Mechanical characterization of thin film, water-based polymer gels through simple tension testing of laminated bilayers.

We present a method of characterizing the nonlinear stress-strain behavior of thin films of extremely soft, water-based polymer gels using uniaxial tension testing of bilayer laminates, in conjunction with methods of membrane nonlinear elasticity. A custom tensile testing apparatus is used to conduct quasi-static, uniaxial extension tests of narrow strips of thin, laminated sheets of bonded hydrogel and silicone rubber, submerged in a saline bath. The tensile load is measured via sensitive load cell and the position of material markers, at a central test-section of the sample, is optically tracked via digital image tracking methods. Stress-strain relationships are calculated for the hydrogel component of the bilayer, considered hyperelastic, homogeneous, isotropic, and incompressible, using membrane theories of finite hyperelasticity. We present the stress response for strains up to about 35% for poly(ethylene glycol) (PEG)-based hydrogels (>90% water) with polymer concentrations by weight of 5% to 10%. Polynomial functions are fit to the data for each formulation, whereby the one-dimensional strain-energy function for each formulation is determined by taking the indefinite integral.

A single cell bioengineering approach to elucidate mechanisms of adult stem cell self-renewal.

The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Adult stem cells, present in tissues such as skeletal muscle, comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Muscle stem cells, known as satellite cells, reside in a quiescent state in an anatomically distinct compartment, or niche, ensheathed between the membrane of the myofiber and the basal lamina. Recently, procedures for isolating satellite cells were developed and experiments testing their function upon transplantation into muscles revealed an extraordinary potential to contribute to muscle fibers and access and replenish the satellite cell compartment. However, these properties are rapidly lost once satellite cells are plated in culture. Accordingly, elucidating the role of extrinsic factors in controlling muscle stem cell fate, in particular self-renewal, is critical. Through careful design of bioengineered culture platforms, analysis of specific proteins presented to stem cells is possible. Critical to the success of the approach is single cell analysis, as more rapidly proliferating progenitors may mask the behavior of stem cells that proliferate slowly. Bioengineering approaches provide a potent means of gaining insight into the role of extrinsic factors in the stem cell microenvironment on stem cell function and the mechanisms that control their diverse fates. Ultimately, the multidisciplinary approach presented here will lead to novel therapeutic strategies for degenerative diseases.

Perturbation of single hematopoietic stem cell fates in artificial niches.

Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet are in limited supply as in culture this self-renewal capacity is lost. Using an approach at the interface of stem cell biology and bioengineering, here we describe a novel platform of hydrogel microwell arrays for assessing the effects of either secreted or tethered proteins characteristic of the in vivo microenvironment, or niche, on HSC fate in vitro. Time-lapse microscopic analyses of single cells were crucial to overcoming inevitable heterogeneity of FACS-enriched HSCs. A reduction in proliferation kinetics or an increase in asynchronous division of single HSCs in microwells in response to specific proteins (Wnt3a and N-Cadherin) correlated well with subsequent serial long-term blood reconstitution in mice in vivo. Single cells that divided once in the presence of a given protein were capable of in vivo reconstitution, providing evidence of self-renewal divisions of HSCs in vitro. These results validate the hydrogel microwell platform as a broadly applicable paradigm for dissecting the regulatory role of specific signals within a complex stem cell niche.

An active wound dressing for controlled convective mass transfer with the wound bed.

Conventional wound dressings-gauze, plastic films, foams, and gels-do not allow for spatial and temporal control of the soluble chemistry within the wound bed, and are thus limited to a passive role in wound healing. Here, we present an active wound dressing (AWD) designed to control convective mass transfer with the wound bed; this mass transfer provides a means to tailor and monitor the chemical state of a wound and, potentially, to aid the healing process. We form this AWD as a bilayer of porous poly(hydroxyethyl methacrylate) (pHEMA) and silicone; the pHEMA acts as the interface with the wound bed, and a layer of silicone provides a vapor barrier and a support for connecting to external reservoirs and pumps. We measure the convective permeability of the pHEMA sponge, and use this value to design a device with a spatially uniform flow profile. We quantify the global coefficient of mass transfer of the AWD on a dissolvable synthetic surface, and compare it to existing theories of mass transfer in porous media. We also operate the AWD on model wound beds made of calcium alginate gel to demonstrate extraction and delivery of low molecular weight solutes and a model protein. Using this system, we demonstrate both uniform mass transfer over the entire wound bed and patterned mass transfer in three spatially distinct regions. Finally, we discuss opportunities and challenges for the clinical application of this design of an AWD.

Functional antibody immobilization on 3-dimensional polymeric surfaces generated by reactive ion etching.

Reactive ion etching (RIE) was used to pattern antibodies onto the surfaces of polymer substrates. A low pressure, inductively coupled oxygen plasma was used to anisotropically etch 25-30 mum deep features into poly(methyl methacrylate) (PMMA), Zeonex, and polycarbonate (PC). Scanning electron microscopy and contact angle measurements show that the resulting surfaces exhibit significant microroughness and enhanced hydrophilicity. Fourier transform infrared spectroscopy suggests that, in addition to enhanced surface area, chemical modifications may contribute to antibody immobilization. Polyclonal antibodies preferentially bind to the etched areas in RIE-patterned PMMA and Zeonex substrates but localize in unetched regions of RIE-patterned PC surfaces. Simple immunoassays were performed to demonstrate a potential application for RIE-modified polymer surfaces. Antibodies specific for the capture of fluorescently labeled cholera toxin, S. aureus enterotoxin B, and B. anthracis protective antigen were immobilized onto etched PMMA surfaces and shown to specifically capture their labeled antigen from solution. This work demonstrates a potentially useful fabrication methodology for constructing antibody microarrays on plastic substrates.

Antibody microarrays for native toxin detection.

We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.