The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra-Web) whose three-dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self-renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self-renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3-kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three-dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self-renewal. (+info)
A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS).
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OBJECTIVE: The orientation of collagen molecules is an important determinant of their functionality in connective tissues. The objective of the current study is to establish a method to determine the alignment of collagen molecules in histological sections of cartilage by polarized Fourier transform infrared imaging spectroscopy (FT-IRIS), a method based on molecular vibrations. METHODS: Polarized FT-IRIS data obtained from highly oriented tendon collagen were utilized to calibrate the derived spectral parameters. The ratio of the integrated areas of the collagen amide I/II absorbances was used as an indicator of collagen orientation. These data were then applied to FT-IRIS analysis of the orientation of collagen molecules in equine articular cartilage, in equine repair cartilage after microfracture treatment, and in human osteoarthritic cartilage. Polarized light microscopy (PLM), the most frequently utilized technique to evaluate collagen fibril orientation in histological sections, was performed on picrosirius red-stained sections for comparison. RESULTS AND CONCLUSION: Thicknesses of each zone of normal equine cartilage (calculated based on differences in collagen orientation) were equivalent as determined by PLM and FT-IRIS. Comparable outcomes were obtained from the PLM and FT-IRIS analyses of repair and osteoarthritis tissues, whereby similar zonal variations in collagen orientation were apparent for the two methods. However, the PLM images of human osteoarthritic cartilage showed less obvious zonal discrimination and orientation compared to the FT-IRIS images, possibly attributable to the FT-IRIS method detecting molecular orientation changes prior to their manifestation at the microscopic level. (+info)
Influence of fibril taper on the function of collagen to reinforce extracellular matrix.
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Collagen fibrils provide tensile reinforcement for extracellular matrix. In at least some tissues, the fibrils have a paraboloidal taper at their ends. The purpose of this paper is to determine the implications of this taper for the function of collagen fibrils. When a tissue is subjected to low mechanical forces, stress will be transferred to the fibrils elastically. This process was modelled using finite element analysis because there is no analytical theory for elastic stress transfer to a non-cylindrical fibril. When the tissue is subjected to higher mechanical forces, stress will be transferred plastically. This process was modelled analytically. For both elastic and plastic stress transfer, a paraboloidal taper leads to a more uniform distribution of axial tensile stress along the fibril than would be generated if it were cylindrical. The tapered fibril requires half the volume of collagen than a cylindrical fibril of the same length and the stress is shared more evenly along its length. It is also less likely to fracture than a cylindrical fibril of the same length in a tissue subjected to the same mechanical force. (+info)
Control of the collagen fibril diameter in the equine superficial digital flexor tendon in horses by decorin.
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The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin. (+info)
An x-ray diffraction study of corneal structure in mimecan-deficient mice.
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PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea. (+info)
Neonatal development of the corneal stroma in wild-type and lumican-null mice.
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PURPOSE: Between days 8 and 14 of neonatal development, the corneal stroma of the mouse undergoes critical changes in tissue thickness, cell density, and light scattering. The authors investigate the stromal matrix structure in wild-type and lumican-deficient corneas in this developmental phase. METHODS: Wild-type (n = 44) and lumican-deficient (n = 42) mouse corneas at neonatal days 8, 10, 12, and 14 were investigated by synchrotron x-ray diffraction to establish the average collagen fibril spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal matrix. RESULTS: Collagen interfibrillar spacing in the normal mouse cornea became more closely packed between days 8 and 14, though not significantly so. In lumican-null mice, interfibrillar spacing was significantly elevated at days 8, 10, and 12, but not day 14, compared with that in wild-type mice. At all stages investigated, collagen fibrils were, on average, marginally thinner than normal in lumican-null mutants, and the spatial distribution of the fibrils was less well organized. CONCLUSIONS: Transient thickening of the corneal stroma of the normal mouse at eye opening is probably not caused by widespread, homogeneous rearrangement of collagen fibrils but more likely by a temporary increase in cell or stromal "lake" volume. Lumican, structurally influential in adult mouse corneas, is also a key molecule in the neonatal development of the stromal matrix. (+info)
Structure and component alteration of rabbit Achilles tendon in tissue culture.
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The aim of this study was to investigate alterations of cultured tendon tissues to determine whether tissue culture is a useful method for biological analyses of the tendon. Tendon tissues for tissue culture were isolated from Achilles tendons of rabbits. The tendon segments were placed one segment per well and incubated in growth medium consisting of Dullbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 37 degrees C in a humidified atmosphere with 5% CO(2) for various periods. The alignment of collagen fibrils was preserved for 48 h, but tendon structure has disintegrated at 96 h. Alcian blue staining and gelatine zymography revealed that proteoglycan markedly diminished and that matrix metalloproteinase (MMPs) activity was upregulated sharply at 72 and 96 h. The ratio of collagen fibrils with large diameter had increased and the mean diameter and mass average diameter value had reached maximum at 48 h. The values then decreased and mean diameters at 72 and 96 h were significantly different from that at 48 h. At 96 h, the ratio of collagen fibrils with small diameters had increased and collagen fibrils with large diameters had disappeared. These findings indicate that structural alteration is possible to be induced by disintegration of collagen fibrils and disappearance of glycosaminoglycans from extracellular matrix (ECM), subsequent of upregulation of MMPs activity. Although the study period is limited, the tissue culture method is available for investigating cell-ECM interaction in tendons. (+info)
Co-expression and functional interaction of silicatein with galectin: matrix-guided formation of siliceous spicules in the marine demosponge Suberites domuncula.
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Sponges (phylum Porifera) of the class of Demospongiae are stabilized by a siliceous skeleton. It is composed of silica needles (spicules), which provide the morphogenetic scaffold of these metazoans. In the center of the spicules there is an axial filament that consists predominantly of silicatein, an enzyme that catalyzes the synthesis of biosilica. By differential display of transcripts we identified additional proteins involved in silica formation. Two genes were isolated from the marine demosponge Suberites domuncula; one codes for a galectin and the other for a fibrillar collagen. The galectin forms aggregates to which silicatein molecules bind. The extent of the silicatein-mediated silica formation strongly increased if associated with the galectin. By applying a new and mild extraction procedure that avoids hydrogen fluoride treatment, native axial filaments were extracted from spicules of S. domuncula. These filaments contained, in addition to silicatein, the galectin and a few other proteins. Immunogold electron microscopic studies underscored the role of these additional proteins, in particular that of galectin, in spiculogenesis. Galectin, in addition to silicatein, presumably forms in the axial canal as well as on the surface of the spicules an organized net-like matrix. In the extraspicular space most of these complexes are arranged concentrically around the spicules. Taken together, these additional proteins, working together with silicatein, may also be relevant for potential (nano)-biotechnological applications of silicatein in the formation of surface coatings. Finally, we propose a scheme that outlines the matrix (galectin/silicatein)-guided appositional growth of spicules through centripetal and centrifugal synthesis and deposition of biosilica. (+info)