Tunable biomimetic materials elaborated by ice templating and self-assembly of collagen for tubular tissue engineering.

Synthetic tubular grafts currently used in clinical context fail frequently, and the expectations that biomimetic materials could tackle these limitations are high. However, developing tubular materials presenting structural, compositional and functional properties close to those of native tissues remains an unmet challenge. Here we describe a combination of ice templating and topotactic fibrillogenesis of type I collagen, the main component of tissues' extracellular matrix, yielding highly concentrated yet porous tubular collagen materials with controlled hierarchical architecture at multiple length scales, the hallmark of native tissues' organization. By modulating the thermal conductivity of the cylindrical molds, we tune the macroscopic porosity defined by ice. Coupling the aforementioned porosity patterns with two different fibrillogenesis routes results in a new family of tubular materials whose textural features and the supramolecular arrangement of type I collagen are achieved. The resulting materials present hierarchical elastic properties and are successfully colonized by human endothelial cells and alveolar epithelial cells on the luminal side, and by human mesenchymal stem cells on the external side. The proposed straightforward protocol is likely to be adapted for larger graft sizes that address ever-growing clinical needs, such as peripheral arterial disease or tracheal and bronchial reconstructions.

Comprehensive Assessment of Cyclic Fatigue Strength in Five Multiple-File Nickel-Titanium Endodontic Systems.

The resistance of nickel-titanium endodontic instruments against cyclic fatigue failure remains a significant concern in clinical settings. This study aimed to assess the cyclic fatigue strength of five nickel-titanium rotary systems, while correlating the results with the instruments' geometric and metallurgical characteristics. A total of 250 new instruments (sizes S1/A1, S2/A2, F1/B1, F2/B2, F3/B3) from ProTaper Gold, ProTaper Universal, Premium Taper Gold, Go-Taper Flex, and U-Files systems underwent mechanical testing. Prior to experimental procedures, all instruments were meticulously inspected to identify irregularities that could affect the investigation. Using a stereomicroscope, design characteristics such as the number of spirals, length, spirals per millimeter, and average helical angle of the active blade were determined. The surface finishing characteristics of the instruments were examined using a scanning electron microscope. Differential scanning calorimetry was employed to establish the instruments' phase transformation temperatures, while energy-dispersive X-ray spectroscopy was utilized to analyze the elemental composition of the alloy. The instruments were subjected to cyclic fatigue testing within a stainless steel non-tapered artificial canal featuring a 6 mm radius and 86 degrees of curvature. Appropriate statistical tests were applied to compare groups, considering a significance level of 0.05. The assessed design characteristics varied depending on the instrument type. The least irregular surface finishing was observed in U-Files and Premium Taper Gold files, while the most irregular surface was noted in Go-Taper Flex. All instruments exhibited near-equiatomic proportions of nickel and titanium elements, whereas ProTaper Universal and U-Files instruments demonstrated lower phase transformation temperatures compared to their counterparts. Larger-sized instruments, as well as ProTaper Universal and U-Files, tended to display lower cyclic fatigue strength results. Overall, the design, metallurgical, and cyclic fatigue outcomes varied among instruments and systems. Understanding these outcomes may assist clinicians in making more informed decisions regarding instrument selection.

Comparative analysis of reciprocating and flat-side heat-treated rotary single-file systems: Design, metallurgical characteristics, and mechanical performance.

AIM: To compare two flat-side single-file rotary instruments with three single-file reciprocating systems through a multimethod assessment. METHODOLOGY: A total of 290 new NiTi single-file rotary (AF F One Blue 25/0.06 and Platinum V.EU 25/0.06) and reciprocating (One Files Blue R25, Reciproc Blue R25, Reciproc R25) instruments were selected, carefully examined for any major deformations, and evaluated regarding their macroscopic and microscopic design, nickel and titanium elements ratio, phase transformation temperatures, and mechanical performance (time/rotation to fracture, maximum torque, angle of rotation, microhardness, maximum bending, and buckling strengths). One-way anova post hoc Tukey, T-test, and nonparametric Mood's median tests were used for statistical comparisons (α = 5%). RESULTS: Tested instruments had identical blade counts and near-identical helical angles of approximately 24° (rotary instruments) and 151° (reciprocating instruments). The flat-side analysis revealed a few inconsistencies, such as discontinuity segments, different orientations, and gaps in the homogeneity of the bluish colour. Microscopically, flat-side instruments exhibited blade discontinuity and an incomplete S-shaped cross-section. The surface finish was smoother for One Files Blue and more irregular for both rotary instruments. There were distinct phase transformation temperatures amongst all instruments. All heat-treated instruments were in R-phase arrangement, and Reciproc was in R-phase plus austenite at test temperature (20°C). Compared with the reciprocating instruments, both flat-side instruments exhibited lower results in the cyclic fatigue tests using two different clockwise kinematics, maximum torque, angle of rotation, and maximum buckling strength (p < .05). The rotary systems also exhibited low flexibility (p < .05). AF F One Blue had the lowest microhardness, whilst Reciproc had the highest value. CONCLUSION: This multimethod investigation revealed that the flat-side rotary instruments underperformed the reciprocating instruments regarding cyclic fatigue (with two different clockwise kinematics), maximum torque, angle of rotation, maximum buckling strength, and flexibility. Manufacturing inconsistencies were also observed in some of the flat-side instruments, including discontinuity segments, different orientations, and in the homogeneity of their bluish colour given by the heat treatment.

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization.

Circulating tumor cells (CTCs) represent an interesting source of biomarkers for diagnosis, prognosis, and the prediction of cancer recurrence, yet while they are extensively studied in oncobiology research, their diagnostic utility has not yet been demonstrated and validated. Their scarcity in human biological fluids impedes the identification of dangerous CTC subpopulations that may promote metastatic dissemination. In this Perspective, we discuss promising techniques that could be used for the identification of these metastatic cells. We first describe methods for isolating patient-derived CTCs and then the use of 3D biomimetic matrixes in their amplification and analysis, followed by methods for further CTC analyses at the single-cell and single-molecule levels. Finally, we discuss how the elucidation of mechanical and morphological properties using techniques such as atomic force microscopy and molecular biomarker identification using nanopore-based detection could be combined in the future to provide patients and their healthcare providers with a more accurate diagnosis.

Porous yet dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance.

Standard in vitro cell cultures are one of the pillars of biomedical sciences. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement with in vivo observations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrient exchange, as well as dense collagen walls, which mimic locally the extracellular matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for faster and more extensive colonisation when compared with equivalent non-porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towards in vitro systems applicable to other cell types and with higher physiological relevance.

A Multimethod Assessment of a New Customized Heat-Treated Nickel-Titanium Rotary File System.

This study aimed to compare three endodontic rotary systems. The new Genius Proflex (25/0.04), Vortex Blue (25/0.04), and TruNatomy (26/0.04v) instruments (n = 41 per group) were analyzed regarding design, metallurgy, and mechanical performance, while shaping ability (untouched canal walls, volume of removed dentin and hard tissue debris) was tested in 36 anatomically matched root canals of mandibular molars. The results were compared using one-way ANOVA, post hoc Tukey, and Kruskal−Wallis tests, with a significance level set at 5%. All instruments showed symmetrical cross-sections, with asymmetrical blades, no radial lands, no major defects, and almost equiatomic nickel−titanium ratios. Differences were noted in the number of blades, helical angles, cross-sectional design, and tip geometry. The Genius Proflex and the TruNatomy instruments had the highest and lowest R-phase start and finish temperatures, as well as the highest and lowest time and cycles to fracture (p < 0.05), respectively. The TruNatomy had the highest flexibility (p < 0.05), while no differences were observed between the Genius Proflex and the Vortex Blue (p > 0.05). No differences among tested systems were observed regarding the maximum torque, angle of rotation prior to fracture, and shaping ability (p > 0.05). The instruments showed similarities and differences in their design, metallurgy, and mechanical properties. However, their shaping ability was similar, without any clinically significant errors. Understanding these characteristics may help clinicians to make decisions regarding which instrument to choose for a particular clinical situation.

Multimethod Assessment of Design, Metallurgical, and Mechanical Characteristics of Original and Counterfeit ProGlider Instruments.

A multimethod study was conducted to assess the differences between original (PG-OR) and counterfeit (PG-CF) ProGlider instruments regarding design, metallurgical features, and mechanical performance. Seventy PG-OR and PG-CF instruments (n = 35 per group) were evaluated regarding the number of spirals, helical angles, and measuring line position by stereomicroscopy, while blade symmetry, cross-section geometry, tip design, and surface were assessed by scanning electron microscopy. Energy-dispersive X-ray spectroscopy and differential scanning calorimetry were used to identify element ratio and phase transformation temperatures, while cyclic fatigue, torsional, and bending testing were employed to assess their mechanical performance. An unpaired t-test and nonparametric Mann−Whitney U test were used to compare instruments at a significance level of 5%. Similarities were observed in the number of spirals, helical angles, blade symmetry, cross-sectional geometries, and nickel−titanium ratios. Measuring lines were more reliable in the original instrument, while differences were noted in the geometry of the tips (sharper tip for the original and rounded for the counterfeit) and surface finishing with PG-CF presenting more surface irregularities. PG-OR showed significantly more time to fracture (118 s), a higher angle of rotation (440°), and a lower maximum bending load (146.3 gf) (p < 0.05) than PG-CF (p < 0.05); however, maximum torque was similar for both instruments (0.4 N.cm) (p > 0.05). Although the tested instruments had a similar design, the original ProGlider showed superior mechanical behavior. The results of counterfeit ProGlider instruments were unreliable and can be considered unsafe for glide path procedures.

Biomimetic Silk Macroporous Materials for Drug Delivery Obtained via Ice-Templating.

Silk from Bombyx mori is one of the most exciting materials in nature. The apparently simple arrangement of its two major components─two parallel filaments of silk fibroin (SF) coated by a common sericin (SS) sheath─provides a combination of mechanical and surface properties that can protect the moth during its most vulnerable phase, the pupal stage. Here, we recapitulate the topology of native silk fibers but shape them into three-dimensional porous constructs using an unprecedented design strategy. We demonstrate, for the first time, the potential of these macroporous silk foams as dermal patches for wound protection and for the controlled delivery of Rifamycin (Rif), a model antibiotic. The method implies (i) removing SS from silk fibers; (ii) shaping SF solutions into macroporous foams via ice-templating; (iii) stabilizing the SF macroporous foam in a methanolic solution of Rif; and (iv) coating Rif-loaded SF foams with a SS sheath. The resulting SS@SF foams exhibit water wicking capacity and accommodate up to ∼20% deformation without detaching from a skin model. The antibacterial behavior of Rif-loaded SS@SF foams against Staphylococcus aureus on agar plates outperforms that of SF foams (>1 week and 4 days, respectively). The reassembly of natural materials as macroporous foams─illustrated here for the reconstruction of silk-based materials─can be extended to other multicomponent natural materials and may play an important role in applications where controlled release of molecules and fluid transport are pivotal.

Sepiolite-Hydrogels: Synthesis by Ultrasound Irradiation and Their Use for the Preparation of Functional Clay-Based Nanoarchitectured Materials.

Sepiolite and palygorskite fibrous clay minerals are 1D silicates featuring unique textural and structural characteristics useful in diverse applications, and in particular as rheological additives. Here we report on the ability of grinded sepiolite to generate highly viscous and stable hydrogels by sonomechanical irradiation (ultrasounds). Adequate drying of such hydrogels leads to low-density xerogels that show extensive fiber disaggregation compared to the starting sepiolite-whose fibers are agglomerated as bundles. Upon re-dispersion in water under high-speed shear, these xerogels show comparable rheological properties to commercially available defibrillated sepiolite products, resulting in high viscosity hydrogels that minimize syneresis. These colloidal systems are thus very interesting as they can be used to stabilize many diverse compounds as well as nano-/micro-particles, leading to the production of a large variety of composites and nano/micro-architectured solids. In this context, we report here various examples showing how colloidal routes based on sepiolite hydrogels can be used to obtain new heterostructured functional materials, based on their assembly to solids of diverse topology and composition such as 2D and 1D kaolinite and halloysite aluminosilicates, as well as to the 2D synthetic Mg,Al-layered double hydroxides (LDH).

Colonization versus encapsulation in cell-laden materials design: porosity and process biocompatibility determine cellularization pathways.

Seeding materials with living cells has been-and still is-one of the most promising approaches to reproduce the complexity and the functionality of living matter. The strategies to associate living cells with materials are limited to cell encapsulation and colonization, however, the requirements for these two approaches have been seldom discussed systematically. Here we propose a simple two-dimensional map based on materials' pore size and the cytocompatibility of their fabrication process to draw, for the first time, a guide to building cellularized materials. We believe this approach may serve as a straightforward guideline to design new, more relevant materials, able to seize the complexity and the function of biological materials. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Preservation of biomaterials and cells by freeze-drying: Change of paradigm.

Freeze-drying is the most widespread method to preserve protein drugs and vaccines in a dry form facilitating their storage and transportation without the laborious and expensive cold chain. Extending this method for the preservation of natural biomaterials and cells in a dry form would provide similar benefits, but most results in the domain are still below expectations. In this review, rather than consider freeze-drying as a traditional black box we "break it" through a detailed process thinking approach. We discuss freeze-drying from process thinking aspects, introduce the chemical, physical, and mechanical environments important in this process, and present advanced biophotonic process analytical technology. In the end, we review the state of the art in the freeze-drying of the biomaterials, extracellular vesicles, and cells. We suggest that the rational design of the experiment and implementation of advanced biophotonic tools are required to successfully preserve the natural biomaterials and cells by freeze-drying. We discuss this change of paradigm with existing literature and elaborate on our perspective based on our new unpublished results.

Biomimetic Tough Gels with Weak Bonds Unravel the Role of Collagen from Fibril to Suprafibrillar Self-Assembly.

Biological tissues rich in type I collagen exhibit specific hierarchical fibrillar structures together with remarkable mechanical toughness. However, the role of collagen alone in their mechanical response at different structural levels is not fully understood. Here, it is proposed to rationalize such challenging interplay from a materials science perspective through the subtle control of this protein self-assembly in vitro. It is relied on a spray-processing approach to readily use the collagen phase diagram and set a palette of biomimetic self-assembled collagen gels in terms of suprafibrillar organization. Their mechanical responses unveil the involvement of mechanisms occurring either at fibrillar or suprafibrillar scales. Noticeably, both modulus at early stage of deformations and tensile toughness probe the suprafibrillar organization, while durability under cyclic loading and stress relaxation reflect mechanisms at the fibril level. By changing the physicochemical environment, the interfibrillar interactions are modified toward more biomimetic mechanical responses. The possibility of making tissue-like materials with versatile compositions and toughness opens perspectives in tissue engineering.

Recent advances in ice templating: from biomimetic composites to cell culture scaffolds and tissue engineering.

Ice templating - or freeze casting - has flourished in multiple domains as a straightforward process to shape solutions and particle suspensions into macroporous materials. Longtime used as a process to shape colloidal suspensions into lightweight ceramics, the use of ice templating has evolved to fabricate materials that mimic the architecture of biological tissues such as nacre and bone. Recently, the technique has been used to shape biopolymers for cell culture systems and tissue engineering applications and eventually to allow the fabrication of biomaterials containing living cells. Here we review how ice templating has progressed to cope with intrinsically labile biological matter and how these advances may shape the future 3D cell culture, tissue engineering and ultimately, cryobiology.

