Hierarchical structure formation by crystal growth-front instabilities during ice templating.

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.

Excellent Specific Mechanical and Electrical Properties of Anisotropic Freeze-Cast Native and Carbonized Bacterial Cellulose-Alginate Foams.

Native and carbonized freeze-cast bacterial cellulose-alginate (BC-ALG) foams possess an ice-templated honeycomb-like architecture with remarkable properties. Their unique pore morphology consists of two levels of porosity: 20-50 µm diameter pores between, and 0.01-10 µm diameter pores within the cell-walls. The mechanical properties of the BC-ALG foams, a Young's modulus of up to 646.2 ± 90.4 kPa and a compressive yield strength of up to 37.1 ± 7.9 kPa, are high for their density and scale as predicted by the Gibson-Ashby model for cellular materials. Carbonizing the BC-ALG foams in an inert atmosphere at 1000-1200 °C in a second processing step, both pore morphology and mechanical properties of the BC-ALG remain well preserved with specific mechanical properties that are higher than those reported in the literature for similar foams. Also the electrical conductivity of the BC-ALG foams is high at 1.68 ± 0.04 S cm-1 at a density of only 0.055 g cm-3, and is found to increase with density as predicted, and as a function of the degree of carbonization determined by both carbonization temperature and atmosphere. The property profile makes freeze-cast BC-ALG foams and their carbonized foams attractive for energy applications and as a sorbent.

Heterogeneous Ice Nucleation Studied with Single-Layer Graphene.

Control of heterogeneous ice nucleation (HIN) is critical for applications that range from iceophobic surfaces to ice-templated materials. HIN on 2D materials is a particular interesting topic that still lacks extensive experimental investigations. Here, we focus on the HIN on single-layer graphene (SLG) transferred onto different substrates, including silicon, silica, and thermal oxide on silicon. Complemented by other samples without SLG, we obtain a large range of wetting contact angles (WCAs) from 2° to 95°. All pristine SLG samples exhibit a large contact angle of ∼95°, which is close to the theoretical value of 96° for free-standing SLG, irrespective of the substrate and even in the presence of nanoscale wrinkles on SLG, which are due to the transfer process, indicating that the topographical features have little impact on the wetting behavior. Interestingly, SLG displays changes in hydrophobicity upon repeated water droplet freezing-melting-drying cycles due to a shift in Fermi level and/or enhanced water-substrate polar molecular interactions, likely induced by residual adsorption of H2O molecules. We found that a 0.04 eV decrease in SLG Fermi level reduces the SLG/water interface energy by ∼6 mJ/m2, thereby making SLG less hydrophobic. Counterintuitively, the reduction in SLG/water interface energy and the enhanced hydrophilicity after repeated freezing-melting-evaporation cycles actually decreases the freezing temperature by ∼3-4 °C, thereby slightly retarding rather than enhancing HIN. We also found that the water droplet freezing temperature differed by only ∼1 °C on different substrates with WCAs from 2° to 95°, an intriguing and yet reasonable result that confirms that wettability alone is not a good indicator of HIN capability. The HIN rate is rather determined by the difference between substrate/water and substrate/ice interface energies, which was found to stay almost constant for substrates weakly interacting with water/ice via van der Waals or hydrogen bonds, irrespective of hydrophilicity.

Plant-Derived Nanocellulose as Structural and Mechanical Reinforcement of Freeze-Cast Chitosan Scaffolds for Biomedical Applications.

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.

Bioinspired structural materials.

Natural structural materials are built at ambient temperature from a fairly limited selection of components. They usually comprise hard and soft phases arranged in complex hierarchical architectures, with characteristic dimensions spanning from the nanoscale to the macroscale. The resulting materials are lightweight and often display unique combinations of strength and toughness, but have proven difficult to mimic synthetically. Here, we review the common design motifs of a range of natural structural materials, and discuss the difficulties associated with the design and fabrication of synthetic structures that mimic the structural and mechanical characteristics of their natural counterparts.

Desert locusts (Schistocerca gregaria) feed with self-sharpening, scissor-like mandibles.

The mandibles of the desert locust Schistocerca gregaria (Forsskål, 1775) are digger-shovel-shaped mouthparts that are part of the locust's exoskeleton formed by the insect cuticle. The cuticle is a polymer-fibre composite, which supports, encases and protects the entire body. Mandibles experience heavy loading and wear due to direct contact with hard and abrasive food, just like teeth, their mineralized analogues in vertebrates. With dual-energy X-ray tomography, we image well-defined regions of zinc (Zn)-enriched cuticle at the mandible cutting edges and quantify the Zn concentrations in these regions. Zn is known to increase stiffness, hardness and wear resistance of the otherwise purely polymeric insect cuticle. In S. gregaria, the position of the Zn-enriched cutting-edge regions relative to one another suggests that the mandibles form a scissor-like cutting tool, which sharpens itself as the mouthparts shear past one another during feeding. Comparing the architecture of these purely polymeric mandibles with the mineralized incisors of rodents, we find fundamental design differences in cutting-tool structure and performance. Locusts' scissors and rodents' carving knives perform different functions, because they act on food that differs significantly in properties and shape: softer, sheet-like material in the case of locusts and harder bulk material in the case of rodents.

Tensile properties of freeze-cast collagen scaffolds: How processing conditions affect structure and performance in the dry and fully hydrated states.

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.

High-plex expression profiling reveals that implants drive spatiotemporal protein production and innate immune activation for tissue repair.

Surprisingly little clarity exists concerning effects of biomaterial properties on spatially localized protein expression, which drives implant success. Wound healing and tissue regeneration must be optimally supported by the implant, adsorbed proteins, immune cells, and fibroblasts; cells determine repair and functional recovery through protein production and regulation. However, not yet fully understood is how implants differentially drive spatial quantities of individual proteins both within the implant interior and the tissue surrounding it. Here we apply GeoMxⓇ digital spatial profiling to site-specifically investigate protein production in porous implants. Data is collected on the location and quantity of 40+ proteins from formalin-fixed, paraffin-embedded tissue slides of anisotropic tissue scaffolds (n = 18) with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days); matching bulk gene expression data (700+ genes) is measured for identical implants. Notably, we discover fundamental spatial relationships in protein localization that in both the implant interior and the exterior are either uniquely independent or dependent of implant microstructure: dendritic cell marker CD11c and fibronectin significantly dominate the scaffold interior, while cell-to-cell adhesion marker CD34 and anti-inflammatory M2 polarization marker CD163 localize in the exterior. Lastly, collating spatial and bulk information, unique spatiotemporal expression patterns are identified for markers such as fibronectin, which are only uncoverable through spatial profiling and are otherwise hidden in bulk expression results. Together, these discoveries illustrate the critical importance of quantifying spatial expression patterns for implants, facilitating a paradigm shift in the iterative design, mechanistic understanding, and rapid assessment of biomaterials. STATEMENT OF SIGNIFICANCE: Spatial localization and expression of proteins, which determine implant success, are not fully understood because quantitative high-plex profiling is challenging. Applying GeoMxⓇ digital spatial profiling to site-specifically investigate protein production in porous implants, data is collected on the location and quantity of 40+ protein targets from tissue scaffolds with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days). Collecting in parallel matched bulk gene expression data (700+ genes) for identical implants, we discover significant spatiotemporal expression patterns that remain otherwise hidden in differential bulk results. This new approach for the rapid assessment of biomaterials offers an enhanced mechanistic understanding and enables the tailoring of implants for superior regenerative outcomes.

Nanotechnology approaches to improve dental implants.

The requirements imposed by the enormous scale and overall complexity of designing new implants or complete organ regeneration are well beyond the reach of present technology in many dimensions, including nanoscale, as researchers do not yet have the basic knowledge required to achieve these goals. The need for a synthetic implant to address multiple physical and biologic factors imposes tremendous constraints on the choice of suitable materials. There is a strong belief that nanoscale materials will produce a new generation of implant materials with high efficiency, low cost, and high volume. The nanoscale in materials processing is truly a new frontier. Metallic dental implants have been used successfully for decades, but they have serious shortcomings related to their osseointegration and the fact that their mechanical properties do not match those of bone. This paper reviews recent advances in the fabrication of novel coatings and nanopatterning of dental implants. It also provides a general summary of the state of the art in dental implant science and describes possible advantages of nanotechnology for future improvements. The ultimate goal is to produce materials and therapies that will bring state-of-the-art technology to the bedside and improve quality of life and current standards of care.

Structure-property-processing correlations of longitudinal freeze-cast chitosan scaffolds for biomedical applications.

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.

Directionally Solidified Biopolymer Scaffolds: Mechanical Properties and Endothelial Cell Responses.

Vascularization is a primary challenge in tissue engineering. To achieve it in a tissue scaffold, an environment with the appropriate structural, mechanical, and biochemical cues must be provided enabling endothelial cells to direct blood vessel growth. While biochemical stimuli such as growth factors can be added through the scaffold material, the culture medium, or both, a well-designed tissue engineering scaffold is required to provide the necessary local structural and mechanical cues. As chitosan is a well-known carrier for biochemical stimuli, the focus of this study was on structure-property correlations, to evaluate the effects of composition and processing conditions on the three-dimensional architecture and properties of freeze-cast scaffolds; to establish whether freeze-cast scaffolds are promising candidates as constructs promoting vascularization; and to conduct initial tissue culture studies with endothelial cells on flat substrates of identical compositions as those of the scaffolds to test whether these are biocompatible and promote cell attachment and proliferation.

Bamboo-inspired tubular scaffolds with functional gradients.

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.

2D and 3D graphical datasets for bamboo-inspired tubular scaffolds with functional gradients: micrographs and tomograms.

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.

Quantitative evaluation of the in vivo biocompatibility and performance of freeze-cast tissue scaffolds.

Quantitative methods are little used for the in vivo assessment of tissue scaffolds to evaluate biocompatibility. To complement current histological techniques, we introduce as a measure of biocompatibility a straightforward, geometric analysis for the quantitative assessment of encapsulation thickness, cross-sectional area, and biomaterial shape. Advantages of this new technique are that it enables, on the one hand, a more complete and objective comparison of scaffolds with differing compositions, architectures, and mechanical properties, and, on the other, a more objective approach to their selection for a given application. In this contribution, we focus on freeze-cast polymeric scaffolds for tissue regeneration and their subcutaneous implantation in mice for biocompatibility testing. Initially, seven different scaffold types are screened. Of these, three are selected for systematic biocompatibility studies based on histopathological criteria: EDC-NHS-crosslinked bovine collagen, EDC-NHS-crosslinked bovine collagen-nanocellulose, and chitin. Geometric models developed to quantify scaffold size, ovalization, and encapsulation thickness are tested, evaluated, and found to be a powerful and objective metric for the in vivo assessment of biocompatibility and performance of tissue scaffolds.

Anisotropic freeze-cast collagen scaffolds for tissue regeneration: How processing conditions affect structure and properties in the dry and fully hydrated states.

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.

Freeze-casting porous chitosan ureteral stents for improved drainage.

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.

Values and property charts for anisotropic freeze-cast collagen scaffolds for tissue regeneration.

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.

Design, Manufacture, and In vivo Testing of a Tissue Scaffold for Permanent Female Sterilization by Tubal Occlusion.

Current FDA-approved permanent female sterilization procedures are invasive and/or require the implantation of non-biodegradable materials. These techniques pose risks and complications, such as device migration, fracture, and tubal perforation. We propose a safe, non-invasive biodegradable tissue scaffold to effectively occlude the Fallopian tubes within 30 days of implantation. Specifically, the Fallopian tubes are mechanically de-epithelialized, and a tissue scaffold is placed into each tube. It is anticipated that this procedure can be performed in less than 30 minutes by an experienced obstetrics and gynaecology practitioner. Advantages of this method include the use of a fully bio-resorbable polymer, low costs, lower risks, and the lack of general anaesthesia. The scaffold devices are freeze-cast allowing for the custom-design of structural, mechanical, and chemical cues through material composition, processing parameters, and functionalization. The performance of the biomaterial and de-epithelialization procedure was tested in an in vivo rat uterine horn model. The scaffold response and tissue-biomaterial interactions were characterized microscopically post-implantation. Overall, the study resulted in the successful fabrication of resilient, easy-to-handle devices with an anisotropic scaffold architecture that encouraged rapid bio-integration through notable angiogenesis, cell infiltration, and native collagen deposition. Successful tubal occlusion was demonstrated at 30 days, revealing the great promise of a sterilization biomaterial.

Fluorescent Reporter Mice for Nerve Guidance Conduit Assessment: A High-Throughput in vivo Model.

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.

Freeze-cast Porous Chitosan Conduit for Peripheral Nerve Repair.

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.