Going wireless and booth-less for hearing testing in industry.

OBJECTIVE: To assess the test-retest variability of hearing thresholds obtained with an innovative, mobile wireless automated hearing-test system (WAHTS) with enhanced sound attenuation to test industrial workers at a worksite as compared to standardised automated hearing thresholds obtained in a mobile trailer sound booth. DESIGN: A within-subject repeated-measures design was used to compare air-conducted threshold tests (500-8000 Hz) measured with the WAHTS in six workplace locations, and a third test using computer-controlled audiometry obtained in a mobile trailer sound booth. Ambient noise levels were measured in all test environments. STUDY SAMPLE: Twenty workers served as listeners and 20 workers served as operators. RESULTS: On average, the WAHTS resulted in equivalent thresholds as the mobile trailer audiometry at 1000, 2000, 3000 and 8000 Hz and thresholds were within ±5 dB at 500, 4000 and 6000 Hz. CONCLUSIONS: Comparable performance may be obtained with the WAHTS in occupational audiometry and valid thresholds may be obtained in diverse test locations without the use of sound-attenuating enclosures.

White light-informed optical properties improve ultrasound-guided fluorescence tomography of photoactive protoporphyrin IX.

Subsurface fluorescence imaging is desirable for medical applications, including protoporphyrin-IX (PpIX)-based skin tumor diagnosis, surgical guidance, and dosimetry in photodynamic therapy. While tissue optical properties and heterogeneities make true subsurface fluorescence mapping an ill-posed problem, ultrasound-guided fluorescence-tomography (USFT) provides regional fluorescence mapping. Here USFT is implemented with spectroscopic decoupling of fluorescence signals (auto-fluorescence, PpIX, photoproducts), and white light spectroscopy-determined bulk optical properties. Segmented US images provide a priori spatial information for fluorescence reconstruction using region-based, diffuse FT. The method was tested in simulations, tissue homogeneous and inclusion phantoms, and an injected-inclusion animal model. Reconstructed fluorescence yield was linear with PpIX concentration, including the lowest concentration used, 0.025 µg/ml. White light spectroscopy informed optical properties, which improved fluorescence reconstruction accuracy compared to the use of fixed, literature-based optical properties, reduced reconstruction error and reconstructed fluorescence standard deviation by factors of 8.9 and 2.0, respectively. Recovered contrast-to-background error was 25% and 74% for inclusion phantoms without and with a 2-mm skin-like layer, respectively. Preliminary mouse-model imaging demonstrated system feasibility for subsurface fluorescence measurement in vivo. These data suggest that this implementation of USFT is capable of regional PpIX mapping in human skin tumors during photodynamic therapy, to be used in dosimetric evaluations.

Highly sensitive single-fibril erosion assay demonstrates mechanochemical switch in native collagen fibrils.

It has been established that the enzyme susceptibility of collagen, the predominant load-bearing protein in vertebrates, is altered by applied tension. However, whether tensile force increases or decreases the susceptibility to enzyme is a matter of contention. It is critical to establish a definitive understanding of the direction and magnitude of the force versus catalysis rate (k C ) relationship if we are to properly interpret connective tissue development, growth, remodeling, repair, and degeneration. In this investigation, we examine collagen/enzyme mechanochemistry at the smallest scale structurally relevant to connective tissue: the native collagen fibril. A single-fibril mechanochemical erosion assay with nN force resolution was developed which permits detection of the loss of a few layers of monomer from the fibril surface. Native type I fibrils (bovine) held at three levels of tension were exposed to Clostridium histolyticum collagenase A. Fibrils held at zero-load failed rapidly and consistently (20 min) while fibrils at 1.8 pN/monomer failed more slowly (35-55 min). Strikingly, fibrils at 23.9 pN/monomer did not exhibit detectable degradation. The extracted force versus k C data were combined with previous single-molecule results to produce a "master curve" which suggests that collagen degradation is governed by an extremely sensitive mechanochemical switch.

Molecular mechanism of force induced stabilization of collagen against enzymatic breakdown.

Collagen cleavage, facilitated by collagenases of the matrix metalloproteinase (MMP) family, is crucial for many physiological and pathological processes such as wound healing, tissue remodeling, cancer invasion and organ morphogenesis. Earlier work has shown that mechanical force alters the cleavage rate of collagen. However, experimental results yielded conflicting data on whether applying force accelerates or slows down the degradation rate. Here we explain these discrepancies and propose a molecular mechanism by which mechanical force might change the rate of collagen cleavage. We find that a type I collagen heterotrimer is unfolded in its equilibrium state and loses its triple helical structure at the cleavage site without applied force, possibly enhancing enzymatic breakdown as each chain is exposed and can directly undergo hydrolysis. Under application of force, the naturally unfolded region refolds into a triple helical structure, potentially protecting the molecule against enzymatic breakdown. In contrast, a type I collagen homotrimer retains a triple helical structure even without applied force, making it more resistant to enzyme cleavage. In the case of the homotrimer, the application of force may directly lead to molecular unwinding, resulting in a destabilization of the molecule under increased mechanical loading. Our study explains the molecular mechanism by which force may regulate the formation and breakdown of collagenous tissue.

Molecular mechanochemistry: low force switch slows enzymatic cleavage of human type I collagen monomer.

In vertebrate animals, fibrillar collagen accumulates, organizes, and persists in structures which resist mechanical force. This antidissipative behavior is possibly due to a mechanochemical force-switch which converts collagen from enzyme-susceptible to enzyme-resistant. Degradation experiments on native tissue and reconstituted fibrils suggest that collagen/enzyme kinetics favor the retention of loaded collagen. We used a massively parallel, single molecule, mechanochemical reaction assay to demonstrate that the effect is derivative of molecular mechanics. Tensile loads higher than 3 pN dramatically reduced (10×) the enzymatic degradation rate of recombinant human type I collagen monomers by Clostridium histolyticum compared to unloaded controls. Because bacterial collagenase accesses collagen at multiple sites and is an aggressive cleaver of the collagen triple helical domain, the results suggest that collagen molecular architecture is generally more stable when mechanically strained in tension. Thus the tensile mechanical state of collagen monomers is likely to be correlated to their longevity in tissues. Further, strain-actuated molecular stability of collagen may constitute the fundamental basis of a smart structural mechanism which enhances the ability of animals to place, retain, and load-optimize material in the path of mechanical forces.

Mechanical strain stabilizes reconstituted collagen fibrils against enzymatic degradation by mammalian collagenase matrix metalloproteinase 8 (MMP-8).

BACKGROUND: Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain. METHODOLOGY/PRINCIPAL FINDINGS: The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded. CONCLUSIONS/SIGNIFICANCE: In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.

Probing collagen/enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep.

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance, and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially loaded strips of bovine corneal tissue (0.1, 0.25, or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at approximately 5% strain) reduces the maximum cutting rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective "cutoff" occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is nonlinear (initially steep between 4.5 and 6.5% and then flattens out from 6.5 to 9.5%). The high sensitivity to strain at low strain implies that even lightly loaded collagenous tissue may exhibit significant strain protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in many different tissue types.

Mechanical strain enhances survivability of collagen micronetworks in the presence of collagenase: implications for load-bearing matrix growth and stability.

There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed 'quickly', while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue.