Bacterial cellulose matrix and acellular dermal matrix seeded with fibroblasts grown in platelet-rich plasma supplemented medium, compared to free gingival grafts: a randomized animal study.

PURPOSE: Mucogingival defects (MGDs), such as dental root recessions, decreased vestibular depth, and absence of keratinized tissues, are commonly seen in dental clinics. MGDs may result in functional, aesthetic, and hygienic concerns. In these situations, autogenous soft tissue grafts are considered the gold-standard treatment. This study compares the healing process of free gingival grafts (FGGs) to bacterial cellulose matrix (BCM) and human acellular dermal matrix (ADM) seeded with fibroblasts from culture supplemented with platelet-rich plasma in a rat model. METHODS: Surgical defects were made in rats, which received the following treatments in a randomized manner: group I, negative control (defect creation only); group II, positive control (FGG); group III, BCM; group IV, BCM + fibroblasts; group V, ADM; and group VI, ADM + fibroblasts. Clinical, histological, and immunological analyses were performed 15 days after grafting. Clinical examinations recorded epithelium regularity and the presence of ulcers, erythema, and/or edema. RESULTS: The histological analysis revealed the degree of reepithelization, width, regularity, and presence of keratin. The Fisher exact statistical test was applied to the results (P<0.05). No groups showed ulcers except for group I. All groups had regular epithelium without erythema and without edema. Histologically, all groups exhibited regular epithelium with keratinization, and myofibroblasts were present in the connective tissue. The groups that received engineered grafts showed similar clinical and histological results to the FGG group. CONCLUSIONS: Within the limitations of this study, it was concluded that BCM and ADM can be used as cell scaffolds, with ADM yielding the best results. This study supports the use of this technical protocol in humans.

Building Mathematical Models for Vascular Growth and Inhibition.

Microvascular channel growth and inhibition, such as what occurs in vasculogenic mimicry, are generally represented in tables or shown in bar graphs. Although informative, those representations lack accurate predictions on dosage or the opportunity to report an unbiased metric when one wants to compare different signal dependence, for instance, the concentration of different drugs or enzymes or expression levels of particular genes.Mathematical model building is an exercise that makes you think of which are the key variables of a particular phenomenon and how they affect the targeting experimental output.Starting from early blood vessel formation and regression (number of vessels) due to an inducer/inhibitor effect, we show how a conceptual mathematical model may be built. As an example, the model was used to parameterize aloin bioactivity on a chick yolk sac membrane (YSM) assay with respect to its vasculogenic and vessel regression properties. A separable functional form where vessel formation and cell death occur as mutually exclusive concentration or signal-dependent functions showed that there was a good correlation with experimental data. Although an analytical solution for that simple case is presented, parameter determination and parametric analysis may be carried out numerically by solving the system of ordinary differential equations that represents the model using nonlinear regression for parameter determinations. Such model formulation thus allows for a more objective evaluation concentration dependence and is suggested as a novel method to evaluate blood vessel formation and inhibition as well as a general model for quantitative balance between chemical stimulation and toxicity.

Melanoma growth in non-chemically modified translucid bacterial nanocellulose hollow and compartimentalized spheres.

