Topographic Cues Guiding Cell Polarization via Distinct Cellular Mechanosensing Pathways.

Cell polarization exists in a variety of tissues to regulate cell behaviors and functions. Space constraint (spatially limiting cell extension) and adhesion induction (guiding adhesome growth) are two main ways to induce cell polarization according to the microenvironment topographies. However, the mechanism of cell polarization induced by these two ways and the downstream effects on cell functions are yet to be understood. Here, space constraint and adhesion induction guiding cell polarization are achieved by substrate groove arrays in micro and nano size, respectively. Although the morphology of polarized cells is similar on both structures, the signaling pathways to induce the cell polarization and the downstream functions are distinctly different. The adhesion induction (nano-groove) leads to the formation of focal adhesions and activates the RhoA/ROCK pathway to enhance the myosin-based intracellular force, while the space constraint (micro-groove) only activates the formation of pseudopodia. The enhanced intracellular force caused by adhesion induction inhibits the chromatin condensation, which promotes the osteogenic differentiation of stem cells. This study presents an overview of cell polarization and mechanosensing at biointerface to aid in the design of novel biomaterials.

Engineering Nanofiber Scaffolds with Biomimetic Cues for Differentiation of Skin-Derived Neural Crest-like Stem Cells to Schwann Cells.

Our laboratory reported the derivation of neural crest stem cell (NCSC)-like cells from the interfollicular epidermis of the neonatal and adult epidermis. These keratinocyte (KC)-derived Neural Crest (NC)-like cells (KC-NC) could differentiate into functional neurons, Schwann cells (SC), melanocytes, and smooth muscle cells in vitro. Most notably, KC-NC migrated along stereotypical pathways and gave rise to multiple NC derivatives upon transplantation into chicken embryos, corroborating their NC phenotype. Here, we present an innovative design concept for developing anisotropically aligned scaffolds with chemically immobilized biological cues to promote differentiation of the KC-NC towards the SC. Specifically, we designed electrospun nanofibers and examined the effect of bioactive cues in guiding KC-NC differentiation into SC. KC-NC attached to nanofibers and adopted a spindle-like morphology, similar to the native extracellular matrix (ECM) microarchitecture of the peripheral nerves. Immobilization of biological cues, especially Neuregulin1 (NRG1) promoted the differentiation of KC-NC into the SC lineage. This study suggests that poly-ε-caprolactone (PCL) nanofibers decorated with topographical and cell-instructive cues may be a potential platform for enhancing KC-NC differentiation toward SC.

Topographically Conductive Butterfly Wing Substrates for Directed Spiral Ganglion Neuron Growth.

Spiral ganglion neuron (SGN) degeneration can lead to severe hearing loss, and the directional regeneration of SGNs has shown great potential for improving the efficacy of auditory therapy. Here, a novel 3D conductive microstructure with surface topologies is presented by integrating superaligned carbon-nanotube sheets (SA-CNTs) onto Morpho Menelaus butterfly wings for SGN culture. The parallel groove-like topological structures of M. Menelaus wings induce the cultured cells to grow along the direction of its ridges. The excellent conductivity of SA-CNTs significantly improves the efficiency of cellular information conduction. When integrating the SA-CNTs with M. Menelaus wings, the SA-CNTs are aligned in parallel with the M. Menelaus ridges, which further strengthens the consistency of the surface topography in the composite substrate. The SA-CNTs integrated onto butterfly wings provide powerful physical signals and regulate the behavior of SGNs, including cell survival, adhesion, neurite outgrowth, and synapse formation. These features indicate the possibility of directed regeneration after auditory nerve injury.

Time-dependent combinatory effects of active mechanical loading and passive topographical cues on cell orientation.

