In vitro effect of curcumin-mediated antimicrobial photodynamic therapy on fibroblasts: viability and cell signaling for apoptosis.

Although it was demonstrated that curcumin-mediated antimicrobial photodynamic therapy (aPDT) is effective for reducing the viability of microbial cells and the vitality of oral biofilms, the cytotoxicity of this therapeutic approach for host cells has not been yet elucidated. Hence, the aim of this study was to evaluate the cytotoxicity and apoptotic effects of curcumin-mediated aPDT on mouse fibroblasts. Cells were treated with 0.6 or 6 µmol.L-1 curcumin combined with 0.075 or 7.5 J.cm-2 LED at 455 nm. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while quantitative reverse transcriptase-PCR (qRT-PCR) was used to assess the expression of Bax, Bad, Bcl-2, VDAC-1, cytochrome C, and Fas-L genes for apoptosis. The differences between groups were detected by Kruskal-Wallis and post hoc Dunn's tests for MTT and CV assays and by ANOVA and post hoc Tukey test for qRT-PCR (P < 0.05). The effect of 0.6 µmol.L-1 curcumin plus 0.075 J.cm-2 LED (minimum parameter) did not differ statistically from control group; however, the combination of 0.6 µmol.L-1 curcumin plus 7.5 J.cm-2 LED reduced viable cells in 34%, while the combinations of 6 µmol.L-1 curcumin plus 0.075 and 7.5 J.cm-2 LED reduced viable cells in 47% and 99%, respectively. aPDT increased significantly the relative expression of Bax/Bcl-2, cytochrome C, VDAC-1, and Fas-L genes, without influence on the ratio Bad/Bcl-2. Therefore, curcumin-mediated aPDT activated Bcl-2 apoptosis signaling pathways in mouse fibroblasts regarding present conditions, reducing the viability of cells with the increase of curcumin concentrations and light energies.

"Aroeira" (Myracrodruon urundeuva) methanol extract: the relationship between chemical compounds and cellular effects.

CONTEXT: "Aroeira" [Myracrodruon urundeuva Allemão (Anacardiaceae)] is a tree whose leaves have been studied for therapeutic purposes in medicine and dentistry. OBJECTIVE: The study chemically identifies the leaf extract of aroeira and determines its effect on human gingival fibroblasts. MATERIALS AND METHODS: An 80% methanol leave extract was obtained by maceration and chemically identified through flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MSn). Cytotoxicity of the aroeira's methanol extract was evaluated in lineage of fibroblasts. Adherent cells were treated with different concentrations of aroeira's methanol extract in the medium: 0.1, 1, 10, 100 and 1000 µg/mL. Control cells were cultivated in the medium only. Analyses were done at 24, 48, 72 and 96 h of culture by neutral red assay; and at 24, 48 and 96 h by crystal violet assay. RESULTS: FIA-ESI-IT-MS analysis determined the presence of compounds, for the first time in the species: quercetin-O-glucuronide and quercetin-O-deoxyhexose-O-glucose in the extract. On one hand, neutral red and crystal violet assay showed a reduction (to 50% up until 100%) of cellular viability of groups of 100 and 1000 µg/mL compared with control at 96 h (p < 0.05). On the other hand, lower concentrations (0.1; 1 and 10 µg/mL) of the extract were similar to that of the control at 96 h (p < 0.05), in general. CONCLUSIONS: In view of the results, we can conclude that the extract of aroeira presents tannins and flavonoids. Furthermore, the extract is capable of modulating the viability of human gingival fibroblasts according to its concentration.

Fluoride and trimetaphosphate association as a novel approach for remineralization and antiproteolytic activity in dentin tissue.

