Mechanical Stability and Proteolytic Activity of Resin-dentin Bonds Using the Cross-linked Dry Bonding Technique.

OBJECTIVE: To evaluate the mechanical stability and the proteolytic activity of bonds created by a two-step, etch-and-rinse adhesive applied to cross-linked and air-dried etched dentin. METHODS: Flat dentin surfaces were produced in 64 extracted sound human molars. The dentin was etched with 35% phosphoric acid for 15 seconds, and then the teeth were divided into groups according to the cross-linking solution applied on the etched dentin. Group 1: 5% grape seed extract (GSE), Group 2: 5% glutaraldehyde, Group 3: Gluma Desensitizer, or Group 4: deionized water (control). Solutions were applied for 60 seconds, followed by rinse and blot drying. Then, the teeth were separated into two subgroups where the etched dentin was kept moist or air-dried. The adhesive was applied followed by a composite resin buildup. After 24 hours, the teeth were cut into beams (0.81 mm²) that were tested for microtensile strength immediately or after 12 months of aging in a 37°C saliva-like buffer. Additional teeth (n=32) were bonded as described and cut into 0.5-mm-thick slabs. The slabs were prepared for nanoleakage (scanning electron microscopy) and in situ zymography (EnzChek Protease Assay Kit). Bond strength data were submitted to ANOVA and Tukey tests (α =0.05). RESULTS: Significant reduction in immediate bond strength (ca 65%) and increase in proteolytic activity was seen when the etched dentin was air dried without previous cross-linking biomodification. Conversely, bond strengths did not differ from those produced on wet dentin when collagen was cross-linked before air drying, irrespective of the solution applied. For both moist and air-dried etched dentin, collagen cross-linking resulted in mechanically stable bonds and reduced proteolytic activity after 12 months of storage. CONCLUSION: Bonds produced by the application of a two-step, etch-and-rinse adhesive to cross-linked, air-dried, etched dentin were mechanically stable and revealed reduced proteolytic activity after 1 year of aging.

Effects of Enzymatic Activation of Bleaching Gels on Hydrogen Peroxide Degradation Rates, Bleaching Effectiveness, and Cytotoxicity.

OBJECTIVES: The aim of this study was to evaluate the effect of horseradish peroxidase (HRP) on the release of free radicals, bleaching effectiveness, and indirect cytotoxicity of a 35% hydrogen peroxide (HP) bleaching gel. METHODS AND MATERIALS: First, HP degradation rates and free radical release were evaluated for 35% HP in contact or not with HRP (10 mg/mL). The bleaching gel associated or not with HRP was then applied (3 × 15 minutes) to enamel/dentin discs adapted to artificial pulp chambers, and the culture medium in contact with dentin surfaces (extract) was collected and exposed to cultured odontoblast-like cells. Membrane damage and viability of cells as well as oxidative stress were evaluated. Residual HP/free radical diffusion was quantified, and bleaching effectiveness (ΔE) was assessed. Unbleached discs served as negative controls. RESULTS: The addition of HRP to the 35% HP bleaching gel enhanced the release of free radicals in comparison with plain HP gel. The 35% HP-mediated cytotoxicity significantly decreased with HRP in the bleaching gel and was associated with reduced HP/free radical diffusion through the enamel/dentin discs. ΔE values increased every bleaching session for HRP-containing gel relative to positive control, accelerating the whitening outcome. CONCLUSION: The enzymatic activation of a 35% HP bleaching gel with HRP accelerated HP degradation mediated by intensification of free radical release. This effect optimized whitening outcome as well as minimized residual HP and free radical diffusion through enamel and dentin, decreasing the harmful effects on odontoblast-like cells.

Effects of homolactic bacterial inoculant on the performance of lactating dairy cows.

The objective of this experiment was to determine the effect of applying a homofermentative bacterial inoculant to corn silage on the performance of dairy cows. After harvesting, corn forage was treated with nothing (CON) or with an inoculant containing a mixture of Lactococcus lactis, Lactobacillus plantarum, and Enterococcus faecium at 1.5 × 105 cfu/g of fresh forage (MC; SiloSolve MC, Chr. Hansen A/S, Hørsholm, Denmark). After 186 d of storage in Ag-Bags (A Miller-St. Nazianz Inc., St. Nazianz, WI), silages were fed as part of a total mixed ration containing 55% concentrates, 10% alfalfa hay, and 35% CON or MC corn silage. Sixty early-lactation Holstein dairy cows (30 multiparous and 30 primiparous) housed in a freestall barn with Calan gates (American Calan Inc., Northwood, NH) were assigned to the dietary treatments from 20 to 100 d in milk. Silage inoculated with MC had a more homofermentative pattern evidenced by greater lactic acid concentration (3.83 vs. 4.48% of DM) and lower concentrations of acetic (2.34 vs. 1.68% of DM) and propionic (0.37 vs. 0.10% of DM) acids and ammonia (9.11 vs. 7.82% of N) for CON and MC, respectively. Dry matter intake (23.1 vs. 23.2 kg/d) did not differ among treatments, but the MC silage had greater apparent digestibility of DM (68.8 vs. 70.8%), which led to greater yields of milk (37.7 vs. 38.5 kg/d), fat-corrected milk (37.6 vs. 38.4 kg/d), milk fat (1.30 vs. 1.33 kg/d), and lactose (1.83 vs. 1.92 kg/d) for CON and MC cows, respectively. Milk from cows fed MC silage had higher lactose (4.86 vs. 4.93%), lower protein (2.93 vs. 2.83%), and similar contents of fat (3.47 vs. 3.44%) compared with CON cows. Feed efficiency (fat-corrected milk/dry matter intake) was not affected by treatment (1.69 vs. 1.72 for CON and MC, respectively). Inoculation of corn silage with the homofermentative inoculant increased digestibility of the total mixed ration and increased milk yield by lactating dairy cows.

Influence of enamel/dentin thickness on the toxic and esthetic effects of experimental in-office bleaching protocols.

OBJECTIVES: This paper aims to assess the whitening effectiveness and toxicity of tooth-bleaching protocols applied to enamel/dentin disks simulating mandibular incisors (ICs) and premolars (PMs). MATERIALS AND METHODS: A 10% hydrogen peroxide (H2O2) gel was applied for 3 × 15, 1 × 15, or 1 × 5 min to enamel/dentin disks simulating mandibular ICs and PMs, and the trans-enamel and trans-dentinal diffusion products were applied to human dental pulp cells (1 h). Professional therapy (35% H2O2-3 × 15 min) was used as positive control, and non-bleached samples were used as negative control. Cell viability and morphology, oxidative stress generation, and odontoblastic marker expression were assessed. The H2O2 diffusion and enamel color change (ΔE) were also analyzed. RESULTS: The 10% H2O2 gel induced significant cell viability reduction only when applied 3 × 15 min, with the intensity of oxidative stress and down-regulation of odontoblastic markers being higher in the IC group. The other experimental bleaching protocols caused slight alterations regarding the cell parameters evaluated, with intensity being related to enamel/dentin thickness. These effects were also correlated with higher H2O2 diffusion in the IC group. ΔE values similar as positive control were found for the 10% 3 × 15 and 1 × 15 protocols on IC group, after 4 and 6 sessions. CONCLUSION: Application of a 10% H2O2 bleaching gel for 15 or 45 min to thin dental substrate significantly minimizes cell toxicity in comparison with highly concentrated gels associated with similar esthetic outcomes by increasing the number of bleaching sessions. CLINICAL RELEVANCE: Bleaching gels with 10% H2O2 applied in small teeth for short periods may be an interesting alternative to obtain whitening effectiveness without causing toxicity to pulp cells, which may be able to reduce the tooth hypersensitivity claimed by patients.

Development of an oral mucosa equivalent using a porcine dermal matrix.

We evaluated the suitability of a porcine acellular dermal matrix for the development of a 3-dimensional oral mucosal equivalent using an ex vivo-produced oral mucosal equivalent (EVPOME). Oral keratinocytes were seeded in a submerged model, and then in an air-liquid interphase model, using Transwell® inserts. EVPOME showed good cell viability and increased glucose consumption over time. Histological evaluation showed that stratified differentiated epithelium had formed in all matrices.

Transdentinal photobiostimulation of stem cells from human exfoliated primary teeth.

