Characterization of levan produced by a Paenibacillus sp. isolated from Brazilian crude oil.

A levan-type fructooligosaccharide was produced by a Paenibacillus strain isolated from Brazilian crude oil, the purity of which was 98.5% after precipitation with ethanol and dialysis. Characterization by FTIR, NMR spectroscopy, GC-FID and ESI-MS revealed that it is a mixture of linear ß(2 â†’ 6) fructosyl polymers with average degree of polymerization (DP) of 18 and branching ratio of 20. Morphological structure and physicochemical properties were investigated to assess levan microstructure, degradation temperature and thermomechanical features. Thermal Gravimetric Analysis highlighted degradation temperature of 218 °C, Differential Scanning Calorimetry (DSC) glass transition at 81.47 °C, and Dynamic Mechanical Analysis three frequency-dependent transition peaks. These peaks, corresponding to a first thermomechanical transition event at 86.60 °C related to the DSC endothermic event, a second at 170.9 °C and a third at 185.2 °C, were attributed to different glass transition temperatures of oligo and polyfructans with different DP. Levan showed high morphological versatility and technological potential for the food, nutraceutical, and pharmaceutical industries.

Bromelain immobilization in cellulose triacetate nanofiber membranes from sugarcane bagasse by electrospinning technique.

Cellulose triacetate (CTAB) synthesized by cellulose extracted from sugarcane bagasse, and commercial cellulose acetate (CA) were used to produce nanofiber membranes contained bromelain by electrospinning technique. About 1.3 g of cellulose acetate per gram of bagasse were obtained, and both CTAB and CA was characterized by analysis of Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC). The nanofiber membranes were produced by electrospinning process testing the following conditions: voltage 25 kV, flow rate 4 mL/h and distance 10 cm, using acetone/ dimethylformamide (DMF) (85:15 m/ m) to 15% cellulose triacetate (70% CA + 30% CTAB) or CA solutions. Scanning Electron Microscopy (SEM) was used to nanofiber membranes characterization. Bromelain was immobilized on the nanofiber membranes by crosslinking with glutaraldehyde and directly in the electrospinning step, the highest activity recovery was about 675% and in vitro controlled release tests were performed to semi-quantitatively evaluate the release of the enzyme bromelain thus demonstrating complete release process in 3 days.

Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers.

Bone demineralization with citric acid enhances adhesion and spreading of preosteoblasts.

BACKGROUND: Previous studies have demonstrated that bone demineralization can improve consolidation in bone grafts. The biologic mechanisms underlying this phenomenon remain unclear. METHODS: Twelve adult male guinea pigs were used in this experiment. Forty-five bone samples removed from the calvaria of nine animals were divided in groups (n = 9) according to the time of demineralization with citric acid (50%, pH 1): 15, 30, 90, and 180 seconds and non-demineralized samples (control). Preosteoblasts (MC3T3-E1) were cultured on the bone samples for 24, 48, and 72 hours (n = 3). Fifteen samples removed from the remaining three animals were analyzed by scanning electron microscopy/energy dispersive spectrometry (SEM/EDS) after demineralization (n = 3). RESULTS: The number of preosteoblasts increased significantly with time in all groups. The bone surface area covered by these cells increased with time, except in the control group. Intragroup differences occurred between 24 and 72 hours (P < 0.05). Samples demineralized for 30 seconds showed greater area covered by preosteoblast cells than for the other times of demineralization in all periods of cell culture (P < 0.05) without a statistically significant difference compared with 15 seconds. SEM/EDS showed diminished content of calcium (Ca) after 15 seconds of demineralization, but the Ca content increased after 180 seconds of demineralization (P < 0.05). The phosphorus (P) amount increased significantly only after 30 seconds of demineralization (P < 0.5). The sulfur (S) content was increased in demineralized samples in relation to non-demineralized ones, reaching the highest level after 90 seconds, when the difference became significant in relation to all the other times of demineralization (P < 0.05). Magnesium (Mg) content did not differ significantly between demineralized and non-demineralized samples. CONCLUSIONS: Bone surfaces demineralized for 30 seconds increased the spreading of preosteoblasts as well as the surface area covered by these cells. Bone demineralization deserves to be studied in periodontal and maxillofacial regenerative procedures.

