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Biochemical and biomechanical extracellular matrix (ECM) cues have recently been shown to play a role in stimulating stem cell differentiation towards several lineages, though how they combine to induce adipogenesis has been less well studied. The objective of this study was to recapitulate both the ECM composition and mechanical properties of adipose tissue in vitro to stimulate adipogenesis of human adipose-derived stem cells (ASCs) in the absence of exogenous adipogenic growth factors and small molecules. Adipose specific ECM biochemical cues have been previously shown to influence adipogenic differentiation; however, the ability of biomechanical cues to promote adipogenesis has been less defined. Decellularized human lipoaspirate was used to functionalize polyacrylamide gels of varying stiffness to allow the cells to interact with adipose-specific ECM components. Culturing ASCs on gels that mimicked the native stiffness of adipose tissue (2 kPa) significantly upregulated adipogenic markers, in the absence of exogenous adipogenic growth factors and small molecules. As substrate stiffness increased, the cells became more spread, lost their rounded morphology, and failed to upregulate adipogenic markers. Together these data imply that as with other lineages, mechanical cues are capable of regulating adipogenesis in ASCs.

Cancer stem cells (CSCs) are crucial for tumorigenesis, metastasis, and therapy resistance in esophageal squamous cell carcinoma (ESCC). To further elucidate the mechanism underlying characteristics of CSCs and develop CSCs-targeted therapy, an efficient culture system that could expand and maintain CSCs is needed. CSCs reside in a complex tumor microenvironment, and three-dimensional (3D) culture systems of biomimetic scaffolds are expected to better support the growth of CSCs by recapitulating the biophysical properties of the extracellular matrix (ECM). Here, we established gelatin-based 3D biomimetic scaffolds mimicking the stiffness and collagen content of ESCC, which could enrich ESCC CSCs efficiently. Biological changes of ESCC cells laden in scaffolds with three different viscoelasticity emulating physiological stiffness of esophageal tissues were thoroughly investigated in varied aspects such as cell morphology, viability, cell phenotype markers, and transcriptomic profiling. The results demonstrated the priming effects of viscoelasticity on the stemness of ESCC. The highly viscous scaffolds (G': 6–403 Pa; G": 2–75 Pa) better supported the enrichment of ESCC CSCs, and the TGF-beta signaling pathway might be involved in regulating the stemness of ESCC cells. Compared to two-dimensional (2D) cultures, highly viscous scaffolds significantly promoted the clonal expansion of ESCC cells in vitro and tumor formation ability in vivo. Our findings highlight the crucial role of biomaterials' viscoelasticity for the 3D culture of ESCC CSCs in vitro , and this newly-established culture system represents a valuable platform to support their growth, which could facilitate the CSCs-targeted therapy in the future. [Display omitted] [ABSTRACT FROM AUTHOR]

Cells in their native microenvironment interact with three-dimensional (3D) nanofeatures. Despite many reports on the effects of substrate nanotopography on cells, the independent effect of 3D parameters has not been investigated. Recent advances in nanofabrication for precise control of nanostructure pattern, periodicity, shape, and height enabled this systematic study of cell interactions with 3D nanotopographies. Two distinct nanopatterns (posts and grates) with varying three-dimensionalities (50-600 nm in nanostructure height) were created, while maintaining the pattern periodicity (230 nm in pitch) and tip shape (needle- or blade-like sharp tips). Human foreskin fibroblasts exhibited significantly smaller cell size and lower proliferation on needle-like nanoposts, and enhanced elongation with alignment on blade-like nanogrates. These phenomena became more pronounced as the nano topographical three-dimensionality (structural height) increased. The nanopost and nanograte architectures provided the distinct contact guidance for both filopodia extension and the formation of adhesion molecules complex, which was believed to lead to the unique cell behaviors observed. (c) 2006 Elsevier Ltd. All rights reserved.

A number of experimental studies have established the critical role of electric field stimulation on cell functionality modulation of various cell types on a wide range of biomaterial platforms in vitro. In particular, cell fate processes, morphological changes and electroporation are significantly modulated over a narrow range of electric field stimulation conditions. Although a few studies using electrical network theory, first principle simulations and electrohydrodynamic models are reported in the literature, the present study establishes a theoretical foundation to a new perspective that bioelectric stress can significantly influence cell morphological changes/electroporation and in a broader sense, the cell response to electric field on biomaterial substrates. A single cell is modelled as a spherical membrane separating the culture medium and the cytoplasm having different dielectric properties. The analytical solutions to the Laplace equation and Poisson equation for the system are adopted to quantitatively capture the potential distribution in the cellular microenvironment for different cases. These include a cell on a conducting substrate, on an insulating substrate and a cell with surface charge density, in electric field stimulated cellular microenvironment. The biophysical significance of the normal stress distribution has been discussed in terms of the variation in the cellular deformation, depending on the frequency of the electric field and substrate conductivity. A significant difference has been observed in the deformation behavior at higher frequencies (> 10 9 Hz) compared to low frequency and DC electric fields. This theoretical study therefore unravels the significance of substrate conductivity in synergy with electric field parameters to modulate cell response. In addition, the tangential component of the Maxwell stress tensor (shear stress), a measure of the stretching force on the membrane, has been used to obtain estimates of the critical electric field required for membrane rupture. It has been predicted that a cell with surface charge density requires electric fields of the order of 10 kV/mm in order to undergo membrane rupture, which is in line with the experimental observations reported in the literature. Taken together, the presented analysis is expected to provide guidelines to develop next generation biomaterials and biomedical devices for regenerative medicine and cancer treatments. • Theoretical model describing cell-electric field interaction in terms of Maxwell stress. • Takes into account the effect of substrate conductivity on the cell response. • Describes the cell shape deformation due to electric field on substrates. • Shows the significance of frequency in cell deformation and rupture phenomena. [ABSTRACT FROM AUTHOR]

