Single Collagen Fiber Research Articles

Three-dimensional fine structures in deep fascia revealed by combined use of cryo-fixed histochemistry and low-vacuum scanning microscopy

Recent physiological studies have shown that the deep fascia has received much attention concerning clinical medicine; however, histological examination of the deep fascia has not been well established. In this study, we aimed to clarify and visualize the structure of the deep fascia by taking advantage of cryofixation techniques and low-vacuum scanning electron microscopy. As a result, the ultrastructural observations revealed three-dimensional stratification of the deep fascia composed of three layers: the first superficial layer consisting of collagen fibers extending in various directions with blood vessels and peripheral nerves; the second intermediate layer formed by single straight and thick collagen fibers with flexibility; and the third deepest layer, consisting of relatively straight and thin collagen fibers. We explored the use of two hooks to hold a piece of deep fascia in place through the course of cryo-fixation. A comparative observation with or without the hook-holding procedure would indicate the morphological adaptation to physiological stretch and contraction of the deep fascia. The present morphological approach paves the way to visualize three-dimensional ultrastructures for future biomedical studies including clinical pathophysiology.

The influence of glutaraldehyde on the microscopic structure of human pericardium

ObjectivesIn our study, we investigate the collagen structure of human pericardium microscopically in dependence of glutaraldehyde (GA) concentration and fixation time. MethodsPericardial samples were taken from 9 patients aged 40+ years who underwent cardiac surgery, either coronary artery bypass surgery or valve implantation/reconstruction. Specimens were cut in 5 equal pieces and treated with GA at fixed concentrations (0.3125%, 0.625%, or 1.25%) but different exposer times (5 min, 10 min, 20 min, 30 min, and 60 min). Elastica van Gieson (EvG) staining was used for microscopic examination of pericardial collagen structure. ResultsThe collagen structure studied microscopically depended on both GA incubation time and GA concentration. At low GA concentrations (0.3125%, 0.625%) and short incubation times, individual collagen fibers appeared separately. After one hour incubation period, single collagen fibers could not be distinguished at any GA concentration. For fixed incubation times no differences were seen in the collagen structure when 0.3125% and 0.625% GA were used. However, at a concentration of 1.25% GA fusion of individual collagen fibers was already observed at low incubation times. ConclusionPericardial collagen structure changes with increasing incubation time and increasing GA concentration by raising fusion of single fibers. For GA concentrations of ≤0.625%, fiber fusion depends plainly on incubation time. That is relevant as this concentration is used in cardiac surgery. At a concentration of 1.25% GA, single collagen fibers could not be separated, even at short incubation times. Fusion of individual collagen fibers and changes in appearance (less undulating) were assumed to be responsible for stiffening of GA-fixed pericardium.

The Nucleus Bypasses Obstacles by Deforming Like a Drop with Surface Tension Mediated by Lamin A/C.

Migrating cells must deform their stiff cell nucleus to move through pores and fibers in tissue. Lamin A/C is known to hinder cell migration by limiting nuclear deformation and passage through confining channels, but its role in nuclear deformation and passage through fibrous environments is less clear. Cell and nuclear migration through discrete, closely spaced, slender obstacles which mimic the mechanical properties of collagen fibers are studied. Nuclei bypass slender obstacles while preserving their overall morphology by deforming around them with deep local invaginations of little resisting force. The obstacles do not impede the nuclear trajectory and do not cause rupture of the nuclear envelope. Nuclei likewise deform around single collagen fibers in cells migrating in 3D collagen gels. In contrast to its limiting role in nuclear passage through confining channels, lamin A/C facilitates nuclear deformation and passage through fibrous environments; nuclei in lamin‐null (Lmna−/− ) cells lose their overall morphology and become entangled on the obstacles. Analogous to surface tension‐mediated deformation of a liquid drop, lamin A/C imparts a surface tension on the nucleus that allows nuclear invaginations with little mechanical resistance, preventing nuclear entanglement and allowing nuclear passage through fibrous environments.

Label-free characterization of collagen fibers in cancerous esophagus tissues using ratiometric nonlinear optical microscopy.

The issue of classifying esophageal cancer at various developmental stages is crucial for determining the optimized treatment protocol for the patients, as well as the prognosis. Precision improvement in staging esophageal cancer keeps seeking quantitative and analytical imaging methods that could augment histopathological techniques. In this work, we used nonlinear optical microscopy for ratiometric analysis on the intrinsic signal of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) from single collagen fibers only in submucosa of esophageal squamous cell carcinoma (ESCC). The blind tests of TPEF/SHG and forward (F)/backward (B) SHG were demonstrated to compare with the histology conclusion. The discussion of sensitivity and specificity was provided via statistical comparison between the four stages of esophageal cancer. To the best of our knowledge, this is the first study of using these two ratios in combination for staging ESCC.

Influence of osmolarity and hydration on the tear resistance of the human amniotic membrane

The amnion is considered to be the load-bearing part of the fetal membranes. We investigated the influence of osmolarity of the testing medium and hydration on its fracture toughness. Mode I fracture tests revealed that physiological variations in the bath osmolarity do not influence the tear resistance of amnion, while larger changes, i.e. from physiological saline solution to distilled water, lead to a significant reduction of the fracture toughness. Uniaxial tensile tests on collagen hydrogels confirmed the reduction in toughness, suggesting that lower bath osmolarity triggers changes in the failure properties of single collagen fibers. Prenatal surgeries, in particular fetoscopic procedures with partial amniotic carbon dioxide insufflation, might result in dehydration of the amnion. Dehydration induced a brittle behavior; however, subsequent rehydration for 15 min resulted in a similar tear resistance as for the fresh tissue.

Dissolution of Collagen Fibers from Tannery Solid Wastes in 1-Allyl-3-methylimidazolium Chloride and Modulation of Regenerative Morphology

Collagen fibers from leather-making solid wastes were successfully dissolved in 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). Polarized optical microscopy, temperature-dependent Fourier-transformed infrared spectroscopy (FTIR), and dynamic light scattering were applied to characterize the structural changes of collagen fibers during the dissolution process. The results demonstrated that a single collagen fiber could be totally dissolved in 40 min, at a maximum of 100 °C. During the dissolution process, the average molecular weight of the collagen chains decreased, suggesting that the structure of collagen was destroyed to some degree. Collagen was regenerated from the solution by treatment with alcohol reagents. The regenerated collagen had the characteristic structure of the initial collagen in FTIR spectra. The morphologies of regenerated collagen included nanospheres aggregation, nanosphere dispersion, and networking, which could be adjusted by changing the alcohol type, alcohol temperature, and col...

Multi-scale modeling of soft fibrous tissues based on proteoglycan mechanics

Collagen in the form of fibers or fibrils is an essential source of strength and structural integrity in most organs of the human body. Recently, with the help of complex experimental setups, a paradigm change concerning the mechanical contribution of proteoglycans (PGs) took place. Accordingly, PG connections protect the surrounding collagen fibrils from over-stretching rather than transmitting load between them. In this paper, we describe the reported PG mechanics and incorporate it into a multi-scale model of soft fibrous tissues. To this end, a nano-to-micro model of a single collagen fiber is developed by taking the entropic-energetic transition on the collagen molecule level into account. The microscopic damage occurring inside the collagen fiber is elucidated by sliding of PGs as well as by over-stretched collagen molecules. Predictions of this two-constituent-damage model are compared to experimental data available in the literature.

Minimum wound size for clotting: flowing blood coagulates on a single collagen fiber presenting tissue factor and von Willebrand factor.

It is unknown if a lower size limit exists for human blood coagulation under flow over physiological vessel wall triggers as small as a single collagen fiber. Prior determinations of the smallest sized surface stimuli necessary for clotting of human blood, defined as the patch size threshold, have not deployed whole blood, hemodynamic flow, and platelet adhesive stimuli. For whole blood perfused in microfluidic devices, we report that steady venous flow (wall shear rate, 100 s(-1)) was sufficient to drive platelet deposition on 20 micron long zones of collagen fibers or on a single fiber. With tissue factor (TF)-coated collagen, flowing blood generated robust platelet deposits, platelet-localized thrombin, and fibrin on a single collagen fiber, thus demonstrating the absence of a physiological patch size threshold under venous flow. In contrast, at arterial wall shear rate (1000 s(-1)) with TF present, essentially no platelet or fibrin deposition occurred on 20 micron collagen zones or on a single collagen fiber, demonstrating a patch threshold, which was overcome by pre-coating the collagen with von Willebrand factor (vWF). For venous flows, human blood can clot on one of the smallest biological units of a single collagen fiber presenting TF. For arterial flows, vWF together with TF allows human blood to generate thrombin and fibrin on a patch stimulus as limited as a single collagen fiber. vWF-dependent platelet adhesion represents a particle-based sensing mechanism of micron-scale stimuli that then allows amplification of the molecular components of TF-driven thrombin and fibrin production under arterial flow.

Mechanics of electrospun collagen and hydroxyapatite/collagen nanofibers

Single collagen fibers and nanohydroxyapatite/collagen nanocomposite fibers with 6.4–18.5wt% hydroxyapatite (HA) content were prepared by electrospinning. Their mechanical properties were systematically investigated at ambient conditions by means of nanotensile tests. A narrow range of fiber diameters, 250–350nm, was selected for these tests as size effects are observed for the mechanical properties of all the fibers types, namely a decrease as the fiber diameter increases. The pure collagen fibers are found to exhibit tensile properties comparable to natural collagen fibril. Young's modulus of the HA-filled nanocomposite fibers is found to be only slightly higher than that of the pure collagen fibers, but significant improvements in strength, strain and toughness, are obtained. Optimal mechanical properties arise in the 6.4–11.6wt% HA range.

Elastic and viscoelastic properties of a type I collagen fiber

A new mathematical model is presented to describe the elastic and viscoelastic properties of a single collagen fiber. The model is formulated by accounting for the mechanical contribution of the collagen fiber's main constituents: the microfibrils, the interfibrillar matrix and crosslinks. The collagen fiber is modeled as a linear elastic spring, which represents the mechanical contribution of the microfibrils, and an arrangement in parallel of elastic springs and viscous dashpots, which represent the mechanical contributions of the crosslinks and interfibrillar matrix, respectively. The linear elastic spring and the arrangement in parallel of elastic springs and viscous dashpots are then connected in series. The crosslinks are assumed to gradually break under strain and, consequently, the interfibrillar is assumed to change its viscous properties. Incremental stress relaxation tests are conducted on dry collagen fibers reconstituted from rat tail tendons to determine their elastic and viscoelastic properties. The elastic and total stress–strain curves and the stress relaxation at different levels of strain collected by performing these tests are then used to estimate the parameters of the model and evaluate its predictive capabilities.

An engineering, multiscale constitutive model for fiber‐forming collagen in tension

This work proposes a nonlinear constitutive model for a single collagen fiber. Fiber-forming collagen can exhibit different hierarchies of basic units, called fascicles, bundles, fibrils, microfibrils, and so forth, down to the molecular (tropocollagen) level. Exploiting the fact that at each hierarchy level the microstructure can be seen, at least approximately, as that of a wavy, or crimped, extensible cable, the proposed stress-strain model considers a given number of levels, each of which contributes to the overall mechanical behavior according to its own geometrical features (crimp, or waviness), as well as to the basic mechanical properties of the tropocollagen. The crimp features at all levels are assumed to be random variables, whose statistical integration furnishes a stress-strain curve for a collagen fiber. The soundness of this model-the first, to the Authors' knowledge, to treat a single collagen fiber as a microstructured nonlinear structural element-is checked by its application to collagen fibers for which experimental results are available: rat tail tendon, periodontal ligament, and engineered ones. Here, no attempt is made to obtain a stress-strain law for generic collagenous tissues, which exhibit specific features, often much more complex than those of a single fiber. However, it is trivial to observe that the availability of a sound, microstructurally based constitutive law for a single collagen fiber (but applicable at any sub-level, or to any other material with a similar microstructure) is essential for assembling complex constitutive models for any collagenous fibrous tissue.

Mechanical properties of single electrospun collagen type I fibers

The mechanical properties of single electrospun collagen fibers were investigated using scanning mode bending tests performed with an AFM. Electrospun collagen fibers with diameters ranging from 100 to 600 nm were successfully produced by electrospinning of an 8% w/v solution of acid soluble collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP). Circular dichroism (CD) spectroscopy showed that 45% of the triple helical structure of collagen molecules was denatured in the electrospun fibers. The electrospun fibers were water soluble and became insoluble after cross-linking with glutaraldehyde vapor for 24 h. The bending moduli and shear moduli of both non- and cross-linked single electrospun collagen fibers were determined by scanning mode bending tests after depositing the fibers on glass substrates containing micro-channels. The bending moduli of the electrospun fibers ranged from 1.3 to 7.8 GPa at ambient conditions and ranged from 0.07 to 0.26 MPa when immersed in PBS buffer. As the diameter of the fibrils increased, a decrease in bending modulus was measured clearly indicating mechanical anisotropy of the fiber. Cross-linking of the electrospun fibers with glutaraldehyde vapor increased the shear modulus of the fiber from ∼30 to ∼50 MPa at ambient conditions.

Changes in light-induced fluorescence of cervical collagen in guinea pigs during gestation and after sodium nitroprusside treatment.

Light-induced fluorescence (LIF) of collagen was used to investigate in vivo changes in cervical collagen in guinea pigs during gestation and following sodium nitroprusside treatment. Natural fluorescence of collagen is due to collagen cross-linking molecules that connect single collagen fibers and therefore provide rigidity of the cervical stroma. LIF of cervical collagen was measured from the surface of the exocervix in anesthetized nonpregnant and timed pregnant guinea pigs at different times of gestation with an instrument designed in our lab (Collascope). Measurements were also performed in guinea pigs at midgestation before and 8 hours after intracervical treatment with sodium nitroprusside. Collagen fluorescence decreased significantly as pregnancy progressed, reached lowest values at delivery, and increased gradually postpartum. Treatment with sodium nitroprusside, but not with the vehicle, caused a significant decrease in LIF (p = 0.007). We conclude, that LIF changes in the cervix reflect the gradual cervical softening (ripening) during pregnancy and the return to the rigid state of the cervix postpartum. Cervical softening during pregnancy, and after sodium nitroprusside treatment, is associated with a decrease in collagen cross-links. Measurements of LIF can be used to investigate cervical softening in vivo.

Lymphatic vessels in lungfishes (Dipnoi)

Lymphatic capillaries are distributed throughout the body of lepidosirenid and protopterid Dipnoi, except in the central nervous system. They form small, interconnected units which are individually evacuated into nearby blood capillaries by lymphatic micropumps. The number of lymphatic micropumps varies considerably in different parts of the body. In fin areas, 30–50 per mm3 tissue may be considered normal in Protopterus annectens, but up to 105 per mm3 have been counted in an anterior fin of Lepidosiren paradoxa. Lymphatic capillaries are formed by thin endothelial cells with fine processes into the surrounding interstitial space. Occasionally there is a faint, discontinuous basal lamina. Pericytes, however, are completely absent. Microfibrils establish contact between endothelial cells and surrounding connective tissue fibers. The lymphatic micropumps are essentially spherical, contractile organs of 35–55 μm in diameter. Their central lumen is lined by extensions of a single endothelial cell. Additional endothelial cells form inflow and outflow valves. The endothelial layer is surrounded by a single large, highly specialized muscle cell. This spherical muscle cell has many perforations, allowing the passage of thin outward processes of the endothelial cell which form part of the suspension apparatus of the lymphatic micropump. The muscle cell establishes a specialized end-to-end contact between opposing parts of its own cell membrane. This contact is very similar to an intercalated disc in vertebrate heart muscle. Each lymphatic micropump is suspended within a cell-free tissue area by microfibrils which radiate from the lymphatic micropump into the surrounding connective tissue. The microfibrils are occasionally reinforced by single collagen fibers. The cell-free area around each lymphatic micropump appears as a bright halo in both light and electron micrographs. No type of lymphatic vessel other than lymphatic capillaries could be detected in the Dipnoi studied. Lepidosireniform Dipnoi are the only Vertebrata besides the Tetrapoda in which lymphatic vessels and characteristic lymphatic pumps have been documented. In addition, these Dipnoi and all Tetrapoda share the same overall design of blood circulation, which is not divided into a primary and a secondary system of vessels, as it is in Actinopterygii, Chondrichthyes, and Agnatha. Since there are primary and secondary blood vessels in the gills of Latimeria chalumnae, while the existence of lymphatic vessels has not been confirmed, general angioarchitecture should be taken into account as an important character when phylogenetic relationships among extant Sarcopterygii are discussed.

Guidance and Activation of Murine Macrophages by Nanometric Scale Topography

We studied the guidance and activation of macrophages from the P388D1 cell line and rat peritoneum by topographic features on a nanometric scale. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves and steps, 30–282 nm deep. The contact of cells with the patterned surface activated cell spreading and adhesion and increased the number of protrusions of the cell membrane. These changes were accompanied by an increase in the amount of F-actin in cells. The accumulation of F-actin and vinculin in cells was observed along the edges of single steps or grooves. Formation of focal contacts along discontinuities in the substratum was accompanied by the phosphorylation of tyrosine colocalized with F-actin and vinculin. A similar pattern of staining was seen in cells stained for vitronectin receptor, αV integrin, but not for integrins α5β1 or α3β1. Cells cultured on nanogrooves showed a higher phagocytotic activity than cells cultured on plain substrata. We show that macrophages can react to ultrafine features of topography of a size comparable to that of a single collagen fiber and become activated by the contact with topographic features.

Dependence of stress-strain nonlinearity of connective tissues on the geometry of collagen fibres

Collagen fibers in connective tissues have a periodic, “crimped” shape. In this work, they are modelled as long, sinusoidal beams. Constitutive equations in uniaxial tension are obtained by geometric considerations for a single collagen fiber as well as for bundles of fibers embedded in a matrix. The effect of fiber orientation is briefly discussed.