Tailoring the Microarchitectures of 3D Printed Bone-like Scaffolds for Tissue Engineering Applications.

Material extrusion (MEX), commonly referred to as fused deposition modeling (FDM) or fused filament fabrication (FFF), is a versatile and cost-effective technique to fabricate suitable scaffolds for tissue engineering. Driven by a computer-aided design input, specific patterns can be easily collected in an extremely reproducible and repeatable process. Referring to possible skeletal affections, 3D-printed scaffolds can support tissue regeneration of large bone defects with complex geometries, an open major clinical challenge. In this study, polylactic acid scaffolds were printed resembling trabecular bone microarchitecture in order to deal with morphologically biomimetic features to potentially enhance the biological outcome. Three models with different pore sizes (i.e., 500, 600, and 700 µm) were prepared and evaluated by means of micro-computed tomography. The biological assessment was carried out seeding SAOS-2 cells, a bone-like cell model, on the scaffolds, which showed excellent biocompatibility, bioactivity, and osteoinductivity. The model with larger pores, characterized by improved osteoconductive properties and protein adsorption rate, was further investigated as a potential platform for bone-tissue engineering, evaluating the paracrine activity of human mesenchymal stem cells. The reported findings demonstrate that the designed microarchitecture, better mimicking the natural bone extracellular matrix, favors a greater bioactivity and can be thus regarded as an interesting option for bone-tissue engineering.

Filling ability of three variants of the single-cone technique with bioceramic sealer: a micro-computed tomography study.

The present study evaluated the quality of single-cone root canal fillings with bioceramic (BC) sealer using three different techniques by means of micro-computed tomography (micro-CT). The canals of 30 extracted single-rooted permanent teeth were shaped with R40 Reciproc blue files and filled with the single-cone technique (SCT). BioRoot RCS BC sealer was placed inside the canals with one of the following master cones: R40 cone to working length (RWL, n = 10); R40 cone trimmed 1 mm short of working length (RWL-1, n = 10); non-standardized gutta-percha cone to working length (NSWL, n = 10). A quantitative and qualitative micro-CT analysis assessed the filling quality and internal/external voids formation. Collected data underwent statistical analysis by multivariate one-way analysis of variance (α = 0.05). In all groups, the voids were minimal and prevalently external. The NSWL and RWL-1 groups had increased sealer ratios in the whole canal and the apical canal portion, respectively. The lowest amounts of voids were found in the RWL group; the void volumes were slightly greater in the RWL-1 mm and NSWL groups, especially at the apical level. Two alternative SCTs showed satisfactory filling ability, uniform distribution of the BC sealer, and a minimally increased voids formation compared to the standard SCT with dedicated cone. The two tested alternative SCTs could take advantage of the beneficial characteristics of the BC sealer, which evenly filled the endodontic space, ideally sealing both the major and the accessory communications with the periodontium.

3D printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and characterization.

Additive manufacturing for tissue engineering applications offers the possibility to design scaffolds characterized by a fine and detailed microarchitecture. Several fabrication technologies are currently available which allow to prepare tailored structures with a large selection of materials for restoring and healing tissues. However, 3D printed scaffolds are generally collected by assembling repetitive geometrical units or reproducing specific patterns in the layering direction, leading to a highly ordered architecture that does not mimic the morphology of the natural extracellular matrix (ECM), one of the main goals to be reached for an effective therapeutic approach. It is usually stated in the tissue engineering field that a scaffold has to be considered a temporary ECM, resembling all the peculiar features as close as possible and, in this regard, an ordered microstructure cannot be usually observed within biological tissues and organs. With the aim to overcame this limitation and offer a potential approach for bone tissue applications, the present study proposes a design methodology to fabricate 3D printed scaffolds characterized by a random microarchitecture which can be repeatedly reproduced thanks to the intrinsic controllable process of additive manufacturing. In this framework, four different models in polylactic acid were fabricated by means of fused deposition modelling, including a three-dimensional random distribution of spherical pores of 400, 500, and 600 µm for the first three cases, and a randomly varied distribution in the range 400-600 µm for the fourth case. A detailed assessment by means of microcomputed tomography and mechanical evaluation was then carried out in order to fully analyse the resulting scaffolds, providing both morphological and quantitative data.

Spiculosiphon oceana (Foraminifera) a new bio-indicator of acidic environments related to fluid emissions of the Zannone Hydrothermal Field (central Tyrrhenian Sea).