Recent advances in regenerative medicine have confirmed the potential to manufacture viable and effective tissue engineering 3D constructs comprising living cells for tissue repair and augmentation. Cell printing has shown promising potential in cell patterning in a number of studies enabling stem cells to be precisely deposited as a blueprint for tissue regeneration guidance. Such manufacturing techniques, however, face a number of challenges including; (i) post-printing cell damage, (ii) proliferation impairment and, (iii) poor or excessive final cell density deposition. The use of hydrogels offers one approach to address these issues given the ability to tune these biomaterials and subsequent application as vectors capable of delivering cell populations and as extrusion pastes. While stem cell-laden hydrogel 3D constructs have been widely established in vitro , clinical relevance, evidenced by in vivo long-term efficacy and clinical application, remains to be demonstrated. This review explores the central features of cell printing, cell-hydrogel properties and cell-biomaterial interactions together with the current advances and challenges in stem cell printing. A key focus is the translational hurdles to clinical application and how in vivo research can reshape and inform cell printing applications for an ageing population. Cell printing - capacity and limitations. Cell type and density are important factors in the printing of living cells. Post-printing consequences including: (i) impaired cell proliferation, (ii) maintenance of phenotype and genotype, (iii) preservation of cell integrity and morphology are issues that need to be considered. Stem cell printing aims to deposit cells in three dimensions within an environment conducive to proliferation and differentiation. Therefore, long-term investigations of cell viability and proliferation in vitro and in vivo are required to elucidate construct maturation and effective tissue regeneration and integration. Importantly, the properties of the hydrogel cell carrier (biocompatibility, bioactivity, physical characteristics) for the select 3D print approach envisaged are crucial for cell encapsulation, protection and support during differentiation and proliferation. Image 1 [ABSTRACT FROM AUTHOR]

Abstract The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC phenotype remains to be a major challenge. Cell morphology has been shown to regulate MSC differentiation into various lineages, including SMCs. We engineered substrates with microgrooves to induce cell elongation to study the mechanism underlying the MSC shape modulation in SMC differentiation. In comparison to those on flat substrates, MSCs cultured on engineered substrates were elongated with increased aspect ratios for both cell body and nucleus, as well as augmented cytoskeletal tensions. Biochemical studies indicated that the microgroove-elongated cells expressed significantly higher levels of SMC markers. MicroRNA analyses showed that up-regulation of miR-145 and the consequent repression of KLF4 in these elongated cells promoted MSC-to-SMC differentiation. Rho/ROCK inhibitions, which impair cytoskeletal tension, attenuated cell and nuclear elongations and disrupted the miR-145/KLF4 regulation for SMC differentiation. Furthermore, cell traction force measurements showed that miR-145 is essential for the functional contractility in the microgroove-induced SMC differentiation. Collectively, our findings demonstrate that, through a Rho-ROCK/miR-145/KLF4 pathway, the elongated cell shape serves as a decisive geometric cue to direct MSC differentiation into functional SMCs. [ABSTRACT FROM AUTHOR]

Human stem cells, including pluripotent, embryonic and mesenchymal, stem cells play pivotal roles in cell-based therapies. Over the past decades, various methods for expansion and differentiation of stem cells have been developed to satisfy the burgeoning clinical demands. One of the most widely endorsed technologies for producing large cell quantities is using microcarriers (MCs) in bioreactor culture systems. In this review, we focus on microcarriers properties that can manipulate the expansion and fate of stem cells. Here, we provide an overview of commercially available MCs and focus on novel stimulus responsive MCs controlled by temperature, pH and field changes. Different features of MCs including composition, surface coating, morphology, geometry/size, surface functionalization, charge and mechanical properties, and their cellular effects are also highlighted. We then conclude with current challenges and outlook on this promising technology. [ABSTRACT FROM AUTHOR]

Cells form hollow, spheroidal structures during the development of many tissues, including the ocular lens, inner ear, and many glands. Therefore, techniques for in vitro formation of hollow spheroids are valued for studying developmental and disease processes. Current in vitro methods require cells to self-organize into hollow morphologies; we explored an alternative strategy based on cell growth in predefined, spherical scaffolds. Our method uses sacrificial, gelatin microbeads to simultaneously template spherical chambers within a hydrogel and deliver cells into the chambers. We use mouse lens epithelial cells to demonstrate that cells can populate the internal surfaces of the chambers within a week to create numerous hollow spheroids. The platform supports manipulation of matrix mechanics, curvature, and biochemical composition to mimic in vivo microenvironments. It also provides a starting point for engineering organoids of tissues that develop from hollow spheroids. [ABSTRACT FROM AUTHOR]

Human stem cells are vulnerable to unfavorable conditions, and their transportation relies on costly and inconvenient cryopreservation. We report here that human mesenchymal stem cells (MSC) in spheroids survived ambient conditions (AC) many days longer than in monolayer. Under AC, the viability of MSC in spheroids remained >90% even after seven days, whereas MSC in monolayer mostly died fast. AC-exposed MSC spheroids, after recovery under normal monolayer culture conditions with controlled carbon dioxide and humidity contents, resumed typical morphology and proliferation, and retained differentiating and immunosuppressive capabilities. RNA-sequencing and other assays demonstrate that reduced cell metabolism and proliferation correlates to the enhanced survival of AC-exposed MSC in spheroids versus monolayer. Moreover, AC-exposed MSC, when injected as either single cells or spheroids, retained therapeutic effects in vivo in mouse colitis models. Spheroidal formation also prolonged survival and sustained pluripotency of human embryonic stem cells kept under AC. Therefore, this work offers an alternative and relatively simple method termed spheropreservation versus the conventional method cryopreservation. It shall remarkably simplify long-distance transportation of stem cells of these and probably also other types within temperature-mild areas, and facilitate therapeutic application of MSC as spheroids without further processing. [ABSTRACT FROM AUTHOR]

Cell morphology has been identified as a potential indicator of stem cell response to biomaterials. However, determination of cell shape phenotype in biomaterials is complicated by heterogeneous cell populations, microenvironment heterogeneity, and multi-parametric definitions of cell morphology. To associate cell morphology with cell-material interactions, we developed a shape phenotyping framework based on support vector machines. A feature selection procedure was implemented to select the most significant combination of cell shape metrics to build classifiers with both accuracy and stability to identify and predict microenvironment-driven morphological differences in heterogeneous cell populations. The analysis was conducted at a multi-cell level, where a “supercell” method used average shape measurements of small groups of single cells to account for heterogeneous populations and microenvironment. A subsampling validation algorithm revealed the range of supercell sizes and sample sizes needed for classifier stability and generalization capability. As an example, the responses of human bone marrow stromal cells (hBMSCs) to fibrous vs flat microenvironments were compared on day 1. Our analysis showed that 57 cells (grouped into supercells of size 4) are the minimum needed for phenotyping. The analysis identified that a combination of minor axis length, solidity, and mean negative curvature were the strongest early shape-based indicator of hBMSCs response to fibrous microenvironment. [ABSTRACT FROM AUTHOR]

