Collagen fibril diameters increase and fibril densities decrease in skin subjected to repetitive compressive and shear stresses

Understanding microstructural changes that occur in skin subjected to repetitive mechanical stress is crucial towards the development of therapies to enhance skin adaptation and load tolerance in patients at risk of skin breakdown (e.g. prosthesis users, wheelchair users). To determine if collagen fibril diameter, collagen fibril density, dermal thickness, epidermal thickness, basement membrane length, and dermal cell density changed in response to repetitive stress application, skin subjected to moderate cyclic compressive and shear stresses for 1 h/d, 5 d/week, for 4 week was compared with skin from an unstressed contralateral control. The lateral aspects of the hind limbs of 12 Landrace/Yorkshire pigs were used. Skin from under the stressed site and a contralateral control site was processed for electron microscopy and light microscopy analysis. Electron microscopy results demonstrated significant (p<0.01) increases in collagen fibril diameter of 15.9%, 22.4%, and 22.9% for the upper, mid, and lower layers of the dermis, respectively, for the stressed skin compared with the control skin. Collagen fibril density (fibrils/unit cross-sectional area) decreased significantly for stressed vs. control by 19.8%, 29.2%, and 31.8% for the upper, mid, and lower layers, respectively. Light microscopy results demonstrated trends of a decrease in dermal thickness and an increase in cell density for stressed vs. control samples, but the differences were not significant. Differences in epidermal thickness and basement membrane length were not significant. These results demonstrate that quantifiable changes occur in collagen fibril architecture but not in the gross tissue morphology following in vivo cyclic loading of pig skin.     

Control of collagen fibril diameters in tissues.

It is proposed that radial growth of collagen fibrils, which takes place in all connective tissues to varying extents, according to the tensile stresses exerted on them, proceeds mainly by aggregation of protofibrils (approximately 10 nm) and existing fibrils. In young tissues, fibrils are prevented from making frequent intimate contacts which would lead to aggregation by abundant interfibrillar proteoglycan, that keeps the fibrils apart. Collagen fibrils are probably unable to fuse except when the molecules within them are packed in the same sense, i.e. fusing fibrils are parallel. The roughly equal numbers of parallel and antiparallel fibrils seen in several tissues must limit radial fibril growth in older tissues, where proteoglycan is usually less abundant. Possible origins of the balance of fibril polarities, which must be conserved after fibril nucleation on cell or non-cell templates, are analysed. The two controlling factors, ambient proteoglycan and fibril polarity, working against the tendency of fibrils to fuse, account for many features of the observed distributions of collagen fibril diameters in diverse tissues and at different ages.     

Collagen fibril formation. A new target to limit fibrosis

We present a concept for reducing formation of fibrotic deposits by inhibiting self-assembly of collagen molecules into fibrils, a main component of fibrotic lesions. Employing monoclonal antibodies that bind to the telopeptide region of a collagen molecule, we found that blocking telopeptide-mediated collagen/collagen interactions reduces the amount of collagen fibrils accumulated in vitro and in keloid-like organotypic constructs. We conclude that inhibiting extracellular steps of the fibrotic process provides a novel approach to limit fibrosis in a number of tissues and organs.     

Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice.

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).     

Collagen fibril formation in the presence of sodium dodecyl sulphate.

Sodium dodecyl sulphate (SDS) was used to weaken both the electrostatic and the hydrophobic interactions during collagen fibrillogenesis in vitro. The rate and extent of fibril formation as well as fibril morphology were affected by SDS concentration. Both the formation of large fibrils at 0.3 mM-SDS and the complete cessation of fibril formation at 0.5 mM-SDS were considered to be the result of SDS-induced conformational changes in the non-helical telopeptides. A possible mechanism of SDS interaction with the N-terminal and the distal region of the C-terminal telopeptides is offered.     

Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons.