The new record of a shallow-water submarine hydrothermal field (<150 m w.d.) in the western Mediterranean Sea (Tyrrhenian Sea, Italy) allows us to study CO2 fluid impact on benthic foraminifers. Benthic foraminifers calcification process is sensitive to ocean acidification and to local chemical and physical parameters of seawater and pore water. Thus, foraminifers can record specific environmental conditions related to hydrothermal fluids, but at present their response to such activity is poorly defined. The major outcome of this study is the finding of a very uncommon taxon for the Mediterranean Sea, i.e., the Spiculosiphon oceana, a giant foraminifer agglutinating spicules of sponges. This evidence, along with the strong decrease of calcareous tests in the foraminiferal assemblages associated to hydrothermal activity, provides new insights on the meiofauna living in natural stressed environment. In particular, observations obtained from this study allow us to consider S. oceana a potential tolerant species of high CO2 concentrations (about 2-4 times higher than the normal marine values) and a proxy of acidic environments as well as of recent ocean acidification processes.

Galectin-3 is essential for proper bone cell differentiation and activity, bone remodeling and biomechanical competence in mice.

OBJECTIVE: Galectin-3 is constitutively expressed in bone cells and was recently shown to modulate osteogenic transdifferentiation of vascular smooth muscle cells and atherosclerotic calcification. However, the role of galectin-3 in bone physiology is largely undefined. To address this issue, we analyzed (1) the skeletal features of 1-, 3- and 6-month-old galectin-3 null (Lgals3-/-) and wild type (WT) mice and (2) the differentiation and function of osteoblasts and osteoclasts derived from these animals. METHODS: Long bone phenotype, gene expression profile, and remodeling were investigated by micro-computed tomography, real time-PCR, static and dynamic histomorphometry, and assessment of biochemical markers of bone resorption and formation. Bone competence was also evaluated by biomechanical testing at 3 months. In vitro, the effects of galectin-3 deficiency on bone cell differentiation and function were investigated by assessing (a) gene expression of osteoblast markers, alkaline phosphatase activity, mineralization assay, and WNT/ß-catenin signaling (of which galectin-3 is a known regulator) in osteoblasts; and (b) tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts. RESULTS: Lgals3-/- mice revealed a wide range of age-dependent alterations including lower bone formation and higher bone resorption, accelerated age-dependent trabecular bone loss (p < 0.01 vs. WT at 3 months) and reduced bone strength (p < 0.01 vs. WT at 3 months). These abnormalities were accompanied by a steady inflammatory state, as revealed by higher bone expression of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 (p < 0.001 vs. WT at 3 months), increased content of osteal macrophages (p < 0.01 vs. WT at 3 months), and reduced expression of markers of alternative (M2) macrophage activation. Lgals3-/- osteoblasts and osteoclasts showed impaired terminal differentiation, reduced mineralization capacity (p < 0.01 vs. WT cells) and resorption activity (p < 0.01 vs. WT cells). Mechanistically, impaired differentiation and function of Lgals3-/- osteoblasts was associated with altered WNT/ß-catenin signaling (p < 0.01 vs. WT cells). CONCLUSIONS: These data provide evidence for a contribution of galectin-3 to bone cell maturation and function, bone remodeling, and biomechanical competence, thus identifying galectin-3 as a promising therapeutic target for age-related disorders of bone remodeling.

Implant-Abutment Contact Surfaces and Microgap Measurements of Different Implant Connections Under 3-Dimensional X-Ray Microtomography.

PURPOSE: The presence of a microgap between implant and abutment could produce a bacterial reservoir which could interfere with the long-term health of the periimplant tissues. The aim of this article was to evaluate, by x-ray 3-dimensional microtomography, implant-abutment contact surfaces and microgaps at the implant-abutment interface in different types of implant-abutment connections. MATERIALS AND METHODS: A total of 40 implants were used in this in vitro study. Ten implants presented a screw-retained internal hexagon abutment (group I), 10 had a Morse Cone taper internal connection (group II), 10 another type of Morse Cone taper internal connection (group III), and 10 had a screwed trilobed connection (group IV). RESULTS: In both types of Morse Cone internal connections, there was no detectable separation at the implant-abutment in the area of the conical connection, and there was an absolute congruity without any microgaps between abutment and implant. No line was visible separating the implant and the abutment. On the contrary, in the screwed abutment implants, numerous gaps and voids were present. CONCLUSIONS: The results of this study support the hypothesis that different types of implant-abutment joints are responsible for the observed differences in bacterial penetration.

Mechanical Reliability Evaluation of an Oral Implant-Abutment System According to UNI EN ISO 14801 Fatigue Test Protocol.

PURPOSE: The aim of this in vitro study was to evaluate the mechanical reliability of a dental implant system by testing its maximum fracture load and mechanical performance under cyclic fatigue stress. METHODS: An experimental study according to the international standards (UNI EN ISO 14801: 2008) was performed using 13 implants (3.80 mm in diameter and 12 mm in length) with straight titanium abutments tightened to 30 N. Five samples were subjected to compression stress at break. Based on the mean fracture load value obtained in this test, the levels of dynamic loading range were set and were carried on at a frequency of 15 Hz for 5 × 10 cycles. RESULTS: The compression stress at break mean value of the tested implants was 430 N (SD ± 35.66 N). In the mechanical fatigue stress test, the fatigue limit for 5 × 10 load cycles was 172 N. CONCLUSIONS: The evaluated implant system proved to withstand considerable mechanical loads under the "worst-case" loading situation performed according to UNI EN ISO 14801 standard. The reliability of this test protocol makes it suitable to be accomplished for understanding and comparing mechanical properties of implant systems.

Evaluation of Microgap With Three-Dimensional X-Ray Microtomography: Internal Hexagon Versus Cone Morse.

BACKGROUND: Microgap is defined as the microscopic space that exists between the implant body and abutment. The main mechanism proposed for microgap-related crestal bone loss is the role of this space as a trap for bacteria and thus, as a putative etiological factor for inflammatory reaction in the peri-implant soft tissues. The aim of this paper was to evaluate, with X-ray 3D microtomography, the microscopic space that exists between the implant body and abutment with internal hexagon versus cone morse. METHODS: A total of 20 implants were used in this in vitro study. Ten implants per group were used. Ten implants presented a screw retained internal hexagon abutment (Group I) and 10 had a Cone Morse taper internal connection (Group II). RESULTS: In both types of Cone Morse internal connection implants there was no detectable separation at the implant/abutment in the area of the conical connection, and there was an absolute congruity without any microgaps between abutment and implant. No line was visible separating the implant and the abutment. On the contrary, in the internal hexagon screwed abutment numerous gaps and voids were present between the implant body and abutment. CONCLUSION: The results of the present study seem, then, to support the hypothesis that the length and the characteristics of the implant-abutment joint could be a reason for the observed differences in mechanical stability.

Evaluation of an endosseous oral implant system according to UNI EN ISO 14801 fatigue test protocol.

PURPOSE: The aim of this in vitro study was to evaluate the maximum fracture load and the mechanical performance to cyclic fatigue stress of a dental implant system. METHODS: An experimental study according to the international standards (UNI EN ISO 14801: 2008) was carried out using 15 implants (3.80 mm of diameter and 13 mm of length) with applied straight titanium abutment tightened to 30 N. Five samples were subjected to compression stress at break. Based on the mean fracture load value obtained in this test, the levels of dynamic loading range were set that were carried on at a frequency of 15 Hz for 5 × 10 cycles. RESULTS: The compression stress at break mean value of the tested implants was 499.40 N (SD ±50.1 N). In the mechanical fatigue stress test, the fatigue limit for 5 × 10 load cycles for all tested samples was 250 N. CONCLUSIONS: The evaluated implant system proved to withstand considerable mechanical loads under the "worst-case" loading situation performed according to UNI EN ISO 14801 standard. The reliability of this test protocol makes it suitable to be accomplished for understanding and comparing mechanical properties of other implant systems.

Comparative evaluation of the accuracy of linear measurements between cone beam computed tomography and 3D microtomography.

OBJECTIVE: The aim of this study was to evaluate the influence of artifacts on the accuracy of linear measurements estimated with a common cone beam computed tomography (CBCT) system used in dental clinical practice, by comparing it with microCT system as standard reference. MATERIALS AND METHODS: Ten bovine bone cylindrical samples containing one implant each, able to provide both points of reference and image quality degradation, have been scanned by CBCT and microCT systems. Thanks to the software of the two systems, for each cylindrical sample, two diameters taken at different levels, by using implants different points as references, have been measured. Results have been analyzed by ANOVA and a significant statistically difference has been found. RESULTS AND DISCUSSION: Due to the obtained results, in this work it is possible to say that the measurements made with the two different instruments are still not statistically comparable, although in some samples were obtained similar performances and therefore not statistically significant. CONCLUSION: With the improvement of the hardware and software of CBCT systems, in the near future the two instruments will be able to provide similar performances.

Technique for bone volume measurement from human femur head samples by classification of micro-CT image histograms.