BACKGROUND: Bacterial nanocellulose (BNC) has been used as cell support in numerous tissue engineering studies. Its use can be explained based on the fact its structure allows the creation of a required microenvironment for an ideal material, which supports 3D cell culture. Its structure and interconnected pores lead to animal cells adhesion and proliferation, also allowing oxygen and nutrients transportation. METHODS: We developed a new methodology to produce spherical platforms synthesized by Komagataebacter hansenii (ATCC 23769) under dynamic culture conditions in minimal medium. The chemical composition and physical properties of the platforms were evaluated. Then, human melanoma cells (SK-MEL-28) were encapsulated into the platforms and evaluated by metabolic activity, morphology and their ability on adhering to the Hollow Translucid BNC Spheres (BNC-TS-H) and Compartmentalized Translucid BNC Spheres (BNC-TS-C) up to 3 days. RESULTS: BNC-TS-H and BNC-TS-C platforms were produced as translucid spheroid platforms with distinct microenvironment under dynamic fermentation. The chemical and physical characterizations confirmed the platforms composition as BNC. The produced internal microenvironments in spherical platforms are relevant to determine tumor cell fate. In the first 12 h of culture, cells could adhere to nanocellulose microfibers assuming their typical tumorous phenotype in 72 h of culture. CONCLUSION: The dynamic fermentation in minimal medium produced distinct microstructured platforms of BNC-TS-H and BNC-TS-C. The platforms microstructure resulted in microenvironments that enabled distinct cell-cell and cell-matrix interactions. This behavior suggests several applications in tissue engineering. GENERAL SIGNIFICANCE: The method produced translucid BNC sphere platforms with distinct microenvironments for 3D cell culture.

A novel antimicrobial-containing nanocellulose scaffold for regenerative endodontics.

OBJECTIVES: The aim of this study was to evaluate bacterial nanocellulose (BNC) membranes incorporated with antimicrobial agents regarding cytotoxicity in fibroblasts of the periodontal ligament (PDLF), antimicrobial activity, and inhibition of multispecies biofilm formation. MATERIALS AND METHODS: The tested BNC membranes were BNC + 1% clindamycin (BNC/CLI); BNC + 0.12% chlorhexidine (BNC/CHX); BNC + nitric oxide (BNC/NO); and conventional BNC (BNC; control). After PDLF culture, the BNC membranes were positioned in the wells and maintained for 24 hours. Cell viability was then evaluated using the MTS calorimetric test. Antimicrobial activity against Enterococcus faecalis, Actinomyces naeslundii, and Streptococcus sanguinis (S. sanguinis) was evaluated using the agar diffusion test. To assess the antibiofilm activity, BNC membranes were exposed for 24 hours to the mixed culture. After sonicating the BNC membranes to remove the remaining biofilm and plating the suspension on agar, the number of colony-forming units (CFU)/mL was determined. Data were analyzed by 1-way analysis of variance and the Tukey, Kruskal-Wallis, and Dunn tests (α = 5%). RESULTS: PDLF metabolic activity after contact with BNC/CHX, BNC/CLI, and BNC/NO was 35%, 61% and 97%, respectively, compared to BNC. BNC/NO showed biocompatibility similar to that of BNC (p = 0.78). BNC/CLI showed the largest inhibition halos, and was superior to the other BNC membranes against S. sanguinis (p < 0.05). The experimental BNC membranes inhibited biofilm formation, with about a 3-fold log CFU reduction compared to BNC (p < 0.05). CONCLUSIONS: BNC/NO showed excellent biocompatibility and inhibited multispecies biofilm formation, similarly to BNC/CLI and BNC/CHX.

Efeito antibiofilme e citotoxicidade do hidróxido de cálcio associado ao óleo essencial de Melaleuca alternifolia / Antibiofilm effect and cytotoxicity of calcium hydroxide associated with the Melaleuca alternifolia essential oil

