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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).)

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide and represent a pressing clinical need. Vascular occlusions are the predominant cause of CVD and necessitate surgical interventions such as bypass graft surgery to replace the damaged or obstructed blood vessel with a synthetic conduit. Synthetic small-diameter vascular grafts (sSDVGs) are desired to bypass blood vessels with an inner diameter <6 mm yet have limited use due to unacceptable patency rates. The incorporation of biophysical cues such as topography onto the sSDVG biointerface can be used to mimic the cellular microenvironment and improve outcomes. In this review, the utility of surface topography in sSDVG design is discussed. First, the primary challenges that sSDVGs face and the rationale for utilizing biomimetic topography are introduced. The current literature surrounding the effects of topographical cues on vascular cell behavior in vitro is reviewed, providing insight into which features are optimal for application in sSDVGs. The results of studies that have utilized topographically-enhanced sSDVGs in vivo are evaluated. Current challenges and barriers to clinical translation are discussed. Based on the wealth of evidence detailed here, substrate topography offers enormous potential to improve the outcome of sSDVGs and provide therapeutic solutions for CVDs., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

The expansion of pluripotent stem cells (PSCs) in vitro remains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markers Oct4, Nanog , and Sox2 and the differentiation marker LmnA as well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1 µ m pillars (1 µ m spacing, square arrangement), 2 µ m wells (c-c ( x, y ) = 4, 4 µ m), and 5 µ m pillars (c-c ( x, y ) = 7.5, 7.5 µ m). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion., (Creative Commons Attribution license.)

Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. Polyvinyl alcohol (PVA) nerve conduits have been clinically approved, but the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neuritogenesis and axon guidance. PVA was conjugated with extracellular matrix proteins and fucoidan, a bioactive sulfated polysaccharide, to improve cell adhesion. Micro-sized topographies, including 1.8 μm convex lenses, 2 μm gratings, and 10 μm gratings were successfully fabricated on PVA by nanofabrication, and the synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0% to 2.8% and further increased to 5% by presenting laminin on the surface. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. The incorporation of 2 μm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration., (Creative Commons Attribution license.)

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.

Small diameter synthetic vascular grafts have high failure rate due to the thrombosis and intimal hyperplasia formation. Compliance mismatch between the synthetic graft and native artery has been speculated to be one of the main causes of intimal hyperplasia. However, changing the compliance of synthetic materials without altering material chemistry remains a challenge. Here, we used poly(vinyl alcohol) (PVA) hydrogel as a graft material due to its biocompatibility and tunable mechanical properties to investigate the role of graft compliance in the development of intimal hyperplasia and in vivo patency. Two groups of PVA small diameter grafts with low compliance and high compliance were fabricated by dip casting method and implanted in a rabbit carotid artery end-to-side anastomosis model for 4 weeks. We demonstrated that the grafts with compliance that more closely matched with rabbit carotid artery had lower anastomotic intimal hyperplasia formation and higher graft patency compared to low compliance grafts. Overall, this study suggested that reducing the compliance mismatch between the native artery and vascular grafts is beneficial for reducing intimal hyperplasia formation.

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.

Stem cell differentiation can be modulated by the substrate topographies. Focal adhesion kinase (FAK) has been identified as a key regulator in topography-induced stem cell mechanotransduction. This chapter will describe a protocol to study the effect of FAK phosphorylation inhibition on topography-mediated stem cell differentiation. The FAK phosphorylation was inhibited using a FAK inhibitor and the effects on stem cell differentiation were examined using western blot and immunofluorescence staining., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Cell sheet technology (CST) is a fascinating scaffoldless tissue engineering technique to generate a physiologically representative tissue replacement from autologous sources. As compared to conventional enzymatic cell harvesting methods, CST enables the preservation of important cell-to-cell junctions and extracellular matrix (ECM) components. However, covalent grafting methods are often employed for CST. In this study, a series of triblock copolymers with a hydrophobic and biocompatible poly[( R )-3-hydroxybutyrate] (PHB) central block flanked by varying lengths of terminal poly( N -isopropylacrylamide) (PNIPAAm) blocks (PNIPAAm-PHB-PNIPAAm) was synthesized via atom transfer radical polymerization of NIPAAm. The thermoresponsive triblock copolymers were explored as a non-covalent surface coating for culturing and detaching bovine corneal endothelial cell (BCEC) sheets. Aqueous solutions of the triblock copolymers produced thermosensitive micelles which can be drop-casted on glass substrates, resulting in a temperature-responsive surface. Importantly, incorporating a central hydrophobic PHB block enabled the anchoring of the coating to the bare substrate and enhanced the proliferation rate of the BCECs studied. Effective detachment of an intact cell sheet was also demonstrated via a cooling treatment at 4 °C for 20 min, and the viability of the detached cell sheet was found to be unaffected by the cooling. This work may potentially inspire more studies involving the non-covalent thermoresponsive polymer coatings for corneal tissue engineering applications.

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.)

Conventional ultrasound with frequency (2-15 MHz) has been a global diagnostic and therapeutic tool in clinical medicine, and high-frequency ultrasound (>30 MHz) has been a powerful investigative device for preclinical studies such as cardiovascular research. In this chapter, we describe the use of conventional ultrasound with a 2.5-10 MHz transducer as an investigative device for the measurement/detection of blood flow in rabbit model. The chapter will describe the procedures for the preparation of sonographer, imaging locations, and the details of the rabbits used as well as detailed imaging steps for the preoperative, immediately after operation, and postoperative follow-up ultrasound for vascular surgery, using a vascular graft implantation as an example. We also provide useful notes to avoid pitfalls for successful imaging. The overall goal of this chapter is to deliver the steps in using low-cost, non-invasive, and highly versatile clinical ultrasound imaging in preclinical small animal testing., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Preclinical testing in animal model is a required stage of vascular device development. Among small animal models, rabbits provide vasculature with relative larger caliber for anastomotic implantation of vascular grafts as preclinical testing before conducting large animal studies. Rabbits have similar hemostatic mechanism with human and can accommodate vascular grafts with various diameters at different locations, and thus provide a valid model to assess small-diameter vascular grafts. This chapter will describe the procedures and materials required to conduct survival surgery in rabbit carotid artery models for implantation of small-diameter tubular grafts with an end-to-side and end-to-end anastomotic technique., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fucoidan is a sulfated polysaccharide with various bioactivities. The application of fucoidan in cancer treatment, wound healing, and food industry has been extensively studied. However, the therapeutic value of fucoidan in cardiovascular diseases has been less explored. Increasing number of investigations in the past years have demonstrated the effects of fucoidan on cardiovascular system. In this review, we will focus on the bioactivities related to cardiovascular applications, for example, the modulation functions of fucoidan on coagulation system, inflammation, and vascular cells. Factors mediating those activities will be discussed in detail. Current therapeutic strategies and future opportunities and challenges will be provided to inspire and guide further research., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Nonviral direct neuronal reprogramming holds significant potential in the fields of tissue engineering and regenerative medicine. However, the issue of low reprogramming efficiency poses a major barrier to its application. We propose that topographical cues, which have been applied successfully to enhance lineage-directed differentiation and multipotent stem cell transdifferentiation, could improve nonviral direct neuronal reprogramming efficiency. To investigate, we used a polymer-BAM (Brn2, Ascl1, Myt1l) factor transfection polypex to reprogram primary mouse embryonic fibroblasts. Using a multiarchitecture chip, we screened for patterns that may improve transfection and/or subsequent induced neuron reprogramming efficiency. Selected patterns were then investigated further by analyzing β-tubulin III (TUJ1) and microtubule-associated protein 2 (MAP2) protein expression, cell morphology and electrophysiological function of induced neurons. Certain hierarchical topographies, with nanopatterns imprinted on micropatterns, significantly improved the percentage of TUJ1+ and MAP2+ cells. It is postulated that the microscale base pattern enhances initial BAM expression while the nanoscale sub-pattern promotes subsequent maturation. This is because the base pattern alone increased expression of TUJ1 and MAP2, while the nanoscale pattern was the only pattern yielding induced neurons capable of firing multiple action potentials. Nanoscale patterns also produced the highest fraction of cells showing spontaneous synaptic activity. Overall, reprogramming efficiency with one dose of polyplex on hierarchical patterns was comparable to that of five doses without topography. Thus, topography can enhance nonviral direct reprogramming of fibroblasts into induced neurons.

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).)

Endothelial dysfunction has been implicated in atherosclerosis, ischemic heart disease, and stroke. Endothelial progenitor cells (EPCs), found in the bone marrow and peripheral blood as rare cell population, demonstrated a high proliferation and differentiation capacity. Understanding how such diseases influence the quantity and functionality of EPCs is essential for the development of novel therapies. This study aims to investigate the factors that affect the quantity and functionality of circulating EPCs in stroke patients and healthy controls. Blood samples were collected once from healthy donors (n = 30) and up to 3 times (within 7 days (baseline), 3 and 12 months post-stroke) from stroke patients (n = 207). EPC subpopulations were isolated with flow cytometry for characterization. The Matrigel tubular formation assay was performed as a measure of functionality. An increased amount of circulating EPCs was observed in stroke patients over 45 years when compared to age-matched healthy individuals. EPCs showed a rising trend in stroke patients over the 12-month post-stroke period, reaching statistical significance at 12 months post-stroke. Isolated CD34 + KDR + cells from stroke patients showed impairment in tubular formation capability when compared to cells from healthy donors. The quantity and vasculogenic function of circulating EPCs in peripheral blood have been effectively evaluated in stroke patients and healthy control donors in this study. Age and stroke are found to be 2 influencing factors on the angiogenic capacity. It is suggested that the increase in EPC number is triggered by the recovery response following ischemic stroke. Graphical abstract.

Topography mediated contact guidance affects multiple cell behaviors such as establishment of cellular morphology and migration. The direction of cell migration is associated with the establishment of cell polarity, which also affects the primary cilia in migrating cells. POPX2, a partner of PIX2, is involved in pathways essential to primary cilium formation, while over-expression of POPX2 has been reported to cause a loss of cell polarity during migration. This study aims to examine how topographical cues direct morphological changes, and how topography affects the process of cellular migration and primary cilium architecture, in the context of POPX2 over-expression. Thus, the effect of anisotropic topography, 2 μm grating pattern on tissue-culture polystyrene, was used as a contact guidance cue to investigate the migration and cell polarity of POPX2 overexpressing cells, in comparison to control NIH3T3 fibroblast cells. We report that POPX2 overexpressing NIH3T3 cells were more sensitive to surface topographical cues as the cells became more elongated. In addition, these cues also affected focal adhesion alignment of POPX2 overexpressing cells. Cell migration was further studied using wound closure assays, in which the 2 μm gratings were designed to be either perpendicular or parallel to wound-induced cell migration direction, which would be agonistic or antagonistic to cell migration, respectively. We observed that both POPX2 overexpressing cells' migration direction and migration rate were more significantly influenced by gratings direction compared to control NIH3T3 cells. The migration paths of POPX2 overexpressing cells become more direct in the presence of anisotropic topographical cues. Further, cilia and centrosome alignment, which is important in cell migration, was also affected by the direction of gratings during this migration process. Collectively, enhancement of NIH3T3 cell sensitivity towards surface topography through POPX2 overexpression might reflect one of the mechanisms that combine biochemical and mechanical cues for directional cell migration.

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.)

Gelatin methacryloyl (GelMA) hydrogel scaffolds and GelMA-based bioinks are widely used in tissue engineering and bioprinting due to their ability to support cellular functions and new tissue development. Unfortunately, while terminal sterilization of the GelMA is a critical step for translational tissue engineering applications, it can potentially cause thermal or chemical modifications of GelMA. Thus, understanding the effect of terminal sterilization on GelMA properties is an important, though often overlooked, aspect of material design for translational tissue engineering applications. To this end, we characterized the effects of FDA-approved terminal sterilization methods (autoclaving, ethylene oxide treatment, and gamma (γ)-irradiation) on GelMA prepolymer (bioink) and GelMA hydrogels in terms of the relevant properties for biomedical applications, including mechanical strength, biodegradation rate, cell culture in 2D and 3D, and printability. Autoclaving and ethylene oxide treatment of the GelMA decreased the stiffness of the hydrogel, but the treatments did not modify the biodegradation rate of the hydrogel; meanwhile, γ-irradiation increased the stiffness, reduced the pore size and significantly slowed the biodegradation rate. None of the terminal sterilization methods changed the 2D fibroblast or endothelial cell adhesion and spreading. However, ethylene oxide treatment significantly lowered the fibroblast viability in 3D cell culture. Strikingly, γ-irradiation led to significantly reduced ability of the GelMA prepolymer to undergo sol-gel transition. Furthermore, printability studies showed that the bioinks prepared from γ-irradiated GelMA had significantly reduced printability as compared to the GelMA bioinks prepared from autoclaved or ethylene oxide treated GelMA. These results reveal that the choice of the terminal sterilization method can strongly influence important properties of GelMA bioink and hydrogel. Overall, this study provides further insight into GelMA-based material design with consideration of the effect of terminal sterilization.