INTRODUCTION: Micro-CT analysis is a powerful technique for a non-invasive evaluation of the morphometric parameters of trabecular bone samples. This elaboration requires a previous binarization of the images. A problem which arises from the binarization process is the partial volume artifact. Voxels at the external surface of the sample can contain both bone and air so thresholding operates an incorrect estimation of volume occupied by the two materials. AIM: The aim of this study is the extraction of bone volumetric information directly from the image histograms, by fitting them with a suitable set of functions. METHODS: Nineteen trabecular bone samples were extracted from femoral heads of eight patients subject to a hip arthroplasty surgery. Trabecular bone samples were acquired using micro-CT Scanner. Hystograms of the acquired images were computed and fitted by Gaussian-like functions accounting for: a) gray levels produced by the bone x-ray absorption, b) the portions of the image occupied by air and c) voxels that contain a mixture of bone and air. This latter contribution can be considered such as an estimation of the partial volume effect. RESULTS: The comparison of the proposed technique to the bone volumes measured by a reference instrument such as by a helium pycnometer show the method as a good way for an accurate bone volume calculation of trabecular bone samples.

Synthetic bone substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation.

BACKGROUND: Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. AIM: In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study. MATERIAL AND METHODS: Two blocks of synthetic bone substitute (∼0.14 cm(3)), alone or engineered with 1×10(6) ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses. RESULTS AND CONCLUSIONS: The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues.

Morphological comparison of PVA scaffolds obtained by gas foaming and microfluidic foaming techniques.

In this article, we have exploited a microfluidic foaming technique for the generation of highly monodisperse gas-in-liquid bubbles as a templating system for scaffolds characterized by an ordered and homogeneous porous texture. An aqueous poly(vinyl alcohol) (PVA) solution (containing a surfactant) and a gas (argon) are injected simultaneously at constant flow rates in a flow-focusing device (FFD), in which the gas thread breaks up to form monodisperse bubbles. Immediately after its formation, the foam is collected and frozen in liquid nitrogen, freeze-dried, and cross-linked with glutaraldehyde. In order to highlight the superior morphological quality of the obtained porous material, a comparison between this scaffold and another one, also constituted of PVA but obtained with a traditional gas foaming technique, was carried out. Such a comparison has been conducted by analyzing electron microscopy and X-ray microtomographic images of the two samples. It turned out that the microfluidic produced scaffold was characterized by much more uniform porous texture than the gas-foaming one as witnessed by narrower pore size, interconnection, and wall thickness distributions. On the other side, scarce pore interconnectivity, relatively low pore volume, and limited production rate represent, by now, the principal disadvantages of microfluidic foaming as scaffold fabrication method, emphasizing the kind of improvement that this technique needs to undergo.

Role of X-ray microtomography in tissue engineering.

The structure and architecture of scaffolds are crucial factors in scaffolds-based tissue engineering since they affect the functionality of the tissue engineering construct and the eventual application in health care. Therefore, effective scaffold assessment techniques are required right at the initial stages of research and development so as to select or design scaffolds with suitable properties. Furthermore, since the biological performances of a scaffold is evaluated with respect to its capacity of favouring cell adhesion, proliferation as well as production of extracellular matrix, it is important to have an analytical technique able to monitor the various stages of cell culture both in vitro and especially in vivo. Finally, the development of a vascular network inside the cell scaffold construct is a fundamental requisite for achieving a full integration of the developing tissue with the host tissue. Also in this respect it is mandatory to assess the propensity of the scaffold to be permeated by blood vessels. In the review, it will be shown how X-ray microtomography (micro-CT) can give fundamental information regarding all the three aspects outlined above.

Present and future in the use of micro-CT scanner 3D analysis for the study of dental and root canal morphology.

The goal of the present article is to illustrate and analyze the applications and the potential of microcomputed tomography (micro-CT) in the analysis of tooth anatomy and root canal morphology. The authors performed a micro-CT analysis of the following different teeth: maxillary first molars with a second canal in the mesiobuccal (MB) root, mandibular first molars with complex anatomy in the mesial root, premolars with single and double roots and with complicated apical anatomy. The hardware device used in this study was a desktop X-ray microfocus CT scanner (SkyScan 1072, SkyScan bvba, Aartselaar, Belgium). A specific software ResolveRT Amira (Visage Imaging) was used for the 3D analysis and imaging. The authors obtained three-dimensional images from 15 teeth. It was possible to precisely visualize and analyze external and internal anatomy of teeth, showing the finest details. Among the 5 upper molars analyzed, in three cases, the MB canals joined into one canal, while in the other two molars the two mesial canals were separate. Among the lower molars two of the five samples exhibited a single canal in the mesial root, which had a broad, flat appearance in a mesiodistal dimension. In the five premolar teeth, the canals were independent; however, the apical delta and ramifications of the root canals were quite complex. Micro-CT offers a simple and reproducible technique for 3D noninvasive assessment of the anatomy of root canal systems.

