Effects of repeated biaxial loads on the creep properties of cardinal ligaments.

The cardinal ligament (CL) is one of the major pelvic ligaments providing structural support to the vagina/cervix/uterus complex. This ligament has been studied mainly with regards to its important function in the treatment of different diseases such as surgical repair for pelvic organ prolapse and radical hysterectomy for cervical cancer. However, the mechanical properties of the CL have not been fully determined, despite the important in vivo supportive role of this ligament within the pelvic floor. To advance our limited knowledge about the elastic and viscoelastic properties of the CL, we conducted three consecutive planar equi-biaxial tests on CL specimens isolated from swine. Specifically, the CL specimens were divided into three groups: specimens in group 1 (n = 7) were loaded equi-biaxially to 1 N, specimens in group 2 (n = 8) were loaded equi-biaxially to 2N, and specimens in group 3 (n = 7) were loaded equi-biaxially to 3N. In each group, the equi-biaxial loads of 1N, 2N, or 3N were applied and kept constant for 1200s three times. The two axial loading directions were selected to be the main in-vivo loading direction of the CL and the direction that is perpendicular to it. Using the digital image correlation (DIC) method, the in-plane Lagrangian strains in these two loading directions were measured throughout the tests. The results showed that CL was elastically anisotropic, as statistical differences were found between the mean strains along the two axial loading directions for specimens in group 1, 2, or 3 when the equi-biaxial load reached 1N, 2N, or 3N, respectively. For specimens in group 1 and 2, no statistical differences were detected in the mean normalized strains (or, equivalently, the increase in strain over time) between the two axial loading directions for each creep test. For specimens in group 3, some differences were noted but, by the end of the 3rd creep test, there were no statistical differences in the mean normalized strains between the two axial loading directions. These findings indicated that the increase in strain over time by the end of the 3rd creep test were comparable along these directions. The greatest mean normalized strain (or, equivalently, the largest increase in strain over time) was measured at the end of the 1st creep test (t=1200s), regardless of the equi-biaxial load magnitude or loading direction. Mean normalized strains during the 2nd and 3rd creep tests (t = 100, 600, and 1200s), along each loading direction, were not statistically different. Isochronal data collected at 1N, 2N, or 3N equi-biaxial loads indicated that the CL may be a nonlinear viscoelastic material. Overall, this experimental study offers new knowledge of the mechanical properties of the CL that can guide the development of better treatment methods such as surgical reconstruction for pelvic organ prolapse and radical hysterectomy for cervical cancer.

Mechanical recruitment of N- and C-crosslinks in collagen type I.

Collagen type I is an extracellular matrix protein found in connective tissues such as tendon, ligament, bone, skin, and the cornea of the eyes, where it functions to provide tensile strength; it also serves as a scaffold for cells and other extracellular matrix components. A single collagen type I molecule is composed of three amino acid chains that form a triple helix for most of the molecule's length; non-triple-helical extensions called N- and C-telopeptides are located at the amino/N-terminal and carboxy/C-terminal ends of the molecule, respectively. In two of the three chains, the C-telopeptide has been reported to possess a hair-pin/hook conformation, while the three N-telopeptides display a more extended structure. These telopeptides are crucial for the formation of enzymatic covalent crosslinks that form in collagens near their N- and C-ends. Such crosslinks provide structural integrity, strength, and stiffness to collagenous tissues. However, deformation mechanisms of N- and C-crosslinks and functional roles for the N- and C-telopeptide conformations are not yet well known. By performing molecular dynamics simulations, we demonstrated that two dehydro-hydroxylysino-norleucine crosslinks, positioned at the N- and C-crosslinking sites, exhibited a two-stage response to the mechanical deformation of their parent molecules. We observed that the N-crosslink served as the first responder to mechanical deformation, followed by the C-crosslink. The results of our simulations suggest a mechanical recruitment mechanism for N- and C-crosslinks. Understanding this mechanism will be crucial for the development of larger-scale predictive models of the mechanical behavior of native collagenous tissues, engineered tissues, and collagen-based materials.

The cytoskeletal organization of breast carcinoma and fibroblast cells inside three dimensional (3-D) isotropic silicon microstructures.

Studying the cytoskeletal organization as cells interact in their local microenvironment is interest of biological science, tissue engineering and cancer diagnosis applications. Herein, we describe the behavior of cell lines obtained from metastatic breast tumor pleural effusions (MDA-MB-231), normal fibrocystic mammary epithelium (MCF10A), and HS68 normal fibroblasts inside three dimensional (3-D) isotropic silicon microstructures fabricated by a single-mask, single-isotropic-etch process. We report differences in adhesion, mechanism of force balance within the cytoskeleton, and deformability among these cell types inside the 3-D microenvironment. HS68 fibroblasts typically stretched and formed vinculin-rich focal adhesions at anchor sites inside the etched cavities. In contrast, MCF10A and MDA-MB-231 cells adopted the curved surfaces of isotropic microstructures and exhibited more diffuse vinculin cytoplasmic staining in addition to vinculin localized in focal adhesions. The measurement of cells elasticity using atomic force microscopy (AFM) indentation revealed that HS68 cells are significantly stiffer (p < 0.0001) than MCF10A and MDA-MB-231 cells. Upon microtubule disruption with nocodazole, fibroblasts no longer stretched, but adhesion of MCF10A and MDA-MB-231 within the etched features remained unaltered. Our findings are consistent with tensegrity theory. The 3-D microstructures have the potential to probe cytoskeletal-based differences between healthy and diseased cells that can provide biomarkers for diagnostics purposes.