Orientation Of Collagen Bundles Research Articles

Influence of Fibronectin Immobilization on the Orientation of Collagen Bundles in Soft Tissue around Zirconia Implants

Influence of Fibronectin Immobilization on the Orientation of Collagen Bundles in Soft Tissue around Zirconia Implants

Fourier analysis of collagen bundle orientation in myocardial infarction scars

Collagen bundle orientation (CBO) in myocardial infarct scars plays a major role in scar mechanics and complications after infarction. We aim to compare four histopathological methods for CBO measurement in myocardial scarring. Myocardial infarction was induced in 21 pigs by balloon coronary occlusion. Scar samples were obtained at 4 weeks, stained with Masson’s trichrome, Picrosirius red, and Hematoxylin–Eosin (H&E), and photographed using light, polarized light microscopy, and confocal microscopy, respectively. Masson’s trichrome images were also optimized to remove non-collagenous structures. Two observers measured CBO by means of a semi-automated, Fourier analysis protocol. Interrater reliability and comparability between techniques were studied by the intraclass correlation coefficient (ICC) and Bland–Altman (B&A) plots and limits of agreement. Fourier analysis showed an almost perfect interrater reliability for each technique (ICC ≥ 0.95, p < 0.001 in all cases). CBO showed more randomly oriented values in Masson’s trichrome and worse comparability with other techniques (ICC vs. Picrosirius red: 0.79 [0.47–0.91], p = 0.001; vs. H&E-confocal: 0.70 [0.26–0.88], p = 0.005). However, optimized Masson’s trichrome showed almost perfect agreement with Picrosirius red (ICC 0.84 [0.6–0.94], p < 0.001) and H&E-confocal (ICC 0.81 [0.54–0.92], p < 0.001), as well as these latter techniques between each other (ICC 0.84 [0.60–0.93], p < 0.001). In summary, a semi-automated, Fourier-based method can provide highly reproducible CBO measurements in four different histopathological techniques. Masson’s trichrome tends to provide more randomly oriented CBO index values, probably due to non-specific visualization of non-collagenous structures. However, optimization of Masson’s trichrome microphotographs to remove non-collagenous components provides an almost perfect comparability between this technique, Picrosirius red and H&E-confocal.

Clinicomorphological correlation of psoriasis and psoriasiform dermatitis

Introduction: Psoriasis is a chronic inflammatory skin disorder. Psoriasiform dermatitis on other hand is a frequently encountered terminology in a variety of inflammatory dermatoses. It often poses challenges to both dermatologists and pathologists alike. Clinical features when considered alone may not be reliable to differentiate psoriasis from psoriasiform dermatitis. Aims: (a)To correlate the clinicomorphological features of psoriasis and psoriasiform dermatitis. (b)To identify determinants that may contribute to the diagnosis of psoriasis and psoriasiform dermatitis. Methods: This is a prospective study of 40 cases. Cases clinically diagnosed as psoriasis and psoriasiform dermatitis were screened microscopically and as per morphological criterias 20 cases from each entity were selected and included in the study. Detailed histopathological analysis and correlation was done. Results: The clinical features of typical scales (p= 0.0012) and Auspitz's sign (p = 0.058) morphological evidence of regular acanthosis (p= 0.0005), absent granular layer (p = 0.0001) and presence of micromunros abscess (p=0.0001) were found to be statistically highly significant contributors to the diagnosis of psoriasis in comparison to psoriasiform hyperplasia. Other morphological features like suprapapillary thinning (p= 0.051) was also found to be morphologically significant. Vertical orientation of collagen bundles (p = 0.002) was found to be significantly associated with diagnosis of psoriasiform hyperplasia when compared to psoriasis. Conclusion: The present study reconfirms the diagnostic accuracy of scales, Auspitz’s sign as clinically reliable signs of psoriasis. However, in their absence, morphological presence of regular acanthosis, absent granular layer and Munro microabscess may contribute to the diagnosis of psoriasis. Similarly, vertical orientation of collagen bundles points toward a diagnosis of psoriasiform dermatitis. This may help clinicians not to miss the diagnosis of clinically insignificant psoriasis in order to start early treatment and prevent the poor prognosis in these patients.

