High-Resolution Phase-Contrast Tomography on Human Collagenous Tissues: A Comprehensive Review.

Phase-contrast X-ray imaging is becoming increasingly considered since its first applications, which occurred almost 30 years ago. Particular emphasis was placed on studies that use this technique to investigate soft tissues, which cannot otherwise be investigated at a high resolution and in a three-dimensional manner, using conventional absorption-based settings. Indeed, its consistency and discrimination power in low absorbing samples, unified to being a not destructive analysis, are pushing interests on its utilization from researchers of different specializations, from botany, through zoology, to human physio-pathology research. In this regard, a challenging method for 3D imaging and quantitative analysis of collagenous tissues has spread in recent years: it is based on the unique characteristics of synchrotron radiation phase-contrast microTomography (PhC-microCT). In this review, the focus has been placed on the research based on the exploitation of synchrotron PhC-microCT for the investigation of collagenous tissue physio-pathologies from solely human samples. Collagen tissues' elasto-mechanic role bonds it to the morphology of the site it is extracted from, which could weaken the results coming from animal experimentations. Encouraging outcomes proved this technique to be suitable to access and quantify human collagenous tissues and persuaded different researchers to approach it. A brief mention was also dedicated to the results obtained on collagenous tissues using new and promising high-resolution phase-contrast tomographic laboratory-based setups, which will certainly represent the real step forward in the diffusion of this relatively young imaging technique.

Multidisciplinary evaluation of the remineralization potential of three fluoride-based toothpastes on natural white spot lesions.

OBJECTIVES: This in vitro study aimed assessing the remineralization potential of three commercial fluoride-based toothpastes in permanent teeth with natural white spot lesions (WSLs). A multidisciplinary approach based on Raman microspectroscopy (RMS), Scanning electron microscopy (SEM), Energy-dispersive x-ray spectroscopy (EDS), and Vickers microhardness (VMH) was exploited. METHODS: N = 12 human molars with natural WSLs in the proximal-vestibular zone were selected and divided into 4 groups (n = 3) according to the different treatments: HAF (hydroxyapatite with fluoride ions); SMF (sodium monofluorophosphate with arginine); SF (sodium fluoride with enzymes), and CTRL (untreated group). All toothpastes tested contained 1450 ppm of fluoride. Teeth samples were submitted to the following protocol: a 7-day pH cycling treatment, with two daily exposures (2 min each time) to the commercial toothpastes described above. The surface micromorphology (SEM), the chemical/elemental composition (RMS and EDS), and the Vickers microhardness (VMH) were evaluated. Statistical analysis was performed. RESULTS: A remarkable remineralization of WSLs in SEM images was observed in all treated groups compared to CTRL. In particular, HAF and SF displayed higher values of VMH, phosphates amount (I960), crystallinity (FWHM960), and lower ones of C/P (I1070/I960) with respect to CTRL. Intermediate values were found in SMF, higher than CTRL but lower with respect to HAF and SF. As regards the Ca/P ratio, statistically significant differences (p < 0.05) were found between SF and the other groups. CONCLUSIONS: All the tested dentifrices have shown to remineralize the WSLs. SF and HAF have comparable capability in hardness recovery and crystallinity; however, SF shows the best remineralizing potential according to both micromorphological and chemical analyses. Clinical relevance The daily use of toothpastes containing hydroxyapatite partially replaced with fluoride, sodium monofluorophosphate with arginine and sodium fluoride toothpaste associated with enzymes represents a preventive, therapeutic, effective, and non-invasive tool for remineralize WSLs.

Combined Effects of HA Concentration and Unit Cell Geometry on the Biomechanical Behavior of PCL/HA Scaffold for Tissue Engineering Applications Produced by LPBF.

This experimental study aims at filling the gap in the literature concerning the combined effects of hydroxyapatite (HA) concentration and elementary unit cell geometry on the biomechanical performances of additively manufactured polycaprolactone/hydroxyapatite (PCL/HA) scaffolds for tissue engineering applications. Scaffolds produced by laser powder bed fusion (LPBF) with diamond (DO) and rhombic dodecahedron (RD) elementary unit cells and HA concentrations of 5, 30 and 50 wt.% were subjected to structural, mechanical and biological characterization to investigate the biomechanical and degradative behavior from the perspective of bone tissue regeneration. Haralick's features describing surface pattern, correlation between micro- and macro-structural properties and human mesenchymal stem cell (hMSC) viability and proliferation have been considered. Experimental results showed that HA has negative influence on scaffold compaction under compression, while on the contrary it has a positive effect on hMSC adhesion. The unit cell geometry influences the mechanical response in the plastic regime and also has an effect on the cell proliferation. Finally, both HA concentration and elementary unit cell geometry affect the scaffold elastic deformation behavior as well as the amount of micro-porosity which, in turn, influences the scaffold degradation rate.

Influence of abutment macro- and micro-geometry on morphologic and morphometric features of peri-implant connective tissue.

OBJECTIVES: The aim of the present human observational study is to provide morphologic and morphometric analysis of peri-implant connective tissue next to abutments with divergent or convergent macro-geometry and different surface micro-characteristics. MATERIALS AND METHODS: Thirty patients were rehabilitated with single implants in the posterior area and one out of three different healing abutments with a one-stage technique: machined divergent abutment (DIV-MAC), machined convergent abutment (CONV-MAC) or convergent abutment with ultrathin threaded surface (CONV-UTM). At 3 months postimplant insertion, peri-implant soft tissue was harvested; the following outcomes were investigated: histomorphometry (vertical width of connective and epithelial components) as detected by histology and polarized light; and connective tissue vertical width and 3D organization as detected by synchrotron-based high-resolution phase-contrast-based tomography (PhC-µCT). RESULTS: Significant differences in connective tissue vertical dimension (aJE-AM) were found between DIV-MAC and both CONV-MAC and CONV-UTM, both by histology and PhC-µCT, with significantly higher values for the last two groups. Moreover, 2D histological analysis did not find significant differences in the junctional epithelium vertical dimension (PM-aJE). Importantly, PhC-µCT analysis revealed, at 3D level, significant greater amount and density of collagen bundles for CONV-UTM compared with the other two groups. CONCLUSIONS: Convergent abutment profiles, regardless of their surface micro-geometry, seem to favor axial development of peri-implant connective tissue. Moreover, ultrathin threaded surfaces seem associated with denser and greater connective tissue organization, which might improve peri-implant soft tissue seal.

Influence of Trabecular Geometry on Scaffold Mechanical Behavior and MG-63 Cell Viability.

In a scaffold-based approach for bone tissue regeneration, the control over morphometry allows for balancing scaffold biomechanical performances. In this experimental work, trabecular geometry was obtained by a generative design process, and scaffolds were manufactured by vat photopolymerization with 60% (P60), 70% (P70) and 80% (P80) total porosity. The mechanical and biological performances of the produced scaffolds were investigated, and the results were correlated with morphometric parameters, aiming to investigate the influence of trabecular geometry on the elastic modulus, the ultimate compressive strength of scaffolds and MG-63 human osteosarcoma cell viability. The results showed that P60 trabecular geometry allows for matching the mechanical requirements of human mandibular trabecular bone. From the statistical analysis, a general trend can be inferred, suggesting strut thickness, the degree of anisotropy, connectivity density and specific surface as the main morphometric parameters influencing the biomechanical behavior of trabecular scaffolds, in the perspective of tissue engineering applications.

Micro-computed tomography for assessing the internal and external voids of bulk-fill composite restorations: A technical report.

Purpose: This technical report aims to describe and detail the use of micro-computed tomography for a reliable evaluation of the bulk-fill composite/tooth interface. Materials and Methods: Bulk-fill composite restorations in tooth cavities were scanned using micro-computed tomography to obtain qualitatively and quantitatively valuable information. Two-dimensional information was processed using specific algorithms, and ultimately a 3-dimensional (3D) specimen reconstruction was generated. The 3D rendering allowed the visualization of voids inside bulk-fill composite materials and provided quantitative measurements. The 3D analysis software VG Studio MAX was used to perform image analysis and assess gap formation within the tooth-restoration interface. In particular, to evaluate internal adaptation, the Defect Analysis add-on module of VG Studio Max was used. Results: The data, obtained with the processing software, highlighted the presence and the shape of gaps in different colours, representing the volume of porosity within a chromatic scale in which each colour quantitatively represents a well-defined volume. Conclusion: Micro-computed tomography makes it possible to obtain several quantitative parameters, providing fundamental information on defect shape and complexity. However, this technique has the limit of not discriminating materials without radiopacity and with low or no filler content, such as dental adhesives, and hence, they are difficult to visualise through software reconstruction.

