Microcrimped collagen fiber-elastin composites.

Adv. Mater. 2010, 22, 2041–2044 2010 WILEY-VCH Verlag G T IO N Emerging biomaterials based upon analogues of native extracellular matrix proteins provide an opportunity to create protein scaffolds that mimic tissue mechanical behavior and guide cellular responses. However, in order to reproduce macroscale tissue properties, protein analogues must be endowed with appropriate microstructural features. In particular, the crimped or wavymicrostructure of native collagen fibers, with a periodicity of 10–200mm, contributes in a significant manner to the compliance, strength, and durability of soft tissues. In this report, we describe a templating strategy based upon the application of micropatterned elastomeric substrates, which yields dense, aligned arrays of synthetic collagen microfibers that display a well-defined microcrimped pattern. Following crosslinking with glutaraldehyde vapor, fiber arrays were embedded in a recombinant elastin protein polymer, which contributes to the resilience of the composite structure by bearing tensile loads at low strains, analogous to a native elastin fiber network. We demonstrate the preservation of fiber crimp after repetitive cyclic loading, as well as the assembly of hierarchical microcrimped multilamellar composites with mechanical responses similar to native tissues. The periodic waviness of fibrous collagen is observed in nearly all human tissues, including blood vessels, valve leaflets, intestine, tendon, and intervertebral discs. Themorphological features of crimp structure has been characterized as planar zig-zag, sinusoid, or helical with wavelengths between 10 to 200mm. Crimp ensures that at low levels of tensile strain, loads are sustained both by the surrounding matrix and the fiber network. Typically, fibers straighten as a load is imposed with an observed transition from low to high tissue stiffness. These mechanisms serve to enhance compliance at low strain while generating greater strength as load increases. Since physiologic strains are imposed at levels of stress where fibers are often not fully extended, the propensity for fatigue-related fiber damage is minimized. All told, fiber crimp has evolved as an important bioengineering principle that affords a favorable combination of compliance, strength, and durability. A set of techniques using soft, contracting substrates to shape thin coatings of high modulus materials into crimped, wrinkled, and wavy structures has recently emerged. For example, Bowden and colleagues deposited metal films on heated PDMS and noted that, upon cooling, the contraction of the PDMS buckled the metal layer into sophisticated patterns of wrinkles. However, in these cases the extent of waviness is limited by the extent of inducible thermal shrinkage. Alternatively, an elastomeric substrate may be mechanically stretched prior to the application of a thin film, array of nanoribbons, integrated circuit, or carbon nanotubes, with relaxation of stretch producing defined wavy structures. Collectively, these studies have lead to the fabrication of controlled microand nanoscale waveforms. However, microcrimping techniques have not been developed that are suitable for biological materials, such as collagen fibers. Controlled deformation of a flexible microridged membrane dictates microcrimp fiber morphology. Flexible polyurethane microridges with a buttress-rectangular profile were fabricated following photolithographic and micromolding techniques. Collagen fibers are sandwiched between a smooth base substrate and a microridged membrane, both pre-extended to a desired strain, strain relaxed to induce microcrimp features, and fibers crosslinked by glutaraldehyde vapor (Fig. 1a–d). Scanning electron microscopy revealed a repetitive crimp pattern, resembling native collagen with the degree of crimp directly related to the magnitude of imposed pre-extension strain (Fig. 1h–k). Fiber embedding within an elastin-mimetic protein matrix and subsequent lamination of multiple, individual, fiber-reinforced sheets was accomplished by a thermally controlled sol-gel process. As detailed elsewhere, an aqueous solution of elastin-mimetic triblock protein polymer forms a physically crosslinked gel above 13 8C. Single sheets of embedded microcrimped fibers were fabricated by dispensing a cooled solution of protein over an array of crimped fiber. An acrylic plate was then applied, the assembly warmed, and a fiber-reinforced elastin composite separated from the mold (Fig. 1e and f). Three-dimensional analysis of embedded fibers confirmed preservation of crimp structure within the protein matrix (Fig. 2a–c). Exposure to glutaraldehyde vapor was used to preserve the crimp structure before the fibers were hydrated with the elastin-mimetic protein and embedded within the sheet. Crimp of 3.1% 0.4% and 9.4% 2.9% was observed for fibers templated at pre-extension strains of 15% and 30%, respectively. Observed differences between pre-extension and degree of crimp presumably reflect fiber shortening induced by drying steps and matrix swelling after fiber embedding. Indeed, peak-to-peak periodicity was (143 5) mm for fibers embedded in a hydrated protein matrix, but decreased to (127 5) mm for dry fibers in a non-embedded state.