Mechanical stretching and topographical cues are both effective mechanical stimulations for regulating cell morphology, orientation, and behaviors. The competition of these two mechanical stimulations remains largely underexplored. Previous studies have suggested that a small cyclic mechanical strain is not able to reorient cells that have been pre-aligned by relatively large linear microstructures, but can reorient those pre-aligned by small linear micro/nanostructures if the characteristic dimension of these structures is below a certain threshold. Likewise, for micro/nanostructures with a given characteristic dimension, the strain must exceed a certain magnitude to overrule the topographic cues. There are however no in-depth investigations of such "thresholds" due to the lack of close examination of dynamic cell orientation during and shortly after the mechanical loading. In this study, the time-dependent combinatory effects of active and passive mechanical stimulations on cell orientation are investigated by developing a micromechanical stimulator. The results show that the cells pre-aligned by linear micro/nanostructures can be altered by cyclic in-plane strain, regardless of the structure size. During the loading, the micro/nanostructures can resist the reorientation effects by cyclic in-plane strain while the resistive capability (measured by the mean orientation angle change and the reorientation speed) increases with the increasing characteristic dimension. The micro/nanostructures also can recover the cell orientation after the cessation of cyclic in-plane strain, while the recovering capability increases with the characteristic dimension. The previously observed thresholds are largely dependent on the observation time points. In order to accurately evaluate the combinatory effects of the two mechanical stimulations, observations during the active loading with a short time interval or endpoint observations shortly after the loading are preferred. This study provides a microengineering solution to investigate the time-dependent combinatory effects of the active and passive mechanical stimulations and is expected to enhance our understanding of cell responses to complex mechanical environments. Biotechnol. Bioeng. 2016;113: 2191-2201. © 2016 Wiley Periodicals, Inc.

Periodic Lamellae-Based Nanofibers for Precise Immunomodulation to Treat Inflammatory Bone Loss in Periodontitis.

Periodontitis is a common oral disease accompanied by inflammatory bone loss. The pathological characteristics of periodontitis usually accompany an imbalance in the periodontal immune microenvironment, leading to difficulty in bone regeneration. Therefore, effective treatment strategies are needed to modulate the immune environment in order to treat periodontitis. Here, highly-oriented periodic lamellae poly(ε-caprolactone) electrospun nanofibers (PLN) are developed by surface-directed epitaxial crystallization. The in vitro result shows that the PLN can precisely modulate macrophage polarization toward the M2 phenotype. Macrophages polarized by PLN significantly enhance the migration and osteogenic differentiation of Bone marrow stromal cells. Notably, results suggest that the topographical cues presented by PLN can modulate macrophage polarization by activating YAP, which reciprocally inhibits the NF-κB signaling pathway. The in vivo results indicate that PLN can inhibit inflammatory bone loss and facilitate bone regeneration in periodontitis. The authors' findings suggest that topographical nanofibers with periodic lamellae is a promising strategy for modulating immune environment to treat inflammatory bone loss in periodontitis.

Harnessing the Coil Electrospinning Method for Fabricating Superflexible and Multiscale-Patterned Fibrous Tubular Scaffolds with Topographical Features.

The fibrous tubular scaffold (FTS) has potential as a vascular graft; however, its clinical application is hindered by insufficient mechanical properties. Inadequate mechanical properties of vascular grafts can lead to some serious side effects such as intimal hyperplasia, luminal expansion, and blood thrombogenicity. In this study, we developed a novel fibrous tubular scaffold comprising multiscale fibers to ensure superior mechanical properties. Our novel approach involves a one-step manufacturing method that can fabricate the superflexible fibrous tubular scaffold (SF-FTS) with topographical features via a modified electrospinning setup. We investigated the effect of humidity and temperature during the fabrication process on the formation of multiscale fibers. It was demonstrated that the incorporation of multiscale fibers and topographical features significantly enhances the mechanical properties of FTS. The mechanical advantages of SF-FTS were confirmed through the kinking resistance test, compressive test, and in vivo experiments. Additionally, we explored the interaction between the multiscale fibers and human umbilical vein endothelial cells (HUVECs) behavior. Our results suggest a novel strategy for fabricating FTS with advanced mechanical properties, and the designed SF-FTS holds promise as a potential candidate for clinical applications.

Hierarchically structured nanofibrous scaffolds spatiotemporally mediate the osteoimmune micro-environment and promote osteogenesis for periodontitis-related alveolar bone regeneration.