Synchrotron tomography of intervertebral disc deformation quantified by digital volume correlation reveals microstructural influence on strain patterns

The intervertebral disc (IVD) has a complex and multiscale extracellular matrix structure which provides unique mechanical properties to withstand physiological loading. Low back pain has been linked to degeneration of the disc but reparative treatments are not currently available. Characterising the disc’s 3D microstructure and its response in a physiologically relevant loading environment is required to improve understanding of degeneration and to develop new reparative treatments. In this study, techniques for imaging the native IVD, measuring internal deformation and mapping volumetric strain were applied to an in situ compressed ex vivo rat lumbar spine segment. Synchrotron X-ray micro-tomography (synchrotron CT) was used to resolve IVD structures at microscale resolution. These image data enabled 3D quantification of collagen bundle orientation and measurement of local displacement in the annulus fibrosus between sequential scans using digital volume correlation (DVC). The volumetric strain mapped from synchrotron CT provided a detailed insight into the micromechanics of native IVD tissue. The DVC findings showed that there was no slipping at lamella boundaries, and local strain patterns were of a similar distribution to the previously reported elastic network with some heterogeneous areas and maximum strain direction aligned with bundle orientation, suggesting bundle stretching and sliding. This method has the potential to bridge the gap between measures of macro-mechanical properties and the local 3D micro-mechanical environment experienced by cells. This is the first evaluation of strain at the micro scale level in the intact IVD and provides a quantitative framework for future IVD degeneration mechanics studies and testing of tissue engineered IVD replacements. Statement of SignificanceSynchrotron in-line phase contrast X-ray tomography provided the first visualisation of native intact intervertebral disc microstructural deformation in 3D. For two annulus fibrosus volumes of interest, collagen bundle orientation was quantified and local displacement mapped as strain. Direct evidence of microstructural influence on strain patterns could be seen such as no slipping at lamellae boundaries and maximum strain direction aligned with collagen bundle orientation. Although disc elastic structures were not directly observed, the strain patterns had a similar distribution to the previously reported elastic network. This study presents technical advances and is a basis for future X-ray microscopy, structural quantification and digital volume correlation strain analysis of soft tissue.

Synchrotron Tomographic Measurement of Strain in Soft Tissue: Native Intervertebral Disc Deformation at Histological Resolution

The intervertebral disc (IVD) has a complex and multiscale extracellular matrix structure which provides unique mechanical properties to withstand physiological loading. Low back pain has been linked to degeneration of the disc but reparative treatments are not currently available. Characterising the disc’s 3D microstructure and its response in a physiologically relevant loading environment is required to improve understanding of degeneration and to develop new reparative treatments. In this study, techniques for imaging the native IVD, measuring internal deformation and mapping volumetric strain were applied to a compressed spine segment. Synchrotron X-ray micro-tomography (sCT) was used to resolve IVD structures at histological resolution. These image data enabled 3D quantification of collagen bundle orientation and measurement of local displacement in the annulus fibrosus between sequential scans using digital volume correlation (DVC). The volumetric strain mapped from sCT provided a detailed insight into the micromechanics of native IVD tissue. The DVC findings showed that there was no slipping at lamella boundaries, and local strain patterns were of a similar distribution to the previously reported elastic network with some heterogeneous areas and maximum strain direction aligned with bundle orientation, suggesting bundle stretching and sliding. This method has the potential to bridge the gap between measures of macro-mechanical properties and the local 3D micro-mechanical environment experienced by cells. This is the first evaluation of strain at the micro scale level in the intact IVD and provides a quantitative framework for future IVD degeneration mechanics studies and testing of tissue engineered IVD replacements.

Matrix metalloproteinase-1 (MMP-1) and (MMP-8) gene polymorphisms promote increase and remodeling of the collagen III and V in posterior tibial tendinopathy.