AIM: To evaluate the effects of infrared light-emitting diode (LED) irradiation on stem cells from human exfoliated deciduous teeth (SHEDs). METHODOLOGY: Exfoliated primary teeth were obtained (n = 3), and SHEDs obtained from the teeth were seeded on the pulpal surface of 0.2-mm-thick dentine discs produced from permanent molars. The cells were incubated for 24 h by placing the discs in plain Dulbecco's modified Eagle's medium (DMEM). The DMEM was then replaced with new culture medium formulated for odontoblast differentiation. After 12 h in the second medium, SHEDs were irradiated through the dentine discs using an infrared LED (850 nm) with a power density of 80 mW cm-2 . Energy doses (EDs) delivered to the occlusal surface of the dentine discs were 0 (control), 2 and 4 J cm-2 (n = 6). Subsequent tests were performed 72 h after irradiation. These tests included cell viability (MTT), alkaline phosphatase activity (ALP), total protein production (TP), scanning electron microscopy (SEM), as well as gene expression for ALP, Col I, DSPP and DMP-1. Data were analysed using Kruskal-Wallis and Mann-Whitney t-tests (α = 0.05). RESULTS: Both EDs (2 and 4 J cm-2 ) significantly increased cell viability and ALP activity. For TP, ALP and Col I gene expression, only the 4 J cm-2 group had significantly higher values compared to the control group. Cell morphology was not affected by irradiation. CONCLUSION: Infrared LED irradiation was capable of biostimulating SHEDs through a 0.2 mm thickness of dentine, especially at the 4 J cm-2 level.

Nutritional deprivation and LPS exposure as feasible methods for induction of cellular - A methodology to validate for vitro photobiomodulation studies.

Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10µg/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies.

In vivo photodynamic inactivation of Candida albicans using chloro-aluminum phthalocyanine.

This study evaluated the photoinactivation of Candida albicans in a murine model of oral candidiasis using chloro-aluminum phthalocyanine (ClAlP) encapsulated in cationic nanoemulsions (NE) and chloro-aluminum phthalocyanine (ClAlP) diluted in DMSO (DMSO) as photosensitizer (PS). Seventy-five 6-week-old female Swiss mice were immunosuppressed and inoculated with C. albicans to induce oral candidiasis. PDT was performed on the tongue by the application of the photosensitizers and LED light (100 J cm(-2) -660 nm). Twenty-four hours and 7 days after treatments, microbiological evaluation was carried out by recovering C. albicans from the tongue of animals (CFU ml(-1) ). Then, mice were sacrificed and the tongues were surgically removed for histological and biomolecular analysis of pro- and anti-inflammatory cytokines. Data were analyzed by ANOVA followed by Tukey's post hoc test. ClAlP-NE-mediated PDT reduced 2.26 log10 of C. albicans recovered from the tongue when compared with the control group (P-L-) (P < 0.05). PDT did not promote adverse effects on the tongue tissue. Seven days after treatment, all animals were completely healthy. In summary, PDT mediated by chloro-aluminum phthalocyanine entrapped in cationic nanoemulsions was effective in reducing C. albicans recovered from the oral lesions of immunocompromised mice.

Influence of Restoration Type on the Cytotoxicity of a 35% Hydrogen Peroxide Bleaching Gel.

OBJECTIVES: The tooth/restoration interface may act as a pathway for hydrogen peroxide (H2O2) diffusion into the pulp chamber. Therefore, the influence of resin-modified glass ionomer cement (RMGIC) and resin composite simulated restorations on the cytotoxicity of an in-office bleaching gel was assessed in vitro. MATERIALS AND METHODS: Cavities in enamel/dentin discs restored with RMGIC Vitremer (3M ESPE) or Single Bond/Filtek Z350 (3M ESPE) resin composite (RC) were subjected or not subjected to hydrolytic degradation (HD). A 35%-H2O2 bleaching gel was applied to simulated restored and nonrestored enamel surfaces, and culture medium in contact with the dentin substrate (extract) was collected and applied to MDPC-23 cells. Nonrestored discs subjected or not subjected to bleaching were used as positive and negative controls, respectively. Cell viability, oxidative stress, interleukin (IL)-1ß expression, alkaline phosphatase (ALP) activity, and mineralized nodule deposition were evaluated. The H2O2 in the extracts was quantified. Data were subjected to statistical analysis. RESULTS: Higher oxidative stress associated with reduced cell viability, ALP activity, and mineralized nodule deposition was observed for all bleached groups compared with the negative control group. The RMGIC/HD group, which presented the highest H2O2 diffusion, had the lowest values of cell viability, ALP activity, and mineralized nodule deposition, as well as significantly increased IL-1ß expression. CONCLUSIONS: Dental cavities restored with the RMGIC subjected to hydrolytic degradation allowed for more intense diffusion of H2O2 into the pulp chamber, intensifying the toxicity of a 35%-H2O2 bleaching gel to pulp cells.

