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Encapsulation of a single (bio)particle into individual droplets (referred to as single encapsulation) presents tremendous potential for precise biological and chemical reactions at the single (bio)particle level. Previously demonstrated successful strategies often rely on the use of high flow rates, gel, or viscoelastic materials for initial cell ordering prior to encapsulation into droplets, which could potentially challenge the system's operation. We propose to enhance the single encapsulation rate by using a stratified flow structure to focus and pre-order the (bio)particles before encapsulation. The stratified flow structure is formed using two simple aqueous Newtonian fluids with a viscosity contrast, which together serve as the dispersed phase. The single encapsulation rate is influenced by many parameters, including fluid viscosity contrast, geometric conditions, flow conditions and flow rate ratios, and dimensionless numbers such as the capillary number. This study focuses on investigating the influences of these parameters on the focused stream of the stratified flow, which is key for single encapsulation. The results allow the proposal of a simple guideline that can be adopted to design droplet microfluidic chips with an improved single encapsulation rate demanded by a wide range of applications. The guideline was validated by performing the single encapsulation of mouse embryonic stem cells suspended in a gelatin-methacryloyl solution in individual droplets of phosphate buffer saline, achieving a single encapsulation efficiency of up to 70%., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Tissues and organs consist of cells organized in specified patterns that support their function, as exemplified by tissues such as skin, muscle, and cornea. It is, therefore, important to understand how external cues, such as engineered surfaces or chemical contaminants, can influence the organization and morphology of cells. In this work, we studied the impact of indium sulfate on human dermal fibroblast (GM5565) viability, production of reactive oxygen species (ROS), morphology, and alignment behavior on tantalum/silicon oxide parallel line/trench surface structures. The viability of cells was measured using the alamarBlue™ Cell Viability Reagent probe, while the ROS levels in cells were quantified using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate. Cell morphology and orientation on the engineered surfaces were characterized using fluorescence confocal and scanning electron microscopy. When cells were cultured in media containing indium (III) sulfate, the average cell viability decreased by as much as ~32% and the concentration of cellular ROS increased. Cell geometry became more circular and compact in the presence of indium sulfate. Even though actin microfilaments continue to preferentially adhere to tantalum-coated trenches in the presence of indium sulfate, the cells are less able to orient along the line axes of the chips. Interestingly, the indium sulfate-induced changes in cell alignment behavior are pattern dependent-a larger proportion of adherent cells on structures with line/trench widths in the range of 1 μm and 10 μm lose the ability to orient themselves, compared to those grown on structures with line widths smaller than 0.5 μm. Our results show that indium sulfate impacts the response of human fibroblasts to the surface structure to which they adhere and underscores the importance of evaluating cell behaviors on textured surfaces, especially in the presence of potential chemical contaminants.

Biophysical parameters such as substrate topography and stiffness have been shown independently to elicit profound effects on neuronal differentiation and maturation from neural progenitor cells (NPCs) yet have not been investigated in combination. Here, the effects of various micrograting and stiffness combinations on neuronal differentiation and maturation were investigated using a polyacrylamide and N-acryloyl-6-aminocaproic acid copolymer (PAA-ACA) hydrogel with tunable stiffness. Whole laminin was conjugated onto the PAA-ACA surface indirectly or directly to facilitate long-term mouse and human NPC-derived neuron attachment. Three micrograting dimensions (2-10 µm) were patterned onto gels with varying stiffness (6.1-110.5 kPa) to evaluate the effects of topography, stiffness, and their interaction. The results demonstrate that the extracellular matrix (ECM)-modified PAA-ACA gels support mouse and human neuronal cell attachment throughout the differentiation and maturation stages (14 and 28 days, respectively). The interaction between topography and stiffness is shown to significantly increase the proportion of β-tubulin III (TUJ1) positive neurons and microtubule associated protein-2 (MAP2) positive neurite branching and length. Thus, the effects of topography and stiffness cannot be imparted. These results provide a novel platform for neural mechanobiology studies and emphasize the utility of optimizing numerous biophysical cues for improved neuronal yield in vitro.