Altered mechanics of vaginal smooth muscle cells due to the lysyl oxidase-like1 knockout.

The remodeling mechanisms that cause connective tissue of the vaginal wall, consisting mostly of smooth muscle, to weaken after vaginal delivery are not fully understood. Abnormal remodeling after delivery can contribute to development of pelvic organ prolapse and other pelvic floor disorders. The present study used vaginal smooth muscle cells (vSMCs) isolated from knockout mice lacking the expression of the lysyl oxidase-like1 (LOXL1) enzyme, a well-characterized animal model for pelvic organ prolapse. We tested if vaginal smooth muscle cells from LOXL1 knockout mice have altered mechanics including stiffness and surface adhesion. Using atomic force microscopy, we performed nanoindentations on both isolated and confluent cells to evaluate the effect of LOXL1 knockout on in vitro cultures of vSMCs cells from nulliparous mice. The results show that LOXL1 knockout vSMCs have increased stiffness in pre-confluent but decreased stiffness in confluent cultures (p* < 0.05) and significant decreased surface adhesion in pre-confluent cultures (p* < 0.05). This study provides evidence that the weakening of vaginal connective tissue in the absense of LOXL1 changes the mechanical properties of the vSMCs. STATEMENT OF SIGNIFICANCE: Pelvic organ prolapse is a common condition affecting millions of women worldwide, which significantly impacts their quality of life. Alterations in vaginal and pelvic floor mechanical properties can change their ability to support the pelvic organs. This study provides evidence of altered stiffness of vaginal smooth muscle cells from mice resembling pelvic organ prolapse. The results from this study set a foundation to develop pathophysiology-driven therapies focused on the interplay between smooth muscle mechanics and extracellular matrix remodeling.

Nano measurements with micro-devices: mechanical properties of hydrated collagen fibrils.

The mechanical response of a biological material to applied forces reflects deformation mechanisms occurring within a hierarchical architecture extending over several distinct length scales. Characterizing and in turn predicting the behaviour of such a material requires an understanding of the mechanical properties of the substructures within the hierarchy, the interaction between the substructures, and the relative influence of each substructure on the overall behaviour. While significant progress has been made in mechanical testing of micrometre to millimetre sized biological specimens, quantitative reproducible experimental techniques for making mechanical measurements on specimens with characteristic dimensions in the smaller range of 10-1000 nm are lacking. Filling this void in experimentation is a necessary step towards the development of realistic multiscale computational models useful to predict and mitigate the risk of bone fracture, design improved synthetic replacements for bones, tendons and ligaments, and engineer bioinspired efficient and environmentally friendly structures. Here, we describe a microelectromechanical systems device for directly measuring the tensile strength, stiffness and fatigue behaviour of nanoscale fibres. We used the device to obtain the first stress-strain curve of an isolated collagen fibril producing the modulus and some fatigue properties of this soft nanofibril.

Molecular views and measurements of hemostatic processes using atomic force microscopy.

Hemostasis and thrombosis are highly complex and coordinated interfacial responses to vascular injury. In recent years, atomic force microscopy (AFM) has proven to be a very useful approach for studying hemostatic processes under near physiologic conditions. In this report, we review recent progress in the use of AFM for studying hemostatic processes, including molecular level visualization of plasma proteins, protein aggregation and multimer assembly, and structural and morphological details of vascular cells under aqueous conditions. AFM offers opportunities for visualizing surface-dependent molecular and cellular interactions in three dimensions on a nanoscale and for sensitive, picoNewton level, measurements of intermolecular forces. AFM has been used to obtain molecular and sub-molecular, resolution of many biological molecules and assemblies, including coagulation proteins and cell surfaces. Surface-dependent molecular processes including protein adsorption, conformational changes, and subsequent interactions with cellular components have been described. This review outlines the basic principles and utility of AFM for imaging and force measurements, and offers objective perspectives on both the advantages and disadvantages. We focus primarily on molecular level events related to hemostasis and thrombosis, particularly coagulation proteins, and blood platelets, but also explore the use of AFM in force measurements and surface property mapping.

Three dimensional structure of human fibrinogen under aqueous conditions visualized by atomic force microscopy.

Fibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.

Shape and size of isolated bone mineralites measured using atomic force microscopy.

The inorganic phase of bone is comprised primarily of very small mineralites. The size and shape of these mineralites play fundamental roles in maintaining ionic homeostasis and in the biomechanical function of bone. Using atomic force microscopy, we have obtained direct three-dimensional visual evidence of the size and shape of native protein-free mineralites isolated from mature bovine bone. Approximately 98% of the mineralites are less than 2 nm thick displaying a plate-like habit. Distributions of both thickness and width show single peaks. The distribution of lengths may be multimodal with distinct peaks separated by approximately 6 nm. Application of our results is expected to be of use in the design of novel orthopaedic biomaterials. In addition, they provide more accurate inputs to molecular-scale models aimed at predicting the physiological and mechanical behavior of bone.

Interactions of human von Willebrand factor with a hydrophobic self-assembled monolayer studied by atomic force microscopy.

Human von Willebrand Factor (vWF) was studied by atomic force microscopy under physiologic buffer on a hydrophobic octadecyltrichlorosilane self-assembled monolayer. The self-assembled monolayer deposited on glass was sufficiently smooth (root mean square roughness = 0.25 +/- 0.12 nm) to permit identification of adsorbed vWF. Adhesion of the protein to the hydrophobic substrate was sufficient to allow repeated scanning by the atomic force microscope probe, and images of vWF on a submolecular scale were obtained. The frictional force between the surface and the protein was sufficient to withstand an applied lateral force of 19 nN. This result shows that vWF experiences strong interaction with a hydrophobic surface in aqueous media. Statistical analysis of adsorbed vWF shows that the protein is composed of large globular domains with elliptical cross sections of average dimensions 56 +/- 24 nm (major axis) 26 +/- 19 nm (minor axis), and 2.8 +/- 1.0 nm (height). Further analysis of the major axis dimension shows that the molecular chain of vWF contains two statistically different populations of domain size. However, no sequence order of the different domains within the individual molecule was found. On the basis of our analysis of the globular domains, we present a model describing the three-dimensional structure of vWF protomer adsorbed on a hydrophobic surface in a physiologic solution.

Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.

High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.

Size and shape of mineralites in young bovine bone measured by atomic force microscopy.

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm x 6 nm x 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous "channels" across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.

Shear-dependent changes in the three-dimensional structure of human von Willebrand factor.

The three-dimensional tertiary structure of human von Willebrand Factor (vWF) on a hydrophobic surface under aqueous conditions and different shear stress regimes was studied by atomic force microscopy (AFM). vWF was imaged by AFM at molecular level resolution under negligible shear stress, under a local applied shear force (7.4 to 19 nN) using the AFM probe in contact mode scanning, and after subjecting vWF to a range of shear stress (0 to 42.4 dyn/cm2) using a rotating disk system. The results demonstrate that vWF undergoes a shear stress-induced conformational transition from a globular state to an extended chain conformation with exposure of intra-molecular globular domains at a critical shear stress of 35 +/- 3.5 dyn/cm2. The globular vWF conformation (149 nm by 77 nm and height 3.8 nm) is representative of native vWF after simple diffusion to the hydrophobic surface, followed by adhesion and some spreading. In a shear stress field above the critical value, protein unfolding occurs and vWF is observed in extended chain conformations oriented in the direction of the shear stress field with molecular lengths ranging from 146 to 774 nm and 3.4 nm mean height. The shear stress-induced structural changes to vWF suggest a close conformation-function relationship in vWF properties for thrombogenesis in regions of high shear stress.