Indirect cytocompatibility of a low-concentration hydrogen peroxide bleaching gel to odontoblast-like cells.

AIM: To assess the initial cytotoxicity and the late phenotype marker expression of odontoblast-like cells (MDPC-23) subjected to less aggressive in-office bleaching therapies. METHODOLOGY: A 17.5% hydrogen peroxide (H2O2) gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to trans-wells positioned over cultured MDPC-23 cells. No treatment was performed on the negative control. Immediately after bleaching, the cell viability, gene expression of inflammatory mediators and quantification of H2O2 diffusion were evaluated. The ALP activity, DSPP and DMP-1 gene expression and mineralized nodule deposition (MND) were assessed at 7, 14 or 21 days post-bleaching and analysed statistically with Mann-Whitney U-tests (α = 5%). RESULTS: H2O2 diffusion, proportional to treatment time, was observed in all bleached groups. Reductions of approximately 31%, 21% and 13% in cell viability were observed for the 45-, 15- and 5-min groups, respectively. This reduction was significant (P < 0.05) for the 45- and 15-min groups, which also presented significant (P < 0.05) over-expression of inflammatory mediators. The 45-min group was associated with significant (P < 0.05) reductions in DMP-1/DSPP expression at all periods, relative to control. The ALP activity and MND were reduced only in initial periods. The 15-min group had less intense reduction of all markers, with no difference to control at 21 days. CONCLUSIONS: The 17.5% H2O2 applied to tooth specimens for 5 min caused no alteration in the odontoblast-like cells. When this gel was applied for 45 or 15 min, a slight cytotoxicity, associated with alterations in phenotypic markers, was observed. However, cells were able to recover their functions up to 21 days post-bleaching.

Histopathological hoof laminar changes in horses with pituitary pars Intermedia adenoma: cases report / Alterações histopatológicas do tecido laminar do casco em cavalos com adenoma da pars intermedia da hipófise: relato de casos

Laminitis in horses is often associated with endocrine disorders, especially the pituitary pars intermedia dysfunction (PPID) in older animals. Morphologic exams of the laminar tissue of the hoof were performed in two horses with suspected PPID, with no clinical signs of laminitis. Changes compatible with laminitis of endocrine origin were observed, such as rounding of the nuclei of the basal cells, thinning and stretching of the secondary epidermal laminae and tissue proliferation. PPID horses with no clinical signs of laminitis may be affected by lesions of the laminar tissue of the hoof that compromise the integrity of the dermal-epidermal junction and may develop clinical symptoms of the disease. It has been suggested that the development stage of endocrine laminitis is longer, but further studies should be conducted to confirm it.

A laminite em cavalos está frequentemente associada a distúrbios endócrinos, como a disfunção da pars intermedia da pituitária (PPID) em animais mais velhos. Exames morfológicos do tecido laminar do casco foram realizados em dois cavalos com suspeita de PPID após o óbito, os quais não apresentaram sinais clínicos de laminite. Alterações compatíveis com a laminite de origem endócrina foram observadas, como arredondamento da núcleo das células basais, alongamento e afilamento das lâminas epidérmicas secundárias e proliferação tecidual. Cavalos com PPID sem sinais clínicos de laminite podem estar acometidos por lesões do tecido laminar do casco que comprometam a integridade das interdigitações dérmico-epidérmicas e podem desenvolver a sintomatologia clínica da doença. Sugere-se que o período de desenvolvimento da laminite endócrina seja mais longo, porém estudos adicionais devem ser realizados para confirmar essa hipótese.

Effect of LPS treatment on the viability and chemokine synthesis by epithelial cells and gingival fibroblasts.

