Berdan Aydin

Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desorption ionization mass spectrometry

The heptapeptide ARHPHPH was identified from biofilms and planktonic cultures of two different strains of Enterococcus faecalis, V583 and ATCC 29212, using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). ARHPHPH was also imaged at the boundary of cocultured, adjacent E. faecalis and Escherichia coli (ATCC 25922) biofilms, appearing only on the E. faecalis side. ARHPHPH was proteolyzed from κ-casein, a component in the growth media, by E. faecalis microbes. Additionally, top down and bottom up proteomic approaches were combined to identify and spatially locate multiple proteins within intact E. faecalis V583 biofilms by MALDI-MS. The resultant tandem MS data were searched against the NCBInr E. faecalis V583 database to identify thirteen cytosolic and membrane proteins which have functional association with the cell surface. Two of these proteins, enolase and GAPDH, are glycolytic enzymes known to display multiple functions in bacterial virulence in related bacterial strains. This work illustrates a powerful approach for discovering and localizing multiple peptides and proteins within intact biofilms.

Apical microleakage of root-end cavities prepared by Er, Cr: YSGG laser

The aim of this study is to assess the apical microleakage of the composite filled root-end cavities prepared by an Erbium, Chromium: Yttrium-Scandium-Gallium-Garnet laser. Fifty-five maxillary incisor teeth were enlarged and filled. Following the apical resection, root-end cavities were prepared using conventional methods: either using a bur (n = 30) or an ultrasonic retrotip (n = 15). Root-end cavities of the 15 teeth in the bur group were finished with the laser at 3.5 W. All root-end cavities were filled using two-step self-etching primer and composite resin. After 4 months of storage, apical microleakage was measured by a fluid filtration method. Microleakage of composite filled root-end cavities that were prepared by Er, Cr: YSGG was significantly larger than those made by conventional methods (p < 0.05). In conclusion, using the Er, Cr: YSGG laser has no advantages over conventional root-end cavity preparation methods when a composite filling material is used to seal root-end cavities.

Quantification of antibiotic in biofilm-inhibiting multilayers by 7.87 eV laser desorption postionization MS imaging.

The potential of laser desorption postionization mass spectrometry (LDPI-MS) imaging for small molecule quantification is demonstrated here. The N-methylpiperazine acetamide (MPA) of ampicillin was adsorbed into polyelectrolyte multilayer surface coatings composed of chitosan and alginate, both high molecular weight biopolymers. These MPA-ampicillin spiked multilayers were then shown to inhibit the growth of Enterococcus faecalis biofilms that play a role in early stage infection of implanted medical devices. Finally, LDPI-MS imaging using 7.87 eV single-photon ionization was found to detect MPA-ampicillin within the multilayers before and after biofilm growth with limits of quantification and detection of 0.6 and 0.3 nmol, respectively. The capabilities of LDPI-MS imaging for small molecule quantification are compared to those of MALDI-MS. Furthermore, these results indicate that 7.87 eV LDPI-MS imaging should be applicable to quantification of a range of small molecular species on a variety of complex organic and biological surfaces. Finally, while MS imaging for quantification was demonstrated here using LDPI, it is a generally useful strategy that can be applied to other methods.

Collagen-collagen interactions mediated by plant-derived proanthocyanidins: A spectroscopic and atomic force microscopy study.