Variability of morphometric parameters of human trabecular tissue from coxo-arthritis and osteoporotic samples.

Morphometric and architectural bone parameters change in diseases such as osteoarthritis and osteoporosis. The mechanical strength of bone is primarily influenced by bone quantity and quality. Bone quality is defined by parameters such as trabecular thickness, trabecular separation, trabecular density and degree of anisotropy that describe the micro-architectural structure of bone. Recently, many studies have validated microtomography as a valuable investigative technique to assess bone morphometry, thanks to micro-CT non-destructive, non-invasive and reliability features, in comparison to traditional techniques such as histology. The aim of this study is the analysis by micro-computed tomography of six specimens, extracted from patients affected by osteoarthritis and osteoporosis, in order to observe the tridimensional structure and calculate several morphometric parameters.

Analysis of single point and continuous wave of condensation root filling techniques by micro-computed tomography.

The aim of the present microtomographic study was to investigate the quality of root canal filling and the voids formation in canals of extracted teeth instrumented with a simultaneous technique and filled with two different methods. Twenty-four single-rooted teeth were assigned to two experimental groups (no. = 12); canals were shaped with NiTi rotary files, irrigated with NaOCl and filled either with the single point (group 1) or the continuous wave of condensation technique (group 2). Specimens underwent microtomographic scanning. Collected data were statistically analyzed by nonparametric methods. Void mean percentages were found to be limited and similar between the two groups; the single point technique led to greater sealer thickness in partially oval canals.

A new software for dimensional measurements in 3D endodontic root canal instrumentation.

The main issue to be faced to get size estimates of 3D modification of the dental canal after endodontic treatment is the co-registration of the image stacks obtained through micro computed tomography (micro-CT) scans before and after treatment. Here quantitative analysis of micro-CT images have been performed by means of new dedicated software targeted to the analysis of root canal after endodontic instrumentation. This software analytically calculates the best superposition between the pre and post structures using the inertia tensor of the tooth. This strategy avoid minimization procedures, which can be user dependent, and time consuming. Once the co-registration have been achieved dimensional measurements have then been performed by contemporary evaluation of quantitative parameters over the two superimposed stacks of micro-CT images. The software automatically calculated the changes of volume, surface and symmetry axes in 3D occurring after the instrumentation. The calculation is based on direct comparison of the canal and canal branches selected by the user on the pre treatment image stack.

Fixture-abutment connection surface and micro-gap measurements by 3D micro-tomographic technique analysis.

X-ray micro-tomography (micro-CT) is a miniaturized form of conventional computed axial tomography (CAT) able to investigate small radio-opaque objects at a-few-microns high resolution, in a non-destructive, non-invasive, and tri-dimensional way. Compared to traditional optical and electron microscopy techniques, which provide two-dimensional images, this innovative investigation technology enables a sample tri-dimensional analysis without cutting, coating or exposing the object to any particular chemical treatment. X-ray micro-tomography matches ideal 3D microscopy features: the possibility of investigating an object in natural conditions and without any preparation or alteration; non-invasive, non-destructive, and sufficiently magnified 3D reconstruction; reliable measurement of numeric data of the internal structure (morphology, structure and ultra-structure). Hence, this technique has multi-fold applications in a wide range of fields, not only in medical and odontostomatologic areas, but also in biomedical engineering, materials science, biology, electronics, geology, archaeology, oil industry, and semi-conductors industry. This study shows possible applications of micro-CT in dental implantology to analyze 3D micro-features of dental implant to abutment interface. Indeed, implant-abutment misfit is known to increase mechanical stress on connection structures and surrounding bone tissue. This condition may cause not only screw preload loss or screw fracture, but also biological issues in peri-implant tissues.

Microtomography evaluation of dental tissue wear surface induced by in vitro simulated chewing cycles on human and composite teeth.

In this study a 3D microtomography display of tooth surfaces after in vitro dental wear tests has been obtained. Natural teeth have been compared with prosthetic teeth, manufactured by three different polyceramic composite materials. The prosthetic dental element samples, similar to molars, have been placed in opposition to human teeth extracted by paradontology diseases. After microtomography analysis, samples have been subjected to in vitro fatigue test cycles by servo-hydraulic mechanical testing machine. After the fatigue test, each sample has been subjected again to microtomography analysis to obtain volumetric value changes and dental wear surface images. Wear surface images were obtained by 3D reconstruction software and volumetric value changes were measured by CT analyser software. The aim of this work has been to show the potential of microtomography technique to display very clear and reliable wear surface images. Microtomography analysis methods to evaluate volumetric value changes have been used to quantify dental tissue and composite material wear.