OBJECTIVE: Several local factors can affect the wound-healing process, delaying its progression and postponing tissue homeostasis. It is known that local inflammation is related to wound healing; however, the maintenance of the inflammatory reaction can impair the proliferation and migration of oral mucosal cells. The aim of this study was to evaluate the viability and chemokine expression of epithelial cells and gingival fibroblasts exposed to long-term lipopolysaccharide (LPS) treatment. DESIGN: Epithelial cells (HaCaT, Cell Lines Service, 300493) and human gingival fibroblasts (HGFs) were seeded (1×10(5) cells/well) in 24-well plates and incubated for 24h. To simulate the responses of cells to a local chronic oral mucosal inflammation, we added LPS of Escherichia coli (10 µg/ml) to Dulbecco's modified Eagle's medium (DMEM), kept in contact with fibroblasts and epithelial cells for 24, 48, and 72h. Then the cells were assessed for viability (alamarBlue assay), number (trypan blue assay), and expression of CCL2 and CCL5 inflammatory chemokines (enzyme-linked immunosorbent assay (ELISA)). Data were statistically analyzed by nonparametric Kruskal-Wallis and Mann-Whitney tests at a significance level of 5%. RESULTS: Cell treatment with LPS caused significant decrease in viability for both cell lines. No time-dependent effect was observed for epithelial cells. However, reduction in fibroblast viability was greater at 48 and 72 h. CCL2 and CCL5 synthesis was significantly increased for both LPS-treated cells, and this expression decreased with time. CONCLUSION: The maintenance of an inflammatory cell stimulus by LPS decreases the number and viability of cultured oral mucosal cells, which may be related to delayed wound healing.

Increased Durability of Resin-Dentin Bonds Following Cross-Linking Treatment.

OBJECTIVES: This study evaluated the long-term effect of carbodiimide treatments of acid-etched dentin on resin-dentin bond strength of a simplified etch-and-rinse adhesive system. METHODS: Forty-eight sound third molars were divided into three groups (n=16) according to the dentin treatment: G1: deionized water; G2: 0.5 mol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied for 30 seconds; and G3: 0.5 mol/L EDC applied for 60 seconds. Flat dentin surfaces were produced, etched with 37% phosphoric acid for 15 seconds, and then treated with deionized water for 60 seconds or with 0.5 mol/L EDC for 30 or 60 seconds prior to the application of Single Bond 2. Crowns were restored with resin composite, and beam specimens were prepared for microtensile testing. The beams from each group were tested 24 hours or 6 or 12 months after the adhesive procedures. One slab from each tooth was prepared and analyzed for nanoleakage. Bond strength (MPa) data were submitted to analysis of variance and Tukey test (α=0.05). RESULTS: The treatment of dentin with 0.5 mol/L EDC for 30 seconds (24.1±6.2 MPa) and 60 seconds (25.5±5.1 MPa) did not negatively affect the immediate bond strength of Single Bond 2 when compared to the control group (24.6±7.3 MPa). Additionally, EDC prevented resin-dentin bond degradation after 12 months in artificial saliva for both periods of treatment. An increased accumulation of silver ions was seen for the control group over time, while a much lower amount of silver grains was observed for the EDC-treated groups. CONCLUSIONS: 0.5 mol/L EDC was able to prevent resin-dentin bond degradation after 12 months, especially when applied for 60 seconds.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells.

OBJECTIVES: To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. METHODS: Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm(2)) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney's tests (p < 0.05). RESULTS: Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. SIGNIFICANCE: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.

Transdentinal cytotoxicity of carbodiimide (EDC) and glutaraldehyde on odontoblast-like cells.

OBJECTIVE: To evaluate the transdentinal cytotoxicity of three different concentrations of carbodiimide (EDC) or 5% glutaraldehyde (GA) on MDPC-23 cells. METHODS: Seventy 0.4-mm-thick dentin disks obtained from human molars were adapted to artificial pulp chambers. MDPC-23 cells were seeded on the pulpal surface of the disks. After 48 hours, the occlusal dentin was acid-etched and treated for 60 seconds with one of the following solutions (n=10): no treatment (negative control); 0.1 M, 0.3 M, or 0.5 M EDC; 5% GA; Sorensen buffer; or 29% hydrogen peroxide (positive control). Cell viability and morphology were assessed by methyltetrazolium assay and scanning electron microscopy (SEM), respectively. The eluates were collected after the treatments and applied on MDPC-23 seeded in a 24-well plate to analyze cell death, total protein (TP), and collagen production. The last two tests were performed 24 hours and seven days after the challenge. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: EDC at all test concentrations did not reduce cell viability, while 5% GA did increase cell metabolism. Cell death by necrosis was not elicited by EDC or 5% GA. At the 24-hour period, 0.3 M and 0.5 M EDC reduced TP production by 18% and 36.8%, respectively. At seven days, increased TP production was observed in all groups. Collagen production at the 24-hour period was reduced when 0.5 M EDC was used. After seven days, no difference was observed among the groups. SEM showed no alteration in cell morphology or number, except in the hydrogen peroxide group. CONCLUSIONS: Treatment of acid-etched dentin with EDC or GA did not cause transdentinal cytotoxic effects on odontoblast-like cells.

Transdentinal cell photobiomodulation using different wavelengths.

OBJECTIVE: The aim of this study was to investigate the effects of transdentinal irradiation with different light-emitting diode (LED) parameters on odontoblast-like cells (MDPC-23). METHODS AND MATERIALS: Human dentin discs (0.2 mm thick) were obtained, and cells were seeded on their pulp surfaces with complete culture medium (Dulbecco modified Eagle medium). Discs were irradiated from the occlusal surfaces with LED at different wavelengths (450, 630, and 840 nm) and energy densities (0, 4, and 25 J/cm(2)). Cell viability (methyltetrazolium assay), alkaline phosphatase activity (ALP), total protein synthesis (TP), and cell morphology (scanning electron microscopy) were evaluated. Gene expression of collagen type I (Col-I) was analyzed by quantitative polymerase chain reaction (PCR). Data were analyzed by the Mann-Whitney test with a 5% significance level. RESULTS: Higher cell viability (21.8%) occurred when the cells were irradiated with 630 nm LED at 25 J/cm(2). Concerning TP, no statistically significant difference was observed between irradiated and control groups. A significant increase in ALP activity was observed for all tested LED parameters, except for 450 nm at 4 J/cm(2). Quantitative PCR showed a higher expression of Col-I by the cells subjected to infrared LED irradiation at 4 J/cm(2). More attached cells were observed on dentin discs subjected to irradiation at 25 J/cm(2) than at 4 J/cm(2). CONCLUSION: The infrared LED irradiation at an energy density of 4 J/cm(2) and red LED at an energy density of 25 J/cm(2) were the most effective parameters for transdentinal photobiomodulation of cultured odontoblast-like cells.

Effects of feeding corn silage inoculated with microbial additives on the ruminal fermentation, microbial protein yield, and growth performance of lambs.

This study aimed to examine the effects of feeding corn silage inoculated without or with either Lactobacillus buchneri (LB) alone or a combination of LB and Lactobacillus plantarum (LBLP) on the apparent digestibility, ruminal fermentation, microbial protein synthesis, and growth performance of lambs. Thirty Santa Inês×Dorper crossbred intact males lambs weighing 20.4±3.8 kg were blocked by weight into 10 groups. Lambs in each group were randomly assigned to 1 of the following 3 dietary treatments: untreated (Control), LB, and LBLP silage. Lambs were fed experimental diets for 61 d. The apparent digestibility was indirectly estimated from indigestible NDF measured on d 57 to 59. Spot urine samples were collected from all animals on d 59 to estimate microbial protein synthesis. Lambs were slaughtered for carcass evaluation on d 61 when they weighed 32.4±5.2 kg. Six additional ruminally cannulated Santa Inês×Dorper crossbred wethers weighing 40.5±1.8 kg were used to examine dietary effects on ruminal fermentation. Average daily gain was increased when lambs were fed LBLP silage (P<0.05) but not LB silage. The LBLP silage had the highest (P<0.05) lactic acid concentration and both inoculated silages had greater acetic acid concentrations than the Control silage (P<0.05). Inoculation of corn silage increased intakes of DM, OM, CP, NDF, total carbohydrate (CHO), and GE by the lambs but decreased digestibility of DM, OM, CP, total and nonstructural carbohydrates, and concentration of GE and ME. (P<0.05). Nevertheless, lambs fed inoculated silages had greater microbial N supply than those on the Control treatment (P<0.05). The acetate to propionate ratio was lower in ruminal fluid of wethers in LBLP treatment than LB and Control treatment (P<0.05) and ruminal pH tended to be greater in LB lambs than in LBLP and Control wethers (P<0.10). Finally, the inoculation with both bacteria combined enhanced the silage fermentation. The intakes of DM, OM, CP, NDF, and GE were improved in the lambs fed corn silage inoculated with L. buchneri alone or combined with L. plantarum. The microbial N supply was enhanced in the lambs fed corn silage inoculated with L. buchneri. The inoculation of L. buchneri combined with L. plantarum reduced the acetate to propionate ratio in ruminal fluid and improved the ADG of lambs.

