Gianluca Turco

Non-cytotoxic Silver Nanoparticle-Polysaccharide Nanocomposites with Antimicrobial Activity

In this work we study (i) the formation and stabilization of silver nanoparticles in a bioactive chitosan-derived polysaccharide solution, (ii) the antimicrobial properties, either in solution or in 3D hydrogel structures, obtained by mixtures with the polysaccharide alginate, and (iii) the cytotoxicity of the latter nanocomposite materials on different eukaryotic cell lines. Antimicrobial results show that these nanocomposite systems display a very effective bactericidal activity toward both Gram+ and Gram- bacteria. However, the hydrogel does not show any cytotoxic effect toward three different eukaryotic cell lines. This is due to the fact that the nanoparticles, immobilized in the gel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while they can not be uptaken and internalized by eukaryotic cells. This novel finding could advantageously contribute to responding to the growing concerns on the toxicity of nanoparticles and facilitate the use of silver-biopolymer composites in the preparation of biomaterials.

Alginate/Hydroxyapatite biocomposite for bone ingrowth: a trabecular structure with high and isotropic connectivity.

Alginate/hydroxyapatite composite scaffolds were developed using a novel production design. Hydroxyapatite (HAp) was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid hydrolyzing d-gluconic acid delta-lactone (GDL) for the direct release of calcium ions from HAp. Hydrogels were then freeze-casted to produce a three-dimensional isotropic porous network. Scanning electron microscopy (SEM) observations, confocal laser scanning microscopy (CLSM) and microcomputed tomography (micro-CT) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 and 300 microm and over 88% porosity. Proliferation assay and SEM observations demonstrated that human osteosarcoma cell lines were able to proliferate, maintain osteoblast-like phenotype and massively colonize the scaffold structure. Overall, these combined results indicate that the novel alginate based composites efficiently support the adhesion and proliferation of cells showing at the same time adequate structural and physical-chemical properties for being used as scaffolds in bone tissue engineering strategies.

Biological responses of silver-coated thermosets: An in vitro and in vivo study

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are biomaterials commonly employed for orthopedic and dental applications; for both these fields, bacterial adhesion to the surface of the implant represents a major issue for the outcome of the surgical procedures. In this study, the antimicrobial properties of a nanocomposite coating formed by polysaccharide 1-deoxylactit-1-yl chitosan (Chitlac) and silver nanoparticles (nAg) on methacrylate thermosets were studied. The Chitlac-nAg system showed good anti-bacterial and anti-biofilm activity although its biocidal properties can be moderately, albeit significantly, inhibited by serum proteins. In vitro studies on the silver release kinetic in physiological conditions showed a steady metal release associated with a gradual loss of antimicrobial activity. However, after 3weeks there was still effective protection against bacterial colonization which could be accounted for by the residual silver. This time-span could be considered adequate to confer short-term protection from early peri-implant infections. Preliminary in vivo tests in a mini-pig animal model showed good biological compatibility of Chitlac-nAg-coated materials when implanted in bony tissue. The comparison was made with implants of titanium Ti6Al4V alloy and with a Chitlac-coated thermoset. Bone healing patterns and biocompatibility parameters observed for nAg-treated material were comparable with those observed for control implants.

Stabilization of dentin matrix after cross-linking treatments, in vitro

OBJECTIVES To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen. METHODS Dentin beams were obtained from human molars and completely demineralized in 10 wt% H3PO4 for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n=10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal-Wallis or Mann-Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance. RESULTS EDC was able to significantly increase collagen stiffness in 60s. 10% GA groups obtained the highest E values after both 30 and 60s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1M or 2M EDC showed the lowest MMP activity. SIGNIFICANCE Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.

