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Alginate/hydroxyapatite composites, with isotropic or anisotropic porosity, were developed using two different freezing methods. Morphological analysis highlighted the differences in their three-dimensional network. Swelling and in vitro degradation studies revealed that both structures are highly hydrophilic and stable. Static compression tests showed that isotropic scaffolds possess higher modulus and toughness than anisotropic ones. Cyclic compression tests revealed that wet scaffolds are able to withstand high deformations with a peculiar shape-recovery behaviour. In vitro biological tests showed that osteoblast-like cells proliferate on both types of structures in a comparable manner.

The surfaces of the oral cavity are always exposed to a broad variety of microor- ganisms able to form biofilms (Filoche et al, 2010) characterized by microbial com- munities that are organized as a network of cell-to-cell interactions. Streptococci are the predominant bacterial population of the oral environment and S.mitis in particular is the first colonizer of the oral biofilm (Di Giulio et al, 2013). Silver-based medical products have been proven to be effective in retarding and preventing bacterial growth. In order to prevent silver nanoparticles aggregation, a lactose-modified chitosan has been set up and resulted effective in stabilizing colloidal solution of nanoparticles (Chitlac-nAg) (Travan et al, 2009). Since many bacteria are able to internalize into eukaryotic cells, in our study we have investigated both the intracellular signaling governing S. mitis internalization into HGFs and the biological effect of ChitlacnAg on eukaryotic and prokaryotic cells in a co-culture model system. The internalization of S. mitis into HGFs is due to F-actin cytoskeleton reorganization and reduced expression within the cell. Immunofluorescence shows actin polymerization at invasion sites along with vinculin increased expression and spot organization. Vinculin is an adaptor protein that regulates the adhesion of integrin receptors to actin cytoskeleton. In presence of S. mitis an increment of integrin β1 and FAK expression, responsible for the entrance of the microorganism in HGFs is consistent, as revealed by electron microscopy analysis. This adhesion and uptake proteins profile is the same in the presence of saliva as well as bacteria uptake. When Chitlac-nAg is administred to cell culture the expression of all four proteins decreases and Ag nano- particles are recognized within the cells. Further, in presence of Ag nanoparticles the low amount of FAK is almost localized at nuclear level. In presence of Ag and S.mitis, the expression of all four proteins is increased, with respect to control, and F-actin cytoskeleton rearranged, while a raised number of bacteria is shown. This effect is mit- igated by the presence of saliva in cell culture, which probably prevents bacteria entry into the cell. These results let us hypothesize that Chitlac-nAg, developing its bacteri- cidal action could represent a good component of tooth paste and mouthwash., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Since ancient times, silver has been extensively used to control infections. Silver based medical products have been proved to be effective in retarding and preventing bacterial infections (Chen et al., 2007). In order to prevent silver nanoparticles aggregation a lactose-modified chitosan was shown to be effective in stabilizing colloidal solutions of silver nanoparticles: “Chitlac-nAg” (Travan et al., 2009). Silver ions and nanoparticle are capable to destroy the bacterial cell wall by reacting with sulfhydryl groups on membrane proteins (Kruszewski et al., 2003). Since the cells are capable of internalizing nanoparticles there is the risk of a massive uptake by eukaryotic cells, which eventually leads to their death through oxidative DNA damage (Li et al. 2013) In the present work we investigated the effects of Chitlac-nAg on primary human gingival fibroblast (HGFs) co-cultured with Streptococcus mitis in the presence of saliva. HGFs were obtained from fragments of healty marginal gingival tissue, co-cultured with the clinical strain of S. mitis and treated for 24-48h with Chitlac or Chitlac-nAg. Cytotoxicity evaluated by LDH assay showed an increment in LDH release in co-culture in the presence of Chitlac n-Ag and saliva. Oxidative stress detected by means of Reactive Oxygen Species formation highlighted an early ROS presence in samples with Chitlac-nAg and saliva, but this value was similar to control after 48h; apoptotic and necrotic cells were detected by means of Annexin V/PI showing an increase in cell death in HGFs treated with Ag and saliva after 24h, and returned to basal levels after 48h; the uptake of nanoparticles by cells was determined by optical and electronic microscopy revealing the Ag uptake in vesicles. The presence of lysosomes and autophagosomes was verified by Lysotracker and by LC3 respectively. In vitro results showed that in our co-culture model, which mimics the microenvironment of the oral cavity, chitlac n-Ag does not exert cytotoxic effect towards HGFs that are able to execute a homeostasis mechanism through autophagy promoting cell survival., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Major target for prosthetic constructs of bone is the ability to elicit osteoinduction, osteoconduction and osteointegration. Promising constructs are represented by composite scaffolds made of biodegradable natural polysaccharides endowed with biocompatibility and mechanical properties. Alginate is a good candidate when its limits - weak mechanical properties and lack of cellular interactions - are overcome by reinforcing with inorganic components. In the present study an innovative strategy was developed to obtain three-dimensional scaffolds made up of alginate (Alg) and Hydroxyapatite (HAp). Scanning electron microscopy (SEM) observations, confocal laser scanning microscopy (CLSM) and micro-computed tomography (µ-CT) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 µm and 300 µm and over 88% porosity. Proliferation assay and SEM observations demonstrated that human osteosarcoma cell lines seeded onto the scaffolds were able to proliferate, maintain osteoblast-like phenotype and massively colonize the 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. Biocompatibility and bioactivity of the scaffolds were tested both in vitro and in vivo.