Comparison of the effects of PRKAR1A and PRKAR2B depletion on signaling pathways, cell growth, and cell cycle control of adrenocortical cells.

The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression.

Effects of low-level laser therapy on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid.

Low-level laser therapy (LLLT) has been indicated as an adjuvant therapy for bisphosphonate-induced osteonecrosis. However, the effects of LLLT on bisphosphonate-treated cells are not yet clear. This study evaluated the effects of LLLT on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid (ZA). Cells were exposed to ZA at 5µM for 48h. Irradiation was performed using a laser diode prototype (LaserTABLE, InGaAsP; 780nm±3nm, 25mW) at 0.5 or 3J/cm(2), three times every 24h. Cell proliferation and apoptosis were evaluated by fluorescence microscopy. Data were analyzed by Mann-Whitney test at the 5% level of significance. ZA decreased cell proliferation to 47.62% (interquartile range (IQR) 23.80-57.14%; P=0.007) and increased apoptosis of gingival fibroblasts to 27.7% (IQR 20.9-33.4%; P=0.0001). LLLT increased cell proliferation compared with non-irradiated cells, at 0.5J/cm(2) (57.14%, IQR 57.14-71.43%; P=0.003) and at 3J/cm(2) (76.19%, IQR 61.90-76.19%; P=0.0001), but did not increase cell proliferation in ZA-treated cells. Irradiated fibroblasts presented lower apoptosis rates than the ZA-treated cells, but apoptosis was no different in ZA-treated cells compared to those that were ZA-treated and also irradiated.

Infrared LED irradiation photobiomodulation of oxidative stress in human dental pulp cells.

AIM: To investigate the effect of infrared light-emitting diode (LED) irradiation on the oxidative stress induced in human dental pulp cells (HDPCs) by lipopolysaccharide (LPS). METHODOLOGY: Human dental pulp cells (HDPCs) were harvested from sound primary teeth that were near exfoliation. Cells were seeded (10(5)  cells cm(-2) ) using α-MEM supplemented with 10% FBS and after 24 h, were placed in contact with LPS (10 µg mL(-1) of culture medium). Immediately afterwards, HDPCs were subjected to a single irradiation with an infrared LED (855 nm) delivering different doses of energy (0, 2, 4, 8, 15 or 30 J cm(-2) ). For each dose, there was a control group without LPS application. Twenty-four hours after irradiation, groups were tested for nitric oxide (NO) quantification, cell viability (MTT assay) and qualitative assessment of reactive oxygen species (ROS). Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α = 0.05). RESULTS: Lipopolysaccharide (LPS)-induced stress resulted in significant increase in NO production by HDPC without causing damage to cell respiratory metabolism. Irrespective of energy dose delivered, NO production was significantly reduced when LPS-stressed cells were irradiated with infrared LED (2 J cm(-2) , P = 0.003; 95% CI = 5.84-27.71; 4 J cm(-2) , P = 0.001; 95% CI = 7.52-26.39; 8 J cm(-2) , P = 0.0195; 95% CI = -2.86-16.01; 15 J cm(-2) , P = 0.0001; 95% CI = 12.10-30.96; 30 J cm(-2) , P = 0.007; 95% CI = 5.84-24.71). The highest decrease in NO production was observed when 15 J cm(-2) was delivered to cells. Infrared LED irradiation resulted in a decrease in ROS production, whilst HDPC metabolism was not significantly affected. CONCLUSION: Biomodulation of oxidative stress of HPDC can be achieved by irradiation with a single dose of infrared LED. Within the range investigated, 15 J cm(-2) resulted in the least production of NO.