UNLABELLED Collagen cross-linkings are determinant of biological tissue stability and function. Plant-derived proanthocyanidins (PACs) mimic different hierarchical levels of collagen cross-links by non-enzymatic interactions resulting in the enhancement to the biomechanics and biostability of collagen-rich tissues such as dentin. This study investigated the interaction of PACs from Vitis vinifera grape seed extract with type I collagen in solubilized form and in the demineralized dentin matrix (DDM) by fluorescence spectral analysis; collagen-collagen binding forces in presence of cross-linking solutions by atomic force microscopy (AFM); and spectroscopic analysis of the DDM using attenuated total reflectance Fourier transform-infrared spectroscopy (ATR-FTIR). Glutaraldehyde (GA) and carbodiimide hydrochloride (EDC) with known cross-linking mechanisms were selected for comparative analyses. Changes in fluorescence upon interaction of solubilized type I collagen with PACs, EDC and GA reflected pronounced modifications in collagen conformation. PACs also promoted stronger collagen-collagen fibrils interaction than EDC and GA. A new feature was observed using ATR-FTIR spectroscopic analysis in PACs-treated collagen and DDM. The findings suggest covalent interactions between collagen and PACs. The mechanisms of interaction between PACs-collagen hold attractive and promising tissue-tailored biomedical applications and the binding forces that potentially drive such interaction were characterized. STATEMENT OF SIGNIFICANCE Connective tissues such as skin, bone and dentin are mainly composed of type I collagen, which is cross-linked to promote tissue stability, strength and function. Novel therapies using substances that mimic cross-links have been proposed to promote repair of collagen-based-tissues. In dentistry, naturally occurring proanthocyanidins (PACs) have the potential to enhance dentin mechanical properties and reduce its enzymatic degradation, but their mechanisms of cross-linking are unclear. The present study investigated the specific interactions between PACs-type I collagen in purified and dentin collagen and compared to the well described cross-linking mechanisms promoted by synthetic chemical substances. Findings reveal that covalent-like bonds are induced by plant PACs in type I collagen as well as in complex dental native tissue, promoting strong collagen-collagen interactions.

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.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

Effect of storage solutions on microhardness of crown enamel and dentin

Objective: The aim of this study was to determine alterations in microhardness of crown dentin and enamel, after 2 and 12-month storage in de-ionized water, 0.2% glutaraldehyde, Hanks′ Balanced Salt Solution (HBSS), 0.1% sodium hypochlorite (NaOCl) or 0.1% thymol. Materials and Methods: Freshly extracted, nonsterile 60 intact human premolars were distributed to five groups. Six teeth from each group were evaluated after two, and other six teeth were evaluated after 12 months storage. After grinding and polishing of teeth, Vickers hardness was evaluated with making indentations on enamel and dentin, using a pyramid diamond indenter tip exerting 100 g load for 15 s. Results: After 2 months storage in solutions, range of the hardness values (HV) of enamel and dentin were in between 315-357 and 64-67, respectively. However, 12 months storage of the teeth resulted in a statistically significant decrease in microhardness when compared to microhardness of teeth stored for 2 months (P = 0.001). Although the differences were not significant regarding solutions, all solutions decreased the microhardness both in enamel and dentin (P > 0.05). However, decrease in microhardness was relatively less in de-ionized water and thymol solutions while glutaraldehyde decreased microhardness the most: 63% for enamel and 53% for dentin. Conclusions: Microhardness of enamel and dentin was in an acceptable range when teeth were stored for 2 months in de-ionized water, glutaraldehyde, HBSS, NaOCl or in thymol; thus, teeth kept up to 2 months in these solutions can be used for mechanical in vitro tests. However, 12 months storage significantly decreased the microhardness of enamel and dentin.

Assessing collagen and micro-permeability at the proanthocyanidin-treated resin-dentin interface

PURPOSE To establish a fluorescence-based method to simultaneously assess micro-permeability and collagen cross-linking induced by chemical agents at the resin-dentin interface. MATERIALS AND METHODS Three chemical agents were investigated (proanthocyanidin-rich grape seed extract: GSE; carbodiimide hydrochloride/N-hydroxysuccinimide: EDC/NHS; glutaraldehyde: GD) along with a control (distilled water) as primers applied on flat occlusal dentin surfaces of 48 teeth and restored with two commercially available etch-and-rinse adhesives. Resin-dentin interfaces were polished and infiltrated with rhodamine-B solution for confocal laser scanning microscopy analysis. Parameters were chosen that would allow acquisition of a simultaneous appearance of collagen and interfacial micro-permeability (rhodamine-B). Fluorescence emission intensity (FEI) was converted into numerals and values were calculated for each group. Data were statistically analyzed using one-way ANOVA and post-hoc Scheffes and multiple comparisons tests (α = 0.05). T-tests with Pearson correlations were used to investigate correlations between collagen cross-linking and micro-permeability. RESULTS The FEI of collagen was the highest for GD, followed by GSE, with no significant differences between EDC/ NHS and the control group (p > 0.05). Micro-permeability was significantly affected by the adhesives (p < 0.05). Micro- permeability was the lowest for GSE groups, regardless of the adhesives (p < 0.001). Weak correlations were found between micro-permeability and collagen auto-fluorescence. CONCLUSIONS Non-enzymatic collagen cross-linking induced by GSE and GD can be detected by increased collagen auto-fluorescence, and results in reduced interfacial micro-permeability. Increased collagen auto-fluorescence was correlated with fluorescent collagen cross-links and decreased micro-permeability at the resin-dentin interface. Collagen auto-fluorescence is a useful tool to detect auto-fluorescent exogenous cross links and their potential impact on the quality of the resin-dentin interface.