Objetivo: Avaliar e comparar os efeitos antimicrobiano e antibiofilme, e a citotoxicidade promovida pela associação do hidróxido de cálcio ao óleo essencial de Melaleuca alternifolia (MA), em diferentes concentrações, e ao propilenoglicol (PG). Métodos: As seguintes medicações compuseram os grupos experimentais: G1) HC/MA 1%; G2) HC/MA 5%; G3) HC/MA 10%; G4) HC/MA 20%; e G5) HC/PG. Solução salina 0,85% e meio DMEM serviram como controle nos testes antimicrobianos e de citotoxidade em fibroblastos do ligamento periodontal humano (FbLP), respectivamente. A atividade antimicrobiana (n = 12) foi avaliada por meio do teste de difusão em ágar. O efeito antibiofilme (n = 12) imediato das medicações foi avaliado por meio do teste de viabilidade bacteriana em biofilmes de 72 horas de E. faecalis, formados sobre discos de dentina e tratados por sete dias com as medicações. Após a coleta microbiológica do biofilme remanescente, os discos de dentina foram imersos em meio estéril e armazenados por mais sete dias, para a análise do efeito antibiofilme residual das medicações, quando nova coleta microbiológica foi realizada. A atividade metabólica de FbLP foi avaliada por meio do ensaio colorimétrico MTS (n = 9). Os valores médios dos halos de inibição, em mm, das unidades formadoras de colônia, e o percentual de atividade metabólica celular foram analisados pelos testes Kruskal-Wallis e post hoc Dunn (α = 5%). Resultados:Todas as medicações experimentais apresentaram superior ação antimicrobiana e antibiofilme comparadas ao controle, solução salina (p < 0,05), e mantiveram viáveis os FbLP, semelhante ao controle DMEM (p > 0,05). Conclusão: A associação do óleo essencial de Melaleuca alternifolia, nas concentrações de 1%, 5%, 10% e 20%, ao hidróxido de cálcio promoveu excelente ação antimicrobiana, antibiofilme e biocompatibilidade com fibroblastos, de forma semelhante à associação com propilenoglicol.

Aim:To evaluate and compare the antimicrobial and antibiofilm effect, as well as the cytotoxicity of calcium hydroxide (CH) associated with the Melaleuca alternifolia (MA)essential oil, in different concentrations, and with propylene glycol. Methods: The following medications composed the experimental groups: G1) CH/MA 1%; G2) CH/MA 5%; G3) CH/MA 10%; G4) CH/MA 20%; and G5) CH/PG. Saline solution and culture medium DMEM were used as a control in antimicrobial and cytotoxicity tests in human periodontal ligament fibroblasts (PDLF), respectively. The antimicrobial activity (n = 12) was evaluated by the disk-diffusion agar method. The immediate antibiofilm effect (n = 12) of the medications was evaluated for bacterial viability in 72 hours-biofilms of E. faecalis, formed on the dentin disc surface and treated for seven days with medications. After microbiological sampling of the remaining biofilm, the dentin discs were immersed in sterile culture medium and stored for another seven days, for analysis of the residual antibiofilm effect of the medications, when a new microbiological sampling was performed. PDLF viability was evaluated by MTS colorimetric assay (n = 9). The mean values of the inhibition halos, in mm, the colony forming units, and the metabolic cell activity percentage were analyzed by means of Kruskal-Wallis and post hoc Dunn (α = 5%) tests. Results:All of the experimental medications presented higher antimicrobial and antibiofilm effects, when compared to the saline solution control (p < 0.05), and maintained the PDLF feasible, similar to the DMEM control (p > 0.05). Conclusions:The association of the Melaleuca alternifolia essential oil, at concentrations of 1%, 5%, 10%, and 20%, with calcium hydroxide promoted an excellent antimicrobial and antibiofilm activity, and biocompatibility with fibroblasts, similarly to the association with propylene glycol.

Systems metabolic engineering of Escherichia coli for gram scale production of the antitumor drug deoxyviolacein from glycerol.

Deoxyviolacein is a microbial drug with biological activity against tumors, gram-positive bacteria, and fungal plant pathogens. Here, we describe an Escherichia coli strain for heterologous production of this high-value drug from glycerol. Plasmid-based expression of the deoxyviolacein cluster vioABCE was controlled by the araBAD promoter and induction by L-arabinose. Through elimination of L-arabinose catabolism in E. coli, the pentose sugar could be fully directed to induction of deoxyviolacein biosynthesis and was no longer metabolized, as verified by (13) C isotope experiments. Deletion of the araBAD genes beneficially complemented with previously described (i) engineering of the pentose phosphate pathway, (ii) chorismate biosynthesis, (iii) tryptophan biosynthesis, (iv) improved supply of L-serine, (v) elimination of tryptophan repression, and (vi) of tryptophan catabolism. Subsequent screening of the created next-generation producer E. coli dVio-8 identified glycerol as optimum carbon source and a level of 100 mg L(-1) of L-arabinose as optimum for induction. Transferred to a glycerol-based fed-batch process, E. coli dVio-8 surpassed the gram scale and produced 1.6 g L(-1) deoxyviolacein. With straightforward extraction from culture broth and purification by flash chromatography, deoxyviolacein was obtained at >99.5% purity. Biotechnol. Bioeng. 2014;111: 2280-2289. © 2014 Wiley Periodicals, Inc.

