Biostability of the Proanthocyanidins-Dentin Complex and Adhesion Studies.

Oligomeric proanthocyanidins (OPACs) are potent and renewable natural bioactives possible to be refined into chemically standardized mixtures for biological applications. Herein, we found that multiscale interactions of OPACs with the dentin matrix create tight biointerfaces with hydrophobic methacrylate adhesives on wet surfaces. An enriched mixture of OPACs, with a known phytochemical profile, was produced from grape seed crude extract ( Vitis vinifera; enriched grape seed extract [e-GSE]) and applied to dentin matrices to determine changes to the mechanical properties and biodegradability of the dentin matrix and favorable resin adhesion mechanisms. Methods included a 3-point flexural test, quantification of hydroxyproline (collagen solubilization), static and dynamic nanomechanical analyses, resin-dentin microtensile bond strength, and micropermeability at the adhesive interface. The e-GSE-modified dentin matrix exhibited remarkably low collagen solubilization and sustained the bulk elastic properties over 12 mo. Tan δ findings reveal a more elastic-like behavior of the e-GSE-modified dentin matrix, which was not affected by H-bond destabilization by urea. Dentin-methacrylate biointerfaces with robust and stable adhesion were created on e-GSE-primed dentin surfaces, leading to a dramatic decrease of the interfacial permeability. Standardized OPAC mixtures provide a new mechanism of adhesion to type I collagen-rich tissues that does not rely on hydrophilic monomers. The bioadhesion mechanism involves physicochemical modifications to the dentin matrix, reduced tissue biodegradation, and bridging to methacrylate resins.

Phosphoric acid concentration affects dentinal MMPs activity.

OBJECTIVES: To evaluate whether the concentration of phosphoric acid (PA) has an effect on the proteolytic activity of sound human demineralized dentin. It is hypothesized that the activity of matrix-bound and extracted enzymes depends on the PA concentration used to demineralize dentin. METHODS: One-gram aliquots of mid-coronal human dentin powder were demineralized with 1wt%, 10wt% and 37wt% PA. Concentrations of released calcium were measured for each set of demineralization. Extracted MMP-2 was immunologically identified by western blot and its activity was determined by conventional gelatin zymography. Analysis of released hydroxyproline (HYP) and in situ zymography were performed to evaluate the activity of insoluble, bound-matrix enzymes. RESULTS: The amount of released calcium from dentin powder treated with 37wt% PA was significantly higher (p≤0.05) than that obtained by dentin demineralization with 10wt% and 1wt% PA. Expression and activity of endogenous enzymes, extracted from or bound to dentin matrix, were detected for all samples regardless of the PA concentration. However, the expression and activity of extracted MMP-2 were significantly higher when dentin was treated with 10wt% PA (p<0.05), followed by 1wt% and 37wt% PA. Similarly, the highest concentration of released HYP (i.e. meaning higher percentage of collagen degradation) and the highest activity in in situ zymography were observed when dentin samples were treated with 10wt% PA (p<0.05). CONCLUSIONS: It was confirmed that PA does not denature endogenous enzymes of dentin matrices, but it may somehow modulate the expression and activity of these enzymes in a concentration-dependent manner. CLINICAL SIGNIFICANCE: Endogenous proteases have been identified and suggested to be responsible for the digestion of dentin matrix when activated by the acidic components of dental adhesives. Proteolytic activity of dentinal MMPs showed to be dependent on phosphoric acid concentration. The clinically-used concentration (37%) does not inhibit MMPs activity, but slows it.

Dentin biomodification potential depends on polyphenol source.

Although proanthocyanidins (PACs) modify dentin, the effectiveness of different PAC sources and the correlation with their specific chemical composition are still unknown. This study describes the chemical profiling of natural PAC-rich extracts from 7 plants using ultra high pressure/performance liquid chromatography (UHPLC) to determine the overall composition of these extracts and, in parallel, comprehensively evaluate their effect on dentin properties. The total polyphenol content of the extracts was determined (as gallic acid equivalents) using Folin-Ciocalteau assays. Dentin biomodification was assessed by the modulus of elasticity, mass change, and resistance to enzymatic biodegradation. Extracts with a high polyphenol and PAC content from Vitis vinifera, Theobroma cacao, Camellia sinensis, and Pinus massoniana induced a significant increase in modulus of elasticity and mass. The UHPLC analysis showed the presence of multiple types of polyphenols, ranging from simple phenolic acids to oligomeric PACs and highly condensed tannins. Protective effect against enzymatic degradation was observed for all experimental groups; however, statistically significant differences were observed between plant extracts. The findings provide clear evidence that the dentin bioactivities of PACs are source dependent, resulting from a combination of concentration and specific chemical constitution of the complex PAC mixtures.

Abundance of MMPs and cysteine cathepsins in caries-affected dentin.

