Eleonora Marsich

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.

Silver–polysaccharide nanocomposite antimicrobial coatings for methacrylic thermosets

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are receiving increasing attention as biomaterials for dental and orthopedic applications; for both these fields, bacterial adhesion to the surface of the implant represents a major issue for the outcome of the surgical procedure. Moreover, the biological behaviour of these materials is influenced by their ability to establish proper interactions between their surface and the eukaryotic cells of the surrounding tissues, which is important for good implant integration. The aim of this work was to develop an antimicrobial non-cytotoxic coating for methacrylic thermosets by means of a nanocomposite material based on a lactose-modified chitosan and antibacterial silver nanoparticles. The coating was characterized by UV-vis spectrophotometry, optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In vitro tests were employed for a biological characterization of the material: antimicrobial efficacy tests were carried out with both Gram+ and Gram- strains. Osteoblast-like cell-lines, primary human fibroblasts and adipose-derived stem cells, were used for LDH cytotoxicity assays and Alamar blue cell proliferation assays. Cell morphology and distribution were evaluated by SEM and confocal laser scanning microscopy. In vitro results showed that the nanocomposite coating is effective in killing both bacterial strains and that this material does not exert any significant cytotoxic effect towards tested cells, which are able to firmly attach and proliferate on the surface of the coating. Such biocompatible antimicrobial polymeric films containing silver nanoparticles may have good potential for surface modification of medical devices, especially for prosthetic applications in orthopedics and dentistry.

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.

Biological response of hydrogels embedding gold nanoparticles.

A nanocomposite hydrogel based on natural polysaccharides and gold nanoparticles (ACnAu) has been prepared and its biological effects were tested in vitro with both bacteria and eukaryotic cells. Antimicrobial tests showed that AC-nAu gels are effective in killing both gram+ (Staphylococcus aureus) and gram- (Pseudomonas aeruginosa) bacteria. LDH assays pointed at a toxic effect towards eukaryotic cell-lines (HepG2 and MG63), in contrast with the case of silver-based hydrogels; cytofluorimetry studies demonstrated an apoptosis-related mechanism induced by increase of ROS intracellular level which leads to cell death after 24 h of direct contact with AC-nAu gels. In vivo biocompatibility has been evaluated in a rat model, investigating the peri-implant soft tissue reaction after 1 month of implantation. The results show that silver-containing samples induced a fibrotic capsule of the same average thickness of the control sample (devoid of nanoparticles) (∼50 μm), while in the case of gold containing materials the fibrotic capsule was thicker (∼100 μm), confirming a higher biocompatibility for silver-based samples than for gold-based ones.

Polysaccharide-coated thermosets for orthopedic applications: from material characterization to in vivo tests.

The long-term stability and success of orthopedic implants depend on the osseointegration process, which is strongly influenced by the biomaterial surface. A promising approach to enhance implant integration involves the modification of the surface of the implant by means of polymers that mimic the natural components of the extracellular matrix, for example, polysaccharides. In this study, methacrylate thermosets (bisphenol A glycidylmethacrylate/triethyleneglycol dimethacrylate), a widely used composition for orthopedic and dental applications, have been coated by electrostatic deposition of a bioactive chitosan-derivative. This polysaccharide was shown to induce osteoblasts aggregation in vitro, to stimulate cell proliferation and to enhance alkaline phosphatase activity. The coating deposition was studied by analyzing the effect of pH and ionic strength on the grafting of the polysaccharide. Contact angle studies show that the functionalized material displays a higher hydrophilic character owing to the increase of surface polar groups. The mechanical properties of the coating were evaluated by nanoindentation studies which point to higher values of indentation hardness and modulus (E) of the polysaccharide surface layer, while the influence of cyclic stress on the construct was assessed by fatigue tests. Finally, in vivo tests in minipigs showed that the polysaccharide-based implant showed a good biocompatibility and an ability for osseointegration at least similar to that of the titanium Ti6Al4V alloy with roughened surface.

Adhesive and sealant interfaces for general surgery applications.