Unveiling Cells' Local Environment during Cryopreservation by Correlative In Situ Spatial and Thermal Analyses.

Cryopreservation is the only fully established procedure to extend the lifespan of living cells and tissues, a key to activities spanning from fundamental biology to clinical practice. Despite its prevalence and impact, the central aspects of cryopreservation, such as the cell's physicochemical environment during freezing, remain elusive. Here we address that question by coupling in situ microscopic directional freezing to visualize cells and their surroundings during freezing with the freezing-medium phase diagram. We extract the freezing-medium spatial distribution in cryopreservation, providing a tool to describe the cell vicinity at any point during freezing. We show that two major events define the cells' local environment over time: the interaction with the moving ice front and the interaction with the vitreous moving front, a term we introduce here. Our correlative strategy may be applied to cells relevant to clinical research and practice and may help in the design of new cryoprotective media based on local physicochemical cues.

Mechanical Tests, Metallurgical Characterization, and Shaping Ability of Nickel-Titanium Rotary Instruments: A Multimethod Research.

INTRODUCTION: This study aimed to compare the mechanical and metallurgical properties and shaping ability of different rotary systems using a multimethod approach. METHODS: New NeoNiti A1 (Neolix SAS, Châtres-La-Forêt, France), HyFlex EDM One File (Coltène/Whaledent, Altstätten, Switzerland), ProTaper Gold F2 (Dentsply Maillefer, Ballaigues, Switzerland), and ProTaper Universal F2 (Dentsply Maillefer) rotary instruments were tested regarding cyclic fatigue, torsional resistance, design, and morphologic characteristics using scanning electron microscopy, metal alloy characterization using differential scanning calorimetry, and energy-dispersive X-ray spectroscopy. Additionally, their shaping ability on the preparation of 48 canals of maxillary molars was evaluated using micro-computed tomographic technology. Mechanical and metallurgical analyses were compared using analysis of variance post hoc Tukey tests, whereas the independent Student t test was used to compare the shaping ability of the ProTaper systems or the thermomechanically treated instruments. The significance level was set at 5%. RESULTS: The highest cyclic fatigue was observed with the NeoNiti and HyFlex EDM instruments (P > .05), whereas HyFlex EDM had the highest angular rotation to fracture (P < .05). Scanning electron microscopic/energy-dispersive X-ray spectroscopic analyses confirmed similarities in the instruments' design and an almost equiatomic composition of the systems. Differential scanning calorimetry showed that ProTaper Gold had higher transformation temperatures than ProTaper Universal, whereas a similar transformation was observed between NeoNiti and HyFlex. Micro-computed tomographic analysis revealed that, despite the fact that none of the systems was able to prepare all root canal walls, no statistical differences were observed in either ProTaper systems (P > .05) or the thermomechanically treated instruments (P > .05). CONCLUSIONS: Apart from differences in the mechanical tests and metallurgical characterization, systems with comparable instrument dimensions and preparation protocols showed a similar percentage of untouched surface areas in the root canal preparation of maxillary molars.

Unveiling the Interstitial Pressure between Growing Ice Crystals during Ice-Templating Using a Lipid Lamellar Probe.

What is the pressure generated by ice crystals during ice-templating? This work addresses this crucial question by estimating the pressure exerted by oriented ice columns on a supramolecular probe composed of a lipid lamellar hydrogel during directional freezing. This process, also known as freeze-casting, has emerged as a unique processing technique for a broad class of organic, inorganic, soft, and biological materials. Nonetheless, the pressure exerted during and after crystallization between two ice columns is not known, despite its importance with respect to the fragility of the frozen material, especially for biological samples. By using the lamellar period of a glycolipid lamellar hydrogel as a common probe, we couple data obtained from ice-templated-resolved in situ synchrotron small-angle X-ray scattering (SAXS) with data obtained from controlled adiabatic desiccation experiments. We estimate the pressure to vary between 1 ± 10% kbar at -15 °C and 3.5 ± 20% kbar at -60 °C.

