The development of multi-resistant bacteria strains is a serious concern for the use of antibiotics in the design of biomaterials. Therefore, there is a strongly need of novel and highly effective strategies and compounds not susceptible to bacterial resistance development. There is a wide variety of new agents based on inorganic and organic/polymeric materials that have been proved to act as wide spectrum agents; some examples are represented by silver, antimicrobial peptides, materials able to generate reactive oxygen species and carbon-based materials.208
Among those compounds, silver has been known for centuries for its bactericidal activity and has been already used in several topical applications for the prevention and the control of bacterial infections.255 In particular silver nanoparticles (nAg) are one of the most widely used antibacterial compounds in the clinical practice.208 nAg, through the release of silver ions, the generation of reactive-oxygen-species and the interaction with cell membranes, DNA and sulfur containing proteins have shown antibacterial activity both against Gram-positive and Gram-negative bacteria
INTRODUCTION
strains, but also against fungi and viruses.208,256-261 nAg have also proved to be toxic for methicillin-resistant strains of S. aureus and S. epidermidis,262 and to enhance the antimicrobial activity of antibiotics.263-265
nAg toxicity for eukaryotic cells is also strongly debated; the same mechanisms that are active against bacterial cells can result in toxic effects also on eukaryotic cells, in particular membrane damage and reactive-oxygen-species (ROS) production. The latter can be worsened by the damages that nAg can induce on the proteins involved in anti-oxidant defense mechanisms.266-268
Another issue related with the use of nAg is their stability, as the agglomeration in microparticles and aggregates significantly affects and decreases their antimicrobial properties.269
Novel nanotechnological strategies for the development of biocompatible wide spectrum agents involve the preparation of systems, for example silver decorated polymeric nanostructures, which are able to guarantee a long-term stability of nAg, controlling and normalizing their size and shape and reducing their toxicity.208
In this contest the preparation of a stable form of nAg, synthesized, dispersed and stabilized in polysaccharides, is an interesting strategy for the implementation of bioactive and antimicrobial properties in biomaterials. In particular, the process reported by Travan et al. in 2009 enables to synthesize, disperse and stabilize the nAg by the reduction of silver ions in the presence of chitlac (Figure 13).184
Figure 13. Schematic representation of silver nanoparticle stabilization by chitlac.
INTRODUCTION
In this form, the nAg are confined inside a polymer matrix and can be stabilized over time. At the same time, the system guarantees a slow release of silver ions that can exert the antimicrobial activity, without being toxic for eukaryotic cells. The chitlac containing silver nanoparticles system (chitlac-nAg) has been thoroughly investigated for the preparation of antimicrobial coatings,195,196,270 and tridimensional hydrogels and microbeads in combination with alginate.184
2 AIMS OF THE WORK
Nanotechnology represents a fertile ground for the development of novel bioactive biomaterials.
In tissue engineering, such biomaterials can be designed and prepared by taking advantage of engineered polysaccharides. The present work, by using the combination of different polysaccharides and organic/inorganic nanostructures, aims at the development and characterization of novel biomaterials to be employed in bone and neural tissue engineering. This thesis has three main objectives:
2.1 CHARACTERIZATION OF FUNCTIONALIZED CARBON NANOTUBES (f-CNTs) DISPERSIONS AND NANOSYSTEMS
Specific aims:
Evaluation of f-CNTs concentration, dispersibility and aggregation tendency in aqueous and polymeric dispersions by means of Low Field Nuclear Magnetic Resonance (LF-NMR).
Spectroscopical, mechanical and rheological characterization of alginate/f-CNTs solutions and hydrogels by means of LF-NMR, uniaxial compression tests and rheological measurements.
2.2 DEVELOPMENT OF A BRIDGING IMPLANT FOR THE SPINAL CORD INJURY TREATMENT
The second objective is part of PRIN-MIUR project “Spinal injury: towards the development of cell-instructive scaffolds for nerve tissue repair” (2014-2017).
Specific aims:
Preparation morphological and physical characterization of polysaccharide coated glass substrates
AIMS OF THE WORK
Evaluation of biocompatibility and biological effects of the polysaccharide-coated glass substrates on two dimensional neuronal network model and on co-cultures of motoneuron progenitors and engineered mesoangioblasts.
Preparation and characterization of porous alginate scaffolds, functionalized with chitlac, with isotropic or anisotropic pore morphologies.
2.3 DEVELOPMENT OF FILLERS FOR NON-CRITICAL BONE DEFECTS HEALING
Specific aims:
Determination of the morphological differences and of the influence of pore morphology on stability, mechanical performances and biological properties of alginate/hydroxyapatite (HAp) scaffolds.
Implementation of alginate/HAp scaffolds with f-CNTs and biological characterization.
Development of an antimicrobial injectable bone filler based on alginate/HAp microbeads implemented with silver nanoparticles (nAg)
Characterization of stability, morphology, biocompatibility, biological properties and injectability of the microbeads
In vivo evaluation of biocompatibility and osteoconductive properties of the injectable filler on a rabbit model of non-critical bone defects.
Preparation and characterization of alginate/HAp materials implemented with collagen and gelatin.
AIMS OF THE WORK Acknowledgments and collaborations
f-CNTs have been provided by prof. Maurizio Prato research group (Department of Chemical and Pharmaceutical Science, University of Trieste).
Rheological and LF-NMR measurements have been performed in collaboration with prof. Mario Grassi and Michela Abrami (Department of Engineering and Architecture, University of Trieste).
The PRIN-MIUR project involves the laboratories of Prof. Laura Ballerini (SISSA, Trieste) and Dr. Raffaella Scardigli (CNR, Laboratory of Neurotrophic factors and Neurodegenerative Diseases of Prof. Antonino Cattaneo).
The in vivo studies have been performed in collaboration with Prof. Niko Moritz and Dr. Julia Kulkova (Turku Clinical Biomaterials Centre, TCBC, University of Turku).
COST Action MP1301 and Consorzio Interuniversitario per le Biotecnologie, are acknowledged for the financial support during the visiting research period at TCBC.
3 RESULTS AND DISCUSSION
The work described in this thesis deals with the preparation and characterization of polysaccharide-based biomaterials, implemented with silver nanoparticles (nAg) and functionalized carbon nanotubes (f-CNTs), for applications in bone and neural tissue engineering. This work can be divided in three major sections: i) the first one is focused on the determination of the effects of the f-CNTs presence on the spectroscopical, rheological and mechanical properties of dispersions and based nanosystems; ii) the second one describes the preparation of polysaccharide-coated two-dimensional substrates for the evaluation of the biological effects of different polysaccharides on the behavior and function of neural cells, and the preparation of a tridimensional scaffold for neural tissue engineering; iii) the third one describes the preparation and the characterization of polysaccharide-based tridimensional scaffolds and antimicrobial injectable fillers for the bone tissue engineering.