The present study examined the hypothesis that collagen fibril diameter and crimp angle in ruptured human Achilles tendons differed from that of intact ones. Tissue samples were obtained from the central core (distal core) and the posterior periphery (distal superficial) at the rupture site, and the proximally intact (proximal superficial) part of the tendon in 10 subjects (38+/-8 years) with a complete tendon rupture. For comparisons corresponding tissue samples were procured from age (38+/-7 years) and gender matched intact Achilles tendons during routine forensic autopsy. The cross-sectional area density and diameter distribution of fibrils were analyzed using stereological techniques of digitized electron microscopy biopsy cross-sections, while crimp angle was measured by the changing banding pattern of collagen fibers when rotated between crossed polars. Nine of 10 persons with tendon ruptures reported that the injury did not occur during exceedingly large forces, and none experienced any symptoms in the days or months prior to the injury. Fibril diameter distribution showed no region-specific differences in either the ruptured or intact tendons for either group. However, in the distal core there were fewer fibrils in the ruptured compared to the intact tendons in 60-150 nm range, P<0.01. Similarly, in the distal superficial portion there were fewer fibrils in the ruptured compared to the intact tendons in the 90-120 nm range, 2P<0.05, while there were no differences in the proximal superficial tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles tendon rupture site. Moreover, the lack of symptoms prior to the rupture suggests that clinical tendinopathy is not an etiological factor in complete tendon ruptures.     

Collagen type I fibril packing in vivo and in vitro

Reviewed are works concerning the mechanisms of collagen (type I) fibril packing and the influence of macromolecular structure and physicochemical parameters of the medium on the process.     

Collagen fibril formation in vitro. The role of the nonhelical terminal regions.

We showed previously that fibril formation in vitro from rat tail tendon collagen requires a temperature-dependent initiation (Step 1) following which linear assembly to form thin filaments (Step 2) proceeds as rapidly at 4 degrees C as at 26 degrees C. Step 3, lateral assembly of filaments to form fibrils, is again temperature-dependent. We now find that Step 1 is complete in 6 min at 26 degrees C and the time is independent of collagen concentration in the range 0.08 to 0.39 mg/ml. Collagen treated with pepsin, which removes the nonhelical ends but leaves the triple helix intact, forms fibrils by a similar mechanism. However, Step 1 is altered or absent and early temperature changes produce a complex response consistent with an alternate, counterproductive pathway. Assembly is also much slower, particularly Step 2, and the fibrils formed are abnormal in that native banding is often absent and short tactoidal forms are common. These results suggest that in the assembly of fibrils from normal collagen the nonhelical ends are involved in an early conformational change and critically regulate later steps.     

A biomechanical model for fibril recruitment: Evaluation in tendons and arteries

Simulations of soft tissue mechanobiological behaviour are increasingly important for clinical prediction of aneurysm, tendinopathy and other disorders. Mechanical behaviour at low stretches is governed by fibril straightening, transitioning into load-bearing at recruitment stretch, resulting in a tissue stiffening effect. Previous investigations have suggested theoretical relationships between stress-stretch measurements and recruitment probability density function (PDF) but not derived these rigorously nor evaluated these experimentally. Other work has proposed image-based methods for measurement of recruitment but made use of arbitrary fibril critical straightness parameters. The aim of this work was to provide a sound theoretical basis for estimating recruitment PDF from stress-stretch measurements and to evaluate this relationship using image-based methods, clearly motivating the choice of fibril critical straightness parameter in rat tail tendon and porcine artery. Rigorous derivation showed that the recruitment PDF may be estimated from the second stretch derivative of the first Piola-Kirchoff tissue stress. Image-based fibril recruitment identified the fibril straightness parameter that maximised Pearson correlation coefficients (PCC) with estimated PDFs. Using these critical straightness parameters the new method for estimating recruitment PDF showed a PCC with image-based measures of 0.915 and 0.933 for tendons and arteries respectively. This method may be used for accurate estimation of fibril recruitment PDF in mechanobiological simulation where fibril-level mechanical parameters are important for predicting cell behaviour.     

A program for computer-aided analyses of ecological field data

A program for IBM-compatible microcomputers is introduced whichcombines several complementary analyses of species-station-tablesgenerated in ecological field investigations. The scope of theprogram encompasses table editing Junctions, routines for communitydelimitation by cluster analysis and procedures for the analysisof properties related both to stations (e.g. diversities) andspecies (e.g. abundance statistics and association indices).The essential reasoning behind the application of communitystudies is presented briefly, as well as the multi-step analyticalapproach implemented in the program.     

Collagen fibril morphology and organization: implications for force transmission in ligament and tendon.