Long-term evaluation of the stability of dentin matrix following treatments with aqueous solutions of titanium tetrafluoride at different concentrations

OBJECTIVE The purpose of this study was to investigate the effects of aqueous solutions of different concentrations of titanium tetrafluoride (TiF4) on dentin matrix stability up to six months. DESIGN Dentin specimens prepared from fifteen nonerupted molars were demineralized and randomly distributed into groups: 2.5% TiF4, 4% TiF4, 1000 ppm NaF, and control (distilled water). The modulus of elasticity (ME) and dry masses of the dentin matrix were determined at baseline, and up to 6 months following treatment. Collagen solubilization was estimated by hydroxyproline (HYP) quantification in the simulated body fluid used to store the dentin specimens. RESULTS The 2.5% TiF4 group presented higher ME immediately after treatment, and at 3 and 6 months storage, whereas 4% TiF4 showed higher means at 3 and 6 months (p < 0.001). No significant differences were observed among the groups over time (p = 0.9325). However, the 2.5% TiF4 group showed significantly higher ME than the control group, immediately after treatment. All the groups presented significantly higher mass change immediately, compared with 3 and 6 months (p < 0.0001). Except for the 4% TiF4 group, HYP release was higher in the first quarter (p = 0.0152), when no significant differences were found among the groups. In the second quarter, the means were significantly higher in the 2.5% TiF4 and 4% TiF4 groups. The group treated with 2.5% TiF4 had a statistically higher HYP release than the control group. CONCLUSION An aqueous solution of 2.5% TiF4 increases the immediate stiffness values, but does not stabilize the collagenous dentin matrix.

Encapsulation of grape seed extract in polylactide microcapsules for sustained bioactivity and time-dependent release in dental material applications

OBJECTIVE To sustain the bioactivity of proanthocyanidins-rich plant-derived extracts via encapsulation within biodegradable polymer microcapsules. METHODS Polylactide microcapsules containing grape seed extract (GSE) were manufactured using a combination of double emulsion and solvent evaporation techniques. Microcapsule morphology, size distribution, and cross-section were examined via scanning electron microscopy. UV-vis measurements were carried out to evaluate the core loading and encapsulation efficiency of microcapsules. The bioactivity of extracts was evaluated after extraction from capsules via solvent partitioning one week or one year post-encapsulation process. Fifteen human molars were cut into 7mm×1.7mm×0.5mm thick mid-coronal dentin beams, demineralized, and treated with either encapsulated GSE, pristine GSE, or left untreated. The elastic modulus of dentin specimens was measured based on three-point bending experiments as an indirect assessment of the bioactivity of grape seed extracts. The effects of the encapsulation process and storage time on the bioactivity of extracts were analyzed. RESULTS Polynuclear microcapsules with average diameter of 1.38μm and core loading of up to 38wt% were successfully manufactured. There were no statistically significant differences in the mean fold increase of elastic modulus values among the samples treated with encapsulated or pristine GSE (p=0.333), or the storage time (one week versus one year storage at room temperature, p=0.967). SIGNIFICANCE Polynuclear microcapsules containing proanthocyanidins-rich plant-derived extracts were prepared. The bioactivity of extracts was preserved after microencapsulation.