On collagen fibril diameter distributions

Distributions of collagen fibril diameters, measured from electron micrographs, are compared using some of the common measures of the mean of the distribution. The role of mixture distributions in modelling these distributions is considered and a method of fitting such distributions to data observed in the form of histograms is described. The method is illustrated with six pairs of fibril diameter distributions used in assessing age and structural changes in various collagen samples from different species of animal. The extent of changes in diameter distributions is assessed in terms of the estimated means, variances and mixing proportions for the component distributions of the histograms in each pair.     

Ultrastructural organization of skin: classification on the basis of mechanical role

In our previous developmental study on skin we reported that correlations existed between the collagen fibril diameter distribution, glycosaminoglycan content and composition, and mechanical role. In this work we present new but related electron microscope data on the ultrastructure of skins from flounder, marlin, cod, frog, toad, caiman, viper, pigeon, whale and opossum. It is shown that mammalian and avian body skins generally contain relatively sharp unimodal distributions of fibril diameter consistent with a "passive" mechanical role. In contrast most reptilian and fish skins have broad, right-skewed (or bimodal) distributions of fibril diameters compatible with the exotendinous attributes required of an "active" skin. A study of the changing form of the collagen fibril diameter distribution with depth in the dermis is also reported.     

Graded Arrangement of Collagen Fibrils in the Equine Superficial Digital Flexor Tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.     

Graded arrangement of collagen fibrils in the equine superficial digital flexor tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.     

An Age-Related Study of Morphology and Cross-Link Composition of Collagen Fibrils in the Digital Flexor Tendons of Young Thoroughbred Horses

The superficial digital flexor tendon is the most commonly injured tendon in the racing Thoroughbred. Despite the clinical significance of this structure, only limited data exist regarding normal age-related morphology of the tensile units, the collagen fibrils. The age at which these collagen fibrils become mature in composition and structure may be of importance. Consequently, the association of age and collagen fibril crosslink composition, diameter distribution and crimp morphology in the superficial and deep digital flexor tendons of Thoroughbreds up to and including three years of age has been studied. Replacement of immature crosslinks, peaking of the collagen fibril mass-average diameter and collagen fibril index, and stabilization of collagen crimp morphology changes supported the hypothesis that both digital flexor tendons become mature in structure by two years of age.     

Investigation of the Collagen Fibril Distribution in the Medial Collateral Ligament in a Rat Knee Model

This study compared the collagen fibril diameter distribution among six anatomical sites of the rat medial collateral ligament (MCL). Ultrathin MCL sections from 4 male Sparague-Dawley rats were examined electron microscopically. With an automated quantitation method, 41,638 fibrils were measured and compared among the periphery and core regions of the femoral, middle, and tibial portions of the MCL. Results demonstrated significant difference ( p < .0033) in mean fibril diameter distribution among the six sites. The mass-averaged diameters of the core and peripheral fibrils were between 175.53 to 190.82 nm and 88.47 to 109.18 nm, respectively, with the peripheral fibrils more homogeneous in size. The fibrils occupied 36.7% to 57.1% of the cross-sectional area of the ligament. About 50% of the fibrils had an oblique factor of 0.8-1.0, implying that most fibrils were aligned longitudinally. This study has provided a detailed profile of the collagen fibril distributions in rat MCL.     

Investigation of the collagen fibril distribution in the medial collateral ligament in a rat knee model

This study compared the collagen fibril diameter distribution among six anatomical sites of the rat medial collateral ligament (MCL). Ultrathin MCL sections from 4 male Sprague-Dawley rats were examined electron microscopically. With an automated quantitation method, 41,638 fibrils were measured and compared among the periphery and core regions of the femoral, middle, and tibial portions of the MCL. Results demonstrated significant difference (p < .0033) in mean fibril diameter distribution among the six sites. The mass-averaged diameters of the core and peripheral fibrils were between 175.53 to 190.82 nm and 88.47 to 109.18 nm, respectively, with the peripheral fibrils more homogeneous in size. The fibrils occupied 36.7% to 57.1% of the cross-sectional area of the ligament. About 50% of the fibrils had an oblique factor of 0.8-1.0, implying that most fibrils were aligned longitudinally. This study has provided a detailed profile of the collagen fibril distributions in rat MCL.     