Sophisticated cell pattern provides unique cellular assay platform for studying cell to cell interaction, cellular differentiation and signaling, high-throughput cell response to chemicals. In this study, we demonstrated reactive oxygen species (ROS) mediated endothelial cell micropatterning on a polydimethylsiloxane (PDMS) substrate. The exposure of UV/O radiation led to the formation of ROS on the surface of PDMS, which could selectively prevent adhesion of endothelial cells. The degree of ROS amount was monitored according to the UV/O irradiation time, and at least 36 μM of ROS resulted in the precise cellular micropattern on the PDMS. The presence of ROS affected not only cellular detachment from the substrate, but also endothelial cell morphology such as cell spreading area, confluence, nuclear area and nuclear inverse aspect ratio. In addition, we could observe that the actin cytoskeleton of cells was also constricted due to ROS, thereby minimizing the focal adhesion area of vinculin. Compared with previously reported methods which use chemical treatment or nano/microstructure on the substrate, the proposed methodology is quite simple, accurate, and harmless to the patterned endothelial cells. [ABSTRACT FROM AUTHOR]

Metals and ceramics are commonly used in orthopaedics, dentistry and other load bearing applications. However, the use of ceramic matrix composites reinforced with biocompatible metals for heavy load-bearing hard tissue replacement applications has not previously been reported. In order to improve the reliability and the mechanical properties of biomedical implants, new zirconia–Nb composites have been recently developed. The aim of this study was to investigate the biological tolerance of these new zirconia/Nb biocermets implants with both in vitro and in vivo approaches. At first, human bone marrow derived mesenchymal stem cells were cultured on sintered biocermet discs with polished surfaces and were compared with responses to niobium metal. In vitro, the biocermets showed no deleterious effect on cell proliferation, extra-cellular matrix production or on cell morphology. Furthermore, the biocermet showed a higher percentage of cell proliferation than Nb metal. On the other hand, the bone response to these new zirconia/Nb biocermets was studied. Cylinders of biocermets, as well as commercially Nb rod were implanted in the tibiae of New Zealand white rabbits. All the animals were euthanatized after 6 months. The specimens were processed to obtain thin ground sections. The slides were observed in normal transmitted light microscope. A newly formed bone was observed in close contact with material surfaces. No inflamed or multinucleated cells were present. This study concluded that zirconia/Nb composites are biocompatible and osteoconductive. The ceramic-metal composite has even better osteointegration ability than pure Nb. In conclusion, zirconia–Nb biocermet is suitable for heavy load-bearing hard tissue replacement from the point of view of both mechanical properties and biocompatibility. [ABSTRACT FROM AUTHOR]

Recent studies suggest that actin filaments are essential in how a cell controls its nuclear shape. However, little is known about the relative importance of membrane tension in determining nuclear morphology. In this study, we used adhesive micropatterned substrates to alter the cellular geometry (aspect ratio, size, and shape) that allowed direct membrane tension or without membrane lateral contact with the nucleus and investigate nuclear shape remodeling and orientation on a series of rectangular shapes. Here we showed that at low cell aspect ratios the orientation of the nucleus was regulated by actin filaments while cells with high aspect ratios can maintain nuclear shape and orientation even when actin polymerization was blocked. A model adenocarcinoma cell showed similar behavior in the regulation of nuclear shape in response to changes in cell shape but actin filaments were essential in maintaining cell shape. Our results highlight the two distinct mechanisms to regulate nuclear shape through cell shape control and the difference between fibroblasts and a model cancerous cell in cell adhesion and cell shape control. [ABSTRACT FROM AUTHOR]

The interactions of cells and surrounding tissues with biomaterials used in tissue engineering, wound healing, and artificial organs ultimately determine their fate in vivo . We have demonstrated the ability to tune fibroblast responses with the use of varied material chemistries. In particular, we examined cell morphology, cytokine production, and collagen fiber deposition angles in response to a library of arginine-based polymeric materials. The data presented here shows a large range of vascular endothelial growth factor (VEGF) secretion (0.637 ng/10 6 cells/day to 3.25 ng/10 6 cells/day), cell migration (∼15 min < persistence time < 120 min, 0.11 μm/min < speed < 0.23 μm/min), and cell morphology (0.039 < form factor (FF) < 0.107). Collagen orientation, quantified by shape descriptor (D) values that ranges from 0 to 1, representing completely random (D = 0) to aligned (D = 1) fibers, exhibited large variation both in vitro and in vivo (0.167 < D < 0.36 and 0.17 < D < 0.52, respectively). These findings demonstrate the ability to exert a certain level of control over cellular responses with biomaterials and the potential to attain a desired cellular response such as, increased VEGF production or isotropic collagen deposition upon exposure to these materials in wound healing and tissue engineering applications. [ABSTRACT FROM AUTHOR]

The chemical composition, structure and surface characteristics of biomaterials/scaffold can affect the adsorption of proteins, and this in turn influences the subsequent cellular response and tissue regeneration. With magnesium/calcium phosphate cements (MCPC) as model, the effects of magnesium (Mg) on the initial adhesion and osteogenic differentiation of bone marrow stromal cells (BMSCs) as well as the underlying mechanism were investigated. A series of MCPCs with different magnesium phosphate cement (MPC) content (0∼20%) in calcium phosphate cement (CPC) were synthesized. MCPCs with moderate proportion of MPC (5% and 10%, referred to as 5MCPC and 10MCPC) were found to effectively modulate the orientation of the adsorbed fibronectin (Fn) to exhibit enhanced receptor binding affinity, and to up-regulate integrin α5β1 expression of BMSCs, especially for 5MCPC. As a result, the attachment, morphology, focal adhesion formation, actin filaments assembly and osteogenic differentiation of BMSCs on 5MCPC were strongly enhanced. Further in vivo experiments confirmed that 5MCPC induced promoted osteogenesis in comparison to ot her CPC/MCPCs. Our results also suggested that the Mg on the underlying substrates but not the dissolved Mg ions was the main contributor to the above positive effects. Based on these results, it can be inferred that the specific interaction of Fn and integrin α5β1 had predominant effect on the MCPC-induced enhanced cellular response of BMSCs. These results provide a new strategy to regulate BMSCs adhesion and osteogenic differentiation by adjusting the Mg/Ca content and distribution in CPC, guiding the development of osteoinductive scaffolds for bone tissue regeneration. [ABSTRACT FROM AUTHOR]

