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La presente invenzione riguarda addotti polimerici solubili tra acido borico o suoi derivati e precursori e derivati oligosaccaridici del chitosano che non formano idrogeli a concentrazioni comprese nell’intervallo tra il 3 % e il 5 %, la loro sintesi e il loro utilizzo nel campo cosmetico, biomedico e farmaceutico, in particolare per le applicazioni come viscosupplementante, elasticizzante ed adesivo.

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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 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.

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 present invention nanocomposite materials in form of three-dimensional structure formed by a polymeric matrix consisting of a polysaccharidic composition of neutral or anionic polysaccharides and a branched cationic polysaccharides, in which metallic nanoparticles are uniformly dispersed and stabilized, are described. Using appropriate techniques of gelification or by means of an appropriate dehydration, the nanocomposite materials are three-dimensional matrices having different shapes in hydrated form as hydrogels, or in non-hydrated form. These nanocomposite materials have a broad-spectrum of strong bactericidal activity, but do not show any cytotoxicity. The particular antibacterial properties associated with metallic particle nano-scale and the presence of biological signals on the polymeric chains along with the lack of cytotoxicity may be exploited in developing new-generation biomaterials provided with antimicrobial properties and for many other applications in biomedical, pharmaceutical and food field.

The present invention provides nanocomposite systems made of metallic nanoparticles stabilized with branched cationic polysaccharides, in particular alditolic or aldonic mono- and oligo-saccharidic derivatives of chitosan, and their preparation obtainable with aqueous solutions of these polysaccharides in the presence or absence of reducing agents. The peculiar chemical and physical-chemical features of these polysaccharides allow to form metallic nanoparticles homogeneously dispersed in the polysaccharidic matrix and an effective stabilization thereof. The properties associated with the nanometric dimensions and the presence of biological signals on polymeric chains may be exploited in applications with antimicrobial activities and of molecular biosensors.

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

Nella presente invenzione è descritta la preparazione di sistemi nanocompositi contenenti nanoparticelle metalliche ottenibili in soluzioni acquose di derivati oligosaccaridici del chitosano. Grazie alle particolari caratteristiche chimiche e chimico-fisiche di tali polisaccaridi è possibile ottenere la formazione di nanoparticelle metalliche omogeneamente disperse e una loro efficace stabilizzazione. Le particolari proprietà associate alla nano-scala delle particelle e la presenza di segnali biologici sulle catene polimeriche possono venire sfruttate nello sviluppo di sistemi con caratteristiche antimicrobiche e per la progettazione di biosensori molecolari.

Nella presente invenzione sono descritti materiali nanocompositi formati da una matrice polimerica consistente in una composizione polisaccaridica tra polisaccaridi neutri o anionici e polisaccaridi cationici ramificati, in cui nanoparticelle metalliche sono disperse uniformemente e stabilizzate. Sfruttando opportune tecniche di gelificazione è possibile ottenere da tali materiali nanocompositi idrogel o, per opportuna disidratazione, matrici tridimensionali non idrate con diverse forme (ad esempio cilindri, microsfere, scaffold, gel a sviluppo superficiale quali film secchi o umidi, fibre). Tali materiali nanocompositi possiedono una marcata attività battericida ad ampio spettro senza però risultare citotossici. Le particolari proprietà antibatteriche associate alla nano-scala delle particelle metalliche e alla presenza di segnali biologici sulle catene polimeriche insieme alla mancanza di citotossicità possono venire sfruttate nello sviluppo di biomateriali di nuova generazione dotati di proprietà antimicrobiche e per svariate altre applicazioni nel campo biomedico, farmaceutico ed alimentare.

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