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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.

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.

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.

Heart regeneration through in vivo cardiac reprogramming has been demonstrated as a possible regenerative strategy. While it has been reported that cardiac reprogramming in vivo is more efficient than in vitro, the influence of the extracellular microenvironment on cardiac reprogramming remains incompletely understood. This understanding is necessary to improve the efficiency of cardiac reprogramming in order to implement this strategy successfully. Here we have identified matrix identity and cell-generated tractional forces as key determinants of the dedifferentiation and differentiation stages during reprogramming. Cell proliferation, matrix mechanics, and matrix microstructure are also important, but play lesser roles. Our results suggest that the extracellular microenvironment can be optimized to enhance cardiac reprogramming.

Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis.

New regenerative paradigms are needed to address the growing global problem of heart failure as existing interventions are unsatisfactory. Outcomes from the current paradigm of cell transplantation have not been stellar but the mechanistic knowledge learned from them is instructive in the development of future paradigms. An emerging biomaterial-based approach incorporating key mechanisms and additional ones scrutinized from the process of natural heart regeneration in zebrafish may become the next evolution in cardiac repair. We highlight, with examples, tested key concepts and pivotal ones that may be integrated into a successful therapy.

We describe the development of a technique for making indentations on the top 5‐20 nm of the surfaces of relatively low modulus materials using a high spatial and force sensitivity atomic force microscope (AFM) whose optical cantilever has been replaced by a quartz crystal resonator (QCR). Unlike conventional optical-cantilever-based AFMs, the accuracy of this technique is not compromised by the compliance of the loading system due to the high stiffness of the QCR. To obtain material modulus values from the indentation results, we find the commonly used Oliver‐Pharr model to be unsuitable because of our use of a sharp tip and relatively deep indentation. Instead, we develop a new analysis that may be more appropriate for the geometry we use as well as the non-linear constitutive behavior exhibited by the materials we examined. We calculated values for the moduli of several different materials, which we find to be consistent with the range of published data.

Imprint lithography has become a potential next generation lithography technique for the microelectronics industry. We have developed new imprint lithography techniques that fabricate three-dimensional polymeric nanostructures so that imprint lithography can create a larger impact in other areas of science and technology. We have also studied the stability of imprinted polymeric nanostructures and found that the stability is reduced significantly as the feature size decreases. The cause is probably the interplay between residual stress and viscosity of the polymer at the relaxation temperature and our preliminary results clamor for more studies in this area.

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.

For effective pulmonary drug delivery of insulin for example, drug particles must be in the range of 1 to 5 microns. A piezoelectrically actuated MEMS atomizer based on Rayleigh instability-driven breakup of filaments has been designed to produce drug particles in this range. Although the formation of droplets from jets have been used extensively in ink-jet printing, the currently presented mode of droplet formation has not yet been demonstrated by any MEMS device. Testing of the vaporiser reveals that the droplets generated lie primarily in the range of 1.0 through 3.0 microns, a range that covers the designed droplet size of 2.5 microns. We thus show that it is possible to implement this mode of droplet generation that will achieve better device efficiency.

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 methodology for the simulation of a reciprocating displacement micro-pump is presented. First a check valve model was analyzed using coupled FEM to obtain the characteristics relationship between flow rate and the pressure as well as the minimum valve opening pressure. Then a model for the micro-pump actuator driven by PZT disks is proposed and simulated. The pump model takes into account the effects of chamber pressure and geometrical parameters. The maximum downward deflection of the actuating membrane is taken as the target parameter to analyze. It was found that the maximum membrane deflection could reach over 10micrometers microns, much larger than the radial displacement. This 'displacement amplification' is the underlying working principle of this kind of micro-pump. Quantitative analyses of the effects of various factors on the deflection are conducted. It is found that the thickness of the membrane has the biggest influence on the deflection. For each membrane thickness, there exists an op[t9kum PZT disk thickness that gives the maximum deflection at a particular electric field. Other factors with less influence on the deflection are also investigated. An optimum set of design parameters for the micro-pump is obtained form the analyses.

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.

In this paper, simulation studies to determine the feasibility of producing filaments using `drop and demand' techniques are presented. These filaments will break up into droplets due to the phenomena caused by Rayleigh instability. In the biomedical applications, for effective pulmonary drug delivery of insulin, for example, the drug particles must be in the range of 1 to 5 microns in size. This stringent requirement is also encountered in gas flow seeding for Laser Doppler Velocimetry studies. A piezoelectrically actuated MEMS atomizer based on Rayleigh instability-driven break-up of filaments has been designed to meet this requirement. Although the formation of droplets from jets has been used extensively in ink-jet printing, the currently presented mode of droplet formulation has yet to be demonstrated in a MEMS device.

In this paper, the feasibility of a self-oscillating anemometer is examined. A 2D numerical study of a novel self-oscillating anemometer that can be fabricated using micromachining techniques is performed. The device is essentially a square cylinder suspended in the fluid flow by a fixed beam. The flow velocity can be easily measured by determining the frequency of oscillation obtained from capacitance sensing. Anemometer with different length scales can be fabricated to enable different ranges of velocities to be measured.

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...