Electron Beam Powder Bed Fusion of Ti-48Al-2Cr-2Nb Open Porous Scaffold for Biomedical Applications: Process Parameters, Adhesion, and Proliferation of NIH-3T3 Cells.

Titanium aluminide (TiAl)-based intermetallics, especially Ti-48Al-2Cr-2Nb, are a well-established class of materials for producing bulky components using the electron beam powder bed fusion (EB-PBF) process. The biological properties of Ti-48Al-2Cr-2Nb alloy have been rarely investigated, specifically using complex cellular structures. This work investigates the viability and proliferation of NIH-3T3 fibroblasts on Ti-48Al-2Cr-2Nb dodecahedral open scaffolds manufactured by the EB-PBF process. A process parameter optimization is carried out to produce a fully dense part. Then scaffolds are produced and characterized using different techniques, including scanning electron microscopy and X-ray tomography. In vitro viability tests are performed with NIH-3T3 cells after incubation for 1, 4, and 7 days. The results show that Ti-48Al-2Cr-2Nb represents a promising new entry in the biomaterial field.

Features of Vat-Photopolymerized Masters for Microfluidic Device Manufacturing.

The growing interest in advancing microfluidic devices for manipulating fluids within micrometer-scale channels has prompted a shift in manufacturing practices, moving from single-component production to medium-size batches. This transition arises due to the impracticality of lab-scale manufacturing methods in accommodating the increased demand. This experimental study focuses on the design of master benchmarks 1-5, taking into consideration critical parameters such as rib width, height, and the relative width-to-height ratio. Notably, benchmarks 4 and 5 featured ribs that were strategically connected to the inlet, outlet, and reaction chamber of the master, enhancing their utility for subsequent replica production. Vat photopolymerization was employed for the fabrication of benchmarks 1-5, while replicas of benchmarks 4 and 5 were generated through polydimethylsiloxane casting. Dimensional investigations of the ribs and channels in both the master benchmarks and replicas were conducted using an optical technique validated through readability analysis based on the Michelson global contrast index. The primary goal was to evaluate the potential applicability of vat photopolymerization technology for efficiently producing microfluidic devices through a streamlined production process. Results indicate that the combination of vat photopolymerization followed by replication is well suited for achieving a minimum rib size of 25 µm in width and an aspect ratio of 1:12 for the master benchmark.

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.

On the Biomechanical Performances of Duplex Stainless Steel Graded Scaffolds Produced by Laser Powder Bed Fusion for Tissue Engineering Applications.

This experimental study aims to extend the know-how on biomechanical performances of duplex stainless steel (DSS) for tissue engineering applications to a graded lattice geometry scaffold based on the F53 DSS (UNS S32750 according to ASTM A182) produced by laser powder bed fusion (LPBF). The same dense-out graded geometry based on rhombic dodecahedral elementary unit cells investigated in previous work on 316L stainless steel (SS) was adopted here for the manufacturing of the F53 DSS scaffold (SF53). Microstructural characterization and mechanical and biological tests were carried out on the SF53 scaffold, using the in vitro behavior of the 316L stainless steel scaffold (S316L) as a control. Results show that microstructure developed as a consequence of different volume energy density (VED) values is mainly responsible for the different mechanical behaviors of SF53 and S316L, both fabricated using the same LPBF manufacturing system. Specifically, the ultimate compressive strength (σUC) and elastic moduli (E) of SF53 are three times and seven times higher than S316L, respectively. Moreover, preliminary biological tests evidenced better cell viability in SF53 than in S316L already after seven days of culture, suggesting SF53 with dense-out graded geometry as a viable alternative to 316L SS for bone tissue engineering applications.

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.

Improved biomechanical behavior of 316L graded scaffolds for bone tissue regeneration produced by laser powder bed fusion.

Graded lattice scaffolds based on rhombic dodecahedral (RD) elementary unit cell geometry were manufactured in 316L stainless steel (SS) by laser powder bed fusion (LPBF). Two different strategies based on varying strut thickness layer-by-layer in the building direction were adopted to obtain the graded scaffolds: a) decreasing strut size from core to edge to produce the dense-in (DI) structure and b) increasing strut size in the same direction to produce the dense-out (DO) structure. Both graded structures (DI and DO) were constructed with specular symmetry with respect to the central horizontal axis. Structural, mechanical, and biological characterizations were carried out to evaluate feasibility of designing appropriate biomechanical performances of graded scaffolds in the perspective of bone tissue regeneration. Results showed that mechanical behavior is governed by graded geometry, while printing parameters influence structural properties of the material such as density, textures, and crystallographic phases. The predominant failure mechanism in graded structures initiates in correspondence of thinner struts, due to high stress concentrations on strut junctions. Biological tests evidenced better proliferation of cells in the DO graded scaffold, which in turn exhibits mechanical properties close to cortical bone. The combined control of grading strategy, printing parameters and elementary unit cell geometry can enable implementing scaffolds with improved biomechanical performances for bone tissue regeneration.

Assessment of the Remineralizing Potential of Biomimetic Materials on Early Artificial Caries Lesions after 28 Days: An In Vitro Study.

This study aimed to evaluate the loss of mineral content in the enamel surface in early artificial lesions and to assess the remineralizing potential of different agents by means of SEM coupled with energy-dispersive X-ray analysis (EDX). The analysis was performed on the enamel of 36 molars divided into six equal groups, in which the experimental ones (3-6) were treated using remineralizing agents for a 28-day pH cycling protocol as follows: Group 1, sound enamel; Group 2, artificially demineralized enamel; Group 3, CPP-ACP treatment; Group 4, Zn-hydroxyapatite treatment; Group 5, NaF 5% treatment; and Group 6, F-ACP treatment. Surface morphologies and alterations in Ca/P ratio were evaluated using SEM-EDX and data underwent statistical analysis (p < 0.05). Compared with the sound enamel of Group 1, the SEM images of Group 2 clearly showed loss of integrity, minerals, and interprismatic substances. Groups 3-6 showed a structural reorganization of enamel prisms, interestingly comprising almost the entire enamel surface. Group 2 revealed highly significant differences of Ca/P ratios compared with other groups, while Groups 3-6 showed no differences with Group 1. In conclusion, all tested materials demonstrated a biomimetic ability in remineralizing lesions after 28 days of treatment.

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.

Beads for Cell Immobilization: Comparison of Alternative Additive Manufacturing Techniques.

The attachment or entrapment of microbial cells and enzymes are promising solutions for various industrial applications. When the traps are beads, they are dispersed in a fluidized bed in a vessel where a pump guarantees fresh liquid inflow and waste outflow without washing out the cells. Scientific papers report numerous types of cell entrapment, but most of their applications remain at the laboratory level. In the present research, rigid polymer beads were manufactured by two different additive manufacturing (AM) techniques in order to verify the economy, reusability, and stability of the traps, with a view toward a straightforward industrial application. The proposed solutions allowed for overcoming some of the drawbacks of traditional manufacturing solutions, such as the limited mechanical stability of gel traps, and they guaranteed the possibility of producing parts of constant quality with purposely designed exchange surfaces, which are unfeasible when using conventional processes. AM proved to be a viable manufacturing solution for beads with complex shapes of two different size ranges. A deep insight into the production and characteristics of beads manufactured by AM is provided. The paper provides biotechnologists with a manufacturing perspective, and the results can be directly applied to transit from the laboratory to the industrial scale.

Silk fibroin films with embedded magnetic nanoparticles: evaluation of the magneto-mechanical stimulation effect on osteogenic differentiation of stem cells.

We report about a biomaterial in the form of film ∼10 µm thick, consisting of a silk fibroin matrix with embedded iron oxide superparamagnetic nanoparticles, for prospective applications as bioactive coating in regenerative medicine. Films with different load of magnetic nanoparticles are produced (nanoparticles/silk fibroin nominal ratio = 5, 0.5 and 0 wt%) and the structural, mechanical and magnetic properties are studied. The nanoparticles form aggregates in the silk fibroin matrix and the film stiffness, as tested by nanoindentation, is spatially inhomogeneous, but the protein structure is not altered. In vitro biological tests are carried out on human bone marrow-derived mesenchymal stem cells cultured on the films up to 21 days, with and without an applied static uniform magnetic field. The sample with the highest nanoparticles/silk fibroin ratio shows the best performance in terms of cell proliferation and adhesion. Moreover, it promotes a faster and better osteogenic differentiation, particularly under magnetic field, as indicated by the gene expression level of typical osteogenic markers. These findings are explained in light of the results of the physical characterization, combined with numerical calculations. It is established that the applied magnetic field triggers a virtuous magneto-mechanical mechanism in which dipolar magnetic forces between the nanoparticle aggregates give rise to a spatial distribution of mechanical stresses in the silk fibroin matrix. The film with the largest nanoparticle load, under cell culture conditions (i.e. in aqueous environment), undergoes matrix deformations large enough to be sensed by the seeded cells as mechanical stimuli favoring the osteogenic differentiation.

Remineralization Efficacy of Four Remineralizing Agents on Artificial Enamel Lesions: SEM-EDS Investigation.

Dental remineralization represents the process of depositing calcium and phosphate ions into crystal voids in demineralized enamel, producing net mineral gain and preventing early enamel lesions progression. The aim of the present study was to qualitatively and quantitatively compare the remineralizing effectiveness of four commercially available agents on enamel artificial lesions using Scanning Electron Microscopy (SEM) combined with Energy Dispersive Spectroscopy (EDS) techniques. Thirty-six extracted third molars were collected and randomly assigned to six groups (n = 6), five of which were suspended in demineralizing solution for 72 h to create enamel artificial lesions, and one serving as control: G1, treated with a mousse of casein phosphopeptide and amorphous calcium−phosphate (CPP-ACP); G2, treated with a gel containing nano-hydroxyapatite; G3, treated with a 5% SF varnish; G4, treated with a toothpaste containing ACP functionalized with fluoride and carbonate-coated with citrate; G5, not-treated artificial enamel lesions; G6, not demineralized and not treated sound enamel. G1−G4 were subjected to pH cycling over a period of seven days. Analyses of the specimens' enamel surfaces morphology were performed by SEM and EDS. Data were statistically analyzed for multiple group comparison by one-way ANOVA/Tukey's test (p < 0.05). The results show that the Ca/P ratio of the G5 (2.00 ± 0.07) was statistically different (p < 0.05) from G1 (1.73 ± 0.05), G2 (1.76 ± 0.01), G3 (1.88 ± 0.06) and G6 (1.74 ± 0.04), while there were no differences (p > 0.05) between G1, G2 and G6 and between G4 (2.01 ± 0.06) and G5. We concluded that G1 and G2 showed better surface remineralization than G3 and G4, after 7 days of treatment.

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.

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.

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.

Topological, Mechanical and Biological Properties of Ti6Al4V Scaffolds for Bone Tissue Regeneration Fabricated with Reused Powders via Electron Beam Melting.

Cellularized scaffold is emerging as the preferred solution for tissue regeneration and restoration of damaged functionalities. However, the high cost of preclinical studies creates a gap between investigation and the device market for the biomedical industry. In this work, bone-tailored scaffolds based on the Ti6Al4V alloy manufactured by electron beam melting (EBM) technology with reused powder were investigated, aiming to overcome issues connected to the high cost of preclinical studies. Two different elementary unit cell scaffold geometries, namely diamond (DO) and rhombic dodecahedron (RD), were adopted, while surface functionalization was performed by coating scaffolds with single layers of polycaprolactone (PCL) or with mixture of polycaprolactone and 20 wt.% hydroxyapatite (PCL/HA). The mechanical and biological performances of the produced scaffolds were investigated, and the results were compared to software simulation and experimental evidence available in literature. Good mechanical properties and a favorable environment for cell growth were obtained for all combinations of scaffold geometry and surface functionalization. In conclusion, powder recycling provides a viable practice for the biomedical industry to strongly reduce preclinical costs without altering biomechanical performance.

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.

Integrated 3D Information for Custom-Made Bone Grafts: Focus on Biphasic Calcium Phosphate Bone Substitute Biomaterials.

PURPOSE: Several studies showed that the sintering temperature of 1250 °C could affect the formation of α-Ca3(PO4)2, which is responsible for the reduction of the hardness value of biphasic calcium phosphate biocomposites, but they did not evaluate the inference of the sintering time at peak temperature on transition of ß-Ca3(PO4)2 to α-Ca3(PO4)2. This analysis explored, in an innovative way, inferences and correlations between volumetric microstructure, mechanical properties, sintering temperature, and time at peak temperature in order to find the best sintering conditions for biphasic calcium phosphate composites grafted in severe alveolar bone defects. METHODS: Sintered biphasic calcium phosphates (30%-hydroxyapatite/70%-tricalcium phosphate) were tested by microCT imaging for the 3D morphometric analysis, by compressive loading to find their mechanical parameters, and by X-ray diffraction to quantify the phases via Rietveld refinement for different sintering temperatures and times at the peak temperature. Data were analysed in terms of statistical inference using Pearson's correlation coefficients. RESULTS: All the studied scaffolds closely mimicked the alveolar organization of the jawbone, independently on the sintering temperatures and times; however, mechanical testing revealed that the group with peak temperature, which lasted for 2 hours at 1250 °C, showed the highest strength both at the ultimate point and at fracture point. CONCLUSION: The good mechanical performances of the group with peak temperature, which lasted for 2 hours at 1250 °C, is most likely due to the absence of the α-Ca3(PO4)2 phase, as revealed by X-ray diffraction. However, we detected its presence after sintering at the same peak temperature for longer times, showing the time-dependence, combined with the temperature-dependence, of the ß-Ca3(PO4)2 to α-Ca3(PO4)2 transition.