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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.

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.

Progress in utilizing inorganic nanoparticles for biomedical applications has advanced rapidly due to the extensive amount of work done in the synthesis and modification of the materials.1 These nanosized materials provide a robust framework in which two or more components can be incorporated to give multifunctional capabilities. An example can be seen in gold nanomaterials.2 Gold nanoparticles are bioinert, nontoxic, and readily synthesized and functionalized.3 They also provide a multifunctional platform for both therapeutic and diagnostic purposes. Indeed, through proper functionalization, these particles can be engineered to accumulate at illness cells using targeting ligands providing a powerful tool, for example, for gene therapy.4 The biophysico-chemical properties of the vehicle, such as size, charge, surface hydrophilicity, and the nature and density of the ligands on their surface, can all impact the circulating half-life of the particles as well as their biodistribution. Innovation may be introduced by controlling the surface properties of the monolayer protecting the gold core. Indeed, recently it has been demonstrated that particles coated with a molecularly ordered ligand shell were able to enter cells directly through the membrane without perforating it basing on a novel physical chemistry phenomenon.6 This property is ideal as it provides the particles with minimal if none genotoxicity. Mixed monolayers composed of mixtures of hydrogenated/fluorurated ligands favor the phase segregation and consequently the ordered morphology of the NP surface. 7 In addition, the introduction in the monolayer of perfluorocarbon ligands might enable, for example, the imaging by 19F MRI techniques of the nanoparticles and, consequently, the tracking in vivo of cell fate. In this communication we will discuss the approaches for the realization of such innovative nanoparticles easy to make, because obtained by self-assembly strategies, but with an unprecedented degree of complexity, with respect to nanotechnology platforms for drug delivery applications know to date, as far as their features is concerned.

The study of bone morphology is of great importance as bone morphology is influenced by factors such as age and underlying comorbidities and is associated with bone mechanical properties and fracture risk. Standard diagnostic techniques used in bone disease, such as Dual-Energy X-ray absorptiometry and ultrasonography do not provide qualitative and quantitative morphological information. In recent years, techniques such as High Resolution Computed Tomography (HR-CT), micro- CT, Magnetic Resonance Imaging (MRI), and Low Field Nuclear Magnetic Resonance (LF-NMR) have been developed for the study of bone structure and porosity. Data obtained from these techniques have been used to construct models to predict bone mechanical properties thanks to finite element analysis. Cortical porosity has been extensively studied and successfully correlated with disease progression and mechanical properties. Trabecular porosity and pore size distribution, however, have increasingly been taken into consideration to obtain a comprehensive analysis of bone pathology and mechanic. Therefore, we have decided to evaluate the ability of micro- CT (chosen for its high spatial resolving power) and LF-NMR (chosen to analyze the behavior of water molecules within trabecular bone pores) to characterize the morphology of trabecular bone in osteoporosis. Trabecular bone samples from human femoral heads collected during hip replacement surgery were from osteoporosis (test group) and osteoarthritis (control group) patients. Our data show that both micro- CT and LF-NMR can detect qualitative changes in trabecular bone (i.e., transition from plate-like to rod-like morphology). Micro- CT failed to detect significant differences in trabecular bone morphology parameters between osteoporotic and osteoarthritic specimens, with the exception of Trabecular Number and Connectivity Density, which are markers of osteoporosis progression. In contrast, LF-NMR was able to detect significant differences in porosity and pore size of trabecular bone from osteoporotic versus osteoarthritic (control) samples. However, only the combination of these two techniques allowed the detection of structural morphometric changes (increase in the larger pore fraction and enlargement of the larger pores) in the trabecular bone of osteoporotic specimens compared to osteoarthritic ones. In conclusion, the combined use of LF-NMR and micro- CT provides a valuable tool for characterizing the morphology of trabecular bone and may offer the possibility for a new approach to the study and modeling of bone mechanics in the context of aging and disease.

