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Tensile properties of directionally freeze-cast biopolymer scaffolds are rarely reported, even though they are of interest from a fundamental science perspective and critical in applications such as scaffolds for the regeneration of nerves or when used as ureteral stents. The focus of this study is on collagen scaffolds freeze-cast with two different applied cooling rates (10 °C/min and 1 °C/min) in two freezing directions (longitudinal and radial). Reported are the results of a systematic structural characterization of dry scaffolds by scanning electron microscopy and the mechanical characterization in tension of both dry and fully hydrated scaffolds. Systematic structure-property-processing correlations are obtained for a comparison of the tensile performance of longitudinally and radially freeze-cast collagen scaffolds with their performance in compression. Collated, the correlations, obtained both in tension in this study and in compression for collagen and chitosan in two earlier reports, not only enable the custom-design of freeze-cast biopolymer scaffolds for biomedical applications but also provide new insights into similarities and differences of scaffold and cell-wall structure formation during the directional solidification of "smooth" and "fibrillar" biopolymers.

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Directional solidification of aqueous solutions and slurries in a temperature gradient is widely used to produce cellular materials through a phase separation of solutes or suspended particles between growing ice lamellae. While this process has analogies to the directional solidification of metallurgical alloys, it forms very different hierarchical structures. The resulting honeycomb-like porosity of freeze-cast materials consists of regularly spaced, lamellar cell walls which frequently exhibit unilateral surface features of morphological complexity reminiscent of living forms, all of which are unknown in metallurgical structures. While the strong anisotropy of ice-crystal growth has been hypothesized to play a role in shaping those structures, the mechanism by which they form has remained elusive. By directionally freezing binary water mixtures containing small solutes obeying Fickian diffusion, and phase-field modeling of those experiments, we reveal how those structures form. We show that the flat side of lamellae forms because of slow faceted ice-crystal growth along the c-axis, while weakly anisotropic fast growth in other directions, including the basal plane, is responsible for the unilateral features. Diffusion-controlled morphological primary instabilities on the solid-liquid interface form a cellular structure on the atomically rough side of the lamellae, which template regularly spaced "ridges" while secondary instabilities of this structure are responsible for the more complex features. Collating the results, we obtain a scaling law for the lamellar spacing,  [Formula: see text] , where [Formula: see text] and [Formula: see text] are the local growth rate and temperature gradient, respectively.

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This work showcases a novel phenomenological method to create predictive simulations of metallic lattice structures. The samples were manufactured via laser powder bed fusion (LPBF). Simulating LPBF-manufactured metamaterials accurately presents a challenge. The printed geometry is different from the CAD geometry the lattice is based on. The reasons are intrinsic limitations of the printing process, which cause defects such as pores or rough surfaces. These differences result in material behavior that depends on the surface/volume ratio. To create predictive simulations, this work introduces an approach to setup a calibrated simulation based on a combination of experimental force data and local displacements obtained via global Digital Image Correlation (DIC). The displacement fields are measured via Finite Element based DIC and yield the true local deformation of the structure. By exploiting symmetries of the geometry, a simplified parametrized simulation model is created. The simulation is calibrated via Response Surface Methodology based on nodal displacements from FE-DIC combined with the experimental force/displacement data. This method is used to create a simulation of an anti-tetrachiral, auxetic structure. The transferability and accuracy are discussed, as well as the possible extension into 3D space.

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Needed for the custom-design of longitudinally freeze-cast chitosan scaffolds for biomedical applications are systematic structure-property-processing correlations. Combining mechanical testing in compression with both scanning electron microscopy and semiautomated confocal microscopy for a quantitative structural characterization of fully hydrated chitosan scaffolds, robust correlations were determined. Decreasing the applied cooling rate from 10 °C/min to 0.1 °C/min, the short and long axes of the pore cross-sections, the pore aspect ratio, and the pore area were found to increase from 68.0 µm to 120.5 µm, from 189.2 µm to 401.2 µm, from 2.64 to 3.52, and from 8,922 µm2 to 35,596 µm2, respectively. Values for the scaffolds' modulus, yield strength, and toughness range from 1,067 kPa to 3,209 kPa, from 37.7 kPa to 75.5 kPa, and from 20.3 kJ/m3 to 35.3 kJ/m3, respectively. Because of additional structural features, such as cell wall stiffening ridges, affecting the mechanical properties, not linear but more complex correlation with modulus, yield strength, and toughness were observed. Contrasting the results of this study with those obtained in an earlier study of dry and fully hydrated collagen scaffolds, we were able to identify features that are important and peculiar to each material system. Highlighted in this study are newly determined robust structure-property-processing correlations as well as processing conditions and features that are critical for the mechanical performance of chitosan and other biopolymer scaffolds made by freeze casting for biomedical applications.

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Bamboo achieves its mechanical efficiency in bending and compression, meaning mechanical performance per unit mass, due to its hierarchical structure. As an orthotropic tube with a higher strength and stiffness parallel to the tube axis and with a density and property gradient across the tube wall, in which fiber bundles are embedded in a porous matrix, the bamboo culm is both stiffer and stronger in bending and less prone to ovalization and catastrophic failure than an orthotropic tube without property gradients would be. Few engineered materials exist that emulate bamboo's mechanical efficiency. The results of the study presented here demonstrate that freeze casting (ice templating) is a manufacturing process with which bamboo-inspired tubular scaffolds with property gradients across the tube wall can be custom-made. A highly aligned, honeycomb-like porosity is generated by ice crystal growth opposite to the direction of heat flow. Using a core-shell mold, the microstructure of the tube wall material, such as the pore size, geometry, and alignment, is defined by the mold materials' properties and applied cooling conditions. These also allow to custom-design the desired property gradient across the section. Further customization of the tube gradient structure and properties is possible through the deposition of additional layers on the freeze-cast scaffolds. Characterizing the pore structures of the tubes using X-ray microtomography, pore morphology and property gradients can be analyzed and correlated to both the processing conditions and the resulting mechanical properties determined in three-point bending, longitudinal and radial compression. The resulting fundamental structure-property-processing correlations support the custom design of tubular scaffolds that are ideally suited for applications that range from conduits for peripheral nerve repair to ureteral stents.

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Presented in this article are 2D and 3D graphical datasets in the form of micrographs and tomograms that were obtained as part of a systematic microstructural characterization by scanning electron microscopy and X-ray microtomography to illustrate freeze-cast bamboo-inspired tubular scaffolds with functional gradients ("Bamboo-inspired Tubular Scaffolds with Functional Gradients" [1]). Four material combinations of the coaxial 'core-shell' molds and their two end pieces were used to freeze cast highly porous tubes (Tube/Rod/Holder): ASA (Aluminum, 316 Stainless Steel, Aluminum), ASP (Aluminum, 316 Stainless Steel, Epoxy (Plastic)), SCA (316 Stainless Steel, Copper, Aluminum), and CSP (Copper, 316 Stainless Steel, Epoxy (Plastic)). Three techniques were used to coat the best performing CSP freeze-cast tubes: spray freezing (SF), spray coating (SC), and brush freezing (BF). The structure and density profile of the uncoated and coated tubes was quantified using X-ray microtomography and their functional gradients, and the resulting mechanical performance in bending were determined and compared. The structure-property-processing correlations determined for the coated and uncoated coaxially freeze cast tubular scaffolds offer strategies for the biomimetic design of bamboo-inspired porous tubes, which emulate bamboo's stiff outer shell supported by a porous, elastic inner layer to delay the onset of ovalization and failure, thereby increasing the tubes' mechanical efficiency.

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Despite considerable recent interest in micro- and nanofibrillated cellulose as constituents of lightweight structures and scaffolds for applications that range from thermal insulation to filtration, few systematic studies have been reported to date on structure-property-processing correlations in freeze-cast chitosan-nanocellulose composite scaffolds, in general, and their application in tissue regeneration, in particular. Reported in this study are the effects of the addition of plant-derived nanocellulose fibrils (CNF), crystals (CNCs), or a blend of the two (CNB) to the biopolymer chitosan on the structure and properties of the resulting composites. Chitosan-nanocellulose composite scaffolds were freeze-cast at 10 and 1 °C/min, and their microstructures were quantified in both the dry and fully hydrated states using scanning electron and confocal microscopy, respectively. The modulus, yield strength, and toughness (work to 60% strain) were determined in compression parallel and the modulus also perpendicular to the freezing direction to quantify anisotropy. Observed were the preferential alignments of CNCs and/or fibrils parallel to the freezing direction. Additionally, observed was the self-assembly of the nanocellulose into microstruts and microbridges between adjacent cell walls (lamellae), features that affected the mechanical properties of the scaffolds. When freeze-cast at 1 °C/min, chitosan-CNF scaffolds had the highest modulus, yield strength, toughness, and smallest anisotropy ratio, followed by chitosan and the composites made with the nanocellulose blend, and that with crystalline cellulose. These results illustrate that the nanocellulose additions homogenize the mechanical properties of the scaffold through cell-wall material self-assembly, on the one hand, and add architectural features such as bridges and pillars, on the other. The latter transfer loads and enable the scaffolds to resist deformation also perpendicular to the freezing direction. The observed property profile and the materials' proven biocompatibility highlight the promise of chitosan-nanocellulose composites for a large range of applications, including those for biomedical implants and devices.

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Presented in this article are systematic microstructural and mechanical property data for anisotropic collagen scaffolds made by freeze casting. Three applied cooling rates (10 °C/min, 1 °C/min, 0.1 °C/min) and two freezing directions (longitudinal and radial) were used during scaffold manufacture. Utilizing a semi-automated image analysis technique applied to confocal micrographs of fully hydrated scaffolds, pore area, long and short pore axes, and pore aspect ratio were determined. Compression testing was performed to determine scaffold modulus, yield strength, and toughness.

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As a new strategy for improved urinary drainage, in parallel to the potential for additional functions such as drug release and self-removal, highly porous chitosan stents are manufactured by radial, bi-directional freeze-casting. Inserting the porous stent in to a silicone tube to emulate its placement in the ureter shows that it is shape conforming and remains safely positioned in place, also during flow tests, including those performed in a peristaltic pump. Cyclic compression tests on fully-hydrated porous stents reveal high stent resilience and close to full elastic recovery upon unloading. The drainage performance of the chitosan stent is evaluated, using effective viscosity in addition to volumetric flow and flux; the porous stent's performance is compared to that of the straight portion of a commercial 8 Fr double-J stent which possesses, in its otherwise solid tube wall, regularly spaced holes along its length. Both the porous and the 8 Fr stent show higher effective viscosities, when tested in the silicone tube. The performance of the porous stent improves considerably more (47.5%) than that of the 8 Fr stent (30.6%) upon removal from the tube, illustrating the effectiveness of the radially aligned porosity for drainage. We conclude that the newly-developed porous chitosan ureteral stent merits further in vitro and in vivo assessment of its promise as an alternative and complement to currently available medical devices. STATEMENT OF SIGNIFICANCE: No papers, to date, report on porous ureteral stents, which we propose as a new strategy for improved urinary drainage. The highly porous chitosan stents of our study are manufactured by radial, bi-directional freeze casting. Cyclic compression tests on fully-hydrated porous stents revealed high stent resilience and close to full recovery upon unloading. The drainage performance of the chitosan is evaluated, using effective viscosity in addition to volumetric flow and flux, and compared to that of the straight portion of a commercial 8 Fr double-J stent. The performance of the porous stent improves considerably more (47.5%) than that of the 8 Fr stent (30.6%) upon removal from the tube, illustrating the effectiveness of the radially aligned porosity for drainage. While further studies are required to explore other potential benefits of the porous stent design such as antimicrobial behavior, drug release, and biodegradability, we conclude that the newly-developed porous chitosan ureteral stent has considerable potential as a medical device.

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Few systematic structure-property-processing correlations for directionally freeze-cast biopolymer scaffolds are reported. Such correlations are critical to enable scaffold design with attractive structural and mechanical cues in vivo. This study focuses on freeze-cast collagen scaffolds with three different applied cooling rates (10, 1, and 0.1 °C/min) and two freezing directions (longitudinal and radial). A semi-automated approach for the structural characterization of fully hydrated scaffolds by confocal microscopy is developed to facilitate an objective quantification and comparison of structural features. Additionally, scanning electron microscopy and compression testing are performed longitudinally and transversely. Structural and mechanical properties are determined on dry and fully hydrated scaffolds. Longitudinally frozen scaffolds have aligned and regular pores while those in radially frozen ones exhibit greater variations in pore geometry and alignment. Lamellar spacing, pore area, and cell wall thickness increase with decreasing cooling rate: in longitudinally frozen scaffolds from 25 µm to 83.5 µm, from 814 µm2 to 8452 µm2, and from 4.21 µm to 10.4 µm, and in radially frozen ones, from 69 µm to 116 µm, from 7679 µm2 to 25,670 µm2, and from 6.18 µm to 13.6 µm, respectively. Both longitudinally and radially frozen scaffolds possess higher mechanical property values, when loaded parallel rather than perpendicular to the ice-crystal growth direction. Modulus and yield strength range from 779 kPa to 4700 kPa and from 38 kPa to 137 kPa, respectively, as a function of cooling rate and freezing direction. Collated, the correlations obtained in this study enable the custom-design of freeze-cast collagen scaffolds, which are ideally suited for a large variety of tissue regeneration applications.

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OBJECTIVE: Use of cell culture and conventional in vivo mammalian models to assess nerve regeneration across guidance conduits is resource-intensive. Herein we describe a high-throughput platform utilizing transgenic mice for stain-free axon visualization paired with rapid cryosection techniques for low-cost screening of novel bioengineered nerve guidance conduit performance. METHODS: Interposition repair of sciatic nerve transection in mice expressing yellow fluorescent protein in peripheral neurons (Thy1.2 YFP-16) was performed with various bioengineered neural conduit compositions using a rapid sutureless entubulation technique under isoflurane anesthesia. Axonal ingrowth was assessed at 3 and 6 weeks using epifluorescent microscopy following cryosectioning. RESULTS: Mean procedure time (incision-to-closure) was less than 2½ minutes. Direct operational costs of a 3-week experiment was calculated at $21.47 per animal. Tissue processing steps were minimized to aldehyde fixation, cryoprotection and sectioning, and rapid fluorescent dye staining for conduit visualization. Fluorescent microscopy readily resolved robust axonal sprouting at 3 weeks, with clear elucidation of ingrowth-permissive, semipermissive, or restrictive nerve guidance conduit environments. CONCLUSION: A rapid and cost-efficient in vivo platform for screening of nerve guidance conduit performance has been described. LEVEL OF EVIDENCE: NA. Laryngoscope, E392-E392, 2018.

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A novel freeze-cast porous chitosan conduit for peripheral nerve repair with highly-aligned, double layered porosity, which provides the ideal mechanical and chemical properties was designed, manufactured, and assessed in vivo. Efficacies of the conduit and the control inverted nerve autograft were evaluated in bridging 10-mm Lewis rat sciatic nerve gap at 12 weeks post-implantation. Biocompatibility and regenerative efficacy of the porous chitosan conduit were evaluated through the histomorphometric analysis of longitudinal and transverse sections. The porous chitosan conduit was found to have promising regenerative characteristics, promoting the desired neovascularization, and axonal ingrowth and alignment through a combination of structural, mechanical and chemical cues.

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Tapered nanopillars with various cross sections, including cone-shaped, stepwise, and pencil-like structures (300 nm in diameter at the base of the pillars and 1.1 µm in height), are prepared from epoxy resin templated by nanoporous anodic aluminum oxide (AAO) membranes. The effect of pillar geometry on the shear adhesion behavior of these nanopillar arrays is investigated via sliding experiments in a nanoindentation system. In a previous study of arrays with the same geometry, it was shown that cone-shaped nanopillars exhibit the highest adhesion under normal loading while stepwise and pencil-like nanopillars exhibit lower normal adhesion strength due to significant deformation of the pillars that occurs with increasing indentation depth. Contrary to the previous studies, here, we show that pencil-like nanopillars exhibit the highest shear adhesion strength at all indentation depths among three types of nanopillar arrays and that the shear adhesion increases with greater indentation depth due to the higher bending stiffness and closer packing of the pencil-like nanopillar array. Finite element simulations are used to elucidate the deformation of the pillars during the sliding experiments and agree with the nanoindentation-based sliding measurements. The experiments and finite element simulations together demonstrate that the shape of the nanopillars plays a key role in shear adhesion and that the mechanism is quite different from that of adhesion under normal loading.