Mechanisms of degradation of the hybrid layer in adhesive dentistry and therapeutic agents to improve bond durability—A literature review

OBJECTIVE Success in adhesive dentistry means long lasting restorations. However, there is substantial evidence that this ideal objective is not always achieved. Current research in this field aims at increasing the durability of resin-dentin bonds. The objective of this paper is to examine the fundamental processes responsible for the aging mechanisms involved in the degradation of resin-bonded interfaces and the potential approaches to prevent and counteract this degradation. METHODS PubMed searches on the hybrid layer degradation were carried out. Keywords were chosen to assess hybrid layer degradation for providing up-dated information on the basis of scientific coherence with the research objective. Approaches to prevent and counteract this degradation were also reviewed. RESULTS 148 peer-review articles in the English language between 1982 and 2015 were reviewed. Literature shows that resin-dentin bond degradation is a complex process, involving the hydrolysis of both the resin and the collagen fibril phases contained within the hybrid layer. Collagen fibers become vulnerable to mechanical and hydraulic fatigue, as well as degradation by host-derived proteases with collagenolytic activity (matrix metalloproteinases and cysteine cathepsins). Inhibition of the collagenolytic activity and the use of cross-linking agents are the two main strategies to increase the resistance of the hybrid layer to enzymatic degradation. SIGNIFICANCE This review analyzes the issues regarding the durability of the adhesive interface, and the techniques to create stable resin-dentin bonds able to resist the collagenolytic hydrolysis that are currently studied.

Kinetics of polymerization and contraction stress development in self-adhesive resin cements.

OBJECTIVES The aim of the study was to evaluate the contraction stress, microhardness and polymerization kinetics of three self-adhesive cements vs. conventional dual-cure resin cement. METHODS Cements tested were: RelyX Unicem (3M ESPE, St. Paul, MN, USA), MaxCem Elite (Kerr, Orange, CA, USA), Clearfil SA Cement (Kuraray, Tokyo, Japan) and Duolink (Bisco Inc., Schaumburg, IL, USA). Cements were irradiated with a LED-curing unit (bluephase, IvoclarVivadent) for 20 or 40 s and the contraction forces (N) generated during polymerization were continuously recorded for 6 h with a universal testing machine. Polymerization kinetics were monitored using micro-Raman spectroscopy and degree of conversion was calculated. Vickers microhardness was also recorded. All measurements were performed at 10 min and 6h. Data were statistically analyzed by three-way ANOVA with repeated measures and Tukeys post hoc test (α=0.05). RESULTS Irrespective of exposure time, stress analysis ranked in the following order: Clearfil SA Cement<MaxCem<RelyX Unicem≤Duolink (p<0.05). Stress was correlated with microhardness values (p<0.05). Kinetic curves showed that maximum degree of conversion was attained more quickly than maximum stress after light activation. SIGNIFICANCE The conventional resin-based cement showed higher stress values than the self-adhesive cements. The results were material-dependent and probably correlated to the composition of each material.

Mechanical Spectroscopy and Relaxometry on Alginate Hydrogels: A Comparative Analysis for Structural Characterization and Network Mesh Size Determination

The structure of calcium-saturated alginate hydrogels has been studied by combining rheological determinations and relaxometry measurements. The mechanical spectroscopy analyses performed on alginate gel cylinders at different polysaccharide concentration allowed estimating their main structural features such as the average mesh size. The calculation was based on the introduction of a front factor in the classical rubber elasticity approach which was correlated to the average length of the Guluronic acid blocks along the polysaccharide chain. Transverse relaxation time (T(2)) determinations performed on the cylinders revealed the presence of two relaxation rates of the water entrapped within the hydrogel network. The cross-correlation of the latter data with the rheological measurements allowed estimating the mesh size distribution of the hydrogel network. The results obtained for the hydrogel cylinders were found to be consistent with the relaxometric analysis performed on the alginate microbeads where, however, only one type of water bound into the network structure was detected. A good correlation was found in the average mesh size determined by means of relaxometric measurements on alginate microbeads and by a statistical analysis performed on TEM micrographs. Finally, the addition of a solution containing calcium ions allowed investigating further the different water relaxation modes within alginate hydrogels.

Inactivation of Matrix-bound Matrix Metalloproteinases by Cross-linking Agents in Acid-etched Dentin

OBJECTIVES Published transmission electron microscopy analysis of in vitro resin-dentin bonds shows that, after 44 months, almost 70% of collagen fibrils from the hybrid layer disappear. Matrix metalloproteinases (MMPs) play an important role in that process and are thought to be the main factor responsible for the solubilization of dentin collagen. Therefore, this study aimed to evaluate the inactivation of matrix-bound MMPs by two different cross-linking agents, carbodiimide (EDC) or proanthocyanidin (PA), or the MMP-inhibitor, chlorhexidine (CHX), on acid-etched dentin using a simplified MMP assay method. MATERIALS AND METHODS Dentin beams (2×1×6 mm) were obtained from mid-coronal dentin of sound third molars and randomly divided into six groups (G) according to the dentin treatment: G1: Deionized water (control); G2: 0.1 M EDC; G3: 0.5 M EDC; G4: 0.5 M EDC + 35% hydroxyethyl methacrylate (HEMA); G5: 5% PA; and G6: 2% CHX. The beams were etched for 15 seconds with 37% phosphoric acid, rinsed, and then immersed for 60 seconds in one of the treatment solutions. The data were expressed both in absorbance values at 412 nm and in MMP-9 activity equivalents. The total MMP activity of dentin was analyzed for one hour by colorimetric assay (Sensolyte). Data were submitted to Wilcoxon nonparametric test and Mann-Whitney tests (p>0.05). RESULTS All experimental cross-linking solutions significantly reduced MMP activity from 79.8% to 95.2% when compared to the control group. No difference was observed among 0.1 M EDC (84.8%), 5% PA (87.6%), and 2% CHX (79.8%). Addition of 35% HEMA to 0.5 M EDC produced inactivation (95.2%) that was similar to that of 0.5 M EDC alone (92.7%). CONCLUSION Dentin treatment with cross-linking agents is effective to significantly reduce MMP activity. Mixing 0.5 M EDC and 35% HEMA did not influence EDC inhibitor potential.

Beeswax as dental filling on a Neolithic human tooth

Evidence of prehistoric dentistry has been limited to a few cases, the most ancient dating back to the Neolithic. Here we report a 6500-year-old human mandible from Slovenia whose left canine crown bears the traces of a filling with beeswax. The use of different analytical techniques, including synchrotron radiation computed micro-tomography (micro-CT), Accelerator Mass Spectrometry (AMS) radiocarbon dating, Infrared (IR) Spectroscopy and Scanning Electron Microscopy (SEM), has shown that the exposed area of dentine resulting from occlusal wear and the upper part of a vertical crack affecting enamel and dentin tissues were filled with beeswax shortly before or after the individual’s death. If the filling was done when the person was still alive, the intervention was likely aimed to relieve tooth sensitivity derived from either exposed dentine and/or the pain resulting from chewing on a cracked tooth: this would provide the earliest known direct evidence of therapeutic-palliative dental filling.

Automated quantitative characterization of alginate/hydroxyapatite bone tissue engineering scaffolds by means of micro-CT image analysis

Accurate image acquisition techniques and analysis protocols for a reliable characterization of tissue engineering scaffolds are yet to be well defined. To this aim, the most promising imaging technique seems to be the X-ray computed microtomography (μ-CT). However critical issues of the analysis process deal with the representativeness of the selected Volume of Interest (VOI) and, most significantly, its segmentation. This article presents an image analysis protocol that computes a set of quantitative descriptors suitable for characterizing the morphology and the micro-architecture of alginate/hydroxyapatite bone tissue engineering scaffolds. Considering different VOIs extracted from different μ-CT datasets, an automated segmentation technique is suggested and compared against a manual segmentation. Variable sizes of VOIs are also considered in order to assess their representativeness. The resulting image analysis protocol is reproducible, parameter-free and it automatically provides accurate quantitative information in addition to the simple qualitative observation of the acquired images.