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast in situ endothelialization of polyvinyl alcohol, which is not endothelial cell-adhesive. Among three different immobilization approaches compared, conjugation of aminated-fucoidan promoted endothelial monolayer formation while minimizing thrombogenicity in both in vitro platelet rich plasma testing and ex vivo non-human primate shunt assay. Screening of six topographical patterns showed that 2 μm gratings increased endothelial cell migration without inducing inflammation responses of endothelial cells. Mechanistic studies demonstrated that fucoidan could attract fibronectin, enabling integrin binding and focal adhesion formation and activating focal adhesion kinase (FAK) signaling, and 2 μm gratings further enhanced FAK-mediated cell migration. In a clinically relevant rabbit carotid artery end-to-side anastomosis model, 60% in situ endothelialization was observed throughout the entire lumen of 1.7 mm inner diameter modified grafts, compared to 0% of unmodified graft, and the four-week graft patency also increased. This work presents a promising strategy to stimulate in situ endothelialization on synthetic materials for improving long-term performance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Small-diameter synthetic vascular grafts that have improved hemocompatibility and patency remain an unmet clinical need due to thrombosis. A surface modification that has potential to attenuate these failure mechanisms while promoting an endothelial layer is the micropatterning of luminal surfaces. Anisotropic features have been shown to downregulate smooth muscle cell proliferation, direct endothelial migration, and attenuate platelet adhesion and activation. However, the effect of micropatterning feature size and orientation relative to whole blood flow has yet to be investigated within a systematic study. In this work, hemocompatibility of micropattern grating sizes of 2, 5, and 10 µm were investigated. The thrombogenicity of the micropattern surface modifications were characterized by quantifying FXIIa activity, fibrin formation, and static platelet adhesion in vitro . Additionally, dynamic platelet attachment and end-point fibrin formation were quantified using an established, flowing whole blood ex vivo non-human primate shunt model without antiplatelet or anticoagulant therapies. We observed a higher trend in platelet attachment and significantly increased fibrin formation for larger features. We then investigated the orientation of 2 µm gratings relative to whole blood flow and found no significant differences between the various orientations for platelet attachment, rate of linear platelet attachment, or end-point fibrin formation. MicroCT analysis of micropatterned grafts was utilized to quantify luminal patency. This work is a significant step in the development of novel synthetic biomaterials with improved understanding of hemocompatibility for use in cardiovascular applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fallon, Le, Bates, Yao, Yim, Hinds and Anderson.)

Hydrogel has been an attractive biomaterial for tissue engineering, drug delivery, wound healing, and contact lens materials, due to its outstanding properties, including high water content, transparency, biocompatibility, tissue mechanical matching, and low toxicity. As hydrogel commonly possesses high surface hydrophilicity, chemical modifications have been applied to achieve the optimal surface properties to improve the performance of hydrogels for specific applications. Ideally, the effects of surface modifications would be stable, and the modification would not affect the inherent hydrogel properties. In recent years, a new type of surface modification has been discovered to be able to alter hydrogel properties by physically patterning the hydrogel surfaces with topographies. Such physical patterning methods can also affect hydrogel surface chemical properties, such as protein adsorption, microbial adhesion, and cell response. This review will first summarize the works on developing hydrogel surface patterning methods. The influence of surface topography on interfacial energy and the subsequent effects on protein adsorption, microbial, and cell interactions with patterned hydrogel, with specific examples in biomedical applications, will be discussed. Finally, current problems and future challenges on topographical modification of hydrogels will also be discussed., (© 2021 Author(s).)

Poly(vinyl alcohol) hydrogel, PVA, is a suitable material for small-diameter vascular grafting. However, the bioinert properties of the material do not allow for in situ endothelialization, which is needed to combat common graft failure mechanisms, such as intimal hyperplasia and thrombosis. In this work, the surface of planar and tubular PVA was covalently modified with a collagen-mimicking peptide, GFPGER. The surface of modified PVA was characterized by measuring contact angle and x-ray photoelectron spectroscopy. Endothelial cell attachment to GFPGER-modified PVA was quantified and qualitatively examined using immunohistochemical staining. Then, in vitro hemocompatibility testing was performed by quantifying platelet attachment, coagulation factor XII activation, and initiation of fibrin formation. Finally, an established ex vivo , non-human primate model was employed to examine platelet attachment and fibrin formation under non-anticoagulated, whole blood flow conditions. GFPGER-modified PVA supported increased EC attachment. In vitro initiation of fibrin formation on the modified material was significantly delayed. Ex vivo thrombosis assessment showed a reduction in platelet attachment and fibrin formation on GFPGER-modified PVA. Overall, GFPGER-modified PVA encouraged cell attachment while maintaining the material's hemocompatibility. This work is a significant step toward the development and characterization of a modified-hydrogel surface to improve endothelialization while reducing platelet attachment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Bates, Heidenreich, Fallon, Yao, Yim, Hinds and Anderson.)