The behavior of cancer cells is strongly influenced by the properties of extracellular microenvironments, including topology, mechanics and composition. As topological and mechanical properties of the extracellular matrix are hard to access and control for in-depth studies of underlying mechanisms in vivo , defined biomimetic in vitro models are needed. Herein we show, how pore size and fibril diameter of collagen I networks distinctively regulate cancer cell morphology and invasion. Three-dimensional collagen I matrices with a tight control of pore size, fibril diameter and stiffness were reconstituted by adjustment of concentration and pH value during matrix reconstitution. At first, a detailed analysis of topology and mechanics of matrices using confocal laser scanning microscopy, image analysis tools and force spectroscopy indicate pore size and not fibril diameter as the major determinant of matrix elasticity. Secondly, by using two different breast cancer cell lines (MDA-MB-231 and MCF-7), we demonstrate collagen fibril diameter – and not pore size – to primarily regulate cell morphology, cluster formation and invasion. Invasiveness increased and clustering decreased with increasing fibril diameter for both, the highly invasive MDA-MB-231 cells with mesenchymal migratory phenotype and the MCF-7 cells with amoeboid migratory phenotype. As this behavior was independent of overall pore size, matrix elasticity is shown to be not the major determinant of the cell characteristics. Our work emphasizes the complex relationship between structural-mechanical properties of the extracellular matrix and invasive behavior of cancer cells. It suggests a correlation of migratory and invasive phenotype of cancer cells in dependence on topological and mechanical features of the length scale of single fibrils and not on coarse-grained network properties. [ABSTRACT FROM AUTHOR]

The development of xeno-free, chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard, various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal, i.e. , proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly, when surface chemistry of the substrates was uniformly controlled by collagen conjugation, the stiffness of substrate was inversely related to the sphericity, a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture, implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall, we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal. [ABSTRACT FROM AUTHOR]

Critical morphological requirements for pulp regeneration are tissues replete with vascularisation, neuron formation, and dentin deposition. Autophagy was recently shown to be related to angiogenesis, neural differentiation, and osteogenesis. The present study aimed to investigate the involvement of autophagy in stromal cell-derived factor-1α (SDF-1α)-mediated dental pulp stem cell (DPSC) migration and pulp regeneration, and identify its presence during pulp revascularisation of pulpectomised dog teeth with complete apical closure. In vitro studies showed that SDF-1α enhanced DPSCs migration and optimised focal adhesion formation and stress fibre assembly, which were accompanied by autophagy. Moreover, autophagy inhibitors significantly suppressed, whereas autophagy activator substantially augmented SDF-1α-stimulated DPSCs migration. Furthermore, after ectopic transplantation of tooth fragment/silk fibroin scaffold with DPSCs into nude mice, pulp-like tissues with vascularity, well-organised fibrous matrix formation, and new dentin deposition along the dentinal wall were generated in SDF-1α-loaded samples accompanied by autophagy. More importantly, in a pulp revascularisation model in situ , SDF-1α-loaded silk fibroin scaffolds improved the de novo ingrowth of pulp-like tissues in pulpectomised mature dog teeth, which correlated with the punctuated LC3 and Atg5 expressions, indicating autophagy. Our findings provide novel insights into the pulp regeneration mechanism, and SDF-1α shows promise for future clinical application in pulp revascularisation. [ABSTRACT FROM AUTHOR]

Abstract: The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces. [Copyright &y& Elsevier]

Abstract: Dedifferentiation of chondrocytes is common in culturing, and seriously affects the restorative efficacy of cartilage repair. The present study examines the effect of initial cell shapes on dedifferentiation of chondrocytes in vitro. The cell shape was controlled with a unique material micropatterning technique. With this technique, a series of microarrays of cell-adhesive peptide arginine-glycine-aspartate (RGD) were generated on a persistent non-fouling poly(ethylene glycol) (PEG) hydrogel. After culturing chondrocytes derived from rats on the micropatterned surfaces, the cell shapes were adapted by the geometries of adhesive microislands with pre-defined diameters (10, 15, 20 and 30 μm) for round ones and aspect ratios (1, 1.2, 1.5, 2, 4 and 6) for elliptical ones. After 10 days, collagen II staining was demonstrated to identify normal chondrocytes and dedifferentiated cells for those single cells on microislands. Furthermore, the gene expression of collagen II, collagen I, aggrecan and SOX9 were detected by qRT-PCR. The statistical results illustrated that dedifferentiation of chondrocytes happened more probably in the cases of larger sizes and higher aspect ratios. The conclusions stand under circumstances of both normoxia (21% O2) and hypoxia (5% O2) atmospheres. [Copyright &y& Elsevier]

Abstract: Optimizing growth cone guidance through the use of patterned substrates is important for designing regenerative substrates to aid in recovery from neuronal injury. Using laser ablation, we designed micron-scale patterns capable of confining dissociated mouse cerebellar granule neuron growth cones to channels of different widths ranging from 1.5 to 12 μm. Growth cone dynamics in these channels were observed using time-lapse microscopy. Growth cone area was decreased in channels between 1.5 and 6 μm as compared to that in 12 μm and unpatterned substrates. Growth cone aspect ratio was also affected as narrower channels forced growth cones into a narrow, elongated shape. There was no difference in the overall rate of growth cone advance in uniform channels between 1.5 and 12 μm as compared to growth on unpatterned substrates. The percentage of time growth cones advanced, paused, and retracted was also similar. However, growth cones did respond to changes in confinement: growth cones in narrow lanes rapidly sped up when encountering a wide region and then slowed down as they entered another narrow region. Our results suggest that the rate of neurite extension is not affected by the degree of confinement, but does respond to changes in confinement. [Copyright &y& Elsevier]

Abstract: In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500 μm2 to 160,000 μm2. The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30 μm and 80 μm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes. [Copyright &y& Elsevier]