Connective tissue mechanical behavior is primarily determined by the composition and organization of collagen. In ligaments and tendons, type I collagen is the principal structural element of the extracellular matrix, which acts to transmit force between bones or bone and muscle, respectively. Therefore, characterization of collagen fibril morphology and organization in fetal and skeletally mature animals is essential to understanding how tissues develop and obtain their mechanical attributes. In this study, tendons and ligaments from fetal rat, bovine, and feline, and mature rat were examined with scanning electron microscopy. At early fetal developmental stages, collagen fibrils show fibril overlap and interweaving, apparent fibril ends, and numerous bifurcating/fusing fibrils. Late in fetal development, collagen fibril ends are still present and fibril bundles (fibers) are clearly visible. Examination of collagen fibrils from skeletally mature tissues, reveals highly organized regions but still include fibril interweaving, and regions that are more randomly organized. Fibril bifurcations/fusions are still present in mature tissues but are less numerous than in fetal tissue. To address the continuity of fibrils in mature tissues, fibrils were examined in individual micrographs and consecutive overlaid micrographs. Extensive microscopic analysis of mature tendons and ligaments detected no fibril ends. These data strongly suggest that fibrils in mature ligament and tendon are either continuous or functionally continuous. Based upon this information and published data, we conclude that force within these tissues is directly transferred through collagen fibrils and not through an interfibrillar coupling, such as a proteoglycan bridge.     

A morphometric filter improves the diagnostic value of morphometric analyses of frozen histopathological sections from mammary tumours.

Frozen sections of 202 consecutive breast tumour cases were analyzed by morphometric quantitation of nuclear features. Nuclei were selected at random. Conventional light microscope examination of the paraffin-embedded specimens revealed 144 cases of cancer and 56 benign tumours. Using multivariate discriminant analysis of morphometric features, all but two of the benign cases and 79% of the malignant tumours were correctly classified. When a morphometrically based dynamic filter set to exclude 'non-diagnostic' nuclei was used, the correctly classified malignant cases rose to 86% Morphometry is a fast, reproducible and efficient method that can be used in conjunction with the histomorphological diagnosis of mammary frozen sections. The combination of systematic sampling and an objective dynamic filter may be a powerful approach to quantitative analyses of tumours from other sites. However, it is also likely that efficiency can be improved by combining nuclear morphometric features with structural, histochemical and molecular biological data. The combination of traditional histomorphological examination with quantitative information may well increase the diagnostic accuracy in individual patients.     

Collagen fibril biosynthesis in tendon: a review and recent insights

The development and evolution of multicellular animals relies on the ability of certain cell types to synthesise an extracellular matrix (ECM) comprising very long collagen fibrils that are arranged in very ordered 3-dimensional scaffolds. Tendon is a good example of a highly ordered ECM, in which tens of millions of collagen fibrils, each hundreds of microns long, are synthesised parallel to the tendon long axis. This review highlights recent discoveries showing that the assembly of collagen fibrils in tendon is hierarchical, and involves the formation of fairly short "collagen early fibrils" that are the fusion precursors of the very long fibrils that occur in mature tendon.     

Caliber and media thickness of intrahepatic arteries in a normal human liver. A morphometric study

Morphometrical data on the intrahepatic arterial wall were studied over a wide range of vascular diameters (100-1708 microns) in one human liver. The liver tissue containing Araldite-filled blood vessels was embedded in the water-soluble glycolmethacrylate (GMA). The calculation of the correction factor for the morphometric data obtained from 3 microns sections is described. The influence of formaldehyde fixation on the volume of the liver proves to be negligible. During dehydration a linear shrinkage of about 3% occurs. After infiltration and embedding in GMA only 1% of this shrinkage remains. During the drying phase about 4.3% further linear stretching occurs. No significant "residual compression factor" was found. Araldite plays a significant role in the retention of the dimensions of the liver specimen during histologic processing, while the dimensional changes of the Araldite itself are negligible. A positive linear correlation was found between the media thickness and the radius of the vessel. The physiological consequences are discussed. It is concluded that in the morphometric analysis of the arterial wall it is essential to apply a standardized procedure in histologic processing and in the measuring of the inner vascular diameter. The advantages of our method, in which blood vessels are perfused with Araldite under physiological pressure, are discussed.     