Self-Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials.

Extracellular matrices (ECM) rich in type I collagen exhibit characteristic anisotropic ultrastructures. Nevertheless, working in vitro with this biomacromolecule remains challenging. When processed, denaturation of the collagen molecule is easily induced in vitro avoiding proper fibril self-assembly and further hierarchical order. Here, an innovative approach enables the production of highly concentrated injectable collagen microparticles, based on collagen molecules self-assembly, thanks to the use of spray-drying process. The versatility of the process is shown by performing encapsulation of secretion products of gingival mesenchymal stem cells (gMSCs), which are chosen as a bioactive therapeutic product for their potential efficiency in stimulating the regeneration of a damaged ECM. The injection of collagen microparticles in a cell culture medium results in a locally organized fibrillar matrix. The efficiency of this approach for making easily handleable collagen microparticles for encapsulation and injection opens perspectives in active tissue regeneration and 3D bioprinted scaffolds.

Topotactic Fibrillogenesis of Freeze-Cast Microridged Collagen Scaffolds for 3D Cell Culture.

Type I collagen is the main component of the extracellular matrix (ECM). In vitro, under a narrow window of physicochemical conditions, type I collagen self-assembles to form complex supramolecular architectures reminiscent of those found in native ECM. Presently, a major challenge in collagen-based biomaterials is to couple the delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions. In this work, an ice-templating approach promoting the structuration of collagen into macroporous monoliths is used. Instead of common solvent removal procedures, a new topotactic conversion approach yielding self-assembled ordered fibrous materials is implemented. These collagen-only, non-cross-linked scaffolds exhibit uncommon mechanical properties in the wet state, with a Young's modulus of 33 ± 12 kPa, an ultimate tensile stress of 33 ± 6 kPa, and a strain at failure of 105 ± 28%. With the help of the ice-patterned microridge features, normal human dermal fibroblasts and C2C12 murine myoblasts successfully migrate and form highly aligned populations within the resulting three-dimensional (3D) collagen scaffolds. These results open a new pathway to the development of new tissue engineering scaffolds ordered across various organization levels from the molecule to the macropore and are of particular interest for biomedical applications where large-scale 3D cell alignment is needed such as for muscular or nerve reconstruction.

Tetramethylguanidine-based gels and colloids of cellulose.

Novel and stable gels of cellulose were produced. These gels are prepared at room temperature by combination of cellulose and tetramethylguanidine (TMG) in different ratios (1:1, 1:2, 1:3 in equivalents of alcohol groups of cellulose per number of molecules of TMG). Detailed NMR, SEM, rheological and XRD studies of these gels were carried out. The concentration of cellulose in the gel, temperature, frequency of oscillation and shear rate were used as variables in order to understand the fundamentals and optimize operational conditions, considering their possible use as matrices for CO2 capture. Cellulose recovery from a specific gel was performed using ethanol as precipitating agent, leading to a lower crystallinity, which permits to consider this polymer in further studies associated to physical/chemical modification of cellulose.

Water/ice phase transition: The role of zirconium acetate, a compound with ice-shaping properties.

Few compounds feature ice-shaping properties. Zirconium acetate is one of the very few inorganic compounds reported so far to have ice-shaping properties similar to that of ice-shaping proteins, encountered in many organisms living at low temperature. When a zirconium acetate solution is frozen, oriented and perfectly hexagonal ice crystals can be formed and their growth follows the temperature gradient. To shed light on the water/ice phase transition while freezing zirconium acetate solution, we carried out differential scanning calorimetry measurements. From our results, we estimate how many water molecules do not freeze because of their interaction with Zr cations. We estimate the colligative properties of the Zr acetate on the apparent critical temperature. We further show that the phase transition is unaffected by the nature of the base which is used to adjust the pH. Our results provide thus new hints on the ice-shaping mechanism of zirconium acetate.