Periodontitis suffer from inflammation-induced destruction of periodontal tissues, resulting in the serious loss of alveolar bone. Controlling inflammation and promoting bone regeneration are two crucial aspects for periodontitis-related alveolar bone defect treatment. Herein, we developed a hierarchically structured nanofibrous scaffold with a nano-embossed sheath and a bone morphogenetic protein 2-loaded core to match the periodontitis-specific features that spatiotemporally modulated the osteoimmune environment and promoted periodontal bone regeneration. We investigated the potential of this unique scaffold to treat periodontitis-related alveolar bone defects in vivo and in vitro. The results demonstrated that the hierarchically structured scaffold effectively reduced the inflammatory levels in macrophages and enhanced the osteogenic potential of bone mesenchymal stem cells in an inflammatory microenvironment. Moreover, in vivo experiments revealed that the hierarchically structured scaffold significantly ameliorated inflammation in the periodontium and inhibited alveolar bone resorption. Notably, the hierarchically structured scaffold also exhibited a prolonged effect on promoting alveolar bone regeneration. These findings highlight the significant therapeutic potential of hierarchically structured nanofibrous scaffolds for the treatment of periodontitis, and their promising role in the field of periodontal tissue regeneration. STATEMENT OF SIGNIFICANCE: We present a novel hierarchically structured nanofibrous scaffold of coupling topological and biomolecular signals for precise spatiotemporal modulation of the osteoimmune micro-environment. Specifically, the scaffold was engineered via coaxial electrospinning of the poly(ε-caprolactone) sheath and a BMP-2/polyvinyl alcohol core, followed by surface-directed epitaxial crystallization to generate cyclic nano-lamellar embossment on the sheath. With this unique hierarchical structure, the cyclic nano-lamellar sheath provided a direct nano-topographical cue to alleviate the osteoimmune environment, and the stepwise release of BMP-2 from the core provided a biological cue for bone regeneration. This research underscores the potential of hierarchically structured nanofibrous scaffolds as a promising therapeutic approach for periodontal tissue regeneration and highlights their role in advancing periodontal tissue engineering.

Piezoelectrically and Topographically Engineered Scaffolds for Accelerating Bone Regeneration.

Bone regeneration remains a critical concern across diverse medical disciplines, because it is a complex process that requires a combinatorial approach involving the integration of mechanical, electrical, and biological stimuli to emulate the native cellular microenvironment. In this context, piezoelectric scaffolds have attracted considerable interest owing to their remarkable ability to generate electric fields in response to dynamic forces. Nonetheless, the application of such scaffolds in bone tissue engineering has been limited by the lack of a scaffold that can simultaneously provide both the intricate electromechanical environment and the biocompatibility of the native bone tissue. Here, we present a pioneering biomimetic scaffold that combines the unique properties of piezoelectric and topographical enhancement with the inherent osteogenic abilities of hydroxyapatite (HAp). Notably, the novelty of this work lies in the incorporation of HAp into polyvinylidene fluoride-co-trifluoro ethylene in a freestanding form, leveraging its natural osteogenic potential within a piezoelectric framework. Through comprehensive in vitro and in vivo investigations, we demonstrate the remarkable potential of these scaffolds to accelerate bone regeneration. Moreover, we demonstrate and propose three pivotal mechanisms─(i) electrical, (ii) topographical, and (iii) paracrine─that collectively contribute to the facilitated bone healing process. Our findings present a synergistically derived biomimetic scaffold design with wide-ranging prospects for bone regeneration as well as various regenerative medicine applications.

Topographical cues of PLGA membranes modulate the behavior of hMSCs, myoblasts and neuronal cells.

Biomaterial surface modification through the introduction of defined and repeated patterns of topography helps study cell behavior in response to defined geometrical cues. The lithographic molding technique is widely used for conferring biomaterial surface microscale cues and enhancing the performance of biomedical devices. In this work, different master molds made by UV mask lithography were used to prepare poly (D,L-lactide-co-glycolide) - PLGA micropatterned membranes to present different features of topography at the cellular interface: channels, circular pillars, rectangular pillars, and pits. The effects of geometrical cues were investigated on different cell sources, such as neuronal cells, myoblasts, and stem cells. Morphological evaluation revealed a peculiar cell arrangement in response to a specific topographical stimulus sensed over the membrane surface. Cells seeded on linear-grooved membranes showed that this cue promoted elongated cell morphology. Rectangular and circular pillars act instead as discontinuous cues at the cell-membrane interface, inducing cell growth in multiple directions. The array of pits over the surface also highlighted the precise spatiotemporal organization of the cell; they grew between the interconnected membrane space within the pits, avoiding the microscale hole. The overall approach allowed the evaluation of the responses of different cell types adhered to various surface patterns, build-up on the same polymeric membrane, and disclosing the effect of specific topographical features. We explored how various microtopographic signals play distinct roles in different cells, thus affecting cell adhesion, migration, differentiation, cell-cell interactions, and other metabolic activities.

Histone Modification of Osteogenesis Related Genes Triggered by Substrate Topography Promotes Human Mesenchymal Stem Cell Differentiation.

The clinical success of orthopedic implants is closely related to their integration in the bone tissue promoted by rough device surfaces. The biological response of precursor cells to their artificial microenvironments plays a critical role in this process. In this study, we elucidated the relation between cell instructivity and surface microstructure of polycarbonate (PC)-based model substrates. The rough surface structure (hPC) with an average peak spacing (Sm) similar to the trabecular spacing of trabecular bone improved osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), as compared to the smooth surface (sPC) and the surface with a moderate Sm value (mPC). The hPC substrate promoted the cell adhesion and assembling of F-actin and enhanced cell contractile force by upregulating phosphorylated myosin light chain (pMLC) expression. The increased cell contractile force led to YAP nuclear translocation and the elongation of cell nuclei, presenting higher levels of active form of Lamin A/C. The nuclear deformation alternated the histone modification profile, particularly the decrease of H3K27me3 and increase of H3K9ac on the promoter region of osteogenesis related genes (ALPL, RUNX2, and OCN). Mechanism study using inhibitors and siRNAs elucidated the role of YAP, integrin, F-actin, myosin, and nuclear membrane proteins in such a regulatory process of surface topography on stem cell fate. These mechanistical insights on the epigenetic level give a new perspective in understanding of the interaction of substrate and stem cells as well as provide valuable criteria for designing bioinstructive orthopedic implants.

Synergetic Effect of Electrical and Topographical Cues in Aniline Trimer-Based Polyurethane Fibrous Scaffolds on Tissue Regeneration.

Processibility and biodegradability of conductive polymers are major concerns when they are applied to tissue regeneration. This study synthesizes dissolvable and conductive aniline trimer-based polyurethane copolymers (DCPU) and processes them into scaffolds by using electrospinning with different patterns (random, oriented, and latticed). The effects of topographic cue changes on electrical signal transmission and further regulation of cell behaviors concerning bone tissue are researched. Results show that DCPU fibrous scaffolds possessed good hydrophilicity, swelling capacity, elasticity, and fast biodegradability in enzymatic liquid. In addition, the conductivity and efficiency of electrical signal transmission can be tuned by changing the surface's topological structure. Among them, oriented DCPU scaffolds (DCPU-O) showed the best conductivity with the lowest ionic resistance value. Furthermore, the viability and proliferation results of bone mesenchymal stem cells (BMSCs) demonstrate a significant increase on three DCPU scaffolds compared to AT-free scaffolds (DPU-R). Especially, DCPU-O scaffolds exhibit superior abilities to promote cell proliferation because of their unique surface topography and excellent electroactivity. Concurrently, the DCPU-O scaffolds can synergistically promote osteogenic differentiation in terms of osteogenic differentiation and gene expression levels when combined with electrical stimulation. Together, these results suggest a promising use of DCPU-O fibrous scaffolds in the application of tissue regeneration.

Submicron-Grooved Films Modulate the Directional Alignment and Biological Function of Schwann Cells.

Topographical cues on material surfaces are crucial for guiding the behavior of nerve cells and facilitating the repair of peripheral nerve defects. Previously, micron-grooved surfaces have shown great potential in controlling nerve cell alignment for studying the behavior and functions of those cells and peripheral nerve regeneration. However, the effects of smaller-sized topographical cues, such as those in the submicron- and nano-scales, on Schwann cell behavior remain poorly understood. In this study, four different submicron-grooved polystyrene films (800/400, 800/100, 400/400, and 400/100) were fabricated to study the behavior, gene expression, and membrane potential of Schwann cells. The results showed that all submicron-grooved films could guide the cell alignment and cytoskeleton in a groove depth-dependent manner. Cell proliferation and cell cycle assays revealed that there was no significant difference between the submicron groove samples and the flat control. However, the submicron grooves can direct the migration of cells and upregulate the expression of critical genes in axon regeneration and myelination (e.g., MBP and Smad6). Finally, the membrane potential of the Schwann cells was significantly altered on the grooved sample. In conclusion, this study sheds light on the role of submicron-grooved patterns in regulating the behavior and function of Schwann cells, which provides unique insights for the development of implants for peripheral nerve regeneration.

Modeling adult skeletal stem cell response to laser-machined topographies through deep learning.

The response of adult human bone marrow stromal stem cells to surface topographies generated through femtosecond laser machining can be predicted by a deep neural network. The network is capable of predicting cell response to a statistically significant level, including positioning predictions with a probability P < 0.001, and therefore can be used as a model to determine the minimum line separation required for cell alignment, with implications for tissue structure development and tissue engineering. The application of a deep neural network, as a model, reduces the amount of experimental cell culture required to develop an enhanced understanding of cell behavior to topographical cues and, critically, provides rapid prediction of the effects of novel surface structures on tissue fabrication and cell signaling.

The miR-193a-3p-MAP3k3 Signaling Axis Regulates Substrate Topography-Induced Osteogenesis of Bone Marrow Stem Cells.

Substrate topographical features induce osteogenic differentiation of bone marrow stem cells (BMSCs), but the underlying mechanisms are unclear. As microRNAs (miRNAs) play key roles in osteogenesis and bone regeneration, it would be meaningful to elucidate the roles of miRNAs in the intracellular signaling cascade of topographical cue-induced osteogenic differentiation. In this study, the miRNA expression profile of the topographical feature-induced osteogenic differentiation group is different from that of the chemical-factors-induced osteogenic differentiation group. miR-193a-3p is sensitive to substrate topographical features and its downregulation enhances osteogenic differentiation only in the absence of osteogenesis-inducing medium. Also, substrate topographical features specifically activate a nonclassical osteogenetic pathway-the mitogen-activated protein kinase (MAPK) pathway. Loss- and gain-of-function experiments demonstrate that miR-193a-3p regulates the MAPK pathway by targeting the MAP3k3 gene. In conclusion, this data indicates that different osteogenic-lineage-related intracellular signaling cascades are triggered in BMSCs subjected to biophysical or chemical stimulation. Moreover, the miR-193a-3p-MAP3k3 signaling axis plays a pivotal role in the transduction of biophysical cues from the substrate to regulate the osteogenic lineage specification of BMSCs, and hence may be a promising molecular target for bone regenerative therapies.

Contractile behaviors of cardiac muscle cells on mushroom-shaped micropillar arrays.

In this work we propose mushroom-shaped PDMS (Polydimethylsiloxane) µpillar arrays for enhancing the contractile force of cardiomyocytes during cell culturing. Conventional micropillar (µpillar) arrays with flat surfaces were employed as a standard sample to quantitatively recognize experimental data and to conclusively demonstrate the improved performance of mushroom-shaped PDMS µpillar arrays. Cardiomyocytes isolated from experimental animals were cultured on both of the fabricated µpillar arrays and then monitored over a growing period. Deflections of µpillars were precisely measured through a home-built analyzing system quantitatively representing the contractile force of cardiomyocytes. Mushroom-shaped PDMS µpillar arrays exhibited a 20% improved contractile force compared to conventional PDMS µpillar arrays due to their topographical dependency. Preliminary results also show that the proposed mushroom-shaped PDMS µpillar surface positively affects the Z-band width, actin filament polymerization and focal adhesion (FA) of cardiomyocytes. Further, the enhanced performance of mushroom-shaped PDMS µpillar arrays was confirmed by measuring the cardiac sarcomere α-actin length and myofilament width via ICC (immunocytochemistry) staining and western blot experiments.

Nanotopographical regulation of pancreatic islet-like cluster formation from human pluripotent stem cells using a gradient-pattern chip.

Bioengineering approaches to regulate stem cell fates aim to recapitulate the in vivo microenvironment. In recent years, manipulating the micro- and nano-scale topography of the stem cell niche has gained considerable interest for the purposes of controlling extrinsic mechanical cues to regulate stem cell fate and behavior in vitro. Here, we established an optimal nanotopographical system to improve 3-dimensional (3D) differentiation of pancreatic cells from human pluripotent stem cells (hPSCs) by testing gradient-pattern chips of nano-scale polystyrene surface structures with varying sizes and shapes. The optimal conditions for 3D differentiation of pancreatic cells were identified by assessing the expression of developmental regulators that are required for pancreatic islet development and maturation. Our results showed that the gradient chip of pore-part 2 (Po-2, 200-300 nm diameter) pattern was the most efficient setting to generate clusters of pancreatic endocrine progenitors (PDX1+ and NGN3+) compared to those of other pore diameters (Po-1, 100-200 or Po-3, 300-400 nm) tested across a range of pillar patterns and flat surfaces. Furthermore, the Po-2 gradient pattern-derived clusters generated islet-like 3D spheroids and tested positive for the zinc-chelating dye dithizone. The spheroids consisted of more than 30% CD200 + endocrine cells and also expressed NKX6.1 and NKX2.2. In addition, pancreatic ß- cells expressing insulin and polyhormonal cells expressing both insulin and glucagon were obtained at the final stage of pancreatic differentiation. In conclusion, our data suggest that an optimal topographical structure for differentiation to specific cell types from hPSCs can be tested efficiently by using gradient-pattern chips designed with varying sizes and surfaces. STATEMENT OF SIGNIFICANCE: Our study provides demonstrates of using gradient nanopatterned chips for differentiation of pancreatic islet-like clusters. Gradient nanopatterned chips are consisted of two different shapes (nanopillar and nanopore) in three different ranges of nano sizes (100-200, 200-300, 300-400 nm). We found that optimal nanostructures for differentiation of pancreatic islet-like clusters were 200-300 nm nano pores. Cell transplantation is one of the major therapeutic option for type 1 diabetes mellitus (DM) using stem cell-derived ß-like cells. We generated 50 um pancreatic islet-like clusters in size, which would be an optimal size for cell transplantation. Futuremore, the small clusters provide a powerful source for cell therapy. Our findings suggest gradient nanopatterned chip provides a powerful tool to generate specific functional cell types of a high purity for potential uses in cell therapy development.

Probing cellular response to topography in three dimensions.

Biophysical aspects of in vivo tissue microenvironments include microscale mechanical properties, fibrillar alignment, and architecture or topography of the extracellular matrix (ECM). These aspects act in concert with chemical signals from a myriad of diverse ECM proteins to provide cues that drive cellular responses. Here, we used a bottom-up approach to build fibrillar architecture into 3D amorphous hydrogels using magnetic-field driven assembly of paramagnetic colloidal particles functionalized with three types of human ECM proteins found in vivo. We investigated if cells cultured in matrices comprised of fibrils of the same size and arranged in similar geometries will show similar behavior for each of the ECM proteins tested. We were able to resolve spatial heterogeneities in microscale mechanical properties near aligned fibers that were not observed in bulk tissue mechanics. We then used this platform to examine factors contributing to cell alignment in response to topographical cues in 3D laminin-rich matrices. Multiple human cell lines extended protrusions preferentially in directions parallel or perpendicular to aligned fibers independently of the ECM coating. Focal adhesion proteins, as measured by paxillin localization, were mainly diffuse in the cytoplasm, with few puncta localized at the protrusions. Integrin ß1 and fascin regulated protrusion extension but not protrusion alignment. Myosin II inhibition did not reduce observed protrusion length. Instead, cells with reduced myosin II activity generated protrusions in random orientations when cultured in hydrogels with aligned fibers. Similarly, myosin II dependence was observed in vivo, where cells no longer aligned along the abluminal surfaces of blood vessels upon treatment with blebbistatin. These data suggest that myosin II can regulate sensing of topography in 3D engineered matrices for both normal and transformed cells.