The performance of clinical synthetic small diameter vascular grafts remains disappointing due to the fast occlusion caused by thrombosis and intimal hyperplasia formation. Poly(vinyl alcohol) (PVA) hydrogels have tunable mechanical properties and a low thrombogenic surface, which suggests its potential value as a small diameter vascular graft material. However, PVA does not support cell adhesion and thus requires surface modification to encourage endothelialization. This study presents a modification of PVA with fucoidan. Fucoidan is a sulfated polysaccharide with anticoagulant and antithrombotic properties, which was shown to potentially increase endothelial cell adhesion and proliferation. By mixing fucoidan with PVA and co-crosslinked by sodium trimetaphosphate (STMP), the modification was achieved without sacrificing mechanical properties. Endothelial cell adhesion and monolayer function were significantly enhanced by the fucoidan modification. In vitro and ex-vivo studies showed low platelet adhesion and activation and decreased thrombin generation with fucoidan modified PVA. The modification proved to be compatible with gamma sterilization. In vivo evaluation of fucoidan modified PVA grafts in rabbits exhibited increased patency rate, endothelialization, and reduced intimal hyperplasia formation. The fucoidan modification presented here benefited the development of PVA vascular grafts and can be adapted to other blood contacting surfaces., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Extracellular matrix (ECM) influences cell differentiation through its structural and biochemical properties. In nervous system, neuronal behavior is influenced by these ECMs structures which are present in a meshwork, fibrous, or tubular forms encompassing specific molecular compositions. In addition to contact guidance, ECM composition and structures also exert its effect on neuronal differentiation. This short report reviewed the native ECM structure and composition in central nervous system and peripheral nervous system, and their impact on neural regeneration and neuronal differentiation. Using topographies, stem cells have been differentiated to neurons. Further, focussing on engineered biomimicking topographies, we highlighted the role of anisotropic topographies in stem cell differentiation to neurons and its recent temporal application for efficient neuronal differentiation., Competing Interests: None

The effective deployment of arterial (AECs), venous (VECs) and stem cell-derived endothelial cells (PSC-ECs) in clinical applications requires understanding of their distinctive phenotypic and functional characteristics, including their responses to microenvironmental cues. Efforts to mimic the in-vivo vascular basement membrane milieu have led to the design and fabrication of nano- and micro-topographical substrates. Although the basement membrane architectures of arteries and veins are different, investigations into the effects of substrate topographies have so far focused on generic EC characteristics. Thus, topographical modulation of arterial- or venous-specific EC phenotype and function remains unknown. Here, we comprehensively evaluated the effects of 16 unique topographies on primary AECs, VECs and human PSC-ECs using a Multi Architectural (MARC) Chip. Gratings and micro-lenses augmented venous-specific phenotypes and depressed arterial functions in VECs; while AECs did not respond consistently to topography. PSC-ECs exhibited phenotypic and functional maturation towards an arterial subtype with increased angiogenic potential, NOTCH1 and Ac-LDL expression on gratings. Specific topographies could elicit different phenotypic and functional changes, despite similar morphological response in different ECs, demonstrating no direct correlation between the two responses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pluripotent stem cells (PSCs) have great potential to revolutionize the fields of tissue engineering and regenerative medicine as well as stem cell therapeutics. However, the end goal of using PSCs for therapeutic use remains distant due to limitations in current PSC production. Conventional methods for PSC expansion have limited potential to be scaled up to produce the number of cells required for the end-goal of therapeutic use due to xenogenic components, high cost or low efficiency. In this mini review, we explore novel methods and emerging technologies of improving PSC expansion: the use of the two-dimensional mechanobiological strategies of topography and stiffness and the use of three-dimensional (3D) expansion methods including encapsulation, microcarrier-based culture, and suspension culture. Additionally, we discuss the limitations of conventional PSC expansion methods as well as the challenges in implementing non-conventional methods., (Copyright © 2020 Chan, Rizwan and Yim.)

Mechanical cues influence pluripotent stem cell differentiation, but the underlying mechanisms are not well understood. Mouse embryonic stem cells (mESCs) exhibit unusual cytomechanical properties, including low cell stiffness and attenuated responses to substrate rigidity, but the underlying structural basis remains obscure. Using super-resolution microscopy to investigate the actin cytoskeleton in mESCs, we observed that the actin cortex consists of a distinctively sparse and isotropic network. Surprisingly, the architecture and mechanics of the mESC actin cortex appear to be largely myosin II-independent. The network density can be modulated by perturbing Arp2/3 and formin, whereas capping protein (CP) negatively regulates cell stiffness. Transient Arp2/3-containing aster-like structures are implicated in the organization and mechanical homeostasis of the cortical network. By generating a low-density network that physically excludes myosin II, the interplay between Arp2/3, formin, and CP governs the nanoscale architecture of the actin cortex and prescribes the cytomechanical properties of mESCs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Endothelial cells (ECs) are required for a multitude of cardiovascular clinical applications, such as revascularization of ischemic tissues or endothelialization of tissue engineered grafts. Patient derived primary ECs are limited in number, have donor variabilities and their in vitro phenotypes and functions can deteriorate over time. This necessitates the exploration of alternative EC sources. Although there has been a recent surge in the use of pluripotent stem cell derived endothelial cells (PSC-ECs) for various cardiovascular clinical applications, current differentiation protocols yield a heterogeneous EC population, where their specification into arterial or venous subtypes is undefined. Since arterial and venous ECs are phenotypically and functionally different, inappropriate matching of exogenous ECs to host sites can potentially affect clinical efficacy, as exemplified by venous graft mismatch when placed into an arterial environment. Therefore, there is a need to design and employ environmental cues that can effectively modulate PSC-ECs into a more homogeneous arterial or venous phenotype for better adaptation to the host environment, which will in turn contribute to better application efficacy. In this review, we will first give an overview of the developmental and functional differences between arterial and venous ECs. This provides the foundation for our subsequent discussion on the different bioengineering strategies that have been investigated to varying extent in providing biochemical and biophysical environmental cues to mature PSC-ECs into arterial or venous subtypes. The ability to efficiently leverage on a combination of biochemical and biophysical environmental cues to modulate intrinsic arterio-venous specification programs in ECs will greatly facilitate future translational applications of PSC-ECs. Since the development and maintenance of arterial and venous ECs in vivo occur in disparate physio-chemical microenvironments, it is conceivable that the application of these environmental factors in customized combinations or magnitudes can be used to selectively mature PSC-ECs into an arterial or venous subtype.

Plasma-based surface modification is recognized as an effective way to activate biomaterial surfaces, and modulate their interactions with cells, extracellular matrix proteins, and other materials. However, treatment of a luminal surface of a tubular scaffold remains non-trivial to perform in small diameter tubes. Polyvinyl alcohol (PVA) hydrogel, which has been widely used for medical applications, lacks functional groups to mediate cell attachment. This poses an issue for vascular applications, as endothelialization in a vascular graft lumen is crucial to maintain long term graft patency. In this study, a Radio Frequency Glow Discharges (RFGD) treatment in the presence of NH 3 was used to modify the luminal surface of 3-mm diameter dehydrated PVA vascular grafts. The grafted nitrogen containing functional groups demonstrated stability, and in vitro endothelialization was successfully maintained for at least 30 days. The plasma-modified PVA displayed a higher percentage of carbonyl groups over the untreated PVA control. Plasma treatment on PVA patterned with microtopographies was also studied, with only the concave microlenses topography demonstrating a significant increase in platelet adhesion. Thus, the study has shown the possibility of modifying a small diameter hydrogel tubular scaffold with the RFGD plasma treatment technique and demonstrated stability in ambient storage conditions for up to 30 days.

Poly(vinyl alcohol) (PVA) has shown promise as a biomaterial for cardiovascular application. However, its antifouling properties prevent in vivo endothelialization. This work examined the endothelialization and thrombogenicity of modified PVA with different concentrations of proteins and adhesion peptides: collagen, laminin, fibronectin, GFPGER, YIGSR, and cRGD. Material surface properties were quantified, and the endothelialization potential was determined with human endothelial colony forming cells. Additionally, platelet attachment was assessed in vitro with human platelet rich plasma, and promising samples were tested in an ex vivo shunt model. This well-established arteriovenous shunt model was used with and without clinically-relevant antiplatelet therapies, specifically acetylsalicylic acid (ASA) with and without clopidogrel to examine the minimum necessary treatment to prevent thrombosis. Collagen, laminin, and GFPGER biomolecules increased endothelialization, with GFPGER showing the greatest effect at the lowest concentrations. GFPGER-PVA tubes tested under whole blood did exhibit an increase in platelet (but not fibrin) attachment compared to plain PVA and clinical controls. However, application of ASA monotherapy reduced the thrombogenicity of GFPGER-PVA below the clinical control with the ASA. This work is significant in developing cardiovascular biomaterials-increasing endothelialization potential while reducing bleeding side effects by using an antiplatelet monotherapy, typical of clinical patients. STATEMENT OF SIGNIFICANCE: We modified the endothelialization potential of synthetic, hydrogel vascular grafts with proteins and peptides of the vascular tissue matrix. Cell attachment was dramatically increased with the GFPGER peptide, and while some additional platelet attachment was seen under flow with whole blood, this was completely knocked down using clinical antiplatelet monotherapy. This indicates that long-term patency of this biomaterial could be improved without the associated bleeding risk of multiple platelet therapies., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cell elongation and differentiation has been shown to be modulated by topographical cues provided by grating substratum. However, little is known about the mechanisms and forces involved in the grating-induced cell elongation, due to the difficulty in fabricating soft elastic gels that allow 3-dimensional (3D) cell traction stress measurements. In this paper, we present a method to fabricate soft elastic polyacrylamide grating substrates, using an imprinted polyethylene terephthalate mould, for 3D cell traction stress measurements. Fibroblasts were observed to form protrusions in the grating grooves, and elongate and align parallel to the grating direction on the soft polyacrylamide grating substrates. Focal adhesions were also found to be aligned parallel to the grating direction as compared to cells on flat substrates, suggesting that grating grooves restrict focal adhesion growth perpendicular to the grating direction. The 3D traction stress measurements revealed that highly elongated fibroblasts on grating substrates exert anisotropic traction stresses, in the direction parallel to the grating direction. We propose that focal adhesion alignment along the grating direction may result in increased actin stress fibre formation in the direction parallel to the grating, leading to polarized traction stresses which drive cell elongation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Parkinson's Disease is a progressive neurodegenerative disorder attributed to death of mesencephalic dopaminergic (DA) neurons. Pluripotent stem cells have great potential in the study for this late-onset disease, but acquirement of cells that are robust in quantity and quality is still technically demanding. Biophysical cues have been shown to direct stem cell fate, but the effect of different topographies in the lineage commitment and subsequent maturation stages of cells have been less examined. Using human induced pluripotent stem cells (iPSCs), we applied topographical patterns sequentially during differentiation stages and examined their ability to influence derivation yield and functionality of regionalized subtype-specific DA neurons. Gratings showed higher yield of DA neurons and may be beneficial for initial lineage commitment. Cells derived on pillars in the terminal differentiation stage have increased neuronal complexity, and were more capable of firing repetitive action potentials, showing that pillars yielded better network formation and functionality. Our topography platform can be applied to patient-derived iPSCs as well, and that cells harbouring LRRK2 mutation were more functionally mature when optimal topographies were applied sequentially. This will hopefully accelerate development of robust cell models that will provide novel insights into discovering new therapeutic approaches for Parkinson's Disease.

Stem cell differentiation can be regulated by biophysical cues such as nanotopography. It involves sensing and integration of these biophysical cues into their transcriptome with a mechanism that is yet to be discovered. In addition to the cytoskeletal and focal adhesion remodeling, nanotopography has also been shown to modulate nucleus morphology. Here, we studied the effect of nanotopography on the temporal changes in nuclei of human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs). Using a high throughput Multi-architecture (MARC) chip analysis, the circularity of the stem cell nuclei changed significantly on different patterns. Human ESCs and MSCs showed different temporal changes in nucleus morphology, lamin A/C expression and histone methylation during topography-induced neuronal differentiation. In hESCs, the expression of nuclear matrix protein, lamin A/C during neuronal differentiation of hESCs on PDMS samples were weakly detected in the first 7 days of differentiation. The histone 3 trimethylation on Lysine 9 (H3K9me3) decreased after differentiation initiated and showed temporal changes in their expression and organization during neuronal differentiation. In hMSCs, the expression of lamin A/C was significantly increased after the first 24 h of cell culture. The quantitative analysis of histone methylation also showed a significant increase in hMSCs histone methylation on 250 nm anisotropic nanogratings within the first 24 h of seeding. This reiterates the importance of cell-substrate sensing within the first 24 h for adult stem cells. The lamin A/C expression and histone methylation shows a correlation of epigenetic changes in early events of differentiation, giving an insight on how extracellular nanotopographical cues are transduced into nuclear biochemical signals. Collectively, these results provide more understanding into the nuclear regulation of the mechanotransduction of nanotopographical cues in stem cell differentiation.

The topography of the extracellular substrate provides physical cues to elicit specific downstream biophysical and biochemical effects in cells. An example of such a topographical substrate is periodic gratings, where the dimensions of the periodic gratings influence cell morphology and directs cell differentiation. We first develop a novel sample preparation technique using Spurr's resin to allow for cross-sectional transmission electron microscopy imaging of cells on grating grooves, and observed that the plasma membrane on the basal surface of these cells can deform and bend into grooves between the gratings. We postulate that such membrane bending is an important first step in eliciting downstream effects. Thus, we use a combination of image analysis and mathematical modeling to explain the extent of bending of basal membrane into grooves. We show that the extent to which the basal membrane bends into grooves depends on both groove width and angle of the grating ridge. Our model predicts that the basal membrane will bend into grooves when they are wider than 1.9 µm in width. The existence of such a threshold may provide an explanation for how the width of periodic gratings may bring about cellular downstream effects, such as cell proliferation or differentiation.

The endothelial lining of blood vessels is severely affected in type II diabetes. Yet, there is still a paucity on the use of diabetic endothelial cells for study and assessment of implantable devices targeting vascular disease. This critically impairs our ability to determine appropriate topographical cues to be included in implantable devices that can be used to maintain or improve endothelial cell function in vivo. Here, the functional responses of healthy and diabetic human coronary arterial endothelial cells were studied and observed to differ depending on topography. Gratings (2 μm) maintained normal endothelial functions such as adhesiveness, angiogenic capacity and cell-cell junction formation, and reduced immunogenicity of healthy cells. However, a significant and consistent effect was not observed in diabetic cells. Instead, diabetic endothelial cells cultured on the perpendicularly aligned multi-scale hierarchical gratings (250 nm gratings on 2 μm gratings) drastically reduced the uptake of oxidized low-density lipoprotein, decreased immune activation, and accelerated cell migration. Concave microlens (1.8 μm diameter) topography was additionally observed to overwhelmingly deteriorate diabetic endothelial cell function. The results of this study support a new paradigm and approach in the design and testing of implantable devices and biomedical interventions for diabetic patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Neuronal morphology and differentiation have been extensively studied on topography. The differentiation potential of neural progenitors has been shown to be influenced by brain region, developmental stage, and time in culture. However, the neurogenecity and morphology of different neural progenitors in response to topography have not been quantitatively compared. In this study, the correlation between the morphology and differentiation of hippocampal and cortical neural progenitor cells was explored. The morphology of differentiated neural progenitors was quantified on an array of topographies. In spite of topographical contact guidance, cell morphology was observed to be under the influence of regional priming, even after differentiation. This influence of regional priming was further reflected in the correlations between the morphological properties and the differentiation efficiency of the cells. For example, neuronal differentiation efficiency of cortical neural progenitors showed a negative correlation with the number of neurites per neuron, but hippocampal neural progenitors showed a positive correlation. Correlations of morphological parameters and differentiation were further enhanced on gratings, which are known to promote neuronal differentiation. Thus, the neurogenecity and morphology of neural progenitors is highly responsive to certain topographies and is committed early on in development., Competing Interests: The authors declare no conflict of interest.

