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Aim: to assess the fracture resistance of maxillary molars restored using heat-pressed lithium disilicate glass-ceramic (LDG) table-top restorations with two preparation designs. Methods: sixteen maxillary molars were randomly assigned to two groups (n = 8): Group 1 (G1) occlusal preparation for a 1 mm full-coverage table-top restoration with a 90° rounded shoulder margin; Group 2 (G2) 1 mm occlusal reduction with a marginal chamfer. Restorations were obtained by heat-pressed lithium disilicate glass-ceramic (LDG) and luted with Variolink II cement. The specimens were loaded to fracture after thermomechanical aging (1 250 000 cycles). A qualitative analysis on the type of fracture and a SEM analysis of marginal seal were conducted. Collected data were statistically analyzed (Mann Whitney U-test). Results: the mean load to fracture was 1796.58 ± 307.27 N in G1 and 1892.77 ± 379.40 N in G2. No statistical difference was found. Six specimens showed restorable fractures in G1, 4 specimens in G2. Thermomechanical aging did not produce marginal seal degradation. Conclusion: the two preparations designs for LDG table-top restorations exhibited a similar load resistance, type of fracture and marginal adaptation.

Aim: the gold standard in endodontic surgery is characterized by meticulous retrograde procedures that could be not performable in all teeth due to multiple clinical difficulties. The possibility to perform the only apical resection, when feasible, without further retrograde steps might represent a substantial clinical advantage. The present study analyzes the filling quality achieved with standard retrograde technique in comparison with alternative techniques with or without retrograde procedures. Methods: the sample size was determined using with the following parameters: α=0.05; β=0.20; δ=3.0; σ=1.5. Twenty-four single-rooted teeth were selected from a pool of freshly extracted teeth, discarding those with aberrant anatomy. Selected teeth of comparable size were decoronated to obtain 12 mm long roots. The canals were scouted with manual files and the working length acquired. After mechanical glide path establishment, canal shaping was performed with HyFlex EDM rotary files up to size 40.04 taper (500 rpm; 2.5 Ncm). The roots were analyzed with a computed microtomography scanner to identify the possible presence of dentine microcracks. Afterwards, the roots were randomly assigned to two groups: twelve canals were filled with the single cone technique (SCT) with dedicated cones and BioRoot RCS bioceramic sealer; twelve canals were filled with MTA cement (ProRoot MTA) for 6mm in the apical portion with the aid of the MAP System; the empty canal portion was backfilled with thermoplasticized gutta-percha (Obtura III Max System). After 24 h, the roots were apically resected with a carbide bur 3 mm from the apex. At this point, the two groups were randomly subdivided into two further groups of six elements each: G1, SCT and bioceramic sealer without retrograde procedures (n=6); G2, SCT and bioceramic sealer with standard retrograde ultrasonic preparation and MTA filling (n=6); G3, orthograde MTA placement without retrograde filling (n=6); G4, orthograde MTA placement and standard retrograde ultrasonic preparation and MTA filling (n=6). Lastly, the formation of internal and external voids was quantified by means of a second computed microtomographic analysis. The normality of the distribution and the equality of variance of the microtomographic datasets were tested with a Shapiro- Wilk and a Levene test, respectively; then, the volumes measured in the groups were statistically compared with the Kruskal-Wallis test and with the Mann-Withney test with Bonferroni’s correction couple comparison. Results: All groups showed minimal voids volumes formation. In the groups characterized by standard retrograde procedures, the mean voids volumes were 1.16±0.40 % (G4) and 1.87±1.49 % (G2) of the entire canal space, in the groups with the only root-end resection 0.82±0.58 % (G1) and 1.08 ± 0.50% (G3). The difference between the groups was not statistically significant. Analyzing the volume within the apical 3 mm of the canal after root resection (G1=0.06 ± 0.10%; G2=0.76 ± 0.81%; G3=2.06 ± .92%; G4=1.53 ± 1.79%) the difference between G1 and the other groups was statistically significant (p

Aim: To assess the influence of two different placement techniques and powder/liquid mixing ratio of a bioceramic sealer on the quality of root canal filling by single-cone using a micro-CT analysis. Methods: Thirty-two single-rooted elements with a mesio-distal diameter of 5,00±0,50 mm, a buccallingual diameter of 6,50 ±0,50 mm and a root length of 14,50±0,50 mm measured at CEJ level were selected to be included in the study. After creating the coronal access cavity, root canals were scouted with manual files and the working length was acquired. Root canals were shaped with R40 Reciproc Blue and irrigated with NaOCl 5% and EDTA 17% solutions. Prepared samples were randomly assigned into four experimental groups (n=8), according to the sealer placement technique and the powder/liquid mixing ratio: G1, the sealer (BioRoot RCS, Septodont) was mixed in a 1:5 ratio, as suggested by the manufacturer, and carried into the root canal by a dedicated gutta-percha cone up to the working length; G2, sealer placement as group G1, but using 1:6 mixing ratio; G3, the sealer was mixed in a 1:5 ratio and carried into the root canal by a syringe with a cannula (Apexcal, Ivoclar) up to its complete filling, then a dedicated gutta-percha cone was inserted into the canal; G4, sealer placement as group G3, but using 1:6 mixing ratio. All the samples were submitted to micro-CT analysis. Micro-CT scans were acquired after shaping procedures (t0) and after canals obturation (t1) and then compared to evaluate the presence of voids in the gutta-percha, sealer and dentin interfaces. The normality of the distribution and the equality of variance of the microtomographic datasets were tested with a Shapiro-Wilk and Levene test, respectively; the significance of the differences between groups in terms of the formation of voids was ascertained with the Kruskal-Wallis test. The results were considered statistically significant for a p-value < 0.05. Results: Minimal void volumes were observed among groups. G1 performed the best result: total void volumes were 0,253±0,175% of the entire canal space. The other experimental groups follow in order of increasing volume: G3 (0,260±0,254%), G2 (0,532±0,528%), G4 (0,840±0,705%), but no statistically significant difference was observed. All groups showed a similar distribution of voids: in the apical and middle third of the root canal the presence of voids was minimal while the greatest void volumes were detected in the coronal third, due to the canal anatomy and the larger amount of sealer. In this canal portion voids were mainly distributed within the sealer. Concerning the sealer viscosity, the more fluid formulation (1:6) showed an increase of void volumes, although without statistical significance. Likewise, no statistically relevant influence was registered regarding the sealer placement technique. Conclusion: All the examined techniques should be considered effective for the clinician. Under the conditions of the present study, the 1:5 powder/liquid mixing ratio should be preferred when associated with both the sealer placement techniques, according to the clinical situation and the operator preferences. When using the low viscosity formulation, the clinician should insert the sealer slowly and in a controlled way, in order to minimize the formation of voids.

The repair of damaged articular cartilage, which rarely heals spontaneously and develops osteoarthritic complications, is a common clinical issue[1] and none of the available therapies can restore the hyaline cartilage surface beyond just fibrous repair. Over the last few years, polymer-based nanoparticles (NPs) have been in the limelight as a system for the encapsulation and delivery of drugs or active biomolecules, for the treatment of chronic wounds. Water-soluble, biodegradable, polyelectrolyte NPs offer numerous advantages over the conventional delivery forms, particularly so regarding the possibility to tune surface properties, high versatility to control the release profiles of loaded molecules and to interact with membrane receptors of cells, improved efficacy and patient compliance, and reduced toxicity. Polysaccharide-based NPs have, hitherto, been applied in the field of tissue engineering to enhance tissue regeneration by sustained and localized release of appropriate biological molecules, incorporated within the biodegradable nanostructure[2]. Starting from this point, this project is mainly focused on the development and characterization of polymer-based nano-complexes, as a first step in the fulfillment of an injectable composite material for cartilage regeneration. Among different polymers, hyaluronan (HA), Chitosan and its derivatives seem to be very promising for their structural and well established biological role. The two differently charged polymers were used for the preparation of a binary mixture solution by mixing different amounts of a HA solution and a Chitosan/derivatives solution, respectively, hence obtaining different HA weight fractions. The different formulations were prepared in the presence of different concentrations of NaCl to study the influence of ionic strength on the formation of complexes; the variation of pH and the used of HA with higher molecular weight were also investigated to find the best condition for NPs preparation. First, the Transmittance (T) of the different solutions was measured; then the same solutions were investigated by Dynamic Light Scattering (DLS) for the evaluation of Z-average size, zeta-potential, polydispersity index. After that, the best formulation was selected to evaluate the stability of complexes, first in water, and then changing the ionic strength and the pH up to physiological conditions. Overall, these nano-complexes appear as promising biomaterials with interesting biological and physical properties for cartilage regeneration.

Bone tissue damages can derive from diseases such as osteogenesis imperfecta, osteoarthritis, osteomyelitis, and osteoporosis, or from traumatic injury, orthopaedic surgeries and primary tumour resection (Porter et al. 2009 Biotechnol Progress). In particular, non-critical bone defects and voids represent a hot spot for mechanical stresses that can cause fractures, thus it is necessary to accelerate their healing (Jawad et al. 2013 J Orthop Res). Moreover, spinal fusion, maxillary sinus floor elevation, guided bone regeneration, represent other clinical cases in which it is necessary to provide substitutes for bone tissue regeneration. (Mardas et al. 2010 Clin Oral Impl Res) The use of synthetic bone substitutes reduces surgical procedures and the risk of infection or immunogenicity, and decreases the risk of disease transmission (Mottaghitalab et al 2015 J Control Rel; Porter et al. 2009 Biotechnol Progress). Moreover, synthetic scaffolds can be loaded with bioactive molecules or cells before implantation. Polysaccharides offer the opportunity to prepare biocompatible bone substitutes with specific morphological and biological characteristics, such as high interconnectivity and enhanced cell adhesion, which could mimic properly the bone tissue and support and enhance bone healing (Woodruff et al. 2010 Prog Polym Sci; Ward et al. 2010 Oral Dis) In this work, tridimensional scaffolds based on alginate, hydroxyapatite (HAp) and derivatives of chitosan were prepared and characterized. The scaffolds were obtained through freeze casting of alginate/HAp hydrogels. Morphological characterization, obtained by means of micro-computed tomography, revealed that scaffold porosity and interconnectivity are compatible with the requirements for the bone tissue regeneration (Turco et al. 2009 Biomacromolecules). Moreover, the physical-chemical stability displayed by the scaffolds makes them suitable for supporting cell adhesion, colonization and growth. Another important aspect hereby taken in account was the introduction of the chitosan derivatives that showed bioactive properties such as increased osteoblasts proliferation and ALP activity, and increased bone-implant contact in the case of thermosets functionalized with this polysaccharide (Marsich et al. 2013 Acta Biomater). The scaffolds presented in this work were successfully used for the growth and the differentiation of human derived dentin pulp stem cells (hDPSCs). Flow cyotofluorimetry proved that hDPSCs harvested in our laboratory can be maintained in culture with an undifferentiated phenotype and that can be differentiated in osteoblasts under proper stimuli, as demonstrated by enzymatic and biochemical assays. Overall, the combination of hDPSCs and polysaccharide based bioactive scaffolds, represents an effective strategy for the preparation of osteoconductive and osteoinductive cell-loaded biomaterials for the treatment of bone defects and for the clinical practice in oral surgery.

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.

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.