Abstract: Cell behavior such as cell adhesion, spreading, and contraction critically depends on the elastic properties of the extracellular matrix. It is not known, however, how cells respond to viscoelastic or plastic material properties that more closely resemble the mechanical environment cells encounter in the body. In this report, we employ viscoelastic and plastic biomembrane-mimicking cell substrates. The compliance of the substrates can be tuned by increasing the number of polymer-tethered bilayers. This leaves the density and conformation of adhesive ligands on the top bilayer unaltered. We then observe the response of fibroblasts to these property changes. For comparison, we also study the cells on soft polyacrylamide and hard glass surfaces. Cell morphology, motility, cell stiffness, contractile forces and adhesive contact size all decrease on more compliant matrices but are less sensitive to changes in matrix dissipative properties. These data suggest that cells are able to feel and respond predominantly to the effective matrix compliance, which arises as a combination of substrate and adhesive ligand mechanical properties. [Copyright &y& Elsevier]

Abstract: Material features proved to exert a potent influence on cell behaviour in terms of adhesion, migration and differentiation. In particular, biophysical and biochemical signals on material surfaces are able to affect focal adhesion distribution and cytoskeletal assemblies, which are known to regulate signalling pathways that ultimately influence cell fate and functions. However, a general, unifying model that correlates cytoskeletal-generated forces with genetic events has yet to be developed. Therefore, it is crucial to gain a better insight into the material-cytoskeleton crosstalk in order to design and fabricate biomaterials able to govern cell fate more accurately. In this work, we demonstrate that confining focal adhesion distribution and growth dramatically alters the cytoskeleton's structures and dynamics, which in turn dictate cellular and nuclear shape and polarization. MC3T3 preosteoblasts were cultivated on nanograted polydimethylsiloxane substrates and a thorough quantification – in static and dynamic modes – of the morphological and structural features of focal adhesions and cytoskeleton was performed. Nanoengineered surfaces provided well-defined zones for focal adhesions to form and grow. Unique cytoskeletal structures spontaneously assembled when focal adhesions were confined and, in fact, they proved to be very effective in deforming the nuclei. The results here presented provide elements to engineer surfaces apt to guide and control cell behaviour through the material-cytoskeleton-nucleus axis. [Copyright &y& Elsevier]

Abstract: Many scaffold systems have evolved for tissue engineering and in vitro tissue models to provide a 3D (three-dimensional) microenvironment that enables cells to behave more physiologically. We hypothesized that cells would adopt morphologies with more 3D character during culture in scaffolds as compared to planar substrates. Cell shape and function are tightly linked and effects of scaffold niche properties on cell shape and dimensionality are important for directing cell function. Herein, primary human bone marrow stromal cells (hBMSCs) were cultured in 6 different scaffolds and on a planar control substrate. hBMSCs were imaged using 3D confocal microscopy, and 3D image analyses were used to assess hBMSC shape and dimensionality. A characteristic gyration tensor ellipsoid was calculated for hBMSCs in the different scaffolds which enabled hBMSC dimensionality to be classified based on shape. A “Dimensionality Matrix” was developed that showed that hBMSC shape and dimensionality were influenced by scaffold properties, and that scaffolds could drive hBMSCs into 1D, 2D or 3D shapes. In addition, the hBMSC Z-Depth was measured to determine if hBMSCs became less flat during culture in scaffolds. Z-Depth results showed that all 6 scaffolds caused an increase in cell Z-Depth compared to the 2D planar substrate. These results demonstrate that hBMSCs take on morphologies with greater 3D character in scaffolds than on a planar substrate and that scaffold properties can be adjusted to modify cell dimensionality. In addition, biomaterialists can use this measurement approach to assess and compare scaffold design modifications as they strive to create optimal cell niches that provide a 3D microenvironment. [Copyright &y& Elsevier]

Abstract: We used a synthetic biology approach to produce myotubes from mammalian C2C12 myoblasts in non-differentiation growth conditions using the expression of basic helix-loop-helix transcription factors, MyoD and E12, in various combinations and configurations. Our approach not only recapitulated the basics of muscle development and physiology, as the obtained myotubes showed qualities similar to those seen in striated muscle fibers in vivo, but also allowed for the synthesis of populations of myotubes which assumed distinct morphology, myofibrillar development and Ca2+ dynamics. This fashioned class of biomaterials is suitable for the building blocks of soft actuators in micro-scale biomimetic robotics. This production line strategy can be embraced in reparative medicine as synthetic human myotubes with predetermined morphological/functional properties could be obtained using this very approach. This methodology can be adopted beyond striated muscle for the engineering of other tissue components/cells whose differentiation is governed by the principles of basic helix-loop-helix transcription factors, as in the case, for example, of neural or immune cell types. [Copyright &y& Elsevier]

Abstract: Cells environment is increasingly recognized as an important function regulator through cell–matrix interactions. Extracellular matrix (ECM) anisotropy being a key component of heart valves properties, we have devised a method to create highly porous anisotropic nanofibrillar scaffolds and studied their suitability as cell-support and interactions with human adipose derived stem cells (hADSCs) and human valve interstitial cells (hVICs). Anisotropic nanofibrillar scaffolds were produced by a modified jet-spraying method that allows the formation of aligned nanofibres (600 nm) through air-stream diffraction of a polymer solution (poly (ε-caprolactone, PCL) and collection onto a variably rotating drum. The resulting matrices of high porosity (99%) mimicked valve mechanical anisotropy. Dynamically seeded hADSC and hVIC cultured on scaffolds up to 20 days revealed that hADSC and hVIC penetration within the matrices was improved by anisotropic organization. Within 10 days, cells populated the entire scaffolds thickness and produced ECM (collagen I, III and elastin). As a result, mechanical properties of the constructs were improved over culture, while remaining anisotropic. In contrast to isotropic matrices, anisotropy induced elongated hADSCs and hVICs morphology that followed nanofibres orientation. Interestingly, these morphological changes did not induce hADSC differentiation towards the mesoderm lineages while hVIC recovered a physiological phenotype over culture in the biomimetic matrices. Overall, this study indicates that highly porous anisotropic jet-sprayed matrices are interesting candidates for valve tissue engineering, through anisotropic mechanical properties, efficient cell population, conservation of stem cells phenotype and recovery of hVIC physiological phenotype. [Copyright &y& Elsevier]

