of a xenomaterial for grafting: an evaluation in critical-size calvarial defects

Our purpose was to evaluate the osteoconduction potential of mixed bovine bone (MBB) xenografts as an alternative for bone grafting of critical-size defects in the calvaria of rats. After surgery, in the time intervals of 1, 3, 6, and 9 months, rats were killed and their skulls collected, radiographed and histologically prepared for analysis. The data obtained from histological analysis reported that the particles of MBB did not promote an intense immunological response, evidencing its biocompatibility in rats. Our results clearly showed the interesting evidence that MBB was not completely reabsorbed at 9 months while a small amount of newly formed bone was deposited by osteoprogenitor cells bordering the defect. However, this discrete bone-forming stimulation was unable to regenerate the bone defect. Overall, our results suggest that the properties of MBB are not suitable for stimulating intense bone regeneration in critical bone defects in rats.

Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: Possible association with progressive or stable nature of the lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

Expression of vascular endothelian growth factor-C does not predict occult lymphnode metastasis in early oral squamous cell carcinoma

Strong vascular endothelial growth factor-C (VEGF-C) expression has been correlated to occurrence of lymph-node metastases in patients with oral squamous cell carcinoma (OSCC). The incidence of occult lymph-node metastasis remains a decisive factor in the prognosis of patients with early OSCC. The aim of this study was to evaluate VEGF-C expression as a predictor of occult lymph-node metastasis in OSCC. Eighty-seven patients with primary OSCC arising in the tongue or floor of mouth, clinically T1N0M0 or T2N0M0, with (pN+) and without (pN0) occult lymph-node metastases were analyzed for VEGF-C expression by malignant cells. Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection. No statistically significant difference was found between OSCC with and without occult lymph-node metastasis in regard to VEGF-C immunoexpression by malignant cells and clinicopathologic features. Independently of VEGF-C expression, lymph-node metastasis (PN+) was the most significant prognostic factor for overall survival of patients with OSCC (p = 0.030). These findings indicate that isolated VEGF-C expression by malignant cells is not of predictive value for occult lymph-node metastasis in the early stages of OSCC.

SHED Differentiate into Functional Odontoblasts and Endothelium

Studies on mechanisms underlying the differentiation of dental pulp stem cells are critical for the understanding of the biology of odontogenesis and for dental tissue engineering. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) differentiate into functional odontoblasts and endothelial cells. SHED were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. SHED differentiated into functional odontoblasts that generated tubular dentin, as determined by tetracycline staining and confocal microscopy. These cells also differentiated into vascular endothelial cells, as determined by beta-galactosidase staining of LacZ-tagged SHED. In vitro, vascular endothelial growth factor (VEGF) induced SHED to express VEGFR2, CD31, and VE-Cadherin (markers of endothelium) and to organize into capillary-like sprouts. VEGF induced ERK and AKT phosphorylation (indicative of differentiation), while inhibiting phosphorylation of STAT3 (indicative of `stemness`). Collectively, this work demonstrates that SHED can differentiate into angiogenic endothelial cells and odontoblasts capable of generating tubular dentin.

Differential Production of Macrophage Inflammatory Protein-1 alpha, Stromal-Derived Factor-1, and IL-6 by Human Cultured Periodontal Ligament and Gingival Fibroblasts Challenged With Lipopolysaccharide From P. gingivalis

Background: Fibroblasts are considered important cells in periodontitis. When challenged by different agents, they respond through the release of cytokines that participate in the inflammatory process. The aim of this study is to evaluate and compare the expression and production of macrophage inflammatory protein (MIP)-1 alpha, stromal-derived factor (SDF)-1, and interleukin (IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Methods: Fibroblasts were cultured from biopsies of gingival tissue and periodontal ligament of the same donors and used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0.1 to 10 mu g/ml of LPS from P. gingivalis was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells were collected and analyzed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results: MIP-1 alpha, SDF-1, and IL-6 protein production was significantly greater in gingival fibroblasts compared to periodontal ligament fibroblasts. IL-6 was upregulated in a time-dependent manner, mainly in gingival fibroblasts (P<0.05), which secreted more MIP-1 alpha in the lowest concentration of LPS used (0.1 mu g/ml). In contrast, a basal production of SDF-1 that was inhibited with the increase of LPS concentration was detected, especially after 24 hours (P<0.05). Conclusion: The distinct ability of the gingival and periodontal ligament fibroblasts to secrete MIP-1 alpha, SDF-1, and IL-6 emphasizes that these cells may differently contribute to the balance of cytokines in the LPS-challenged periodontium. J Periodontol 2010;81:310-317.