The main functions of biological adhesives and sealants are to repair injured tissues, reinforce surgical wounds, or even replace common suturing techniques. In general surgery, adhesives must match several requirements taking into account clinical needs, biological effects, and material features; these requirements can be fulfilled by specific polymers. Natural or synthetic polymeric materials can be employed to generate three-dimensional networks that physically or chemically bind to the target tissues and act as hemostats, sealants, or adhesives. Among them, fibrin, gelatin, dextran, chitosan, cyanoacrylates, polyethylene glycol, and polyurethanes are the most important components of these interfaces; various aspects regarding their adhesion mechanisms, mechanical performance, and resistance to body fluids should be taken into account to choose the most suitable formulation for the target application. This review aims to describe the main adhesives and sealant materials for general surgery applications developed in the past decades and to highlight the most important aspects for the development of future formulations.

Polysaccharide-based networks from homogeneous chitosan-tripolyphosphate hydrogels: synthesis and characterization.

Polysaccharide networks, in the form of hydrogels and dried membranes based on chitosan and on the cross-linker tripolyphosphate (TPP), were developed using a novel approach. TPP was incorporated into chitosan by slow diffusion to favor a controlled gelation. By varying chitosan, TPP, and NaCl concentration, transition from inhomogeneous to homogeneous systems was achieved. Rheology and uniaxial compression tests enabled to identify the best performing hydrogel composition with respect to mechanical properties. FTIR, (31)P NMR, and spectrophotometric methods were used to investigate the interaction chitosan-TPP, the kinetics of phosphates diffusion during the dialysis and the amount of TPP in the hydrogel. A freeze-drying procedure enabled the preparation of soft pliable membranes. The lactate dehydrogenase assay demonstrated the biocompatibility of the membranes toward fibroblasts. Overall, we devised a novel approach to prepare homogeneous macroscopic chitosan/TPP-based biomaterials with tunable mechanical properties and good biocompatibility that show good potential as novel polysaccharide derivatives.

Surface modification and polysaccharide deposition on BisGMA/TEGDMA thermoset.

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets and composites are well-known examples of biomaterials for dental applications that are receiving growing interest for orthopedic applications. While mechanical bulk properties are guaranteed by the presence of reinforcing fibers, in vitro and in vivo performances of these materials are ultimately driven by their ability to establish proper interactions between their surface and the surrounding tissues. Hence, the development of novel chemical processes enabling the introduction of bioactive molecules on the surface of these methacrylate-based thermosets is of particular interest. In the present work, we have devised a chemical strategy to expose carboxylic groups on the surface of the BisGMA/TEGDMA thermoset. The presence of negative charges was confirmed by Fourier transform infrared-attenuated total reflectance and by UV-vis spectrophotometry. Bulk mechanical properties and surface morphology of the thermoset were only slightly affected upon chemical functionalization. The activated material was further refined by the deposition of a lactose-modified chitosan (chitlac) driven by strong electrostatic interactions. The presence of the bioactive polysaccharide was confirmed by fluorescence spectroscopy and by confocal laser scanning microscopy measurements. Scratch tests were performed to evaluate the mechanical behavior of the coating. Finally, in vitro tests revealed that the presence of chitlac led to a slight enhancement of cell proliferation with respect to the unmodified BisGMA/TEGDMA thermoset. This effect was more pronounced when chitlac decorated with an arginine-glycine-aspartic acid (RGD) peptide was used in the preparation of the coating. In the latter case, the in vitro performance of the coated BisGMA/TEGDMA thermoset became comparable with that of clinically used roughened titanium.

“The PAX6 gene is activated by the basic helix-loop-helix transcription factor NeuroD/BETA2”.

PAX6 is a transcription factor that plays an important role during pancreatic morphogenesis. The aim of the present study is to identify the upstream activator(s) of the PAX6 gene possibly involved in the early stages of pancreatic differentiation. Recently, individual elements regulating PAX6 gene activity in the pancreas have been identified in a 1100 bp Spe / Hin cII fragment 4.6 kb upstream of exon 0. Preliminary sequence analysis of this region revealed some potential DNA-binding sites (E boxes) specific for the binding of basic helix-loop-helix transcription factors. By using electrophoretic mobility shift assays, we demonstrated that both nuclear protein extracts from insulin-secreting RINm5F cells and in vitro -translated NeuroD/BETA2 can bind specifically to these E boxes. Furthermore, by transient transfection experiments we demonstrated that the expression of basic helix-loop-helix transcription factor NeuroD/BETA2 can induce activation of the PAX6 promoter in the NIH-3T3 cell line. Thus we show that NeuroD/BETA2 is involved in the activation of the expression of PAX6 through E boxes in the PAX6 promoter localized in a 1.1 kb sequence within the 4.6 kb untranslated region upstream of exon 0.