Dynamics of Interstitial Fluid Pressure in Extracellular Matrix Hydrogels in Microfluidic Devices.

In order to understand how interstitial fluid pressure and flow affect cell behavior, many studies use microfluidic approaches to apply externally controlled pressures to the boundary of a cell-containing gel. It is generally assumed that the resulting interstitial pressure distribution quickly reaches a steady-state, but this assumption has not been rigorously tested. Here, we demonstrate experimentally and computationally that the interstitial fluid pressure within an extracellular matrix gel in a microfluidic device can, in some cases, react with a long time delay to external loading. Remarkably, the source of this delay is the slight (∼100 nm in the cases examined here) distension of the walls of the device under pressure. Finite-element models show that the dynamics of interstitial pressure can be described as an instantaneous jump, followed by axial and transverse diffusion, until the steady pressure distribution is reached. The dynamics follow scaling laws that enable estimation of a gel's poroelastic constants from time-resolved measurements of interstitial fluid pressure.

Microfluidic models of vascular functions.

In vitro studies of vascular physiology have traditionally relied on cultures of endothelial cells, smooth muscle cells, and pericytes grown on centimeter-scale plates, filters, and flow chambers. The introduction of microfluidic tools has revolutionized the study of vascular physiology by allowing researchers to create physiologically relevant culture models, at the same time greatly reducing the consumption of expensive reagents. By taking advantage of the small dimensions and laminar flow inherent in microfluidic systems, recent studies have created in vitro models that reproduce many features of the in vivo vascular microenvironment with fine spatial and temporal resolution. In this review, we highlight the advantages of microfluidics in four areas: the investigation of hemodynamics on a capillary length scale, the modulation of fluid streams over vascular cells, angiogenesis induced by the exposure of vascular cells to well-defined gradients in growth factors or pressure, and the growth of microvascular networks in biomaterials. Such unique capabilities at the microscale are rapidly advancing the understanding of microcirculatory dynamics, shear responses, and angiogenesis in health and disease as well as the ability to create in vivo-like blood vessels in vitro.

Microfluidic Biomaterials.

Since their initial description in 2005, biomaterials that are patterned to contain microfluidic networks ("microfluidic biomaterials") have emerged as promising scaffolds for a variety of tissue engineering and related applications. This class of materials is characterized by the ability to be readily perfused. Transport and exchange of solutes within microfluidic biomaterials is governed by convection within channels and diffusion between channels and the biomaterial bulk. Numerous strategies have been developed for creating microfluidic biomaterials, including micromolding, photopatterning, and 3D printing. In turn, these materials have been used in many applications that benefit from the ability to perfuse a scaffold, including the engineering of blood and lymphatic microvessels, epithelial tubes, and cell-laden tissues. This article reviews the current state of the field and suggests new areas of exploration for this unique class of materials.

Design principles for lymphatic drainage of fluid and solutes from collagen scaffolds.

In vivo, tissues are drained of excess fluid and macromolecules by the lymphatic vascular system. How to engineer artificial lymphatics that can provide equivalent drainage in biomaterials remains an open question. This study elucidates design principles for engineered lymphatics, by comparing the rates of removal of fluid and solute through type I collagen gels that contain lymphatic vessels or unseeded channels, or through gels without channels. Surprisingly, no difference was found between the fluid drainage rates for gels that contained vessels or bare channels. Moreover, solute drainage rates were greater in collagen gels that contained lymphatic vessels than in those that had bare channels. The enhancement of solute drainage by lymphatic endothelium was more pronounced in longer scaffolds and with smaller solutes. Whole-scaffold imaging revealed that endothelialization aided in solute drainage by impeding solute reflux into the gel without hindering solute entry into the vessel lumen. These results were reproduced by computational models of drainage with a flow-dependent endothelial hydraulic conductivity. This study shows that endothelialization of bare channels does not impede the drainage of fluid from collagen gels and can increase the drainage of macromolecules by preventing solute transport back into the scaffold. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 106-114, 2018.

Microstructured extracellular matrices in tissue engineering and development.

Microscale heterogeneity in the extracellular matrix (ECM) provides spatial information that allows tissues to develop and function properly in vivo. This heterogeneity in composition (chemistry) and structure (geometry) creates distinct microenvironments for the cells that comprise a tissue. In response, populations of cells can coordinate their behaviors across micrometer-to-millimeter length scales to function as a unified whole. We believe techniques to mimic the microscale heterogeneity of the ECM in vitro will revolutionize studies that examine how large groups of cells interact. Micropatterned ECMs used for engineering perfused microvascular networks and functional epidermis and for understanding symmetry-breaking events in epithelial morphogenesis illustrate potential applications in tissue engineering and development.

Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus.

Adherent cells are strongly influenced by the mechanical aspects of biomaterials, but little is known about the cellular effects of spatial variations in these properties. This work describes a novel method to produce polymeric cell culture surfaces containing micrometer-scale regions of variable stiffness. Substrates made of acrylamide or poly(dimethylsiloxane) were patterned with 100- or 10-microm resolution, respectively. Cells were cultured on fibronectin-coated acrylamide having Young's moduli of 34 kPa and 1.8 kPa, or fibronectin-coated PDMS having moduli of 2.5 MPa and 12 kPa. Over several days, NIH/3T3 cells and bovine pulmonary arterial endothelial cells accumulated preferentially on stiffer regions of substrates. The migration, not proliferation, of cells in response to mechanical patterning (mechanotaxis) was responsible for the accumulation of cells on stiffer regions. Differential remodeling of extracellular matrix protein on stiff versus compliant regions was observed by immunofluorescence staining, and may have been responsible for the observed mechanotaxis. These results suggest that mechanically patterned substrates might provide a general means to study mechanotaxis, and a new approach to patterning cells.

Crosslinking of collagen scaffolds promotes blood and lymphatic vascular stability.

The low stiffness of reconstituted collagen hydrogels has limited their use as scaffolds for engineering implantable tissues. Although chemical crosslinking has been used to stiffen collagen and protect it against enzymatic degradation in vivo, it remains unclear how crosslinking alters the vascularization of collagen hydrogels. In this study, we examine how the crosslinking agents genipin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide alter vascular stability and function in microfluidic type I collagen gels in vitro. Under moderate perfusion (∼10 dyn/cm(2) shear stress), tubes of blood endothelial cells (ECs) exhibited indistinguishable stability and barrier function in untreated and crosslinked scaffolds. Surprisingly, under low perfusion (∼5 dyn/cm(2) shear stress) or nearly zero transmural pressure, microvessels in crosslinked scaffolds remained stable, while those in untreated gels rapidly delaminated and became poorly perfused. Similarly, tubes of lymphatic ECs under intermittent flow were more stable in crosslinked gels than in untreated ones. These effects correlated well with the degree of mechanical stiffening, as predicted by analysis of fracture energies at the cell-scaffold interface. This work demonstrates that crosslinking of collagen scaffolds does not hinder normal EC physiology; instead, crosslinked scaffolds promote vascular stability. Thus, routine crosslinking of scaffolds may assist in vascularization of engineered tissues.

Molding of three-dimensional microstructures of gels.

This Communication describes the use of patterned elastomeric stamps to mold, release, and stack hydrogels into three-dimensional microstructures. Molding of gels against stamps derivatized by a hexa(ethylene glycol)-terminated self-assembled monolayer or by an adsorbed monolayer of bovine serum albumin allowed the application of several soft lithographic techniques (replica molding, microtransfer molding, and micromolding in capillaries) to the microfabrication of gels. We describe procedures to generate coplanar or bilayered composites of gels.

Fabrication of aligned microstructures with a single elastomeric stamp.

The fabrication of complex patterns of aligned microstructures has required the use of multiple applications of lithography. Here we describe an approach for microfabrication that encodes the two-dimensional spatial information of several photomasks onto a single elastomeric stamp by mapping each photomask onto distinct heights on the surface of the stamp. Pressing the stamp against a surface collapses the topography of the stamp such that each recessed layer contacts the surface in stepwise sequence; the greater the applied pressure, the larger the area of the stamp that contacts the surface. After contact of each new layer with the surface, we use techniques of soft lithography (microcontact printing, microfluidics, and patterning through membranes) to pattern the surfaces that contact the stamp and those that do not with inorganic, organic, or living materials. Microfabrication through the use of multilevel stamps provides a promising alternative to conventional lithography for the construction of multicomponent, aligned surfaces; these structures may find use as components of microfluidic devices or biological patterns.