Posterior tibial tendinopathy (PTT) can lead to acquired flatfoot in adults. Many patients develop PTT without any identifiable risk factors. Molecular changes in extracellular matrix (ECM) and matrix metalloproteinase (MMP) polymorphism may influence the risk of developing PTT. We aim to investigate the association between matrix metalloproteinase-1 (MMP-1) and (MMP-8) gene polymorphisms with changes in collagen I, III and V in PTT. A case-control study with 22 patients and 5 controls was performed. The MMP-1 (2G/2G) and MMP-8 (T/T) genotypes were determined by PCR-restriction fragment length polymorphism. Tendon specimens were evaluated by a histologic semiquantitative score, immunofluorescence and histomorphometry for collagen I, III and V. Tendon specimens from PTT demonstrated marked distortion of the architecture with necrosis, large basophilic areas with disruption of the normal linear orientation of collagen bundles, infiltration of inflammatory cells, dystrophic calcification and ossification. Under immunofluorescence, PTT tendon specimens showed weak green fluorescence and diffuse distribution of collagen I fibers, but strong fluorescence of collagen III and V. The collagen I fibers were significantly decreased whereas an increase of collagen III and V were found in PTT compared to control groups. In addition, PTT group presented a significant association with MMP-1 and MMP-8 gene polymorphisms. Patients with PTT matrix metalloproteinase-1 (MMP-1) and (MMP-8) gene polymorphisms presented an increase of the collagen III and V ratio, suggesting that the higher proportion in degenerated tendons could contribute to a decrease in the mechanical resistance of the tissue. Still, functional and association studies are needed to elucidate evident roles of MMPs in PTT.

A Novel Nude Mouse Model of Hypertrophic Scarring Using Scratched Full Thickness Human Skin Grafts.

Objective: Hypertrophic scar (HTS) is a dermal form of fibroproliferative disorder that develops following deep skin injury. HTS can cause deformities, functional disabilities, and aesthetic disfigurements. The pathophysiology of HTS is not understood due to, in part, the lack of an ideal animal model. We hypothesize that human skin with deep dermal wounds grafted onto athymic nude mice will develop a scar similar to HTS. Our aim is to develop a representative animal model of human HTS. Approach: Thirty-six nude mice were grafted with full thickness human skin with deep dermal scratch wound before or 2 weeks after grafting or without scratch. The scratch on the human skin grafts was made using a specially designed jig that creates a wound >0.6 mm in depth. The xenografts were morphologically analyzed by digital photography. Mice were euthanized at 1, 2, and 3 months postoperatively for histology and immunohistochemistry analysis. Results: The mice developed raised and firm scars in the scratched xenografts with more contraction, increased infiltration of macrophage, and myofibroblasts compared to the xenografts without deep dermal scratch wound. Scar thickness and collagen bundle orientation and morphology resembled HTS. The fibrotic scars in the wounded human skin were morphologically and histologically similar to HTS, and human skin epithelial cells persisted in the remodeling tissues for 1 year postengraftment. Innovation and Conclusions: Deep dermal injury in human skin retains its profibrotic nature after transplantation, affording a novel model for the assessment of therapies for the treatment of human fibroproliferative disorders of the skin.

The natural behavior of mononuclear phagocytes in HTS formation.

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.

A study of clinicohistopathological correlation in patients of psoriasis and psoriasiform dermatitis

Psoriasis has different clinical variants, which mimic diverse dermatological conditions and may require a histopathological confirmation of the diagnosis. Studies to establish a clinicohistopathological concordance (and its determinants), in psoriasis and psoriasiform dermatitis are lacking. The present study was designed (a) to correlate the clinicohistopathological features of psoriasis and psoriasiform dermatitis, and (b) to identify determinant(s) that may contribute to the diagnosis of psoriasis and psoriasiform dermatitis. This was a prospective study involving 100 patients, with a single clinical diagnosis of psoriasis or with psoriasis as one of the differential diagnoses, and its correlation with histopathological features. The clinical features of typical scale (P = 0.0001) and Auspitz's sign (P = 0.0001), and histological evidence of suprapapillary thinning (P = 0.0001) and absent granular cell layer (P = 0.0001) were found to be statistically significant contributors to the clinicohistological concordance in cases of psoriasis. Vertical orientation of collagen bundles (P = 0.0001) and lymphocytic exocytosis (P = 0.003) were found to be significantly associated with diagnosis of psoriasiform dermatitis. The present study reconfirms the diagnostic accuracy of silvery white scale, Auspitz's sign, and Koebner's phenomenon in a clinical setting suggestive of psoriasis. However, in their absence, histological evidence of suprapapillary thinning and absent granular layer, in addition to the Munro microabscess and Kogoj's abscess, may contribute to the diagnosis of psoriasis. Similarly, vertical orientation of collagen bundles and lymphocytic exocytosis may point toward a diagnosis of psoriasiform dermatitis.