Uterine leiomyoma as useful model to unveil morphometric and macromolecular collagen state and impairment in fibrotic diseases: An ex-vivo human study.

Collagen is one of the main components of the extracellular matrix (ECM), involved, among all, in the maintenance of the structural support of tissues. In fibrotic diseases, collagen is overexpressed, and its production determines the formation of a significantly stiffer ECM. The cross-linking of high-resolution analytical tools, able to investigate both the tridimensional organization and the secondary structure of collagen in fibrotic diseases, could be useful to identify defined markers correlating the status of this protein with specific pathological conditions. To this purpose, an innovative multidisciplinary approach based on Phase-Contrast MicroComputed Tomography, Transmission Electron Microscopy, and Fourier Transform Infrared Imaging Spectroscopy was exploited on leiomyoma samples and adjacent myometrium to characterize microstructural collagen features. Uterine leiomyoma is a common gynecological disorder affecting women in fertile age. It is characterized by a massive collagen production due to the repairing processes occurring at myometrium level, and, hence, it represents a valuable model to investigate collagen self-organization in a pathological condition. Moreover, to evaluate the sensitivity of this multidisciplinary approach, the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids in collagen reduction were also investigated.

Lithography-based Ceramic Manufacturing (LCM) versus Milled Zirconia Blocks under uniaxial compressive loading: An in vitro comparative study.

AIM: This in vitro study aimed to compare the mechanical performance of 3D printed versus milled zirconia blocks, when subjected to uniaxial compression load, and to investigate the microstructural characteristics of the 3D printed samples, before and after the application of the load. METHODS: Twenty zirconia blocks (5 × 5 × 5mm3) were prepared: 10 (tests) were 3D printed with a Lithography-based Ceramic Manufacturing (LCM) printer (Cerafab S65®, Lithoz, Vienna, Austria), and 10 (controls) were milled with a 5-axis milling machine (DWX-52D®, DGShape, a Roland Company, Hamamatsu, Japan). Compression tests were carried out on all samples, using a load cell of 30 kN and crosshead speed of 0.5 mm/min, in according to the ASTM C1424-15. The elastic modulus of the material was calculated from stress/strain curve by taking compressive stress values between 50 MPa and 100 MPa. Compression data obtained were plotted as stress-strain curves. Finally, the 3D printed test samples were also observed by VEGA3 Tescan scanning electron microscope (SEM) to detect the presence of eventual defects on surface before and after compression. A statistical analysis was performed to compare the elastic modulus and the deformation in compression at maximum load of the test samples that did not break and the control samples. RESULTS: Under mechanical compression, four of the test samples reached failure, whereas all the control samples did not reach failure at the limit of the load cell. However, the 3D printed samples that did not break revealed interesting properties, such as a better modulus of elasticity (p = 0.15) and a lower tendency to deformation under compression (p<0.001), when compared to the milled ones. CONCLUSIONS: Within the limits of this study (experimental setting, in vitro design, only one type of force applied) milled zirconia blocks were found more resistant to compression forces than 3D printed ones.

Biomechanical performances of PCL/HA micro- and macro-porous lattice scaffolds fabricated via laser powder bed fusion for bone tissue engineering.

The present experimental study aims to extend know-how on resorbable polycaprolactone/hydroxyapatite (PCL/HA, 70/30 wt%) scaffolds, produced by Laser Powder Bed Fusion (LPBF) technology, to geometrically complex lattice structures and micro porous struts. Using optimized LPBF printing parameters, micro- and macro-porous scaffolds for bone tissue regeneration were produced by regularly repeating in space Diamond (DO) and Rhombic Dodecahedron (RD) elementary unit cells. After production, scaffolds were submitted to structural, mechanical, and biological characterization. The interaction of scaffolds with human Mesenchymal Stem Cells (hMSCs) allowed studying the degradative processes of the PCL matrix. Biomechanical performances and biodegradation of scaffolds were compared to literature results and bone tissue data. Mechanical compression test, biological viability up to 4 days of incubation and degradation rate evidenced strong dependence of scaffold behavior on unit cell geometry as well as on global geometrical features.

Comparison of three different bulk-filling techniques for restoring class II cavities: µCT, SEM-EDS combined analyses for margins and internal fit assessments.