Abstract: Despite the enormous progress in the treatment of coronary artery diseases, they remain the most common cause of heart failure in the Western countries. New translational therapeutic approaches explore cardiomyogenic differentiation of various types of stem cells in combination with tissue-engineered scaffolds. In this study we fabricated PHBHV/gelatin constructs mimicking myocardial structural properties. Chemical structure and molecular interaction between material components induced specific properties to the substrate in terms of hydrophilicity degree, porosity and mechanical characteristics. Viability and proliferation assays demonstrated that these constructs allow adhesion and growth of mesenchymal stem cells (MSCs) and cardiac resident non myocytic cells (NMCs). Immunofluorescence analysis demonstrated that stem cells cultured on these constructs adopt a distribution mimicking the three-dimensional cell alignment of myocardium. qPCR and immunofluorescence analyses showed the ability of this construct to direct initial MSC and NMC lineage specification towards cardiomyogenesis: both MSCs and NMCs showed the expression of the cardiac transcription factor GATA-4, fundamental for early cardiac commitment. Moreover NMCs also acquired the expression of the cardiac transcription factors Nkx2.5 and TBX5 and produced sarcomeric proteins. This work may represent a new approach to induce both resident and non-resident stem cells to cardiac commitment in a 3-D structure, without using additional stimuli. [Copyright &y& Elsevier]

Abstract: The behaviour of cancerous epithelial prostatic cells (PC3) growing on polyelectrolytes (PE) coatings was compared to the behaviour of immortalized normal prostatic cells (PNT-2). The cell behaviour was evaluated and quantified in terms of initial cell attachment, growth, metabolic activity, morphometry, adhesion, apoptosis and stress related gene expression. Both the anionic PSS (poly(sodium 4-styrenesulphonate))-terminated surface and cationic PAH (poly(allylamine hydrochloride))-terminated surfaces were not cytotoxic. The initial attachment of cells was better on the PAH-terminated surface compared to fibronectin. However, the proliferation rate of PC3 cells was reduced on the PAH-terminated surface and slightly increased on the PSS coatings. Only PAH prevented the clustering phenotype of PC3 and reduced the number of focal adhesion points as compared to fibronectin or PSS coatings. In contrast, none of the PE surfaces significantly affected the biological responses of PNT-2 cells. PAH-terminating films provide a tool to preferentially modulate the growth of some cancerous phenotypes, in this case as a micro-environment that reduces the growth of metastatic PC3 cells. [Copyright &y& Elsevier]

Abstract: Cell modulus (stiffness) is a critical cell property that is important in normal cell functions and increasingly associated with disease states, yet most methods to characterize modulus may skew results. Here we show strong evidence indicating that the fundamental nature of free energies associated with cell/substrate interactions regulates adherent cell morphology and can be used to deduce cell modulus. These results are based on a mathematical model of biophysics and confirmed by the measured morphology of normal and cancerous liver cells adhered on a substrate. Cells select their final morphology by minimizing the total free energy in the cell/substrate system. The key mechanism by which substrate stiffness influences cell morphology is the energy tradeoff between the stabilizing influence of the cell-substrate interfacial adhesive energy and the destabilizing influence of the total elastic energies in the system. Using these findings, we establish a noninvasive methodology to determine the intrinsic modulus of cells by observing global changes in cell morphology in response to substrate stiffness. We also highlight the importance of selecting a relevant morphological index, cell roundness, that reflects the interchange between forms of energy governing cell morphology. Thus, cell-substrate interactions can be rationalized by the underlying biophysics, and cell modulus is easily measured. [Copyright &y& Elsevier]

Abstract: Peripheral arterial disease (PAD) is characterized by the altered structure and function of arteries caused by accumulated plaque. There have been many studies on treating this disease by the direct injection of various types of therapeutic cells, however, the low cell engraftment efficiency and diffusion of the transplanted cells have been major problems. In this study, we developed an approach (transfer printing) to deliver monolayer of cells to the hindlimb ischemic tissue using thermosensitive hydrogels, and investigated its efficacy in long term retention upon transplantation and therapeutic angiogenesis. We first investigated the in vitro maintenance of robust cell–cell contacts and stable expression of the ECM proteins in myoblast layer following transfer printing process. In order to confirm the therapeutic effect of the myoblasts in vivo, we cultured a monolayer of C2C12 myoblasts on thermosensitive hydrogels, which was then transferred to the hindlimb ischemia tissue of athymic mice directly from the hydrogel by conformal contact. The transferred myoblast layer was retained for a longer period of time than an intramuscularly injected cell suspension. In addition, the morphology of the mice and laser Doppler perfusion (28 days after treatment) supported that the myoblast layer enhanced the therapeutic effects on the ischemic tissue. In summary, the transplantation of the C2C12 myoblast layer using a tissue transfer printing method could represent a new approach for the treatment of PAD by therapeutic angiogenesis. [Copyright &y& Elsevier]

Although vascular smooth muscle cells (VSMCs) are pivotal in physiology and pathology, there is a lack of detailed morphological data on these cells. The objective of this study was to determine dimensions (width and length) and orientation of swine coronary VSMCs and to develop a microstructural constitutive model of active media. The dimensions, spatial aspect ratio and orientation angle of VSMCs measured at zero-stress state were found to follow continuous normal (or bimodal normal) distributions. The VSMCs aligned off circumferential direction of blood vessels with symmetrical polar angles 18.7° ± 10.9°, and the local VSMC deformation was affine with tissue-level deformation. A microstructure-based active constitutive model was developed to predict the biaxial vasoactivity of coronary media, based on experimental measurements of geometrical and deformation features of VSMCs. The results revealed that the axial active response of blood vessels is associated with multi-axial contraction as well as oblique VSMC arrangement. The present morphological database is essential for developing accurate structural models and is seminal for understanding the biomechanics of muscular vessels. [ABSTRACT FROM AUTHOR]

The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80–500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE. • Substrate rigidity guides the organization of intestinal epithelium (IE) and its differentiation. • Simple method for long-term in vitro cultures of IE on planar hydrogel substrates. • IE monolayers recapitulate features of in vivo intestinal epithelium. • 3D scaffolds with mimetic crypt-villus structure are produced using soft hydrogels. • Ex vivo 3D intestinal model replicates key features of mouse small intestinal epitheliumManuscript # jbmt56984. [ABSTRACT FROM AUTHOR]