A morphometric analysis of craniofacial growth in cleft lip and noncleft mice.

Differences in face shape are considered a factor in cleft lip malformation. The purpose of this study was to analyze craniofacial growth in two strains: A/WySn with 28% cleft lip and C57BL/6J without cleft lip. Standardized photographs of 27 A/WySn and 25 C57BL/6J embryos with 34-46 somites (S) were taken in the superior, frontal, and lateral views. Landmarks were located and digitized for computerized analysis of growth change relative to somite number and at stages of face development before, during, and after primary palate closure. The results showed that both strains had similar overall growth patterns with increases in head width and face width, and decreases in nasal pit width. During early palatal closure in C57BL/6J mice, the nasal pit width was unchanged as brain width increased rapidly; and then later, the nasal pit width decreased as brain width increased slowly. However, during early closure in A/WySn mice, the nasal pit width decreased rapidly as brain width increased slowly; and then later, the nasal pit width was unchanged as brain width increased more rapidly. During early palatal closure, the narrower nasal pit width in A/WySn mice appeared to result from delayed growth of the supporting forebrain as the nasal pits become more medially positioned with normal face development. From the lateral view, the maxillary prominence depth was also smaller in the A/WySn strain during early palatal closure. This deficient forward growth of the maxillary prominences and the narrower positioning of the medial nasal prominences in A/WySn embryos appear to reduce the contact between the prominences and thus predispose this strain to cleft lip malformation.     

Collagen fibril morphology in developing chick metatarsal tendon: 2. Electron microscope studies

Transverse section of embryonic chick metatarsal tendons ranging in age from 11 days to 18 days fetal were examined by electron microscopy to determine both the diameters and the lateral arrangements of the cylindrical collagen fibrils. In early developmental stages, from 11 days to 14 days fetal, sharp unimodal distributions of diameters centred near 32 or 40 nm were observed, but increasingly heterogeneous diameters were seen with increasing age. The heterogeneous diameter distributions were not uniform, but showed discrete populations of preferred diameters. The centre-to-centre distance separating the fibrils in the early developmental stages was about twice the fibril diameter and constant with age. Comparison of X-ray diffraction results with these observations indicated that the saptial relationships of the structures are preserved during the preparative procedures for electron microscopy, but that a transverse shrinkage of 25–30% had occurred relative to the wet dimension.     

Collagen fibril morphology in developing chick metatarsal tendoms: 1. X-ray diffraction studies

The low angle equatorial X-ray diffraction ($\text{R ? 30 μ}\text{m}{}^{\mathrm{?1}}$) from hydrated embryonic chick metatarsal tendon contains minima and maxima that are not seen in mature tendons. This diffraction derives from the disordered array of parallel, cylindrical fibrils of collagen of small, uniform diameter that comprise the major part of this tissue. Comparison of the positions of the minima and maxima with those expected from an array of cylinders allows estimation of the mean diameter of the cylinders and the average centre-to-centre nearest neighbour separation. It was found that in the age range from 13 to 19 days fetal, the mean diameter increased from ～ 46 to ～ 58 nm, whereas the mean nearest neighbour separation remained constant at ～ 90 nm. Detailed analysis of the X-ray intensity profile of a 17 day fetal tendon indicated the presence of a paucidisperse distribution of fibril diameters with two or more discrete populations of preferred diameters separated by 10 to 12 nm.     

A comparison between the inlet and outlet diameters of the normal aortic valve.

Two studies are described comparing the inlet and outlet diameters of the normal aortic valve. Both studies show the valve inlet to be smaller than the valve outlet. The first study is of measurements made on 12 casts of physiologically pressurised human aortic valves. The mean ratio between the diameter of the aortic ring and of the aorta just distal to the sinus ridge was 1.1, and the mean ratio between the diameter of the aortic ring and the maximum diameter of the valve leaflets was 1.18. The second study presents echocardiographic data from normal volunteers. The mean ratio between the diameter of the aortic ring and of the aorta just distal to the sinus ridge was 1.17. It is suggested that stents made to support the leaflets of prosthetic valves are made in conical or hyperbolic form, with the outlet being approximately 20% larger than the inlet.