Extracellular Matrix Alignment Directs Provisional Matrix Assembly and Three Dimensional Fibrous Tissue Closure.

Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. Impact statement Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues.

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues' contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues' Young's moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues' properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships.

Fabrication and Mechanical Properties Measurements of 3D Microtissues for the Study of Cell-Matrix Interactions.

Cell interactions with the extracellular matrix (ECM) are critical to cell and tissue functions involving adhesion, communication, and differentiation. Three-dimensional (3D) in vitro culture systems are an important approach to mimic in vivo cell-matrix interactions for mechanobiology studies and tissue engineering applications. This chapter describes the use of engineered microtissues as 3D constructs in combination with a magnetic tissue gauge (µTUG) system to analyze tissue mechanical properties. The µTUG system is composed of poly(dimethylsiloxane) (PDMS) microwells with vertical pillars in the wells. Self-assembled microtissues containing cells and ECM gel can form between the pillars, and generate mechanical forces that deform the pillars, which provides a readout of those forces. Herein, detailed procedures for microfabrication of the PDMS µTUG system, seeding and growth of cells with ECM gels in the microwells, and measurements of the mechanical properties of the resulting microtissues via magnetic actuation of magnetic sphere-tagged µTUGs are described.

A microfabricated magnetic actuation device for mechanical conditioning of arrays of 3D microtissues.

This paper describes an approach to actuate magnetically arrays of microtissue constructs for long-term mechanical conditioning and subsequent biomechanical measurements. Each construct consists of cell/matrix material self-assembled around a pair of flexible poly(dimethylsiloxane) (PDMS) pillars. The deflection of the pillars reports the tissues' contractility. Magnetic stretching of individual microtissues via magnetic microspheres mounted on the cantilevers has been used to elucidate the tissues' elastic modulus and response to varying mechanical boundary conditions. This paper describes the fabrication of arrays of micromagnetic structures that can transduce an externally applied uniform magnetic field to actuate simultaneously multiple microtissues. These structures are fabricated on silicon-nitride coated Si wafers and contain electrodeposited Ni bars. Through-etched holes provide optical and culture media access when the devices are mounted on the PDMS microtissue scaffold devices. Both static and AC forces (up to 20 µN on each microtissue) at physiological frequencies are readily generated in external fields of 40 mT. Operation of the magnetic arrays was demonstrated via measurements of elastic modulus and dynamic stiffening in response to AC actuation of fibroblast populated collagen microtissues.