OBJECTIVE: The study evaluated the effect of solutions containing fluoride (F) and/or sodium trimetaphosphate (TMP) and F/TMP on the inhibition of MMP-2 and MMP-9, and on dentin remineralization in vitro. DESIGN: Bovine root dentin blocks were prepared, and caries-like lesions were induced in two thirds of the surface. Blocks were then randomly divided into 13 groups/solutions (n = 10): Placebo; 0.3 %, 1 % and 3 % NaOH-hydrolyzed TMP; 0.3 %, 1 % and 3 % TMP; 250, 500 and 1100 ppm F; 250 ppm F + 0.3 % TMP; 500 ppm F + 1 % TMP and 1100 ppm F + 3 % TMP. One third of each specimen was treated with the respective solutions in pH-cycling. The mineral concentration (gHAp × cm-3 × µm) was determined by computed X-ray microtomography, and data submitted to ANOVA and Student-Newman-Keuls' test (p < 0.05). The ability of the solutions to inhibit MMP-2 and MMP-9 activity was assessed by zymography. RESULTS: F/TMP association led to less mineral loss in the deeper region of the lesion and reduced the depth of lesions when compared to its counterpart without TMP (p < 0.001). 3 % TMP (hydrolyzed or not), 500 ppm F and 1100 ppm F completely inhibited MMP-2 activity, while for MMP-9 such effects were only achieved by treatment with 1100 ppm F + 3 % TMP. CONCLUSION: Treatment with 1100 ppm F + 3 % TMP fully inhibits the gelatinolytic activity of MMPs-2 and - 9 and shows greater remineralizing capacity in artificial caries lesions in dentin. However, hydrolyzing TMP does not improve its anti-proteolytic activity and its remineralizing capacity.

Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24pos cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24pos and CD24neg BMSCs identified a limited number of genes with increased expression in CD24pos cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24pos cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.

Residual decontamination chemical agents negatively affect adhesion and proliferation of osteoblast-like cells on implant surface.

PURPOSE: To investigate the influence of implant surface decontaminated and uncontaminated on osteoblast-like cell adhesion and proliferation MATERIALS AND METHODS: Commercially available implants of different brands and surface characteristics were selected: Biomet 3i® Nanotite (NT) and Osseotite (OT), Straumann® SLActive (SLA), and Neodent® Acqua Drive (ACQ) and Neoporos Drive CM (CM). Physical and chemical properties of the implants were investigated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and wettability analysis (WETT). Implants were previously contaminated with Aggregatibacter actinomycetemcomitans strains; after that, samples were decontaminated by different chemical methods. Decontaminated (test group; n = 15/type of implant) and uncontaminated (control group; n = 5/type of implant) samples were analyzed according to the number of human osteoblastic osteosarcoma cells (Saos-2) adhered on the implant surface after 24 h and 72 h in SEM images. RESULTS: ACQ was found to be highly hydrophilic, and NT was the most hydrophobic implant. Increased variation of Saos-2 cell adhesion and proliferation were observed on all test and control groups. Controversially, at the proliferation analysis in 72 h, CM implant was the only implant that showed no significant difference between test and group (p = 0.2833; Tukey's multiple comparisons test). NT implants showed the greater value of cell proliferation when compared with all types of implant surface (p = 0.0002; Tukey's multiple comparisons test). CONCLUSIONS: These findings suggest that decontaminated surfaces were able to impair the counting of osteoblast-like cell adhesion and proliferation.

Analysis of the physicochemical properties, cytotoxicity and volumetric changes of AH Plus, MTA Fillapex and TotalFill BC Sealer.

