Nisarga Naik

Microcrimped Collagen Fiber-Elastin Composites

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 wavy microstructure of native collagen fibers, with a periodicity of 10 – 200 μm, 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,[1] which contributes to the resilience of the composite structure by bearing tensile loads at low strains, analogous to a native elastin fiber network.[2, 3] 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.[4-6] The morphological features of crimp structure has been characterized as planar zig-zag,[7] sinusoid,[8] or helical[9, 10] with wavelengths between 10 to 200 μm. 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.[4, 5, 11, 12] 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.[13-19] 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.[13] 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,[15] array of nanoribbons,[14, 16] integrated circuit,[17] or carbon nanotubes,[18] with relaxation of stretch producing defined wavy structures. Collectively, these studies have lead to the fabrication of controlled micro- and nano-scale waveforms. However, microcrimping techniques have not been developed that are suitable for biological materials, such as collagen fibers.

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 Youngs 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 Youngs 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 Youngs 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.

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.

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.

Mechanosynthesis of three-dimensional replicated nanostructures by nanolithography-based molecular manipulation

This paper reports a nanoscopic mechanosynthesis of three-dimensional (3D) nanostructures by nanolithography-based molecular manipulation (NMM) of target molecules for the applications requiring both physical and chemical anisotropies. The reported molecular manipulators with nanometer-sized patterns and anisotropic surface functionalities are fabricated by exploiting the hybrid nanometer-scale NMM process. They are then utilized for positional nanoassembly of molecules followed by mechanosynthesis producing 3D nanoreplicas. This approach offers “top-down” design and fabrication of morphological features of nanoparticles (NPs). Three types of replicated sub-10nm polystyrene (PS) nanostructures have been successfully demonstrated in this work, namely, “nanomushrooms”, “nanospikes”, and high-aspect-ratio “nanofibers”.

Fabrication and Characterization of Liquid and Gaseous Micro- and Nanojets

This paper reports on the fabrication and characterization of liquid and gaseous jets ejected from microfabricated nozzles with dimensions ranging from 500 nm to 12 μm. Unlike previous work reporting the fabrication of nano-orifices defined within the thickness of the substrates [1-4], the in-plane nanonozzles presented in this paper are designed to sustain the high pressures necessary to obtain substantial nanofluidic jet flows. This approach also allows important three-dimensional features of nozzle, channel and fluidic reservoir to be defined by design and not by fabrication constraints, thereby meeting important fluid-mechanical criteria such as a fully-developed flow. The shrinking jet dimensions demand new metrology tools to investigate their flow behavior. A laser shadowgraphy technique is used to visualize and image the jet flows. Micromachined heated and piezoresistive cantilevers are used to investigate the thrust and heat flux characteristics of the jets.Copyright

Micro-cantilever metrology tool for flow characterization of liquid and micro/nanojets

This paper reports the development of MEMS metrology tools to characterize liquid and gaseous jets ejected from micro/nanofabricated nozzles. To date few highly local measurements have been made on micro/nanojets, due in part to the lack of characterization tools and techniques to investigate their characteristics. Atomic force microscope cantilevers are well-suited for interrogating these flows due to their high spatial and temporal resolution. In this work, cantilever sensors with either integrated heating elements or piezoresistive elements have been fabricated to measure thrust, velocity, and heat flux characteristics of micro/nanojets.Copyright

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.

Fabrication of Glass-Metal Composite Micro/Nanonozzles

This paper reports a fabrication technique for high strength glass-metal composite micro/nanonozzles with orifice diameters ranging from 430 nm to >100 μm. Unlike the conventional methods used to build micro/nanonozzles, the fabrication technique discussed in this paper is a non-lithographic approach. It uses conventional pulled borosilicate micropipettes as a foundation and nickel as a strengthening layer to build high pressure withstanding micro/nanonozzles. Pipettes built using the pulling process offer a smooth transition to the fluid from the reservoir to the tapered part of the nozzle, providing an ideal geometry from fluid flow and stress point of view. The nozzles are tested for high pressure withstanding capacity by integrating them with a high pressure fluidic setup to drive microjets. As an example, a 1.5 μm diameter nozzle, tested with propane as the working fluid to drive a microjet is observed to withstand pressures upto 10.5 MPa. Apart from simplicity of the fabrication process, this approach also offers the ability to incorporate a wireless temperature control system for the nozzles.Copyright

Measurements of microjet cooling and phase change characteristics using microcantilever heaters

This paper reports novel microcantilever metrology tools to investigate free microjets emanating from a micromachined nozzle having 10 μm diameter. Microcantilever sensors are well-suited to interrogate these flows due to their high spatial and temporal resolution. In this work, microcantilevers with integrated piezoresistors have been used to detect the breakup distance of free microjets, and microcantilevers with integrated resistive heaters have been applied to study microjet cooling and phase change characteristics. Measured microjet thrusts were in the range of 10 – 60 μN and heat fluxes were measured in the range of 25 – 350 °C. The convective heat fluxes by microjet impingement boiling were estimated at 2.9 – 7.6 kW/cm2 . The techniques reported herein are promising to characterize microscale flows.© 2007 ASME