This study investigated the effects of terminal sterilization of polyvinyl alcohol (PVA) biomaterials using clinically translatable techniques, specifically ethylene oxide (EtO) and gamma (γ) irradiation. While a few studies have reported the possibility of sterilizing PVA with γ-radiation, the use of EtO sterilization of PVA requires additional study. PVA solutions were chemically crosslinked with trisodium trimetaphosphate and sodium hydroxide. The three experimental groups included untreated control, EtO, and γ-irradiation, which were tested for the degree of swelling and water content, and mechanical properties such as radial compliance, longitudinal tensile, minimum bend radius, burst pressure, and suture retention strength. In addition, samples were characterized with scanning electron microscopy, differential scanning calorimetry, X-ray photoelectron spectroscopy, and water contact angle measurements. Cell attachment was assessed using the endothelial cell line EA.hy926, and the sterilized PVA cytotoxicity was studied with a live/dead stain. Platelet and fibrin accumulation was measured using an ex vivo shunt baboon model. Finally, the immune responses of PVA implants were analyzed after a 21-day subcutaneous implantation in rats and a 30-day implantation in baboon. EtO sterilization reduced the PVA graft wall thickness, its degree of swelling, and water content compared with both γ-irradiated and untreated PVA. Moreover, EtO sterilization significantly reduced the radial compliance and increased Young's modulus. EtO did not change PVA hydrophilicity, while γ-irradiation increased the water contact angle of the PVA. Consequently, endothelial cell attachment on the EtO-sterilized PVA showed similar results to the untreated PVA, while cell attachment significantly improved on the γ-irradiated PVA. When exposing the PVA grafts to circulating whole blood, fibrin accumulation of EtO-sterilized PVA was found to be significantly lower than γ-irradiated PVA. The immune responses of γ-irradiated PVA, EtO-treated PVA, and untreated PVA were compared. Implanted EtO-treated PVA showed the least MAC387 reaction. The terminal sterilization methods in this study changed PVA hydrogel properties; nevertheless, based on the characterizations performed, both sterilization methods were suitable for sterilizing PVA. We concluded that EtO can be used as an alternative method to sterilize PVA hydrogel material. Impact statement Polyvinyl alcohol (PVA) hydrogels have been used for a variety of tissue replacements, including neural, cardiac, meniscal, cartilage, muscle, pancreatic, and ocular applications. In addition, PVA can be made into a tubular shape and used as a small-diameter vascular graft. Ethylene oxide (EtO) is one of the Food and Drug Administration-approved methods for sterilization, but its effect on PVA has not been studied extensively. The outcome of this study provides the effects of EtO and γ-irradiation of PVA grafts on both the material properties and the in vivo responses, particularly for vascular applications. Knowledge of these effects may ultimately improve the success rate of PVA vascular grafts.

Recurrent corneal erosion (RCE) is a condition where the superficial corneal epithelial cells sporadically shed because of poor attachment to the underlying basement membrane, resulting in pain and discomfort. RCE-afflicted corneas exhibit elevated biological factors and enzymes, such as matrix metalloproteinase (MMP)-9. Soft bandage contact lenses (BCLs) are a commonly recommended treatment as they permit continued vision during recovery. However, they do not outperform alternative treatments as they currently lack active ingredients for ocular surface repair. Development of BCLs with a sustained release of active ingredients to promote RCE wound healing over an extended period of time would be clinically valuable. We fabricated gelatin methacrylate (GelMA) hydrogels with MMP-9-triggered controlled release for further BCL development. GelMA disks were characterized to determine the optimal concentration and methacrylation degree for sustained release in the presence of MMP-9. The chosen active ingredient, 60 kDa hyaluronic acid (HA), was verified to promote wound healing in rabbit corneal epithelial cell and human corneal epithelial cell wound assays. The active ingredient was loaded into GelMA hydrogels, and the bioavailability of the vehicle was determined. The GelMA hydrogels revealed a tunable profile for diffusion and enzyme-mediated controlled release of a model molecule. The 60 kDa HA-loaded GelMA disks showed improvement in wound healing at HA loadings of 150 and 250 μg by improving the wound healing rates by 28% (68.7% wound closure) and 26% (67.9% wound closure), respectively, compared to the control (50.5% wound closure) at 24 h. The HA-loaded GelMA disks with 150 and 250 μg HA also exhibited shortened wound closure time of 75 h compared to >147 h with a bolus daily delivery of HA for wound closure. Overall, we demonstrated that the fabricated GelMA hydrogels offered an enzyme-triggered option as a sustained release material for future RCE treatments.

Despite much effort, synthetic small diameter vascular grafts still face limited success due to vascular wall thickening known as intimal hyperplasia (IH). Compliance mismatch between graft and native vessels has been proposed to be one of a key mechanical factors of synthetic vascular grafts that could contribute to the formation of IH. While many methods have been developed to determine compliance both in vivo and in vitro, the effects of compliance mismatch still remain uncertain. This review aims to explain the biomechanical factors that are responsible for the formation and development of IH and their relationship with compliance mismatch. Furthermore, this review will address the current methods used to measure compliance both in vitro and in vivo. Lastly, current limitations in understanding the connection between the compliance of vascular grafts and the role it plays in the development and progression of IH will be discussed.

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.)

Cardiovascular diseases remain the leading cause of death worldwide. Patency rates of clinically-utilized small diameter synthetic vascular grafts such as Dacron® and expanded polytetrafluoroethylene (ePTFE) to treat cardiovascular disease are inadequate due to lack of endothelialization. Sodium trimetaphosphate (STMP) crosslinked PVA could be potentially employed as blood-compatible small diameter vascular graft for the treatment of cardiovascular disease. However, PVA severely lacks cell adhesion properties, and the efforts to endothelialize STMP-PVA have been insufficient to produce a functioning endothelium. To this end, we developed a one-pot method to conjugate cell-adhesive protein via hydroxyl-to-amine coupling using carbonyldiimidazole by targeting residual hydroxyl groups on crosslinked STMP-PVA hydrogel. Primary human umbilical vascular endothelial cells (HUVECs) demonstrated significantly improved cells adhesion, viability and spreading on modified PVA. Cells formed a confluent endothelial monolayer, and expressed vinculin focal adhesions, cell-cell junction protein zonula occludens 1 (ZO1), and vascular endothelial cadherin (VE-Cadherin). Extensive characterization of the blood-compatibility was performed on modified PVA hydrogel by examining platelet activation, platelet microparticle formation, platelet CD61 and CD62P expression, and thrombin generation, which showed that the modified PVA was blood-compatible. Additionally, grafts were tested under whole, flowing blood without any anticoagulants in a non-human primate, arteriovenous shunt model. No differences were seen in platelet or fibrin accumulation between the modified-PVA, unmodified PVA or clinical, ePTFE controls. This study presents a significant step in the modification of PVA for the development of next generation in situ endothelialized synthetic vascular grafts.

Bare metal stenting (BMS) does not adequately address the atheroembolic characteristic of carotid artery stenosis. While simple covered stents (CS) may prevent dislodged fragments of the atherosclerotic plaque from entering the blood stream, they also block blood flow into the major branches of the artery alongside the lesion, which is not desirable. Preferential covered stents (PCS) behave as a covered stent in a tubular part of a vessel but maintain side-branch flow over the bifurcation region by means of slits in the membrane. Stent design, membrane material, and slits configuration are the three main components contributing to stent performance. Optimization of PCS designs was conducted and tested., Methods: A newly designed BMS was developed and compared to a commercially available peripheral stent. Two materials (expanded poly(tetrafluoroethylene)) and silicone polyurethane co-polymers (Elast-eon E2A) were used as stent coverings with slits applied using various cutting methods to form the PCS. These PCS samples were tested for physical resilience, flexibility, ability to preserve side-branch flow, slit edge roughness, and platelet activation., Results: Fabrication of E2A-coated stents required pretreatment of the stent with poly(ethylene glycol) to achieve firm attachment. The newly designed BMS with nine crowns design and larger cell size showed higher flexibility than commercially available stents. A combination of a larger stent cell size, E2A membrane coating, and three slits per stent cell unit configuration resulted in preserved side-branch flow similar to physiological conditions in the flow experiment. Slit edge roughness changed with different cutting methods and laser machine cutting parameters. In vitro studies showed platelet activation was minimal with lower slit edge roughness samples., Conclusion: An optimized PCS prototype was developed consisting of a newly designed stent, E2A membrane, and a three-slit pattern created by specific femtosecond laser cutting.

The skin and cornea are tissues that provide protective functions. Trauma and other environmental threats often cause injuries, infections and damage to these tissues, where the degree of injury is directly correlated to the recovery time. For example, a superficial skin or corneal wound may recover within days; however, more severe injuries can last up to several months and may leave scarring. Thus, therapeutic strategies have been introduced to enhance the wound healing efficiency and quality. Although the skin and cornea share similar anatomic structures and wound healing process, therapeutic agents and formulations for skin and cornea wound healing differ in accordance with the tissue and wound type. In this review, we describe the anatomy and epithelial wound healing processes of the skin and cornea, and summarize the therapeutic molecules that are beneficial to the respective regeneration process. In addition, biomaterial scaffolds that inherently possess bioactive properties or modified with therapeutic molecules for topical controlled release and enhanced wound healing efficiency are also discussed.

The mechanical microenvironment serves as an important factor influencing stem cell differentiation. Mechanobiological responses depend strongly on actomyosin contractility and integrin-based cell-extracellular matrix (ECM) interactions mediated by adhesive structures such as focal adhesions (FAs). While the roles of FAs in mechanobiology have been intensively studied in many mesenchymal and migratory cell types, recently it has been recognized that certain pluripotent stem cells (PSCs) exhibited significantly attenuated FA-mediated mechanobiological responses. FAs in such PSCs are sparsely distributed and much less prominent in comparison to "classical" FAs of typical adherent cells. Despite these differences, insights into how FAs in PSCs are structurally organized to perform their functions are still elusive. Using mouse embryonic stem cells (mESCs) to study PSC-ECM interactions, here we surveyed the molecular composition and nanostructural organization of FAs. We found that, despite being small in size, mESC FAs appeared to be compositionally mature, containing markers such as vinculin, zyxin, and α-actinin, and dependent on myosin II contractility. Using super-resolution microscopy, we revealed that mESC FAs were organized into a conserved multilayer nanoscale architecture. However, the nanodomain organization was compressed in mESCs, with the force transduction layer spanning ∼10 nm, significantly more compact than in FAs of other cell types. Furthermore, we found that the position and orientation of vinculin, a key mechanotransduction protein, were modulated in an ECM-dependent manner. Our analysis also revealed that while most core FA genes were expressed, the expression of LIM domain proteins was comparatively lower in PSCs. Altogether our results suggest that while core structural and mechanosensitive elements are operational in mESC FAs, their structural organization and regulatory aspects may diverge significantly from "classical" FAs, which may account for the attenuated mechanobiological responses of these cell types.

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.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications., (© 2018 Wiley Periodicals, Inc.)

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.)

Bulk material properties and luminal surface interaction with blood determine the clinical viability of vascular grafts, and reducing intimal hyperplasia is necessary to improve their long-term patency. Here, the authors report that the surface of a biocompatible hydrogel material, poly(vinyl alcohol) (PVA) can be altered by exposing it to reactive ion plasma (RIP) in order to increase primary endothelial cell attachment. The power and the carrier gas of the RIP treatment are varied and the resultant surface nitrogen, water contact angle, as well as the ability of the RIP-treated surfaces to support primary endothelial colony forming cells is characterized. Additionally, in a clinically relevant shunt model, the amounts of platelet and fibrin attachment to the surface were quantified during exposure to non-anticoagulated blood. Treatments with all carrier gases resulted in an increase in the surface nitrogen. Treating PVA with O 2 , N 2 , and Ar RIP increased affinity to primary endothelial colony forming cells. The RIP treatments did not increase the thrombogenicity compared to untreated PVA and had significantly less platelet and fibrin attachment compared to the current clinical standard of expanded polytetrafluoroethylene (ePTFE). These findings indicate that RIP-treatment of PVA could lead to increased patency in synthetic vascular grafts., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.)

While clinical vascular grafting uses an end-to-side surgical method, researchers primarily use end-to-end implant techniques in preclinical models. This may be due in part to the limitations of using small animal models in research. The work presented here provides support and evidence for the improvement of vascular graft implant techniques by demonstrating the successful implantation of experimental grafts into both large and small animal models. Specifically, models of aortoiliac baboon (Papio anubis) bypass and common carotid rabbit (Oryctolagus cuniculus) bypass were used to test vascular grafts for thrombosis and vascular healing after 1 month using an end-to-side anastomosis grafting procedure. Patency was evaluated with ultrasound or histological techniques, and neointimal growth was quantified with histology. In the development of this procedure for small animals, both an end-to-end/end-to-side and an end-to-side/end-to-side configuration were tested in rabbits. One hundred percent of rabbit implants (2/2) with an end-to-end/end-to-side configuration were patent at explant. However, with the end-to-side/end-to-side configuration, 66% (6/9) of rabbit implants and 93% (13/14) of baboon implants remained patent at 1 month, suggesting the importance of replicating the end-to-side method for testing vascular grafts for clinical use. This study describes feasible preclinical surgical procedures, which simulate clinical vascular bypass grafts even in small animals. Widespread implementation of these end-to-side surgical techniques in these or other animals should improve the quality of experimental, preclinical testing and ultimately increase the likelihood of translating new vascular graft technologies into clinical applications.

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.

Studies of electrical stimulation therapies for the treatment of neurological disorders, such as deep brain stimulation, have almost exclusively been performed using animal-models. However, because animal-models can only approximate human brain disorders, these studies should be supplemented with an in vitro human cell-culture based model to substantiate the results of animal-based studies and further investigate therapeutic benefit in humans. This study presents a novel approach to analyze the effect of electrical stimulation on the neurogenesis of patient-induced pluripotent stem cell (iPSC) derived neural progenitor cell (NPC) lines, in vitro using a 3D graphene scaffold system. The iPSC-derived hNPCs used to demonstrate the system were collected from patients with Rett syndrome, a debilitating neurodevelopmental disorder. The graphene scaffold readily supported both the wild-type and Rett NPCs. Electrical stimulation parameters were optimized to accommodate both wild-type and Rett cells. Increased cell maturation and improvements in cell morphology of the Rett cells was observed after electrical stimulation. The results of the pilot study of electrical stimulation to enhance Rett NPCs neurogenesis were promising and support further investigation of the therapy. Overall, this system provides a valuable tool to study electrical stimulation as a potential therapy for neurological disorders using patient-specific cells.

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.)

Previous studies have shown that fundamental cell functions such as adhesion, proliferation, and morphology are regulated by the interaction of cells with basement membrane nano- and micron- scale surface topography. By taking the basement membrane as a guiding principle, the surface can be designed with biophysical cues in the form of surface topographies to modulate the cellular functions of vascular endothelial and smooth muscle cells, which are crucial for vascular diseases. Titanium-based materials whose surfaces are covered by a thin layer of titanium dioxide (TiO 2 ) are utilized in several regenerative medicine applications such as vascular prosthetics. The utilization of TiO 2 -covered materials makes it essential to understand the interaction of cells with the TiO 2 layer to control the cell response. While it has been shown by means of patterned micro- and nano-topography that it is important to regulate cell functions on non-metallic materials, it would be of interest in the field of regenerative medicine to study the cell response on patterned TiO 2 surface layers. Previous studies have mostly focused on studying the cell response on random micro- and nano-roughened metallic and metal oxide surfaces as it is challenging to fabricate patterned TiO 2 surface layers. Here, the biocompatibility of a method that is capable of the rational patterning of a continuous TiO 2 surface layer of sub-100 nm resolution scale using thermally curable resin-based nanoimprinting was studied. The responses of human umbilical vein endothelial cell (HUVEC), such as proliferation, morphology and focal adhesions, and smooth muscle cell (SMC) proliferation and morphology to the nano-patterned TiO 2 layer were investigated. Overall, the TiO 2 layer with surface nano-gratings showed enhanced proliferation of HUVECs, while it significantly lowered the proliferation of SMCs as compared to the unpatterned control. The HUVECs and SMCs were shown by topography to be sensitive to the 70 nm gratings as evident by the regulation of proliferation and cell shape. A significantly lower focal adhesion density was found of HUVECs on TiO 2 nano-gratings, while a significantly higher average focal adhesion size of HUVECs was seen on TiO 2 nano-wells and nano-gratings, compared to the unpatterned controls.