BACKGROUND: To evaluate the physicochemical properties and cytotoxicity of AH Plus, MTA Fillapex and TotalFill BC Sealer. Volumetric changes were also evaluating using micro-computed tomography (micro-CT). MATERIAL AND METHODS: Radiopacity and flow were evaluated in accordance with the ISO 6876, while setting time was evaluated in accordance with the ASTM- C266-08 specifications. The release of Ca2+ ions and pH were measured with spectrophotometer and pH meter, respectively, after different time intervals (1h, 3h, 24h, 72h, 168h, and 360h). Cytotoxicity was evaluated by MTT reduction assay to check 3T3 cells viability at 24, 48 and 72 hours. Volumetric change was evaluated by micro-CT, by using 30 acrylic teeth, filled with gutta-percha cones and the tested root canal sealer. The samples were evaluated after 168h, 360h and 720h of immersion in distilled water. Data were statistically analyzed by one-way ANOVA and Tukey test or by Kruskal-Wallis and Dunn tests (P<0.05). RESULTS: MTA Fillapex and TotalFill BC Sealer showed lower radiopacity than AH Plus (P<0.05). The MTA Fillapex showed the highest flow, while AH Plus showed the lowest flow (P<0.05). The initial and final setting time of AH Plus were lower than MTA Fillapex and TotalFill BC Sealer (P<0.05). In general, TotalFill BC Sealer presented higher Ca2+ ion release and pH than the other tested sealers. TotalFill BC Sealer also showed overall lower cytotoxicity when compared to the other sealers. Volumetric change of AH Plus and TotalFill BC Sealer was lower than MTA Fillapex (P<0.05). CONCLUSIONS: AH Plus, MTA Fillapex and TotalFill BC Sealer showed slight differences in the physicochemical properties and cytotoxicity, but all suitable for an endodontic sealer. However, AH Plus and TotalFill BC Sealer showed low volumetric changes when compared to MTA Fillapex. Key words:Calcium silicate, cytotoxicity, physicochemical properties, micro computed tomography.

An extract from Myracrodruon urundeuva inhibits matrix mineralization in human osteoblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Phytotherapy based on plant-derived compounds is an alternative medicinal strategy for the relief of symptoms and the curing of diseases. The leaves of Myracrodruon urundeuva a medicinal plant also known as "aroeira", has been used in traditional medicine as healing, antiulcer and anti-inflammatory to treat skeletal diseases in Brazil, but its role in bone cell toxicity, as well as in bone formation, remains to be established. AIM OF THE STUDY: We sought to determine the in vitro osteogenic effects of a hydroalcoholic M. urundeuva leaves extract in primary human osteoblasts. MATERIALS AND METHODS: Cell viability, reactive oxygen species (ROS) production, alkaline phosphatase (ALP) activity and matrix mineralization were evaluated by MTT assay, DCFH-DA probe, colorimetric-based enzymatic assay and Alizarin Red-staining, respectively. Besides, the matrix metalloproteinase (MMP)-2 and progressive ankylosis protein homolog (ANKH) gene expression were determined by real-time RT-qPCR and MMP-2 activity by zymography. RESULTS: Exposure of osteoblasts to M. urundeuva extract significantly decreased viability and increased reactive oxygen species (ROS) production, regardless of the extract concentration. The M. urundeuva extract at 10 µg/mL also downregulated matrix metalloproteinase (MMP)-2, while upregulating progressive ankylosis protein homolog (ANKH) gene expression. By contrast, the MMP-2 activity was unchanged. The M. urundeuva extract at 10 µg/mL also reduced alkaline phosphatase (ALP) activity and mineralization. CONCLUSIONS: Overall, our findings suggest that the inhibition of osteogenic differentiation and matrix mineralization promoted by M. urundeuva may be due more to an increase in oxidative stress than to the modulation of MMP-2 and ANKH expression.

HOXA cluster gene expression during osteoblast differentiation involves epigenetic control.

The HOXA gene cluster is generally recognized as a pivotal mediator of positional identity in the skeletal system, expression of different orthologues conferring alternative locational phenotype of the vertebrate bone. Strikingly, however, the molecular mechanisms that regulate orthologue-specific expression of different HOXA cluster members in gestating osteoblasts remain largely obscure, but in analogy to the processes observed in acute lymphatic leukemia it is assumed that alternative methylation of HOXA promoter regions drives position specific expression patterns. In an effort to understand HOXA cluster gene expression in osteogenesis we characterize both expression and the epigenetic landscape of the HOXA gene cluster during in vitro osteoblast formation from mesenchymal precursors. We observe that osteoblast formation per se provokes strong upregulation of HOXA gene cluster expression, in particular of midcluster genes, and paradoxal downregulation of HOXA7 and HOXA10. These differences in expression appear related to promoter methylation. LnRNAs HOTAIR and HOTTIP, known to modulate HOXA expression, are also regulated by their promoter methylation processing, but do not correlate with HOXA cluster expression profile. We thus conclude that HOXA expression is profoundly regulated during osteoblast differentiation through canonical methylation-dependent mechanisms but not through the flanking lnRNAs.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