The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging

Changes in arterial wall composition and function underlie all forms of vascular disease. The fundamental structural and functional unit of the aortic wall is the medial lamellar unit (MLU). While the basic composition and organization of the MLU is known, three-dimensional (3D) microstructural details are tenuous, due (in part) to lack of three-dimensional data at micro- and nano-scales. We applied novel electron and confocal microscopy techniques to obtain 3D volumetric information of aortic medial microstructure at micro- and nano-scales with all constituents present. For the rat abdominal aorta, we show that medial elastin has three primary forms: with approximately 71% of total elastin as thick, continuous lamellar sheets, 27% as thin, protruding interlamellar elastin fibers (IEFs), and 2% as thick radial struts. Elastin pores are not simply holes in lamellar sheets, but are indented and gusseted openings in lamellae. Smooth muscle cells (SMCs) weave throughout the interlamellar elastin framework, with cytoplasmic extensions abutting IEFs, resulting in approximately 20° radial tilt (relative to the lumen surface) of elliptical SMC nuclei. Collagen fibers are organized as large, parallel bundles tightly enveloping SMC nuclei. Quantification of the orientation of collagen bundles, SMC nuclei, and IEFs reveal that all three primary medial constituents have predominantly circumferential orientation, correlating with reported circumferentially dominant values of physiological stress, collagen fiber recruitment, and tissue stiffness. This high resolution three-dimensional view of the aortic media reveals MLU microstructure details that suggest a highly complex and integrated mural organization that correlates with aortic mechanical properties.

Force and Geometry Determine Structure and Function of Glaucoma Filtration Capsules

Background/Aims: Glaucoma shunts vent aqueous humor to orbital tissue across a fibrous capsule. The capsule consists of collagen and fibroblasts, and its formation was assumed to be a cicatricial process. Capsule fibrosis remains the leading cause of device failure. We tested the wound healing hypothesis using morphometry and polarization microscopy and found the capsule consists not of scar tissue but rather highly organized extracellular matrix. Methods: Polarization microscopy reveals collagen orientation within the extracellular matrix. Rotation of the specimen in the plane of polarized light reveals collagen bundle orientation with respect to capsule surface geometry. Capsule thickness was measured and correlated with device dimension. Results: Capsule collagen is highly organized and not scar tissue. Relative orientation is predictable from simple engineering principles and shows tissue deposits reinforcing collagen parallel to its principle stress axes along the capsule surface. Capsule thickness and collagen orientation correlate with device dimension and surface geometry. Conclusion: The corollary is true. Geometry can be used to manage the amount, distribution and orientation of collagen and thus control hydraulic conductivity and surface area of the filtration capsule.

Long-term results of a clinical trial on dermal substitution.: A light microscopy and Fourier analysis based evaluation

Although dermal substitution is a main topic of current wound healing research, there is a paucity of clinical trials with a long-term clinical and histopathological evaluation. A clinical trial was conducted to perform an intra-individual comparison of conventional treatment (split-thickness autograft) to a collagen/elastin dermal substitute in combination with an autograft. Promising results with this substitute were obtained with respect to dermal organisation and scar elasticity in animal studies and clinical trials with a short-term follow-up. Twenty-nine of the 42 pairs of the burn wounds and 28 of the 44 pairs of the scar reconstructions enrolled in the study were biopsied after 1 year. Promising but not statistically significant differences were found between substituted groups and control groups for epidermal thickness, basement membrane maturation, rete ridges ( P=0.055), fibroblasts, myofibroblasts, inflammatory cells, vessels and extracellular matrix maturation. An objective and accurate technique, Fourier analysis, was used to evaluate collagen bundle orientation and packing. However, no statistically significant differences were obtained for these parameters. This microscopic evaluation provided no convincing evidence for a long-term effectiveness of a dermal substitute despite promising data over a short-term in in vitro and in vivo studies with the same material.