Abstract: There is a need for an artificial salivary gland as a long-term remedy for patients suffering from salivary hypofunction, a leading cause of chronic xerostomia (dry mouth). Current salivary gland tissue engineering approaches are limited in that they either lack sufficient physical cues and surface area needed to facilitate epithelial cell differentiation, or they fail to provide a mechanism for assembling an interconnected branched network of cells. We have developed highly-ordered arrays of curved hemispherical “craters” in polydimethylsiloxane (PDMS) using wafer-level integrated circuit (IC) fabrication processes, and lined them with electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers, designed to mimic the three-dimensional (3-D) in vivo architecture of the basement membrane surrounding spherical acini of salivary gland epithelial cells. These micropatterned scaffolds provide a method for engineering increased surface area and were additionally investigated for their ability to promote cell polarization. Two immortalized salivary gland cell lines (SIMS, ductal and Par-C10, acinar) were cultured on fibrous crater arrays of various radii and compared with those grown on flat PLGA nanofiber substrates, and in 3-D Matrigel. It was found that by increasing crater curvature, the average height of the cell monolayer of SIMS cells and to a lesser extent, Par-C10 cells, increased to a maximum similar to that seen in cells grown in 3-D Matrigel. Increasing curvature resulted in higher expression levels of tight junction protein occludin in both cell lines, but did not induce a change in expression of adherens junction protein E-cadherin. Additionally, increasing curvature promoted polarity of both cell lines, as a greater apical localization of occludin was seen in cells on substrates of higher curvature. Lastly, substrate curvature increased expression of the water channel protein aquaporin-5 (Aqp-5) in Par-C10 cells, suggesting that curved nanofiber substrates are more suitable for promoting differentiation of salivary gland cells. [Copyright &y& Elsevier]

Abstract: Many stem cells grow into three-dimensional (3D) spheres or colonies, such as neural progenitor cells (NPCs) and embryonic stem cells (ESCs). Sphere morphology helps maintaining the stemness of stem cells. Our previous study demonstrated that forced growth of RT4 and HEK293 cells into 3D sphere on low attachment surface could induce stem cell properties. The close relationship between 3D sphere morphology and stem cell stemness drives us to hypothesize that 3D sphere formation induces fibroblasts reprogramming. The key gene Sox2 for reprogramming fibroblasts into NPCs was found to be overexpressed in 3D sphere cultured mouse fibroblasts. These cells exhibited similar morphological and molecular features to NPCs in vitro, were capable of differentiating into neurons, astrocytes and oligodendrocytes, and could generate long-term expandable neurospheres while maintaining differentiation capability. When engrafted into hippocampus of adult rat brain, the 3D sphere cells differentiated into neural cells. Thus, NPCs can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors. [Copyright &y& Elsevier]

Abstract: Microscale and nanoscale structures can spatially pattern endothelial cells (ECs) into parallel-aligned organization, mimicking their cellular alignment in blood vessels exposed to laminar shear stress. However, the effects of spatial patterning on the function and global transcriptome of ECs are incompletely characterized. We used both parallel-aligned micropatterned and nanopatterned biomaterials to evaluate the effects of spatial patterning on the phenotype of ECs, based on gene expression profiling, functional characterization of monocyte adhesion, and quantification of cellular morphology. We demonstrate that both micropatterned and aligned nanofibrillar biomaterials could effectively guide EC organization along the direction of the micropatterned channels or nanofibrils, respectively. The ability of ECs to sense spatial patterning cues were abrogated in the presence of cytoskeletal disruption agents. Moreover, both micropatterned and aligned nanofibrillar substrates promoted an athero-resistant EC phenotype by reducing endothelial adhesiveness for monocytes and platelets, as well as by downregulating the expression of adhesion proteins and chemokines. We further found that micropatterned ECs have a transcriptional signature that is unique from non-patterned ECs, as well as from ECs aligned by shear stress. These findings highlight the importance of spatial patterning cues in guiding EC organization and function, which may have clinical relevance in the development of vascular grafts that promote patency. [Copyright &y& Elsevier]

Abstract: Upon contact with a biomaterial, cells and surrounding tissues respond in a manner dictated by the physicochemical and mechanical properties of the material. Traditionally, cellular responses are monitored using invasive analytical methods that report the expression of genes or proteins. These analytical methods involve assessing commonly used markers for a predefined readout, masking the actual situation induced in the cells. Hence, a broader expression profile of the cellular response should be envisioned, which technically limits up scaling to higher throughput systems. However, it is increasingly recognized that morphometric readouts, obtained non-invasively, are related to gene expression patterns. Here, we introduced distinct surface roughness to three PLA surfaces, by exposure to oxygen plasma of different duration times. The response of mesenchymal stromal cells was compared to smooth untreated PLA surfaces without the addition of differentiation agents. Morphological and genome wide expression profiles revealed underlying cellular changes which was hidden for the commonly used gene markers for osteo-, chondro- and adipogenesis. Using 3 morphometric parameters, obtained by high content imaging, we were able to build a classifier and discriminate between oxygen plasma-induced modified sheets and non-modified PLA sheets where evaluating classical candidates missed this effect. This approach shows the feasibility to use noninvasive morphometric data in high-throughput systems to screen biomaterial surfaces indicating the underlying genetic biomaterial-induced changes. [Copyright &y& Elsevier]

Abstract: According to conventional understanding regarding dependence of cell behavior on substrate stiffness, tissue cells typically remain round on soft substrates but spread on stiff substrates. The current studies were carried out to learn if the growth factor environment influenced the foregoing relationship. Using standard methods, we prepared planar (2D) polyacrylamide (PA) gels ranging from 0.5 to 40kPa and covalently cross-linked with fibronectin and collagen at concentrations ranging from 2.5 to 50μg/ml. We carried out experiments with fibroblasts varying in their ability to form actin stress fibers and focal adhesions. In fetal bovine serum (FBS) containing medium – the growth factor environment in which most studies on cell spreading and substrate stiffness have been carried out – cell spreading increased with increasing substrate stiffness and adhesion ligand density. However, in platelet-derived growth factor (PDGF) containing medium, cell spreading was relatively independent of substrate stiffness and adhesion ligand density except little cell attachment occurred in the complete absence of cross-linked adhesion ligands. If cell contraction was blocked with blebbistatin, then cell spreading in FBS-containing medium became independent of substrate stiffness. The findings suggest that under growth factor conditions that stimulate global cell contraction (FBS), cell spreading cannot occur unless adhesion ligand density and substrate stiffness result in cell–substrate interactions strong enough to resist and overcome the inward tractional force. Under growth factor conditions that stimulate global cell protrusion (PDGF), such resistance is not required. [Copyright &y& Elsevier]