Ultrastructural localisation and size distribution of collagen fibrils in Glisson's sheath of rat liver: implications for mechanical environment and possible producing cells

The ultrastructure and size distributions of collagen fibrils in Glisson's sheath were investigated in the rat liver to analyse the mechanical environment around the fibrils and their possible cells of origin. Glisson's sheath was found to contain 2 populations of collagen fibrils with different diameters and distinct localisations, namely fibroblast‐associated and bile epithelium‐associated. Fibroblast‐associated collagen was composed of fibrils arranged in bundles and constituted the majority of the collagen in Glisson's sheath. Bile epithelium‐associated collagen was represented by small dispersed groups of fibrils just beneath the basement membrane of the bile duct. The basement membrane of the bile duct was frequently reduplicated into a few or as many as 10 layers of laminae densae, with scattered collagen fibrils between these laminae. The diameters of the fibrils of both groups of collagen increased in relation to the calibre of the bile duct, whereas at any given place in Glisson's sheath bile epithelium‐associated collagen fibrils had a smaller diameter compared with those of the fibroblast‐associated fibrils. The increment in fibril diameter along the bile duct is considered to be correlated with the increase in mechanical stress acting on Glisson's sheath. The difference in diameter between the 2 populations as well as the incorporation of fibrils between the laminae densae of the basement membrane of the bile duct supports the view that the bile epithelium‐associated collagen is produced by the epithelial cells of the bile duct, thus having a different origin from that of fibroblast‐associated collagen. These findings provide the first evidence that the epithelial cells of the interlobular bile duct produce fibril‐forming collagen. Furthermore, it is suggested that cholestasis stimulates the epithelial cells of interlobular bile duct to increased synthesis of fibril‐forming collagen that is also produced by these cells under physiological conditions.     

Collagen fibril arrangement and size distribution in monkey oral mucosa

Collagen fibre organisation and fibril size were studied in the buccal gingival and hard palate mucosa of Macacus rhesus monkey. Light and electron microscopy analysis showed connective papillae exhibiting a similar inner structure in the different areas examined, but varying in distribution, shape and size. Moving from the deep to surface layers of the buccal gingival mucosa (free and attached portions), large collagen fibril bundles became smaller and progressively more wavy with decreasing collagen fibril diameter. This gradual diameter decrease did not occur in the hard palate mucosa (free portion, rugae and interrugal regions) where the fibril diameter remained constant. A link between collagen fibril diameter and mechanical function is discussed.     

Slow stretching that mimics embryonic growth rate stimulates structural and mechanical development of tendon-like tissue in vitro

A distinctive feature of embryonic tendon development is the steady increase in collagen fibril diameter and associated improvement of tissue mechanical properties. A potential mechanical stimulus for these changes is slow stretching of the tendon during limb growth. Testing this hypothesis in vivo is complicated by the presence of other developmental processes including muscle development and innervation. Here we used a cell culture tendon-like construct to determine if slow stretch can explain the increases in fibril diameter and mechanical properties that are observed in vivo. Non-stretched constructs had an ultrastructural appearance and mechanical properties similar to those of early embryonic tendon. However, slowly stretching during 4 days in culture increased collagen fibril diameter, fibril packing volume, and mechanical stiffness, and thereby mimicked embryonic development. 3D EM showed cells with improved longitudinal alignment and elongated nuclei, which raises the hypothesis that nuclear deformation could be a novel mechanism during tendon development.     

Electron microscopic quantification of collagen fibril diameters in the rabbit medial collateral ligament: a baseline for comparison

To establish a normal baseline for comparison, thirty-one thousand collagen fibril diameters were measured in calibrated transmission electron (TEM) photomicrographs of normal rabbit medial collateral ligaments (MCL's). A new automated method of quantitation was used to compare statistically fibril minimum diameter distributions in one midsubstance location in both MCL's from six animals at 3 months of age (immature) and three animals at 10 months of age (mature). Pooled results demonstrate that rabbit MCL's have statistically different (p less than 0.001) mean minimum diameters at these two ages. Interanimal differences in mean fibril minimum diameters were also significant (p less than 0.001) and varied by 20% to 25% in both mature and immature animals. Finally, there were significant differences (p less than 0.001) in mean diameters and distributions from side-to-side in all animals. These mean left-to-right differences were less than 10% in all mature animals but as much as 62% in some immature animals. Statistical analysis of these data demonstrate that animal-to-animal comparisons using these protocols require a large number of animals with appropriate numbers of fibrils being measured to detect small intergroup differences. With experiments which compare left to right ligaments, far fewer animals are required to detect similarly small differences. These results demonstrate the necessity for rigorous control of sampling, an extensive normal baseline and statistically confirmed experimental designs in any TEM comparisons of collagen fibril diameters.     

Collagen fibril diameter and its relation to collagen turnover in three soft connective tissues in the rat

Collagen fibril diameters were measured in electron micrographs of rat skin, gingiva and periodontal ligament. Gingiva was divided into two zones, termed elastin-containing gingiva and attached gingiva, depending on the presence or absence of elastic fibrils. The results revealed that skin had the largest fibrils, followed by elastin-containing gingiva, attached gingiva and periodontal ligament respectively. These differences in fibril diameter were highly significant. The observed trend in fibril diameter was the inverse of that documented for collagen turnover and collagen phagocytosis in the same tissues. A link between fibril diameter and collagen turnover is discussed.     

Analysis of fibrous proteins from electron microscopy images

This paper considers an approach for analyzing fibrillar collagen structures from electron microscopy images. It enables the quantitative comparison between collagen structural data (electron-optical data) and chemical data. The particular objectives of the paper are to model the electron microscopy images according to the periodic structure of collagen, provide methods for extracting periodic features directly from the experimental data and propose schemes for comparing these features with the theoretical amino-acid distributions of the examined collagen tissue. Theoretical models in the form of sequence-generated histograms are used as reference for extracting and analyzing the structural unit in images from collagen fibrils. In this respect, collagen provides a valuable model system for studying the chemical basis of ultra-structure and the mechanisms of various treatments on a protein, as well as detecting the alterations in collagen fibril structure produced by a disorder. The algorithms developed in this study can be applied to any fibrous protein, provided that its amino acid sequences and structural properties are known. Several application examples are presented. The algorithmic results are compared with clinical studies as to verify the applicability and potential of the proposed methodology.     

Structure and Function of the Amino Terminal Propeptide of Type I and III Collagen

Collagen fibril diameters were measured in electron micrographs of rat skin, gingiva and periodontal ligament. Gingiva was divided into two zones, termed elastin-containing gingiva and attached gingiva, depending on the presence or absence of elastic fibrils. The results revealed that skin had the largest fibrils, followed by elastin-containing gingiva, attached gingiva and periodontal ligament respectively. These differences in fibril diameter were highly significant.

The observed trend in fibril diameter was the inverse of that documented for collagen tumover and collagen phagocytosis in the same tissues. A link between fibril diameter and collagen turnover is discussed.     

Imaging of muscle activity-induced morphometric changes in fibril network of myofascia by two-photon microscopy

Myofascia, deep fascia enveloping skeletal muscles, consists of abundant collagen and elastin fibres that play a key role in the transmission of muscular forces. However, understanding of biomechanical dynamics in myofascia remains very limited due to less quantitative and relevant approaches for in vivo examination. The purpose of this study was to evaluate the myofascial fibril structure by means of a quantitative approach using two-photon microscopy (TPM) imaging in combination with intravital staining of Evans blue dye (EBD), a far-red fluorescence dye, which potentially labels elastin. With focus on myofascia of the tibial anterior (TA) muscle, the fibril structure intravitally stained with EBD was observed at the depth level of collagen fibrous membrane above the muscle belly. The EBD-labelled fibril structure and orientation in myofascia indicated biomechanical responses to muscle activity and ageing. The orientation histograms of EBD-labelled fibrils were significantly modified depending upon the intensity of muscle activity and ageing. Moreover, the density of EBD-labelled fibrils in myofascia decreased with habitual exercise but increased with muscle immobilization or ageing. In particular, the diameter of EBD-labelled fibrils in aged mice was significantly higher. The orientation histograms of EBD-labelled fibrils after habitual exercise, muscle immobilization and ageing showed significant differences compared to control. Indeed, the histograms in bilateral TA myofascia of exercise mice made simple waveforms without multiple sharp peaks, whilst muscular immobilization or ageing significantly shifted a histogram with sustaining multiple sharp peaks. Therefore, the dynamics of fibre network with EBD fluorescence in response to the biomechanical environment possibly indicate functional tissue adaptation in myofascia. Furthermore, on the basis of the knowledge that neutrophil recruitment occurs locally in working muscles, we suggested the unique reconstruction mechanism involving neutrophilic elastase in the myofascial fibril structure. In addition to the elastolytic susceptibility of EBD-labelled fibrils, distinct immunoreactivities and activities of neutrophil elastase in the myofascia were observed after electric pulse stimulation-induced muscle contraction for 15 min. Our findings of EBD-labelled fibril dynamics in myofascia through quantitative approach using TPM imaging and intravital fluorescence labelling potentially brings new insights to examine muscle physiology and pathology.     

Collagen Fibrillogenesis and mRNA Levels in the Maturing Rabbit Medial Collateral Ligament and Patellar Tendon

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.     

Collagen fibrillogenesis and mRNA levels in the maturing rabbit medial collateral ligament and patellar tendon

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.     

Topologically defined composites of collagen types I and V as in vitro cell culture scaffolds

Cell fate is known to be triggered by cues from the extracellular matrix, including its chemical, biological and physical characteristics. Specifically, mechanical and topological properties are increasingly recognized as important signals. The aim of this work was to provide an easily accessible biomimetic in vitro platform of topologically defined collagen I matrices to dissect cell behaviour under various conditions in vitro. We reconstituted covalently bound layers of three-dimensional (3-D) networks of collagen type I and collagen type V with a defined network topology. A new erosion algorithm enabled us to analyse the mean pore diameter and fibril content, while the mean fibril diameter was examined by an autocorrelation method. Different concentrations and ratios of collagen I and V resulted in pore diameters from 2.4 to 4.5 μm and fibril diameters from 0.6 to 0.8 μm. A comparison of telopeptide intact collagen I to telopeptide deficient collagen I revealed obvious differences in network structure. The good correlation of the topological data to measurements of network stiffness as well as invasion of human dermal fibroblasts proves that the topological analysis provides meaningful measures of the functional characteristics of the reconstituted 3-D collagen matrices.     

Vascular collagen fibril morphology in type IV Ehlers-Danlos syndrome

Morphometric analysis of transmission electron micrographs of blood vessel, skin and dura mater collagen fibers were performed on postmortem tissues taken from 28-year-old female with Ehlers-Danlos type IV syndrome (EDS IV). Vascular tissue from this patient was compared to 5 age- and sex-matched controls (age range 26-28 years). Our study revealed significant variation in collagen fibril diameter in the walls of almost all the vessels studied. In general, the EDS IV tissue showed a net decrease in average collagen fibril cross sectional area in arterial wall samples. This decrease was significant (p less than .05) across the entire wall of the renal artery, in the media of the carotid artery, and in the media and adventitia of the common iliac artery. Samples from the vena cava show significant increases in collagen fibril cross sectional area across the vessel wall (p less than .005). The only areas studied which did not show significant changes were the intimal and adventitial regions of the common carotid artery. The observed changes may be contributory to the decreased arterial wall strength typical of the syndrome.     

Collagenase degradation decreases collagen fibril diameters--an in vitro study of the rabbit medial collateral ligament

Based on the similarity of fibril diameters in healing and grafted ligaments, it has been speculated that all small fibrils represent newly synthesized collagen. Alternatively, small fibrils in grafts could be due to enzymatic degradation of endogenous large fibrils. This study examined the effect of collagenase on collagen fibril diameters in normal NZW rabbit MCLs. Midsubstance MCL slivers were incubated in buffer for 72 or 144 h for comparison with slivers incubated in buffer containing 4 units/ml bacterial collagenase. The samples were examined under TEM for fibril diameter analysis. Mean fibril diameters of 3-day and 6-day collagenase-treated MCLs were significantly reduced, resembling 40-week scar values. These results suggest that collagenase treatment can alter collagen fibril diameter and shape in normal rabbit MCL, thus it is possible that despite their similarity to ligament scars, that at least some small fibrils in ligament grafts may be enzymatically reduced endogenous fibrils.