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metalloproteases (MMPs) and cysteine cathepsins (CTs). Since this relationship has not been fully established, we hypothesized that the abundance of MMPs and CTs in caries-affected dentin must be higher than in intact dentin. To test this premise, we obtained 5 slices (200 µm) from 5 intact teeth and from 5 caries-affected teeth (1 slice/tooth) and individually incubated them with primary antibodies for CT-B, CT-K, MMP-2, or MMP-9. Negative controls were incubated with pre-immune serum. Specimens were washed and re-incubated with the respective fluorescent secondary antibody. Collagen identification, attained by the autofluorescence capture technique, and protease localization were evaluated by multi-photon confocal microscopy. The images were analyzed with ZEN software, which also quantitatively measured the percentages of collagen and protease distribution in dentin compartments. The abundance of the test enzymes was markedly higher in caries-affected than in intact dentin. CT-B exhibited the highest percentage of co-localization with collagen, followed by MMP-9, MMP-2, and CT-K. The high expression of CTs and MMPs in caries-affected teeth indicates that those host-derived enzymes are intensely involved with caries progression.

Tooth bleaching increases dentinal protease activity.

Hydrogen peroxide is an oxidative agent commonly used for dental bleaching procedures. The structural and biochemical responses of enamel, dentin, and pulp tissues to the in vivo bleaching of human (n = 20) premolars were investigated in this study. Atomic force microscopy (AFM) was used to observe enamel nanostructure. The chemical composition of enamel and dentin was analyzed by infrared spectroscopy (FTIR). The enzymatic activities of dental cathepsin B and matrix metalloproteinases (MMPs) were monitored with fluorogenic substrates. The amount of collagen in dentin was measured by emission of collagen autofluorescence with confocal fluorescence microscopy. The presence of Reactive Oxygen Species (ROS) in the pulp was evaluated with a fluorogenic 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) probe. Vital bleaching of teeth significantly altered all tested parameters: AFM images revealed a corrosion of surface enamel nanostructure; FTIR analysis showed a loss of carbonate and proteins from enamel and dentin, along with an increase in the proteolytic activity of cathepsin-B and MMPs; and there was a reduction in the autofluorescence of collagen and an increase in both cathepsin-B activity and ROS in pulp tissues. Together, these results indicate that 35% hydrogen peroxide used in clinical bleaching protocols dramatically alters the structural and biochemical properties of dental hard and soft pulp tissue.

Chlorhexidine inhibits the activity of dental cysteine cathepsins.

The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2' of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.

[Degradation of naphthalene-2-sulfonate by strains of Micromonospora]. / Degradación de ácido naftalén-2-sulfónico por cepas de Micromonospora.

It was possible to isolate several strains of Micromonospora sp. from waters of Rio Reconquista. They all were filamentous bacteria with lateral spores, highly resistant to Cr(III) and another heavy metal cations. All these strains were able to grow on naphthalene-2-sulphonate as sole carbon source in a mineral medium. The biodegradation of the xenobiotic proceeds via the formation of salicylate and gentisate. These compounds have been isolated and mass spectrometry identified.

Degradación de ácido naftalén-2-sulfónico por cepas de Micromonospora / Naphthalen-2-sulfonate degradation by Micromonospora strains

De aguas del Río Reconquista se han podido aislar cepas de Micromonospora resistentes a altos niveles de cromo y otros metales pesados, capaces de crecer sobre ácido naftalén-2-sulfónico como única fuente de carbono. Este fenotipo es muy estable, lo que hace a este microrganismo interesante desde el punto de vista de la eliminación de residuos industriales que contengan este tipo de sustancias. La biodegradación procede mediante la formación de ácido salicílico y ácido gentísico como intermediarios, metabolitos que han sido aislados mediante cromatografía gaseosa y caracterizados mediante espectroscopía de masa

Degradación de ácido naftalén-2-sulfónico por cepas de Micromonospora / Naphthalen-2-sulfonate degradation by Micromonospora strains

De aguas del Río Reconquista se han podido aislar cepas de Micromonospora resistentes a altos niveles de cromo y otros metales pesados, capaces de crecer sobre ácido naftalén-2-sulfónico como única fuente de carbono. Este fenotipo es muy estable, lo que hace a este microrganismo interesante desde el punto de vista de la eliminación de residuos industriales que contengan este tipo de sustancias. La biodegradación procede mediante la formación de ácido salicílico y ácido gentísico como intermediarios, metabolitos que han sido aislados mediante cromatografía gaseosa y caracterizados mediante espectroscopía de masa(AU)

Degradación de ácido naftalén-2-sulfónico por cepas de Micromonospora. / [Degradation of naphthalene-2-sulfonate by strains of Micromonospora]

It was possible to isolate several strains of Micromonospora sp. from waters of Rio Reconquista. They all were filamentous bacteria with lateral spores, highly resistant to Cr(III) and another heavy metal cations. All these strains were able to grow on naphthalene-2-sulphonate as sole carbon source in a mineral medium. The biodegradation of the xenobiotic proceeds via the formation of salicylate and gentisate. These compounds have been isolated and mass spectrometry identified.