The employment of coaxial fibers for guided tissue regeneration can be extremely advantageous since they allow the functionalization with bioactive compounds to be preserved and released with a long-term efficacy. Antibacterial coaxial membranes based on poly-epsilon-caprolactone (PCL) and rifampicin (Rif) were synthesized here, by analyzing the effects of loading the drug within the core or on the shell layer with respect to non-coaxial matrices. The membranes were, therefore, characterized for their surface properties in addition to analyzing drug release, antibacterial efficacy, and biocompatibility. The results showed that the lower drug surface density in coaxial fibers hinders the interaction with serum proteins, resulting in a hydrophobic behavior compared to non-coaxial mats. The air-plasma treatment increased their hydrophilicity, although it induced rifampicin degradation. Moreover, the substantially lower release of coaxial fibers influenced the antibacterial efficacy, tested against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Indeed, the coaxial matrices were inhibitory and bactericidal only against S. aureus, while the higher release from non-coaxial mats rendered them active even against E. coli. The biocompatibility of the released rifampicin was assessed too on murine fibroblasts, revealing no cytotoxic effects. Hence, the presented coaxial system should be further optimized to tune the drug release according to the antibacterial effectiveness.

To evaluate the filling ability of two orthograde obturation techniques followed by the apical resection with or without retrograde obturation through micro-computed tomography (CT). Thirty-two single-rooted permanent teeth were prepared and randomised into four groups (n = 8) according to the orthograde obturation technique (single cone technique [SCT] and mineral trioxide aggregate placement) combined or not with retrograde obturation. The volume of voids (VoV) within the entire endodontic space, the apical 3 mm, and 1 mm after root resection was calculated by micro-CT. Statistical analysis showed no significant difference among the groups regarding the total VoV in all root canals, as well as within the apical 1 mm after root resection. The SCT and apical resection without retrograde filling showed significantly better results in terms of VoV at the apical 3 mm after root resection. Within the study limitations, SCT associated with apical resection without retrograde preparation exhibited a similar or less amount of voids than the other groups.

Purpose: To analyze and compare the surface properties of commercially available hydrophobic acrylic intraocular lenses (IOLs). Setting: Eye Clinic, University of Trieste, Italy. Design: Experimental study. Methods: The following 6 single-piece hydrophobic acrylic IOL models with the same dioptric power were studied and compared: Clareon SY60WF, Tecnis PCB00, enVista MX60, CT Lucia 601P, Vivinex iSert XY1, and iSert 251. Topography of the IOL surface was analyzed using atomic force microscopy (AFM). Surface contact angle measurements using the sessile drop method were performed to assess IOL wettability. Results: The AFM analysis showed that the Vivinex iSert XY1 IOL and Clareon SY60WF IOL had the lowest surface roughness (P < .05); there was no statistically significant difference in surface roughness between the those 2 IOL models (P > .05). Surface contact angle measurements showed that the iSert 251 IOL had the highest hydrophobicity. The CT Lucia 601P IOL had the lowest contact angle of all IOL models. Conclusions: The AFM analysis and surface contact angle measurements of all IOLs tested showed that the Vivinex iSert XY1 IOL and Clareon SY60WF IOL had the best topographic features. The smoother, more regular surface of these new IOL models might reduce cell adhesion and therefore lower the incidence of posterior capsule opacification.

Background: The blood wettability of titanium implant surfaces favors primary osseointegration and tissue healing in peri-implantitis. The current study aims to evaluate this property on the main different kinds of surfaces, brand new and after treatments. Methods: grade IV titanium disks were used, five machined, five laser-treated, five sandblasted. The experiment was on four steps. Between each step, specimens were sterilized. At the end of each step, a 4 µL blood drop was put on the samples, and contact angles were calculated. The steps consisted of—1: no treatment, 2: surface instrumentation with an ultrasonic titanium tip, 3: platelet-rich fibrin (PRF) coating and drying with sterile gauze, 4: etching with phosphoric acid, rinse and saline solution and air-drying. At the end of each phase, a blood drop was placed on the surfaces. Blood for PRF preparation and for calculation of contact angles was collected from a single source. Results: average wettability decreased after instrumentation, and increased after the PRF application to a superior level both to the first and second steps. The highest wettability was obtained after etching. This trend is statistically significant for machined and sandblasted surfaces. Conclusions: In the clinical environment, PRF and phosphoric acid used for conditioning exposed implant surfaces can be used for the healing of peri-implant tissues.

Background: Neurological alterations are one of the main complications occurring after the third molar extractions. The aim of this prospective multicenter cohort study was to find out Cone Beam Computed Tomography (CBCT) features and distribution of neurological complications in patients undergoing lower third molar surgery and to determine the radiological and patient-related factors that could be correlated to the occurrence of inferior alveolar and lingual nerves injury. Material and Methods: 378 patients who underwent lower third molar extraction from March 2018 to March 2019 were included. Clinical and radiological data were collected. CBCT features were recorded following Maglione et al. classification. Symptoms and characteristics of patients who experienced neurological alterations were evaluated. Results: 193 patients needed a second-level radiological exam (CBCT). In these patients, the most common feature was Maglione class 3: a higher frequency of apical or buccal mandibular canals in direct contact with the tooth was observed. 3.17% of the patients developed a neurological complication. Maglione class 4, increased age, and operative time were all positively correlated with neurological alterations. Conclusions: while the buccal or apical position of the mandibular canal was the more common findings, the lingual position was found to have a higher correlation with a negative outcome. Age and operative time were also found to be risk factors for developing nerve injury in the considered population.

Nanofibre-based membranes or scaffolds exhibit high surface-to-volume ratio, which allows an improved cell adhesion, representing an attractive subgroup of biomaterials due to their unique properties. Among several techniques of nanofiber production, electrospinning is a cost-effective technique that has been, to date, attractive for several medical applications. Among these, guided bone regeneration is a surgical procedure in which bone regeneration, due to bone atrophy following tooth loss, is “guided” by an occlusive barrier. The membrane should protect the initial blood clot from any compression, shielding the bone matrix during maturation from infiltration of soft tissues cells. This review will focus its attention on the application of electrospinning (ELS) in oral surgery bone regeneration. Despite the abundance of published papers related to the electrospinning technique applied in the field of bone regeneration of the jaws, to the authors’ knowledge, no articles report clinical application of these structures. Moreover, only a few records can be found with in vivo application. Therefore, no human studies have to date been detectable. New approaches such as multifunctional multilayering and coupling with bone promoting factors or antimicrobial agents, makes this technology very attractive. However, greater efforts should be made by researchers and companies to turn these results into clinical practice.

Background: Bone substitutes, either from human (autografts and allografts) or animal (xenografts) sources, suffer from inherent drawbacks including limited availability or potential infectivity to name a few. In the last decade, synthetic biomaterials have emerged as a valid alternative for biomedical applications in the field of orthopedic and maxillofacial surgery. In particular, phosphate-based bone substitution materials have exhibited a high biocompatibility due to their chemical similitude with natural hydroxyapatite. Besides the nature of the biomaterial, its porous and interconnected architecture is essential for a correct osseointegration. This performance could be predicted with an extensive characterization of the biomaterial in vitro. Methods: In this study, we compared the biological, chemical, and structural features of four different commercially available bone substitutes derived from an animal or a synthetic source. To this end, &micro, CT and SEM were used to describe the biomaterials structure. Both FTIR and EDS analyses were carried out to provide a chemical characterization. The results obtained by these techniques were correlated with cell adhesion and proliferation of the osteosarcoma MG-63 human cell line cultured in vitro. Results: The findings reported in this paper indicate a significant influence of both the nature and the structure of the biomaterials in cell adhesion and proliferation, which ultimately could affect the clinical performance of the biomaterials. Conclusions: The four commercially available bone substitutes investigated in this work significantly differed in terms of structural features, which ultimately influenced in vitro cell proliferation and may so affect the clinical performance of the biomaterials.

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. Statement of Significance In our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair.