Rapid homogeneous endothelialization of high aspect ratio microvascular networks.

Microvascularization of an engineered tissue construct is necessary to ensure the nourishment and viability of the hosted cells. Microvascular constructs can be created by seeding the luminal surfaces of microfluidic channel arrays with endothelial cells. However, in a conventional flow-based system, the uniformity of endothelialization of such an engineered microvascular network is constrained by mass transfer of the cells through high length-to-diameter (L/D) aspect ratio microchannels. Moreover, given the inherent limitations of the initial seeding process to generate a uniform cell coating, the large surface-area-to-volume ratio of microfluidic systems demands long culture periods for the formation of confluent cellular microconduits. In this report, we describe the design of polydimethylsiloxane (PDMS) and poly(glycerol sebacate) (PGS) microvascular constructs with reentrant microchannels that facilitates rapid, spatially homogeneous endothelial cell seeding of a high L/D (2 cm/35 µm; > 550:1) aspect ratio microchannels. MEMS technology was employed for the fabrication of a monolithic, elastomeric, reentrant microvascular construct. Isotropic etching and PDMS micromolding yielded a near-cylindrical microvascular channel array. A 'stretch - seed - seal' operation was implemented for uniform incorporation of endothelial cells along the entire microvascular area of the construct yielding endothelialized microvascular networks in less than 24 h. The feasibility of this endothelialization strategy and the uniformity of cellularization were established using confocal microscope imaging.

Microablation of collagen-based substrates for soft tissue engineering.

Noting the abundance and importance of collagen as a biomaterial, we have developed a facile method for the production of a dense fibrillar extracellular matrix mimicking collagen-elastin hybrids with tunable mechanical properties. Through the use of excimer-laser technology, we have optimized conditions for the ablation of collagen lamellae without denaturation of protein, maintenance of fibrillar ultrastructure and preservation of native D-periodicity. Strengths of collagen-elastin hybrids ranged from 0.6 to 13 MPa, elongation at break from 9 to 70% and stiffness from 2.9 to 94 MPa, allowing for the design of a wide variety of tissue specific scaffolds. Further, large (centimeter scale) lamellae can be fabricated and embedded with recombinant elastin to generate collagen-elastin hybrids. Exposed collagen in hybrids act as cell adhesive sites for rat mesenchymal stem cells that conform to ablate waveforms. The ability to modulate these features allows for the generation of a class of biopolymers that can architecturally and physiologically replicate native tissue.

Generation of spatially aligned collagen fiber networks through microtransfer molding.

The unique biomechanical properties of native tissue are governed by the organization and composition of integrated collagen and elastin networks. An approach for fabricating spatially aligned, fiber-reinforced composites with adjustable collagen fiber dimensions, layouts, and distribution within an elastin-like protein matrix yielding a biocomposite with controllable mechanical responses is reported. Microtransfer molding is employed for the fabrication of hollow and solid collagen fibers with straight or crimped fiber geometries. Collagen fibers (width: 2-50 µm, thickness: 300 nm to 3 µm) exhibit a Young's modulus of 126 ± 61 MPa and an ultimate tensile strength of 7 ± 3.2 MPa. As fiber networks within composite structures, straight fiber layouts display orthotropic responses with Young's modulus ranging from 0.95 ± 0.35 to 10.4 ± 0.5 MPa and tensile strength from 0.22 ± 0.08 to 0.87 ± 0.5 MPa with increasing fraction of collagen fibers (1-10%, v/v). In contrast, composites based on crimped fiber layouts exhibit strain-dependent stiffness with an increase in Young's modulus from 0.7 ± 0.14 MPa to 3.15 ± 0.49 MPa, at a specific transition strain. Through controlling the microstructure of engineered collagen fiber networks, a facile means is established to control macroscale mechanical responses of composite protein-based materials.

MEMS-assisted spatially homogeneous endothelialization of a high length-to-depth aspect ratio microvascular network.

The endothelialization of an engineered microvascular network is constrained by the mass transport of the endothelial cells through high length-to-depth (l/d) aspect ratio microchannels. This paper presents a deformable, reentrant microvascular scaffold as a microelectromechanical systems (MEMS)-assisted approach for spatially homogeneous endothelial cell seeding of high l/d (>200) aspect ratio microvasculature. Nickel electroplating and micromolding were employed for the fabrication of the polydimethylsiloxane (PDMS) reentrant microvascular scaffold. A 'stretch--seed--seal' ('3S') operation was implemented for uniform incorporation of endothelial cells on the luminal surface of the elastomeric constructs. Confocal microscopy was utilized to establish the uniformity of endothelialization and to demonstrate the feasibility of this strategy.

A TEMPLATE-BASED FABRICATION TECHNIQUE FOR SPATIALLY-DESIGNED POLYMER MICRO/NANOFIBER COMPOSITES.

This paper reports a template-based technique for the fabrication of polymer micro/nanofiber composites, exercising control over the fiber dimensions and alignment. Unlike conventional spinning-based methods of fiber production, the presented approach is based on micro-transfer molding. It is a parallel processing technique capable of producing fibers with control over both in-plane and out-of-plane geometries, in addition to packing density and layout of the fibers. Collagen has been used as a test polymer to demonstrate the concept. Hollow and solid collagen fibers with various spatial layouts have been fabricated. Produced fibers have widths ranging from 2 µm to 50 µm, and fiber thicknesses ranging from 300 nm to 3 µm. Also, three-dimensionality of the process has been demonstrated by producing in-plane serpentine fibers with designed arc lengths, out-of-plane wavy fibers, fibers with focalized particle encapsulation, and porous fibers with desired periodicity and pore sizes.