Dermal Organization in Scleroderma: The Fast Fourier Transform and the Laser Scatter Method Objectify Fibrosis in Nonlesional as well as Lesional Skin

Scleroderma, a chronic, progressive disorder, is characterized by dermal fibrosis with collagen bundles orientated parallel to the epidermis. Simple objective parameters to evaluate disease progression and therapies are needed. We describe two methods, the laser scatter method and the fast Fourier transform (FFT), to measure collagen bundle orientation and spacing. Lesional sclerodermic skin (LS), nonlesional sclerodermic skin (nonLS), and control skin (CS) sections were evaluated for orientation ratio using the laser scatter method. The FFT was used to calculate orientation ratio, variation, and spacing of collagen bundles. Parameters were correlated with local and mean skin score measurements, on a scale of 0 (normal) to 3 (severely sclerotic). With both the laser scatter method and the FFT, orientation ratios of LS (respectively, 2.16 ± 0.33 and 1.83 ± 0.62) were significantly higher than CS (respectively, 1.70 ± 0.35 and 1.38 ± 0.15). NonLS orientation ratios (respectively, 1.92 ± 0.15 and 1.48 ± 0.44) were between LS and CS ratios. Orientation variation and bundle spacing of LS (respectively, 57.3 ± 19.4 and 15.7 ± 5.6 μm) were significantly reduced compared to CS (respectively, 73.8 ± 15.0 and 18.9 ± 1.9 μm). NonLS orientation ratios (respectively, 57.2 ± 29.0 and 15.6 ± 6.1 μm) were similar to LS. Bundles in LS are more parallel, show less variation in orientation, and are more densely packed than in CS. There was a linear correlation between mean skin score and orientation ratio. Local skin score was not linearly correlated to orientation ratio. Our findings suggest that nonLS dermis without clinical sclerosis already shows fibrotic characteristics. Both techniques were easy to use and suitable for objectifying dermal fibrosis in scleroderma lesions. FFT is more accurate and reproducible than the laser scatter method and allows simultaneous pathological evaluation of the location of the analyzed tissue sections. Future studies will need to focus on the correlation between clinical disease severity and collagen bundle characteristics.

Orientation of apatite in single osteon samples as studied by pole figures.

The X-ray diffraction method based on pole figures has been applied to single osteon samples in order to obtain information about the texture of the inorganic bone fraction and the way it changes during calcification. The osteon samples were cylindrically shaped, with axes corresponding to those of the haversian canals. Selection was carried out according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteons were selected: longitudinal, alternate, and transversal. The results indicate that in all three types of osteons, the long axis of the sample is apparently the only direction of orientation because the transversally oriented crystallites give an isotropic diffuse scattering as would be expected if all the inorganic particles were irregularly oriented around the osteon axis. The number of longitudinally oriented crystallites increases progressively from transversally oriented osteons to alternately and longitudinally oriented ones. The crystallite orientation in an axial direction increases in fully calcified osteons. This last result is in agreement with the electron microscopic finding that the long needle-shaped crystallites covering much more than a major collagen period and measuring 40–45 A in width increase in number as calcification proceeds.

X-ray diffraction and electron microscope study of osteons during calcification

To obtain information on the changes in the inorganic bone fraction during calcification, low- and wide-angle X-ray diffraction techniques and electron microscopy have been applied to single osteon samples. The samples were cylindrically shaped and their axes corresponded to the axes of the Haversian canals. The selection was made according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteon were selected, that is, longitudinally structured osteons, transversely structured osteons, and alternately structured osteons. The results indicate that in osteonic lamellar bone there are two types of inorganic particles: (1) granules arranged in linear or needle-shapred entities with maximum width 40-45 A, which are regularly distributed at the level of the main band of the collagen fibrils where their maximum length reaches the length of the main band itself; that is , about 400 A; and (2) very long crystallites, with a diameter of 40-45 A, which grow with their crystallographic c-axis parallel to the collagen fibrils and cover much more than a major collagen period.