Avaliação do efeito antiproliferativo da apocinina e diapocinina em células de osteossarcoma humano / Evaluation of the antiproliferative effect of apocynin and diapocynin on human osteosarcoma cells

O osteossarcoma (OS) é o tumor maligno primário mais comum do tecido ósseo, caracterizado pela formação de osteócitos anormais. Apesar do avanço nas terapias convencionais (quimioterapia e retirada do tumor), essas não conseguem eliminar totalmente as células tumorais e impedir a progressão da doença. Recentemente, agentes derivados de fontes naturais ganharam considerável atenção por causa de sua segurança, eficácia e disponibilidade imediata. Nesse sentido, a apocinina, inibidor do complexo NADPH-oxidase, vem sendo estudada como agente antitumoral em alguns tipos de câncer como: pâncreas, próstata, pulmão e mama. Apocinina é um pró-fármaco e sua ação parece estar relacionada à sua conversão produzindo a diapocinina, a qual se mostrou mais efetiva do que a apocinina. Portanto, o objetivo desse estudo é avaliar, in vitro, o potencial antitumoral da apocinina e diapocinina em células de osteossarcoma humano. Para isso, foram utilizados osteoblastos humanos normais (HOb) e osteossarcoma humano imortalizadas (SaOS-2) tratados ou não com apocinina e diapocinina em diversas concentrações. Foram realizados os ensaios de viabilidade celular, alterações morfológicas, apoptose celular, produção de espécies reativas de oxigênio (EROs), formação de colônias, migração, invasão e expressão do fator indutor de hipóxia-1alfa (HIF-1). Também foram conduzidos ensaios para verificar a atividade de metaloproteinase de matriz (MMP) 2 e 9. Os resultados em SaOS-2 mostraram que o tratamento com apocinina nas concentrações de 1,5 e 3 mM; e diapocinina nas concentrações de 0,75 e 1,5 mM reduziram a viabilidade; aumentaram o número de células em apoptose e diminuíram a produção de EROs; sem causar danos às células HOb. Além disso, essas mesmas concentrações inibiram a migração e invasão celular; diminuíram a expressão de HIF-1; e reduziram a atividade de MMP-2 em SaOS-2. Considerando os resultados obtidos, concluímos que a apocinina e diapocinina podem atuar como possíveis moduladores de células tumorais, sendo que a diapocinina mostrou ser mais efetiva nos parâmetros testados.(AU)

Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, characterized by the formation of abnormal osteocytes. Despite advances in conventional therapies (chemotherapy and surgery) they cannot completely eliminate tumor cells and prevent the progression of the disease. Recently, agents derived from natural sources have achieved considerable attention because of their safety, efficacy and immediate availability of therapies. In this way, apocynin, an inhibitor of the NADPH-oxidase complex, has been studied as an antitumor agent in some types of cancer, such as pancreas, prostate, lung and breast. Apocynin is a prodrug and its action indicate to be related to its conversion to diapocynin, which has been shown to be more efficient than apocynin itself. Thus, the aim of this study is to evaluate, in vitro, the antitumor potential of apocynin and diapocynin in human osteosarcoma cells. For this, normal human osteoblasts (HOb) and immortalized human osteosarcoma cells (SaOS-2) were treated or no-treated with apocynin and diapocynin in various concentrations. Cell viability assay, morphological alterations, cellular apoptosis, reactive oxygen species (ROS) production, colony formation, migration, invasion and expression of hypoxia-inducible factor-1 alpha (HIF-1) were performed. We also performed assays to verify the activity of matrix metalloproteinase (MMP) 2 and 9. The results in SaOS-2 showed that treatment with apocynin at concentrations of 1,5 e 3 mM; and diapocynin at concentrations of 0,75 e 1,5 mM reduced cell viability; increased the number of cells in apoptosis and decreased the production of ROS; without damaging HOb cells. Moreover, these same concentrations inhibited cell migration and invasion; decreased HIF-1 expression; and reduced MMP 2 activity in SaOS-2. Considering the results, we suggest that apocynin and diapocynin may act as possible modulators of tumor cells, and diapocynin has been shown to be more effective.(AU)