OBJECTIVE: Evaluation of the behavior of three different bulk-filling techniques in terms of internal adaptation and external marginal sealing for restoring class II cavities. METHODS: Fifteen extracted sound molar teeth were used. Two standardized class II mesio/disto-occlusal (MO/DO) slot cavities, 4 mm long, 4 mm wide and 3 mm deep were prepared in each tooth, obtaining n=30 cavity preparations. The cavities were randomly assigned into three groups (n=10 per group) according to three bulk filling techniques: Bulk Traditional (BT), Bulk&Go (BG) and Bulk&Flow (BF). The teeth were analyzed by scanning electron microscopy (SEM) to investigate the external marginal seal. Thereafter, the chemical composition of tooth-restoration interface was analyzed by energy-dispersive X-ray spectroscopy (EDS). Complementary information to the SEM and EDS were obtained by micro-computed tomography (µCT) to assess the internal fit. RESULTS: SEM analysis showed a proper external marginal seal for all groups tested as confirmed by the EDS investigation, highlighting the presence of adhesive layer at the tooth-restoration interface. The internal marginal adaptation by means of µCT analysis revealed gaps formation at the tooth-restoration interface only for BT group, while an intimate contact free of gaps were found in the other two groups. Moreover, in BT and BF groups voids were present within the restoration. SIGNIFICANCE: BG and BF techniques can be considered as reliable alternatives to BT technique, as they simplify the class II restoration without transforming it into class I, thus ensuring a successful result.

Biphasic Calcium Phosphate Biomaterials: Stem Cell-Derived Osteoinduction or In Vivo Osteoconduction? Novel Insights in Maxillary Sinus Augmentation by Advanced Imaging.

Maxillary sinus augmentation is often necessary prior to implantology procedure, in particular in cases of atrophic posterior maxilla. In this context, bone substitute biomaterials made of biphasic calcium phosphates, produced by three-dimensional additive manufacturing were shown to be highly biocompatible with an efficient osteoconductivity, especially when combined with cell-based tissue engineering. Thus, in the present research, osteoinduction and osteoconduction properties of biphasic calcium-phosphate constructs made by direct rapid prototyping and engineered with ovine-derived amniotic epithelial cells or amniotic fluid cells were evaluated. More in details, this preclinical study was performed using adult sheep targeted to receive scaffold alone (CTR), oAFSMC, or oAEC engineered constructs. The grafted sinuses were explanted at 90 days and a cross-linked experimental approach based on Synchrotron Radiation microCT and histology analysis was performed on the complete set of samples. The study, performed taking into account the distance from native surrounding bone, demonstrated that no significant differences occurred in bone regeneration between oAEC-, oAFMSC-cultured, and Ctr samples and that there was a predominant action of the osteoconduction versus the stem cells osteo-induction. Indeed, it was proven that the newly formed bone amount and distribution decreased from the side of contact scaffold/native bone toward the bulk of the scaffold itself, with almost constant values of morphometric descriptors in volumes more than 1 mm from the border.

Microleakage Analysis of Different Bulk-Filling Techniques for Class II Restorations: µ-CT, SEM and EDS Evaluations.

This study aimed to compare two different bulk-filling techniques, evaluating the internal and external adaptation of class II resin-composite restorations, by analysing the gap formation using microcomputed tomography (µ-CT) and scanning electronic microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDS). Two standardized mesio/disto-occlusal (MO/DO) cavities were prepared in eight extracted human third molars that were divided, according to the filling technique used, in the following two groups (n = 4): BG (Bulk&Go group) and BT (Bulk Traditional group). After universal bonding application, followed by the light curing, all teeth were restored using a bulk-fill composite. Specimens were scanned with µ-CT to evaluate 3D interfacial gaps. Acquired µ-CT data were analysed to quantify the gap formation. Complementary information to the µ-CT analysis were obtained by SEM. Thereafter, the chemical composition of tooth-restoration interface was analysed using EDS. The µ-CT analysis revealed gaps formation at the tooth-restoration interface for both the BG and BT groups, while within the restoration, only in the BT group there was evidence of microleakage formation. The scanning electron micrographs of both groups showed that the external marginal integrity of the restoration was preserved, while EDS showed the three different structures (tooth surface, adhesive layer and resin composite) of the tooth-restoration interface, highlighting the absence of gap formation. In both BG and BT, the two filling techniques did not show significant differences regarding the internal and external marginal adaptation of the restoration. To achieve a successful restoration, the clinician could be advised to restore a class II cavity using a single increment bulk-filling technique (BG), thus treating it as a class I cavity.