In the broad field of biomaterials, Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are frequently used for dental restoration (Lehtinen et al 2008), but infections due to bacterial adhesion remain the main reason of dental devices failure. In order to avoid biofilm formation on the components used for restoration and to reduce cytotoxicity against eukaryotic cells, a new material with antimicrobial properties was developed. Indeed, silver nanoparticles (n-Ag), which have well-known antimicrobial properties, were stabilized with a polyelectrolyte solution-Chitlac (lactose-modified chitosan) and was used to coating methacrylic thermosets (Travan et al, 2011). This study was aimed at evaluating the in vitro biological response of human gingival fibroblasts (HGFs)/Streptococcus mitis co-colture to this nanocomposite system. HGFs were obtained from fragments of healthy marginal gingival tissue, co-cultured with the clinical strain of S. mitis and treated for 24 -48 h with thermosets (uncoated or coated with Chitlac or Chitlac n-Ag). Cytotoxicity was evaluated by LDH assay; cell morphology and adhesion were verified by means of SEM and optical microscopy; cell migration was studied by a modified Boyden chamber and finally IL-6 and PGE2 secretion were detected by ELISA assays. In vitro results showed that in our co-culture model, which mimics the microenvironment of the oral cavity, the nanocomposite material does not exert cytotoxic effect towards HGFs that are able to adhere and migrate. The secretion of IL-6 is significant, but PGE2 production is minimal suggesting that IL-6 production is not related to an inflammatory response. Basing on its good biocompatibility we suggest this new tool useful for the realization of dental devices., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

2008/2009 Questo lavoro riguarda lo sviluppo di materiali nanocompositi per applicazioni biomediche e si configura all’ interno del progetto europeo “Newbone” (EU-FP6); in particolare, lo scopo principale della tesi era realizzare un rivestimento biocompatibile e dotato di proprietà antibatteriche per protesi ortopediche. Sono stati preparati sistemi nanocompositi basati su un polisaccaride derivato dal chitosano (Chitlac) che permette di ottenere soluzioni colloidali di nanoparticelle (argento e oro) con proprietà antibatteriche. Parallelamente, è stato studiato un particolare meccanismo chimico di riduzione degli ioni argento ad opera dei residui di lattitolo del Chitlac; le proprietà ottiche delle nanoparticelle ottenute attraverso questo meccanismo sono state valutate attraverso spettroscopia Raman, evidenziando la possibilità di avere un incremento del segnale grazie al verificarsi dell’ effetto SERS. Essendo state riscontrate migliori proprietà biologiche del sistema a base di argento (Chitlac-nAg) rispetto a quello a base di oro in termini di efficacia antimicrobica e biocompatibilità, Chitlac-nAg è stato scelto per i successivi studi di realizzazione del rivestimento per la protesi. Test sul meccanismo antimicrobico della soluzione ChitlacnAg hanno dimostrato l’interazione tra le nanoparticelle e la membrana batterica. Allo stesso tempo, poiché la mancanza di barriere fisiche può favorire la diffusione delle nanoparticelle all’ interno delle cellule eucariote con rischio di effetti citotossici causati dalla loro internalizzazione, si è voluto realizzare delle strutture tridimensionali a base di Chitlac in grado di intrappolare le nanoparticelle. A questo scopo, sono state sfruttate le proprietà di gelificazione del polisaccaride alginato in modo da ottenere un sistema semi-solido in miscela con Chitlac-nAg; il materiale ottenuto possiede marcate proprietà antibatteriche senza però risultare tossico per le cellule eucariote, come dimostrato da test in vitro e in vivo. Questo risultato è particolarmente importante in relazione allo stato dell’ arte sull’ argomento. Poiché la parte portante della protesi è costituita da un polimero metacrilico, al fine di rivestire questo materiale di substrato è stata messa a punto una tecnica basata sull’ attivazione della superficie e successiva deposizione del rivestimento a base di Chitlac. Questa tecnica permette di ottenere un rivestimento nanocomposito costituito da nanoparticelle di argento incorporate nella matrice di Chitlac. Grazie a questo strato bioattivo la superficie della protesi acquisisce un’ efficace attività antibatterica che si manifesta quando i batteri entrano in diretto contatto con il materiale. Inoltre, test in vitro hanno dimostrato che le cellule eucariote aderiscono e proliferano sul rivestimento nanocomposito, suggerendo quindi una buona integrazione del materiale nei tessuti attorno all’ impianto. La combinazione di tali proprietà ha determinato la scelta di questo rivestimento per il test in vivo su “minipig” a conclusione del progetto europeo: questo test è al momento in via di svolgimento e da esso ci si può attendere una conferma degli incoraggianti risultati ottenuti dagli studi in vitro. The present work is focused on the development of nanocomposite systems for biomedical applications and has been carried out in the framework of the European Project called “Newbone” (EU-FP6); in particular, the main goal of the thesis was to realize biocompatible coatings for orthopedic prosthesis endowed with antimicrobial properties. Nanocomposite systems based on a chitosan-derived polysaccharide (Chitlac) that stabilizes metal nanoparticles (silver and gold) have been prepared in colloidal solutions which possess broad spectrum antibacterial properties. As a complementary work, it was studied and defined a particular chemical mechanism of silver ions reduction carried out by the lactose moieties of Chitlac; the optical properties of the metallic nanoparticles obtained through this mechanism were tested by means of Raman spectroscopy, thus detecting considerable enhancements of the signal due to the SERS effect (Surface Enhanced Raman Scattering). Given the better biological properties of silver-based systems (Chitlac-nAg) with respect to gold in terms of antimicrobial efficacy and biocompatibility, only the former metal was chosen in the following steps towards the preparation of the nanocomposite coating for the prosthesis. Studies on the biocidal mechanism of the Chitlac-nAg solution ascribed the activity to the interaction metal-bacteria membrane. On the other hand, since the lack of physical barriers to nanoparticle diffusion into eukaryotic cells determines the risk of a massive uptake with cytotoxic outcomes, we focused our attention toward the preparation of Chitlac-based threedimensional structures entrapping silver nanoparticles. To this end, the gel forming properties of the polysaccharide alginate were exploited allowing the production of a semi-solid system in a mixture with Chitlac-nAg: this material displays potent antibacterial properties without showing cytotoxic effects towards eukaryotic cells, as verified by in vitro and in vivo tests. Such result was particularly important in relation to the state of the art in this research field. Since the core material of the prosthesis is made of methacrylic thermosets, in order to coat this substrate material we have devised a technique based on surface activation followed by deposition of the Chitlac-based layer. Such technique allows obtaining a nanocomposite coating where silver nanoparticles are entrapped within the Chitlac matrix. This bioactive layer endows the thermoset surface with considerable antimicrobial properties, as bacteria are rapidly killed upon direct contact with the material. At the same time, in vitro tests proved that eukaryotic cells adhere and proliferate on the nanocomposite coating, which indicates the possibility to have good integration of the material in the tissues surrounding the implant. The combination of these properties determined the choice of our coating for the final in vivo test in a minipig model as a conclusion of the European project; this test is in progress at the moment and it will hopefully confirm the encouraging studies in vitro. XXII Ciclo 1981

An emerging strategy to improve adhesiveness of biomaterials in wet conditions takes inspiration from the adhesive features of marine mussel, which reside in the chemical reactivity of catechols. In this work, a catechol-bearing molecule (dopamine) was chemically grafted onto alginate to develop a polysaccharide-based membrane with improved adhesive properties. The dopamine-modified alginates were characterized by NMR, UV spectroscopy and in vitro biocompatibility. Mechanical tests and in vitro adhesion studies pointed out the effects of the grafted dopamine within the membranes. The release of HA from these resorbable membranes was shown to stimulate fibroblasts activities (in vitro). Finally, a preliminary in vivo test was performed to evaluate the adhesiveness of the membrane on porcine intestine (serosa). Overall, this functionalized membrane was shown to be biocompatible and to possess considerable adhesive properties owing to the presence of dopamine residues grafted on the alginate backbone. Statement of Significance This article describes the development of a mussels-inspired strategy for the development of an adhesive polysaccharide-based membrane for wound healing applications. Bioadhesion was achieved by grafting dopamine moieties on the structural component on the membrane (alginate): this novel biomaterial showed improved adhesiveness to the intestinal tissue, which was demonstrated by both in vitro and in vivo studies. Overall, this study points out how this nature-inspired strategy may be successfully exploited for the development of novel engineered biomaterials with enhanced bioadhesion, thus opening for novel applications in the field of general surgery.

The effect of transient cross-links has been explored on a lactose-modified chitosan, which previously had shown interesting biological features. The presence of galactose side chains and of the polyol spacer resulted particularly appealing for the reticulation by borate ions. The interaction between chitlac and borax was investigated by means of 11B NMR while rheology pointed to a marked non-linear behavior depending on the amount of borax added to the system. The presence of limited amount of cross-linking ion led to dilatant behavior when the steady flow curve was measured. In addition, strain stiffening was noticed on elastic response upon exceeding a critical stress, indicating a transient nature in the formation of the cross-links. The non-linear response of chitlac in the presence of borax compared surprisingly well with the one showed by proteins composing the natural ECM pointing at a possible role of mechanotransduction in the biological significance of the modified chitosan.

Chronic non-healing wounds are a clinically important problem in terms of number of patients and costs. Wound dressings such as hydrogels, hydrocolloids, polyurethane films and foams are commonly used to manage these wounds since they tend to maintain a moist environment which is shown to accelerate re-epithelialization. The use of antibacterial compounds is important in the management of wound infections. A novel wound-dressing material based on a blended matrix of the polysaccharides alginate, hyaluronic acid and Chitlac-silver nanoparticles is here proposed and its application for wound healing is examined. The manufacturing approach to obtain membranes is based on gelling, foaming and freeze-casting of alginate, hyaluronic acid and Chitlac-silver nanoparticles mixtures using calcium ions as the cross-linking agent. Comprehensive evaluations of the morphology, swelling kinetics, permeability, mechanical characteristics, cytotoxicity, capability to inhibit metalloproteinases and of antibacterial property were conducted. Biological in vitro studies demonstrated that hyaluronic acid released by the membrane is able to stimulate the wound healing meanwhile the metal silver exploits an efficient antibacterial activity against both planktonic bacteria and biofilms. Overall, the experimental data evidence that the studied material could be used as antibacterial wound dressing for wound healing promotion.

Sterilization methods such as gamma-irradiation, steam sterilization and ethylene oxide gas treatment can have negative effects on molecular structure and properties of polysaccharide-based biomaterials. In this perspective, the use of supercritical carbon dioxide (scCO(2)) has been proposed as an alternative method for biomaterial sterilization. In this work, chemical, mechanical and biological properties of polysaccharidic membranes for surgical applications were investigated after sterilization by scCO(2). Four sets of sterilizing conditions were considered and SEC analyses were performed in order to identify the one with lower impact on the polysaccharidic matrix of membranes (alginate). Mechanical tests showed that the resistance of membranes was slightly affected after sterilization. Biological analyses proved the biocompatibility of the sterilized membranes both in vitro and in a preliminary in vivo test. Overall, this study points out that this sterilization technique can be successfully employed to achieve an effective and safe sterilization of polysaccharidic membranes for surgical use. (C) 2017 Elsevier Ltd. All rights reserved.

In this work, we studied the antimicrobial properties of a nanocomposite system based on a lactose-substituted chitosan and silver nanoparticles: Chitlac-nAg. Twofold serial dilutions of the colloidal Chitlac-nAg solution were both tested on Streptococcus mitis, Streptococcus mutans, and Streptococcus oralis planktonic phase and biofilm growth mode as well as on saliva samples. The minimum inhibitory and bactericidal concentrations of Chitlac-nAg were evaluated together with its effect on sessile cell viability, as well as both on biofilm formation and on preformed biofilm. In respect to the planktonic bacteria, Chitlac-nAg showed an inhibitory/bactericidal effect against all streptococcal strains at 0.1% (v/v), except for S. mitis ATCC 6249 that was inhibited at one step less. On preformed biofilm, Chitlac-nAg at a value of 0.2%, was able to inhibit the bacterial growth on the supernatant phase as well as on the mature biofilm. For S. mitis ATCC 6249, the biofilm inhibitory concentration of Chitlac-nAg was 0.1%. At sub-inhibitory concentrations, the Streptococcal strains adhesion capability on a polystyrene surface showed a general reduction following a concentration-dependent-way; a similar effect was obtained for the metabolic biofilm activity. From these results, Chitlac-nAg seems to be a promising antibacterial and antibiofilm agent able to hinder plaque formation.