Fluoride modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities.

This study evaluated the influence of fluoride on cell viability and activity of matrix metalloproteinases (MMP) -2 and -9 secreted by preosteoblasts. Preosteoblasts (MC3T3-E1 murine cell line) were cultured in MEM medium supplement with 10% Fetal Bovine Serum (FBS) and nucleosides/ribonucleosides without ascorbic acid. Adherent cells were treated with different concentrations of F (as sodium fluoride-NaF) in medium (5 x 10(-6) M, 10(-5) M, 10(-4) M and 10(-3) M) for 24, 48, 72 and 96 h at 37ºC, 5% CO(2). Control cells were cultivated in MEM only. After each period, preosteoblast viability was assessed by MTT assay. MMP-2 and -9 activities were performed by gel zymography. Also, alkaline phosphatase (ALP) activity was quantified by colorimetry in all experimental groups. It was shown that cultured cells with the highest dose of F (10(-3) M) for 96 h decreased preosteoblast viability while lower doses of F did not alter it, when compared to untreated cells. No differences were observed in ALP activity among groups. Moreover, compared to control, the treatment of cells with F at low dose slightly increased MMP-2 and -9 activities after 24 h. It was concluded that F modulates preosteoblast viability in a dose-dependent manner and also may regulate extracellular matrix remodeling.

Fluoride modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities

This study evaluated the influence of fluoride on cell viability and activity of matrix metalloproteinases (MMP) -2 and -9 secreted by preosteoblasts. Preosteoblasts (MC3T3-E1 murine cell line) were cultured in MEM medium supplement with 10% Fetal Bovine Serum (FBS) and nucleosides/ribonucleosides without ascorbic acid. Adherent cells were treated with different concentrations of F (as sodium fluoride-NaF) in medium (5 x 10-6 M, 10-5 M, 10-4 M and 10-3 M) for 24, 48, 72 and 96 h at 37ºC, 5% CO2. Control cells were cultivated in MEM only. After each period, preosteoblast viability was assessed by MTT assay. MMP-2 and -9 activities were performed by gel zymography. Also, alkaline phosphatase (ALP) activity was quantified by colorimetry in all experimental groups. It was shown that cultured cells with the highest dose of F (10-3 M) for 96 h decreased preosteoblast viability while lower doses of F did not alter it, when compared to untreated cells. No differences were observed in ALP activity among groups. Moreover, compared to control, the treatment of cells with F at low dose slightly increased MMP-2 and -9 activities after 24 h. It was concluded that F modulates preosteoblast viability in a dose-dependent manner and also may regulate extracellular matrix remodeling.

Neste estudo, buscou-se avaliar a influência do fluoreto (F) na viabilidade celular e atividade das metaloproteinases de matriz (MMP) -2 e -9 secretado pelos pré-osteoblastos. Pré-osteoblastos (linhagem celular MC3T3-E1 murina) foram cultivados em meio MEM suplementado com 10% de soro fetal bovino (FBS) e nucleosídeos/ribonucleosídeos sem ácido ascórbico. Células aderidas foram tratadas com diferentes concentrações de F (na forma de fluoreto de sódio-NaF) em meio (5 x 10-6 M, 10-5 M, 10-4 M e 10-3 M) por 24, 48, 72 e 96 h a 37ºC, 5 % de CO2. Células do grupo controle foram cultivadas apenas em MEM. Após cada período, a viabilidade dos pré-osteoblastos foi avaliada por MTT. A atividade das MMP-2 e -9 foram analisadas pela zimografia. Além disso, a atividade da fosfatase alcalina (FA) foi quantificada por colorimetria em todos os grupos experimentais. Foi demonstrado que as células cultivadas com a maior dose de F (10-3 M) no período de 96 h tiveram sua viabilidade comprometida, enquanto doses mais baixas de F não a alteraram significativamente, quando comparado com células não tratadas. Não foi observada diferença na atividade da FA entre os grupos. Além disso, o tratamento de células com F em baixas doses, comparado ao grupo controle, promoveu um pequeno aumento da atividade das MMP-2 e -9 após 24 h. Pode-se concluir que o F modula a viabilidade de pré-osteoblastos de uma maneira dose-dependente e também pode regular a remodelação da matriz extracelular.