Tissue Architectural Cues Drive Organ Targeting of Tumor Cells in Zebrafish.

Tumor cells encounter a myriad of physical cues upon arrest and extravasation in capillary beds. Here, we examined the role of physical factors in non-random organ colonization using a zebrafish xenograft model. We observed a two-step process by which mammalian mammary tumor cells showed non-random organ colonization. Initial homing was driven by vessel architecture, where greater numbers of cells became arrested in the topographically disordered blood vessels of the caudal vascular plexus (CVP) than in the linear vessels in the brain. Following arrest, bone-marrow- and brain-tropic clones exhibited organ-specific patterns of extravasation. Extravasation was mediated by ß1 integrin, where knockdown of ß1 integrin reduced extravasation in the CVP but did not affect extravasation of a brain-tropic clone in the brain. In contrast, silencing myosin 1B redirected early colonization from the brain to the CVP. Our results suggest that organ selectivity is driven by both vessel topography and cell-type-dependent extravasation.

Collagen nanofibre anisotropy induces myotube differentiation and acetylcholine receptor clustering.

To create musculoskeletal tissue scaffolds for functional integration into host tissue, myotubes must be properly aligned with native tissue and spur the formation of neuromuscular junctions. However, our understanding of myoblast differentiation in response to structural alignment is incomplete. To examine how substrate anisotropy mediates myotube differentiation, we studied C2C12 myoblasts grown on aligned collagen substrates in the presence or absence of agrin. Myoblasts grown on microfluidically patterned collagen substrates demonstrated increased multinucleated myotubes and nicotinic acetylcholine receptor (AChR) clusters. However, agrin treatment did not synergistically increase differentiation of myoblasts seeded on these patterned collagen substrates. Myoblasts grown on aligned electrospun collagen nanofibres also demonstrated increased formation of multinucleated myotubes and AChR clusters, and agrin treatment did not increase differentiation of these cells. Using fluorescently labelled collagen nanofibres, we found that AChR clustered in cells grown on nanofibres with significantly higher anisotropy and that this clustering was eliminated with agrin treatment. Interestingly, anisotropy of substrate had no effect on the localization of AChRs along the myotube, suggesting that additional signalling pathways determine the specific location of AChRs along individual myotubes. Taken together, our results suggest a novel role for fibre anisotropy in myotube differentiation, specifically AChR clustering, and that anisotropy may guide differentiation by activating similar pathways to agrin. Our data suggest that agrin treatment is not necessary for differentiation and maturation of myoblasts into myotubes when myoblasts are grown on aligned collagen substrates.

Aligning Synthetic Hippocampal Neural Circuits via Self-Rolled-Up Silicon Nitride Microtube Arrays.

Directing neurons to form predetermined circuits with the intention of treating neurological disorders and neurodegenerative diseases is a fundamental goal and current challenge in neuroengineering. Until recently, only neuronal aggregates were studied and characterized in culture, which can limit information gathered to populations of cells. In this study, we use a substrate constructed of arrays of strain-induced self-rolled-up membrane 3D architectures. This results in changes in the neuronal architecture and altered growth dynamics of neurites. Hippocampal neurons from postnatal rats were cultured at low confluency (∼250 cells mm-2) on an array of transparent rolled-up microtubes (µ-tubes; 4-5 µm diameter) of varying topographical arrangements. Neurite growth on the µ-tubes was characterized and compared to controls in order to establish a baseline for alignment imposed by the topography. Compared to control substrates, neurites are significantly more aligned toward the 0° reference on the µ-tube array. Pitch (20-60 and 100 µm) and µ-tube length (30-80 µm) of array elements were also varied to investigate their impact on neurite alignment. We found that alignment was improved by the gradient pitch arrangement and with longer µ-tubes. Application of this technology will enhance the ability to construct intentional neural circuits through array design and manipulation of individual neurons and can be adapted to address challenges in neural repair, reinnervation, and neuroregeneration.