Computational analysis suggests that virulence of Chromobacterium violaceum might be linked to biofilm formation and poly-NAG biosynthesis.

Groups of genes that produce exopolysaccharide with a N-acetyl-D-glucosamine monomer are in the genome of several pathogenic bacteria. Chromobacterium violaceum, an opportunistic pathogen, has the operon hmsHFR-CV2940, whose proteins can synthesize such polysaccharide. In this work, multiple alignments among proteins from bacteria that synthesize such polysaccharide were used to verify the existence of amino acids that might be critical for pathogen activity. Three-dimensional models were generated for spatial visualization of these amino acid residues. The analysis carried out showed that the protein HmsR preserves the amino acids D135, D228, Q264 and R267, considered critical for the formation of biofilms and, furthermore, that these amino acids are close to each other. The protein HmsF of C. violaceum preserves the residues D86, D87, H156 and W115. It was also shown that these residues are also close to each other in their spatial arrangement. For the proteins HmsH and CV2940 there is evidence of conservation of the residues R104 and W94, respectively. Conservation and favorable spatial location of those critical amino acids that constitute the proteins of the operon indicates that they preserve the same enzymatic function in biofilm synthesis. This is an indicator that the operon hmsHFR-CV2940 is a possible target in C. violaceum pathogenicity.

Computational analysis suggests that virulence of Chromobacterium violaceum might be linked to biofilm formation and poly-NAG biosynthesis

Groups of genes that produce exopolysaccharide with a N-acetyl-D-glucosamine monomer are in the genome of several pathogenic bacteria. Chromobacterium violaceum, an opportunistic pathogen, has the operon hmsHFR-CV2940, whose proteins can synthesize such polysaccharide. In this work, multiple alignments among proteins from bacteria that synthesize such polysaccharide were used to verify the existence of amino acids that might be critical for pathogen activity. Three-dimensional models were generated for spatial visualization of these amino acid residues. The analysis carried out showed that the protein HmsR preserves the amino acids D135, D228, Q264 and R267, considered critical for the formation of biofilms and, furthermore, that these amino acids are close to each other. The protein HmsF of C. violaceum preserves the residues D86, D87, H156 and W115. It was also shown that these residues are also close to each other in their spatial arrangement. For the proteins HmsH and CV2940 there is evidence of conservation of the residues R104 and W94, respectively. Conservation and favorable spatial location of those critical amino acids that constitute the proteins of the operon indicates that they preserve the same enzymatic function in biofilm synthesis. This is an indicator that the operon hmsHFR-CV2940 is a possible target in C. violaceum pathogenicity.

Extension of the IsaViz software for the representation of metabolic and regulatory networks

In this work we developed an extension of IsaViz software, a RDF (Resource Description Framework) authoring tool, designed to be a graphical environment to build models of metabolic and regulatory networks. This environment, called Metabolic IsaViz, was linked to a genomic library of types and was modeled on the basis of ontologies. Biochemical pathways included data at sequence level (e.g., the amino acid sequence of enzymes), besides kinetic and thermodynamic parameters for the reactions. Models created with Metabolic IsaViz could be exported to pathways simulators through SBML (Systems Biology Markup Language), which allowed to analyze the pathway dynamics of target chemicals.