Abstract: Cost-effective and scalable synthetic matrices that support long-term expansion of human pluripotent stem cells (hPSCs) have many applications, ranging from drug screening platforms to regenerative medicine. Here, we report the development of a hydrogel-based matrix containing synthetic heparin-mimicking moieties that supports the long-term expansion of hPSCs (≥20 passages) in a chemically defined medium. HPSCs expanded on this synthetic matrix maintained their characteristic morphology, colony forming ability, karyotypic stability, and differentiation potential. We also used the synthetic matrix as a platform to investigate the effects of various physicochemical properties of the extracellular environment on the adhesion, growth, and self-renewal of hPSCs. The observed cellular responses can be explained in terms of matrix interface-mediated binding of extracellular matrix proteins, growth factors, and other cell-secreted factors, which create an instructive microenvironment to support self-renewal of hPSCs. These synthetic matrices, which comprise of “off-the-shelf” components and are easy to synthesize, provide an ideal tool to elucidate the molecular mechanisms that control stem cell fate. [Copyright &y& Elsevier]

Abstract: The purpose of this study is to demonstrate pH-responsive chitosan is able to be used for cell fractionation under precise adjustment of medium pH. Cells were first seeded to attach on chitosan surface at medium pH 7.20 for 24 h. After raising medium pH to 7.65 for 1 h, cells with elongated morphology possessed rapider detachment rate and cells with round shape detached at a lesser rate. Therefore, successful cell separation has been achieved by choosing appropriate cell combination with different detachment rates without additional antibody or enzyme treatment and extensive washing steps. Furthermore, the method also could be applied to specific manipulation of viable cell populations from tissue specimen. Most importantly and interestingly, the efficiency of cell fractionation of our system could be theoretically predicted according to the individual cell detachment rate on pH-responsive chitosan surface, without considering the presence of heterotypic cells. [Copyright &y& Elsevier]

Abstract: Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine, especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells, but these predominantly rely on biochemical cues. Recently, differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However, the means by which these topographical cues improve neuronal yield remains unknown. In this study, we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 μm gratings increase the rate of neural differentiation, and that an additional culture period of 2 μm gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added, thus generating hPSC-derived neural cells in a more cost effective and efficient manner. [Copyright &y& Elsevier]

Abstract: Oligonucleotide-based membrane inserts can be used as tethers to control attachment of cells to patterned surfaces without interfering with internal cytoskeletal modes of adhesion. Such control can be employed as a means for study of cell–cell interactions or side-by-side co-culture of different cell types without separation/sorting. While there is utility for cell patterning methods decoupled from natural cytoskeletal mechanisms, the consequences of maintaining this artificially induced state of attachment remains unexplored. We present a method for the 2-dimensional patterning of cells via hybridization of membrane-tethered single stranded oligonucleotides to complimentary single stranded oligonucleotides bound to optically transparent glass substrates which allowed us to characterize the long term culture of patterned HEK293 cells. Patterned substrates immersed in FBS-containing media are shown to permit the adsorption of adhesive serum proteins which allowed for the spreading and engagement of natural cytoskeletal adhesion modes in cells initially attached only through DNA hybridization. We show that the coexisting modes of attachment result in competition between membrane-bound tethers and natural cytoskeletal adhesion machinery as cells attempt to migrate away from their initial points of attachment. This competition ends in the escape of cells from their designated patterns and the ‘winning out’ of cytoskeletal migration forces over the affinity of lipid inserts for the cell membrane. [Copyright &y& Elsevier]

Abstract: Discrete micropatterns on biomaterial surfaces can be used to guide the direction of mammalian cell movement by orienting cell morphology. However, guiding cell assembly in three-dimensional scaffolds remains a challenge. Here we demonstrate that the random motions of motile cells can be rectified within continuous microchannels without chemotactic gradients or fluid flow. Our results show that uniform width microchannels with an overhanging zigzag design can induce polarization of NIH3T3 fibroblasts and human umbilical vein endothelial cells by expanding the cell front at each turn. These continuous zigzag microchannels can guide the direction of cell movement even for cells with altered intracellular signals that promote random movement. This approach for directing cell migration within microchannels has important potential implications in the design of scaffolds for tissue engineering. [Copyright &y& Elsevier]

Abstract: Bone tissue shows a highly anisotropic microstructure comprising biological apatite and collagen fibrils produced by the mutual activities of bone cells, which dominates its mechanical function. Accordingly, directional control of osteoblasts is crucial for forming anisotropic bone tissue. A new approach was proposed for controlling cell directionality by using crystallographic slip traces caused by dislocation glide. Dislocations were introduced into α-titanium single crystals by plastic deformation of slip system, inducing a step-like structure with acute angles between the surface normal and the slip plane. Topographical properties of step patterning, including step interval and step height, could be controlled by varying the compressive plastic strain. The step geometry introduced by plastic deformation strongly influenced osteoblast elongation, and it aligned preferentially along slip traces. Ti substrates under 10% plastic strain with step height of approximately 300 nm and step interval of 10 μm induced osteoblast alignment most successfully. Actin stress fibers elongated parallel to slip traces, with polarized vinculin accumulation between steps. [Copyright &y& Elsevier]

Abstract: Developing artificial scaffolding structures in vitro in order to mimic physiological-relevant situations in vivo is critical in many biological and medical arenas including bone and cartilage generation, biomaterials, small-scale biomedical devices, tissue engineering, as well as the development of nanofabrication methods. We focus on using simple physical principles (photolithography) and chemical techniques (liquid vapor deposition) to build non-cytotoxic scaffolds with a nanometer resolution through using silicon substrates as the backbone. This method merges an optics-based approach with chemical restructuring to modify the surface properties of an IC-compatible material, switching from hydrophilicity to hydrophobicity. Through this nanofabrication-based approach that we developed, hydrophobic oxidized silicon nanosponges were obtained. We then probed cellular responses—examining cytoskeletal and morphological changes in living cells through a combination of fluorescence microscopy and scanning electron microscopy—via culturing Chinese hamster ovary cells, HIG-82 fibroblasts and Madin–Darby canine kidney cells on these silicon nanosponges. This study has demonstrated the potential applications of using these silicon-based nanopatterns such as influencing cellular behaviors at desired locations with a micro-/nanometer level. [Copyright &y& Elsevier]