Biodegradación de una mezcla comercial de bifenilos policlorados / Biodegradation of a commercial mixture of polychlorinated byphenyls

De suelos contaminados con bifenilos policlorados (PCBs) se ha aislado una cepa de Klebsiella oxytoca que crece en un medio mineral con una mezcla comercial de PCBs como única fuente de carbono. La mezcla utilizada contiene una variedad de congéneres de bifenilos bi-, tri-, y tetraclorados, siendo los triclorados los constituyentes mayoritarios. La cepa aislada puede crecer también con 6-cloroquinolina, y menos eficientemente, con clorobenceno. En ensayos de consumo en frasco, el microorganismo degrada los PCBs casi completamente en tres días. En estas condiciones se forman compuestos acídicos y/o fenólicos que son degradados en siete días de incubación (AU)

Biodegradación de una mezcla comercial de bifenilos policlorados / Biodegradation of a commercial mixture of polychlorinated byphenyls

De suelos contaminados con bifenilos policlorados (PCBs) se ha aislado una cepa de Klebsiella oxytoca que crece en un medio mineral con una mezcla comercial de PCBs como única fuente de carbono. La mezcla utilizada contiene una variedad de congéneres de bifenilos bi-, tri-, y tetraclorados, siendo los triclorados los constituyentes mayoritarios. La cepa aislada puede crecer también con 6-cloroquinolina, y menos eficientemente, con clorobenceno. En ensayos de consumo en frasco, el microorganismo degrada los PCBs casi completamente en tres días. En estas condiciones se forman compuestos acídicos y/o fenólicos que son degradados en siete días de incubación

[Biodegradation of a commercial mixture of polychlorinated biphenyls]. / Biodegradación de una mezcla comercial de bifenilos policlorados.

A bacterial strain capable of growing on a commercial mixture of polychlorinated biphenyls (PCBs) was isolated from PCBs-contaminated soil. The isolate was identified as a Klebsiella oxytoca strain, able to grow on 6-chloroquinoline and chlorobenzene. The above mentioned PCBs mixture contains several congeners of bi-, tri- and tetra-chloro biphenyls. Xenobiotic consumption could be measured by an assay in glass-stoppered flask. After three days PCBs were almost completely degraded with the production of phenolic and/or acidic products, that were consumed after seven days.

[2-Naphthalene-sulphonic acid degrading microorganisms]. / Microorganismos degradadores de ácido naftalen-2-sulfónico.

Derivatives of anionic surfactants, specially naphthalene sulphonates, when discharged into natural waters are accumulated in waters and sediments because of their poor biodegradability. A four-membered bacterial community, able to degrade the sodium salt of 2-naphthalene-sulphonic acid (2NS), was isolated from Río Reconquista. All the isolated strains consisted of gram-negative, strictly aerobic rods, with a strong proteolytic activity and resistance to high levels of cations like Cr (III), Hg (II), Cd (II) and Pb (II). Some of them were resistant to gentamicin, amikacin and chloramphenicol. These strains appeared to be related to the genera Pseudomonas and Alcaligenes. When isolated, if growing on 2NS, a brown-dark pigment is formed by three of them, resulting in an inhibition of growth. The presence of a Pseudomonas aeruginosa strain avoided the production of pigment and resulting in a complete consume of 2NS.

Microorganismos degradadores de ácido naftalen-2-sulfónico / [2-Naphthalene-sulphonic acid degrading microorganisms]

Derivatives of anionic surfactants, specially naphthalene sulphonates, when discharged into natural waters are accumulated in waters and sediments because of their poor biodegradability. A four-membered bacterial community, able to degrade the sodium salt of 2-naphthalene-sulphonic acid (2NS), was isolated from Río Reconquista. All the isolated strains consisted of gram-negative, strictly aerobic rods, with a strong proteolytic activity and resistance to high levels of cations like Cr (III), Hg (II), Cd (II) and Pb (II). Some of them were resistant to gentamicin, amikacin and chloramphenicol. These strains appeared to be related to the genera Pseudomonas and Alcaligenes. When isolated, if growing on 2NS, a brown-dark pigment is formed by three of them, resulting in an inhibition of growth. The presence of a Pseudomonas aeruginosa strain avoided the production of pigment and resulting in a complete consume of 2NS.

Microorganismos degradadores de ácido naftalen-2-sulfónico. / [2-Naphthalene-sulphonic acid degrading microorganisms]

Derivatives of anionic surfactants, specially naphthalene sulphonates, when discharged into natural waters are accumulated in waters and sediments because of their poor biodegradability. A four-membered bacterial community, able to degrade the sodium salt of 2-naphthalene-sulphonic acid (2NS), was isolated from Río Reconquista. All the isolated strains consisted of gram-negative, strictly aerobic rods, with a strong proteolytic activity and resistance to high levels of cations like Cr (III), Hg (II), Cd (II) and Pb (II). Some of them were resistant to gentamicin, amikacin and chloramphenicol. These strains appeared to be related to the genera Pseudomonas and Alcaligenes. When isolated, if growing on 2NS, a brown-dark pigment is formed by three of them, resulting in an inhibition of growth. The presence of a Pseudomonas aeruginosa strain avoided the production of pigment and resulting in a complete consume of 2NS.