Cell therapy for vascular damage has been showing promises as alternative therapy for endothelial dysfunctions since the discovery of the endothelial progenitor cells (EPCs). However, isolated EPCs from peripheral blood yield low cell amounts and alternative cell source must be explored. The aim of this study was to investigate the influence of topography on the endothelial differentiation of an alternative cell source - human mesenchymal stem cells (hMSCs) from bone marrow. Utilizing the MultiARChitecture (MARC) chip, a systematic screening of variety of patterned surfaces and different medium compositions was performed. While topographical patterns alone induce endothelial differentiation, a synergistic enhancement was observed when topography was combined with a medium enriched with vascular endothelial growth factor (VEGF). The 1.8 μm diameter convex microlens pattern in combination with the VEGF enriched medium was shown to be the most efficient on the endothelial differentiation, yielding up to 10% of CD34 + CD133 + KDR + marker expressing differentiated hMSCs as analyzed by flow cytometry. The quantified tube-like structures in the Matrigel assay in vitro indicated a vasculogenic potential of these endothelial progenitor-like differentiated hMSCs that was investigated further in a Matrigel plug assay in vivo in a rat for seven days. Explanted Matrigel plugs were processed with hematoxylin-eosin (H&E) and anti-Ulex Europaeus agglutinin (UEA-1) staining to visualize the capillaries and to identify the presence of human cells. The hMSCs cultured on the 1.8 μm diameter convex microlens in a medium enriched with VEGF, implanted in a Matrigel plug in a rat, showed the highest capillary density, the highest UEA-1 + capillary density, as well as the highest UEA-1 + cell survival density that were not included in the vasculogenesis. These findings indicate the active participation of the vasculogenic hMSCs in the vasculogenesis. The endothelial differentiation of hMSCs using this synergistic combination of microlens and VEGF enriched medium was also demonstrated in hMSCs from different male and female donors. The culture platform with combination of topography and biochemical cues could generate vasculogenic cell populations that may prove useful in vascular damage or other clinical applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Naturally-bioactive hydrogels like gelatin provide favorable properties for tissue-engineering but lack sufficient mechanical strength for use as implantable tissue engineering substrates. Complex fabrication or multi-component additives can improve material strength, but often compromises other properties. Studies have shown gelatin methacrylate (GelMA) as a bioactive hydrogel with diverse tissue growth applications. We hypothesize that, with suitable material modifications, GelMA could be employed for growth and implantation of tissue-engineered human corneal endothelial cell (HCEC) monolayer. Tissue-engineered HCEC monolayer could potentially be used to treat corneal blindness due to corneal endothelium dysfunction. Here, we exploited a sequential hybrid (physical followed by UV) crosslinking to create an improved material, named as GelMA+, with over 8-fold increase in mechanical strength as compared to regular GelMA. The presence of physical associations increased the subsequent UV-crosslinking efficiency resulting in robust materials able to withstand standard endothelium insertion surgical device loading. Favorable biodegradation kinetics were also measured in vitro and in vivo. We achieved hydrogels patterning with nano-scale resolution by use of oxygen impermeable stamps that overcome the limitations of PDMS based molding processes. Primary HCEC monolayers grown on GelMA+ carrier patterned with pillars of optimal dimension demonstrated improved zona-occludin-1 expression, higher cell density and cell size homogeneity, which are indications of functionally-superior transplantable monolayers. The hybrid crosslinking and fabrication approach offers potential utility for development of implantable tissue-engineered cell-carrier constructs with enhanced bio-functional properties., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Poly(vinyl alcohol) hydrogel (PVA) is a widely used material for biomedical devices, yet there is a need to enhance its biological functionality for in vitro and in vivo vascular application. Significance of surface topography in modulating cellular behaviour is increasingly evident. However, hydrogel patterning remains challenging. Using a casting method, planar PVA were patterned with micro-sized features. To achieve higher patterning resolution, nanoimprint lithography with high pressure and temperature was used. In vitro experiment showed enhanced human endothelial cell (EC) density and adhesion on patterned PVA. Additional chemical modification via nitrogen gas plasma on patterned PVA further improved EC density and adhesion. Only EC monolayer grown on plasma modified PVA with 2 μm gratings and 1.8 μm concave lens exhibited expression of vascular endothelial cadherin, indicating EC functionality. Patterning of the luminal surface of tubular hydrogels is not widely explored. The study presents the first method for simultaneous tubular molding and luminal surface patterning of hydrogel. PVA graft with 2 μm gratings showed patency and endothelialization, while unpatterned grafts were occluded after 20 days in rat aorta. The reproducible, high yield and high-fidelity methods enable planar and tubular patterning of PVA and other hydrogels to be used for biomedical applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)