This PhD thesis deals with the development of bioactive polysaccharide-based biomaterials for bone tissue and neural tissue engineering. Alginate was chosen for its gel forming properties; hyaluronic acid and chitlac (a lactose-modified chitosan) were chosen for their bioactive properties. The properties of these polysaccharides have been implemented by introducing gelatin, functionalized Carbon Nanotubes (f-CNTs) and silver nanoparticles (nAg). In the first part of the work, the dispersibility and aggregation tendency of f-CNTs have been characterized by means of Low Field Nuclear Magnetic Resonance (LF-NMR). It was also possible to correlate the f-CNTs concentration to the proton transversal relaxation rate of water. Alginate/f-CNTs solutions and hydrogels have been analyzed by LF-NMR, rheology and uniaxial compression tests; these investigations showed that the f-CNTs are able to affect nanocomposite properties depending on their concentration and functionalization. In the second part of the work, the preparation of a bioactive (bridging) implant for the treatment of Spinal Cord Injury is described. Neuronal cells and mesoangioblasts (MABs) engineered for the production of neurotrophines have been cultured and co-cultured on polysaccharide-coated glass substrates in order to evaluate the biological effects of chitlac. Chitlac-coated surfaces where shown to possess higher surface energies if compared to chitosan-coated ones and enable the formation of wider neural networks with improved electrical activity. The co-cultures confirmed the higher bioactivity of chitlac/alginate substrates and the biological role of neurotrophines. Porous scaffolds of alginate/chitlac have been prepared; these scaffolds where shown to be stable in simulated body fluid for over a month. The mechanical properties of rehydrated scaffolds where proved to be similar to those of neural tissue. Biological properties of chitlac substrates enriched with f-CNTs are currently under investigation. In the third part of the work, tridimensional scaffolds and injectable fillers were developed for the treatment of non-critical bone defects. Porous scaffolds with different pore morphologies have been prepared by freeze casting of alginate/HAp hydrogels. Isotropic porosity was obtained by freezing the constructs in a cryostat, while anisotropic porosity was obtained by the Ice Segregation Induced Self Assembly process. Physical, mechanical and biological analyses revealed that the differences in pore morphology determine differences in the mechanical properties of the scaffolds. Biocompatible f-CNTs have been used to implement the isotropic scaffolds; the biological analyses showed that the presence of f-CNTs does not affect scaffold properties. Osteoconductive/antimicrobial injectable bone fillers, based on alginate/HAp microbeads dispersed in polysaccharide mixtures, have been developed. Microbeads were enriched with nAg synthesized in chitlac. Antimicrobial assays proved the antibacterial properties of the microbeads towards bacteria in suspension and on pre-formed biofilms. Biological assays showed the biocompatibility of the microbeads and their ability to sustain osteoblast proliferation. The fillers prepared by dispersing microbeads in polysaccharide mixtures were shown to be easily injectable through surgical syringes. In vivo studies on a rabbit model of non-critical bone defect pointed out the biocompatibility and the osteoconductivity of the composite materials. Further studies are ongoing in order to evaluate the possibility to further implement the bioactive properties of the microbeads by addiction of gelatin.

In this work, hydrogen peroxide (H2O2) was employed to improve the adhesion of an alginate-based membrane to intestinal serosa: mechanical adhesion test and SEC-MALLS studies were performed to investigate the contribution of H2O2-induced modifications of both tissue and membrane to the adhesion enhancement. Adhesion tests confirmed that the formation of an adhesive gelatin layer on serosa plays a major role in increasing adhesion forces, while SEC-MALLS studies revealed a slight decrease of alginate molecular weight. However, the latter chain degradation does not seem to significantly impair the adhesion strength. Overall, this study describes a possible strategy to create adhesive interfaces between biomaterials and collagen-containing tissues.

stract Silver-based products have been proven to be effective in retarding and preventing bacterial growth since ancient times. In the field of restorative dentistry, the use of silver ions/nanoparticles has been explored to counteract bacterial infections, as silver can destroy bacterial cell walls by reacting with membrane proteins. However, it is also cytotoxic towards eukaryotic cells, which are capable of internalizing nanoparticles. In this work, we investigated the biological effects of Chitlac-nAg, a colloidal system based on a modified chitosan (Chitlac), administered for 24-48 h to a co-culture of primary human gingival fibroblasts and Streptococcus mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its efficiency to combat oral hygiene-related diseases without affecting eukaryotic cells. Cytotoxicity, reactive oxygen species production, apoptosis induction, nanoparticles uptake, and lysosome and autophagosome metabolism were evaluated. In vitro results show that Chitlac-nAg does not exert cytotoxic effects on human gingival fibroblasts, which seem to survive through a homoeostasis mechanism involving autophagy. That suggests that the novel biomaterial Chitlac-nAg could be a promising tool in the field of dentistry.

The present manuscript addresses the description of binary systems of hyaluronan (HA) and alginate (Alg) in semi-concentrated solution. The two polysaccharides were completely miscible in the entire range of relative weight fraction explored at a total polymer concentration of up to 3 % (w/V). The rheological study encompassed steady flow and mechanical spectra for HA/Alg systems at different weight fractions with hyaluronan at different molecular weights. These extensive analyses allowed us to propose a model for the molecular arrangement in solution that envisages a mutual exclusion between the two polysaccharides even though a clear phase separation does not occur. This result may have profound implications when biomaterials based on the combination of alginate and hyaluronan are proposed in the field of biomedical materials.