Molecular modeling of the axial and circumferential elastic moduli of tubulin.

Microtubules play a number of important mechanical roles in almost all cell types in nearly all major phylogenetic trees. We have used a molecular mechanics approach to perform tensile tests on individual tubulin monomers and determined values for the axial and circumferential moduli for all currently known complete sequences. The axial elastic moduli, in vacuo, were found to be 1.25 GPa and 1.34 GPa for alpha- and beta-bovine tubulin monomers. In the circumferential direction, these moduli were 378 MPa for alpha- and 460 MPa for beta-structures. Using bovine tubulin as a template, 269 homologous tubulin structures were also subjected to simulated tensile loads yielding an average axial elastic modulus of 1.10 +/- 0.14 GPa for alpha-tubulin structures and 1.39 +/- 0.68 GPa for beta-tubulin. Circumferentially the alpha- and beta-moduli were 936 +/- 216 MPa and 658 +/- 134 MPa, respectively. Our primary finding is that that the axial elastic modulus of tubulin diminishes as the length of the monomer increases. However, in the circumferential direction, no correlation exists. These predicted anisotropies and scale dependencies may assist in interpreting the macroscale behavior of microtubules during mitosis or cell growth. Additionally, an intergenomic approach to investigating the mechanical properties of proteins may provide a way to elucidate the evolutionary mechanical constraints imposed by nature upon individual subcellular components.

Differences between collagen morphologies, properties and distribution in diabetic and normal biobreeding and Sprague-Dawley rat sciatic nerves.

Both structural and functional differences between normal and diabetic nerve have been observed, in human patients and animal models. We hypothesize that these structural differences are quantifiable, morphologically and mechanically, with the ultimate aim of understanding the contribution of these differences to permanent nerve damage. The outer collagenous epineurial and perineurial tissues of mammalian peripheral nerves mechanically and chemically shield the conducting axons. We have quantified differences in these collagens, using whole-nerve uniaxial testing, and immunohistochemistry of collagens type I, III, and IV in diabetic and normal nerves. We present results of two studies, on normal and diabetic BioBreeding (BB), and normal, diabetic and weight-controlled Sprague-Dawley (SD) rats, respectively. Overall, we measured slightly higher uniaxial moduli (e.g. 5.9 MPa vs. 3.5 MPa, BB; 10.7 MPa vs. 10.0 MPa, SD at 40% strain) in whole nerves as well as higher peak stresses (e.g. 0.99 MPa vs. 0.74 MPa, BB; 2.16 MPa vs. 1.94 MPa, SD at 40% strain) in the diabetics of both animal models. We measured increased concentrations of types III and IV collagens in the diabetics of both models and mixed upregulation results were observed in type I protein levels. We detected small differences in mechanical properties at the tissue scale, though we found significant structural and morphometric differences at the fibril scale. These findings suggest that whole-tissue mechanical testing is not a sufficient assay for collagen glycation, and that fibrillar and molecular scale assays are needed to detect the earliest stages of diabetic protein glycation.

Nanomanipulation and characterization of structural proteins.

A methodology is presented for simultaneous mechanical testing and atomic force microscopy imaging of single collagen fibrils under load. This method holds the promise for determining single-fibril modulus and strength in various experimental preparations. Examples of this utility include characterization of deformation and failure modes of naturally occurring and engineered structural proteins. Additional promise of this technique is robotic surgery at the submicron scale for repairing neuronal tracts and capillaries with structural proteins. A series of algorithms for tying knots at the nanoscale in single fibrils is also presented.

A mechanical model for collagen fibril load sharing in peripheral nerve of diabetic and nondiabetic rats.

Peripheral neuropathy affects approximately 50% of the 15 million Americans with diabetes. It has been suggested that mechanical effects related to collagen glycation are related to the permanence of neuropathy. In the present paper, we develop a model for load transfer in a whole nerve, using a simple pressure vessel approximation, in order to assess the significant of stiffening of the collagenous nerve sheath on endoneurial fluid pressure. We also develop a fibril-scale mechanics model for the nerve, to model the straightening of wavy fibrils, producing the toe region observed in nerve tissue, and also to interrogate the effects of interfibrillar crosslinks on the overall properties of the tissue. Such collagen crosslinking has been implicated in complications in diabetic tissues. Our fibril-scale model uses a two-parameter Weibull model for fibril strength, in combination with statistical parameters describing fibril modulus, angle, wave-amplitude, and volume fraction to capture both toe region and failure region behavior of whole rat sciatic nerve. The extrema of equal and local load-sharing assumptions are used to map potential differences in diabetic and nondiabetic tissues. This work may ultimately be useful in differentiating between the responses of normal and heavily crosslinked tissue.

Nerve collagens from diabetic and nondiabetic Sprague-Dawley and biobreeding rats: an atomic force microscopy study.

BACKGROUND: Alterations in rat's nerve collagens due to diabetes may be related to the permanence of damage due to diabetic neuropathy. We (1) provide a methodology for determining the diameters of collagen fibers accounting for atomic force microscope (AFM) imaging artifacts, (2) present data on structural differences in sciatic nerve endoneurial, epineurial and tail tendon collagens of control and diabetic Sprague-Dawley and BioBreeding rats, and (3) compare results with literature values. METHODS: We measured collagen diameters and band spacing on endoneurial and epineurial sciatic nerve tissue, and tail tendon, in control and diabetic rats (STZ-induced 12-week diabetic SD and 16-week spontaneously diabetic BB rats). We also developed a model to interpret the raw AFM data. RESULTS: All types of fibrillar collagen diameters studied became larger for diabetic versus control animals. Values for diabetic and control collagen fiber diameters in SD rats were 78 nm and 72 nm for SN epineurium, and 49 nm and 43 nm for SN endoneurium. For diabetic and control BB rats, these values were 83 nm and 77 nm (SN epineurium) and 49 nm and 43 nm (SN endoneurium). Values of 161 nm and 125 nm were found for diabetic and control tail tendon of BB rats. No significant changes were observed in any of the five comparisons made in D-band spacings that ranged from 63 to 69 nm. CONCLUSIONS: The best means we have found to reduce raw AFM data is to measure several diameters with a single scan, using valley-to-valley measurements. Structural, fibrillar collagens of the nerve and tendon become larger in rats exposed to prolonged diabetes.