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Supportive stromal cells of mesenchymal origins regulate vascular morphogenesis in developmental, pathological, and regenerative contexts, contributing to vessel formation, maturation, and long-term stability, in part via the secretion of bioactive molecules. In this work, we adapted a microfluidic lab-on-a-chip system that enables the formation and perfusion of microvascular capillary beds with connections to arteriole-scale endothelialized channels to explore how stromal cell (SC) identity influences endothelial cell (EC) morphogenesis. We compared and contrasted lung fibroblasts (LFs), dermal fibroblasts (DFs), and bone marrow-derived mesenchymal stem cells (MSCs) for their abilities to support endothelial morphogenesis and subsequent perfusion of microvascular networks formed in fibrin hydrogels within the microfluidic device. We demonstrated that while all 3 SC types supported EC morphogenesis, LFs in particular resulted in microvascular morphologies with the highest total network length, vessel diameter, and vessel interconnectivity across a range of SC-EC ratio and density conditions. Not only did LFs support robust vascular morphology, but also, they were the only SC type to support functional perfusion of the resultant capillary beds. Lastly, we identified heightened traction stress produced by LFs as a possible mechanism by which LFs enhance endothelial morphogenesis in 3D compared to other SC types examined. This study provides a unique comparison of three different SC types and their role in supporting the formation of microvasculature that could provide insights for the choice of cells for vascular cell-based therapies and the regulation of tissue-specific vasculature.

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

During normal development, the extracellular matrix (ECM) regulates cell fate mechanically and biochemically. However, the ECM's influence on lineage reprogramming, a process by which a cell's developmental cycle is reversed to attain a progenitor-like cell state followed by subsequent differentiation into a desired cell phenotype, is unknown. Using a material mimetic of the ECM, here we show that ligand identity, ligand density, and substrate modulus modulate indirect cardiac reprogramming efficiency, but were not individually correlated with phenotypic outcomes in a predictive manner. Alternatively, we developed a data-driven model using partial least squares regression to relate short-term cell states, defined by quantitative mechanosensitive responses to different material environments, with long-term changes in phenotype. This model was validated by accurately predicting the reprogramming outcomes on a different material platform. Collectively, these findings suggest a means to rapidly screen candidate biomaterials that support reprogramming with high efficiency, without subjecting cells to the entire reprogramming process.

A major translational challenge in the fields of therapeutic angiogenesis and regenerative medicine is the need to create functional microvasculature. The purpose of this study was to assess whether a potentially autologous endothelial cell (EC) source derived from human induced pluripotent stem cells (iPSC-ECs) can form the same robust, stable microvasculature as previously documented for other sources of ECs. We utilized a well-established in vitro assay, in which endothelial cell-coated (iPSC-EC or HUVEC) beads were co-embedded with fibroblasts in a 3D fibrin matrix to assess their ability to form stable microvessels. iPSC-ECs exhibited a five-fold reduction in capillary network formation compared to HUVECs. Increasing matrix density reduced sprouting, although this effect was attenuated by distributing the NHLFs throughout the matrix. Inhibition of both MMP- and plasmin-mediated fibrinolysis was required to completely block sprouting of both HUVECs and iPSC-ECs. Further analysis revealed MMP-9 expression and activity were significantly lower in iPSC-EC/NHLF co-cultures than in HUVEC/NHLF co-cultures at later time points, which may account for the observed deficiencies in angiogenic sprouting of the iPSC-ECs. Collectively, these findings suggest fundamental differences in EC phenotypes must be better understood to enable the promise and potential of iPSC-ECs for clinical translation to be realized.

Matrix stiffness is a well-established instructive cue in two-dimensional cell cultures. Its roles in morphogenesis in 3-dimensional (3D) cultures, and the converse effects of cells on the mechanics of their surrounding microenvironment , have been more elusive given the absence of suitable methods to quantify stiffness on a length-scale relevant for individual cell-extracellular matrix (ECM) interactions. In this study, we applied traditional bulk rheology and laser tweezers-based active microrheology to probe mechanics across length scales during the complex multicellular process of capillary morphogenesis in 3D, and further characterized the relative contributions of neovessels and supportive stromal cells to dynamic changes in stiffness over time. Our data show local ECM stiffness was highly heterogeneous around sprouting capillaries, and the variation progressively increased with time. Both endothelial cells and stromal support cells progressively stiffened the ECM, with the changes in bulk properties dominated by the latter. Interestingly, regions with high micro-stiffness did not necessarily correlate with remodeled regions of high ECM density as shown by confocal reflectance microscopy. Collectively, these findings, especially the large spatiotemporal variations in local stiffness around cells during morphogenesis in soft 3D fibrin gels, underscore that characterizing ECM mechanics across length scales. provides an opportunity to attain a deeper mechanobiological understanding of the microenvironment's roles in cell fate and tissue patterning.

The fate of adult stem cells can be influenced by physical cues, including nanotopography. However, the response of human embryonic stem cells (hESCs) to dimensionally well-defined nanotopography is unknown. Using imprint lithography, we prepared well-defined nanotopography of hexagonal (HEX) and honeycomb (HNY) configurations with various spacings between the nanostructures. In serum-free hESC culture medium, basic fibroblast growth factor (bFGF) is required to maintain expression of Oct4, a pluripotent gene. Unexpectedly, hESCs cultured on nanotopography could maintain Oct4 expression without bFGF supplementation. With bFGF supplementation, the HEX nanotopography maintained Oct4 expression whereas the HNY configuration caused down-regulation of Oct4 expression. Thus, we observed that the lattice configurations of the nanotopography cause hESCs to respond to bFGF in different ways. This differential response to a biochemical cue by nanotopography was unforeseen, but its discovery could lead to novel differentiation pathways. Consistent with studies of other cells, we observed that nanotopography affects focal adhesion formation in hESCs. We posit that this can in turn affect cell–matrix tension, focal adhesion kinase signaling and integrin–growth factor receptor crosstalk, which eventually modulates Oct4 expression in hESCs.

Relaxation and chain dynamics of polymer nanostructures after release of spatial confinement were studied using line gratings as small as 33 nm on polystyrene surface fabricated by nanoimprint lithography. The temperature for “slumping”—rapid line height relaxation—decreased as the line width diminished for all molecular masses (MWs), but a simple explanation based on enhanced surface mobility fails to explain the results. When MW was low and the structure was large, the line height monitored with an AFM reduced as surface tension overcame the polymer viscosity. Interesting and complex behaviors were observed when the radius of gyration (Rg) of the polymer molecules was not small compared with the dimension of the nanostructure. Careful examination of the surface viscosity shows that confinement of polymer chains into space comparable to or even smaller than its Rg appears to enhance relaxation, which is the major factor for the surprisingly low temperature at which nanostructures of high MW slumps.

This article describes results on using steered molecular dynamics (SMD) simulations and experimental single molecule force spectroscopy (SMFS) to investigate the relationship between hydrogen bonding and mechanical stability of a series of homodimeric β-sheet mimics. The dimers consisting of 4, 6, and 8 H-bonding sites were modeled in explicit chloroform solvent and the rupture force was studied using constant velocity SMD. The role of solvent structuring on the conformation of the dimers was analyzed and showed no significant contribution of chloroform molecules in the rupture event. The simulated stability of the dimers was validated by force data obtained with atomic force microscopy (AFM)-based SMFS in toluene. The computational model for the 8H dimer also offered insight into a possible mismatched dimer intermediate that may contribute to the lower than expected mechanical stability observed by single molecule AFM force studies. In addition, atomic level analysis of the rupture mechanism verified the dependence of mechanical strength on pulling trajectory due to the directional nature of chemical bonding under an applied force. The knowledge gained from this basic study will be used to guide further design of modular polymers having folded nanostructures through strategic programming of weak, non-covalent interactions into polymer backbones.

A soft inkpad imprinting technique to produce stacked micrometer and submicrometer polymer patterns on substrates is presented. A thin soft inkpad is used to coat a polymer film onto the protrusions of a surface treated hard mold. The polymer film on the protrusions of the hard mold is then transferred to a substrate. Simultaneously, a negative pattern of the hard mold is formed on the soft inkpad that may also be transferred to a substrate. Numerical simulations are used to study the mechanisms of pattern transfer by soft inkpad imprinting. With the use of polymer blends, both positive and negative polymeric gratings with 700 nm period were produced. The soft inkpad allows multiple transfers of polymers with similar solubilities to the hard mold since no chemical solution is used for coating. High aspect ratio polymer stacks can be formed without alignment. This capability is an important advantage when forming submicrometer and nanometer multiple-layered polymer structures because current nanoimprint sy...

The chemical, mechanical, and topographical features of the extracellular matrix (ECM) have all been documented to influence cell adhesion, gene expression, migration, proliferation, and differentiation. Topography plays a key role in the architecture and functionality of various tissues in vivo, thus raising the possibility that topographic cues can be instructive when incorporated into biomaterials for regenerative applications. In the literature, there are discrepancies regarding the potential roles of nanotopography to enhance the osteogenic phenotype of mesenchymal stem cells (MSC). In this study, we used thin film substrates of poly(methyl methacrylate) (PMMA) with nanoscale gratings to investigate the influence of nanotopography on the osteogenic phenotype of MSCs, focusing in particular on their ability to produce mineral similar to native bone. Topography influenced focal adhesion size and MSC alignment, and enhanced MSC proliferation after 14 days of culture. However, the osteogenic phenotype was minimally influenced by surface topography. Specifically, alkaline phosphatase (ALP) expression was not increased on nanotopographic films, nor was calcium deposition improved after 21 days in culture. Ca: P ratios were similar to native mouse bone on films with gratings of 415 nm width and 200 nm depth (G415) and 303 nm width and 190 nm depth (G303). Notably, all surfaces had Ca∶P ratios significantly lower than G415 films. Collectively, these data suggest that, PMMA films with nanogratings are poor drivers of an osteogenic phenotype.

We have developed a new method to pattern polymeric materials, including non-thermoplastic polymers, at low temperature and low pressure. In this method, plasticizers are added to increase the chain mobility of the polymers, resulting in lower imprinting temperature and/or pressure. Two established imprinting and transfer techniques were chosen to demonstrate this method, namely, conventional nanoimprint lithography (NIL) and microcontact printing (μCP). These two techniques were used to pattern poly(3,4-ethylenedioxythiophene) (PEDOT). PEDOT was chosen because it is a non-thermoplastic polymer and therefore cannot be easily patterned using conventional NIL. Successful imprint of PEDOT films from the PDMS mold was achieved at a low pressure of 10 kPa and 25°C by controlled addition of glycerol as a plasticizer using conventional NIL; well-defined arrays of 2μm wide, 185 nm high PEDOT dots have also been demonstrated by μCP. In contrast, patterning of PEDOT film without plasticizer requires higher temperature (80°C) and pressure (10 MPa), which could cause severe deformation of the transferred patterns. This method of plasticizer-assisted imprint lithography (PAIL) broadens the applicapability of NIL to a wide range of polymeric materials.

This paper reports a process used for the microfabrication of an array of hollow microneedles. The purpose of the array is for painless transdermal drug delivery. The fabrication process uses wet bulk silicon technology and copper electroplating technology. First, a microneedle array mold on -oriented silicon was fabricated by wet anisotropic etching using KOH solution, then the silicon mold was electroplated with copper. After which, the hollow copper microneedle array was released by a lift-off process or by etching off the silicon mold in KOH solution. The hollow copper microneedle array has been mounted on a polycarbonate platform, which consist of laser ablated cavities and channel for external connection to drug source. In consideration of the contour of human’s skin and the geometry of the microneedle tip, which has walls of sloping gradient corresponding to the (111)-planes, the height of the microneedle array is 200 μm. Two arrays of hollow copper microneedle were fabricated. They have square base of dimensions 390 μm and 400 μm and square tips of size 100 μm and 120 μm with square holes of size 88 μm and 94 μm respectively. Both arrays have microneedle tips at 1900 μm apart from one another and consist of 10 × 10 microneedle tips.

A 3D model of one type of micro pumps was supposed and analyzed using finite element method (FEM). The pump had square shape cavity and was driven by a square shape PZT component. The finite element analysis (FEA) took into consideration of the effects of PZT component dimensions, membrane thickness, pump chamber pressure and other geometric parameters. Modal analyses were also conducted. Compression ratio of the pump chamber was taken as the prime parameter for the analyses. It was found that the membrane thickness and the PZT plate thickness played major roles in determining the compression ratio. For each membrane thickness, there was always an optimum PZT plate thickness that gave the maximum compression ratio. Curves showing the relationship between the optimum PZT plate thickness and the membrane thickness at different chamber pressures were given, based on the FEA results. A set of optimum pump design parameters was proposed.

We have developed a method to pattern polymeric materials, including nonthermoplastic polymers and biomaterials, at low temperature and low pressure. In this method, plasticizers are added to increase the chain mobility of the polymers, resulting in lower imprinting temperature and/or pressure. Three established imprinting and transfer techniques were chosen to demonstrate this method: conventional nanoimprint lithography (NIL), microcontact printing (μCP), and soft ink-pad (SIP). These three techniques were used to pattern poly(3,4-ethylenedioxythiophene) (PEDOT) and chitosan. PEDOT and chitosan were chosen because both of them are nonthermoplastic polymer and therefore cannot be easily patterned using conventional NIL. Imprinting of PEDOT and chitosan films from the poly(dimethylsiloxane) mold was achieved at a low pressure of 10kPa and 25°C by controlled addition of glycerol as a plasticizer using conventional NIL; well-defined arrays of 2μm wide, 185nm high PEDOT dots have also been demonstrated by μC...

Poly(3,4-ethylenedioxythiophene) (PEDOT) and chitosan structures on Si, patterned by plasticizer-assisted imprint lithography (PAIL), are examined under a variety of imprinting conditions. The stabilities of pattern dimension and chemical functionality of these polymers are presented. Thermal annealing for 5min at 80°C is found to be an effective method to stabilize imprinted PEDOT patterns. Biofunctionality in chitosan as a function of imprint temperature and pressure is examined through fluorescence spectroscopy. The accessibility of the amine group of chitosan is observed to decrease for imprint temperatures above 80°C, whereas the chemical functionality is not affected by pressure up to 1MPa. Fluorescence spectra of the chitosan are observed to be strong functions of exposure time to O2 plasma.

We present a new method of imprinting three dimensional (3D) polymeric micro- and nanostructures using a duo-mold process. In this method, two patterned Si molds are surface treated to have different surface energies. A polymer solution is spin-coated onto one of the molds, forming a planarized thin film on the mold. The two molds are pressed together at an appropriate temperature and pressure and then released. The patterned thin film adhering to one of the molds is then pressed onto a substrate at an appropriate temperature and pressure to form supported 3D structures. Alternatively, the patterned thin film on the mold may be released to form freestanding 3D structures. A key success factor in this process is the silane-based surface treatments to enable the patterning of the thin polymer film and final release from the mold to form 3D structures. We demonstrate various polymethyl methacrylate 3D structures with combinations of grating, circular-, and square-patterned molds. The duo-mold process is a po...

A polymer inking technique was developed to form micro- and nanopatterns on a substrate. In this process, a polymer thin film is spin coated on a patterned mold. After contacting the substrate at a suitable temperature and pressure, the polymer on the protruded surfaces of the mold is transferred to the substrate and a positive image of the mold is obtained. A selective surface treatment method has been developed to improve the edge smoothness of the inked pattern. During selective surface treatment, the protruded surfaces of the mold are first treated with a flat poly(dimethylsiloxane) stamp impregnated with a silane that has medium surface energy. The mold is then immersed into the solution of another silane with very low surface energy to treat the trenches of the mold. Because the surface energy of the sidewalls is lower than that on the protrusions, polymer dewetting from the sidewalls is promoted, which makes the polymer film discontinuous along the edges of patterns. Therefore, inked polymer patter...