Interaction Of Collagen Fibrils Research Articles

Orientation-dependent indentation reveals the crosslink-mediated deformation mechanisms of collagen fibrils.

The spatial arrangement and interactions of the extracellular matrix (ECM) components control the mechanical behavior of tissue at multiple length scales. Changes in microscale deformation mechanisms affect tissue function and are often hallmarks of remodeling and disease. Despite their importance, the deformation mechanisms that modulate the mechanical behavior of collagenous tissue, particularly in indentation and compression modes of deformation, remain poorly understood. Here, we develop an integrated computational and experimental approach to investigate the deformation mechanisms of collagenous tissue at the microscale. While the complex deformation arising from indentation with a spherical probe is often considered a pitfall rather than an opportunity, we leverage this orientation-dependent deformation to examine the shear-regulated interactions of collagen fibrils and the role of crosslinks in modulating these interactions. We specifically examine tendon and cervix, two tissues rich in collagen with quite different microstructures and mechanical functions. We find that interacting, crosslinked collagen fibrils resist microscale longitudinal compressive forces, while widely used constitutive models fail to capture this behavior. The reorientation of collagen fibrils tunes the compressive stiffness of complex tissues like cervix. This study offers new insights into the mechanical behavior of collagen fibrils during indentation, and more generally, under longitudinal compressive forces, and illustrates the mechanisms that contribute to the experimentally observed orientation-dependent mechanical behavior. STATEMENT OF SIGNIFICANCE: Remodeling and disease can affect the deformation and interaction of tissue constituents, and thus mechanical function of tissue. Yet, the microscale deformation mechanisms are not well characterized in many tissues. Here, we develop a combined experimental-computational approach to infer the microscale deformation mechanisms of collagenous tissues with very different functions: tendon and cervix. Results show that collagen fibrils resist microscale forces along their length, though widely-used constitutive models do not account for this mechanism. This deformation process partially modulates the compressive stiffness of complex tissues such as cervix. Computational modeling shows that crosslink-mediated shear deformations are central to this unexpected behavior. This study offers new insights into the deformation mechanisms of collagenous tissue and the function of collagen crosslinkers.

An indentation-based framework for probing the glycosaminoglycan-mediated interactions of collagen fibrils

Microscale deformation processes, such as reorientation, buckling, and sliding of collagen fibrils, determine the mechanical behavior and function of collagenous tissue. While changes in the structure and composition of tendon have been extensively studied, the deformation mechanisms that modulate the interaction of extracellular matrix (ECM) constituents are not well understood, partly due to the lack of appropriate techniques to probe the behavior. In particular, the role of glycosaminoglycans (GAGs) in modulating collagen fibril interactions has remained controversial. Some studies suggest that GAGs act as crosslinkers between the collagen fibrils, while others have not found such evidence and postulate that GAGs have other functions. Here, we introduce a new framework, relying on orientation-dependent indentation behavior of tissue and computational modeling, to evaluate the shear-mediated function of GAGs in modulating the collagen fibril interactions at a length scale more relevant to fibrils compared to bulk tests. Specifically, we use chondroitinase ABC to enzymatically deplete the GAGs in tendon; measure the orientation-dependent indentation response in transverse and longitudinal orientations; and infer the microscale deformation mechanisms and function of GAGs from a microstructural computational model and a modified shear-lag model. We validate the modeling approach experimentally and show that GAGs facilitate collagen fibril sliding with minimal crosslinking function. We suggest that the molecular reconfiguration of GAGs is a potential mechanism for their microscale, strain-dependent viscoelastic behavior. This study reveals the mechanisms that control the orientation-dependent indentation response by affecting the shear deformation and provides new insights into the mechanical function of GAGs and collagen crosslinkers in collagenous tissue.

Cryptic binding sites become accessible through surface reconstruction of the type I collagen fibril

Collagen fibril interactions with cells and macromolecules in the extracellular matrix drive numerous cellular functions. Binding motifs for dozens of collagen-binding proteins have been determined on fully exposed collagen triple helical monomers. However, when the monomers are assembled into the functional collagen fibril, many binding motifs become inaccessible, and yet critical cellular processes occur. Here, we have developed an early stage atomic model of the smallest repeating unit of the type I collagen fibril at the fibril surface that provides a novel framework to address questions about these functionally necessary yet seemingly obstructed interactions. We use an integrative approach by combining molecular dynamics (MD) simulations with atomic force microscopy (AFM) experiments and show that reconstruction of the collagen monomers within the complex fibril play a critical role in collagen interactions. In particular, the fibril surface shows three major conformational changes, which allow cryptic binding sites, including an integrin motif involved in platelet aggregation, to be exposed. The observed dynamics and reconstruction of the fibril surface promote its role as a “smart fibril” to keep certain binding sites cryptic, and to allow accessibility of recognition domains when appropriate.

Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

A precise analysis of the mechanical response of collagen fibrils in tendon tissue is critical to understanding the ultrastructural mechanisms that underlie collagen fibril interactions (load transfer), and ultimately tendon structure-function. This study reports a novel experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations. An atomic force microscope was used to characterize diameters and periodic banding (D-period) of individual type-I collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10% macroscopic nominal strain, respectively. D-period banding of the collagen fibrils increased with increasing tendon strain (2.1% increase at 10% applied tendon strain, p<0.05), while fibril diameter decreased (8% reduction, p<0.05). No statistically significant differences between 0% and 5% applied strain were observed, indicating that the onset of fibril (D-period) straining lagged macroscopically applied tendon strains by at least 5%. This confirms previous reports of delayed onset of collagen fibril stretching and the role of collagen fibril kinematics in supporting physiological tendon loads. Fibril strains within the tissue were relatively tightly distributed in unloaded and highly strained tendons, but were more broadly distributed at 5% applied strain, indicating progressive recruitment of collagen fibrils. Using these techniques we also confirmed that collagen fibrils thin appreciably at higher levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon structure-function concepts data revealed that loading of the collagen network is fairly homogenous, with no apparent predisposition for loading of collagen fibrils according to their diameter.

Molecular and structural mapping of collagen fibril interactions

The fibrous collagens form the structural basis of all mammalian connective tissues, including the vasculature, dermis, bones, tendons, cartilage, and those tissues that support organs such as the heart, kindneys, liver, and lungs. The helical structure of collagen has been extensively studied but in addition to its helical character, its molecular packing arrangement (in its aggregated or fibrillar form) and the presence of specific amino acid sequences govern collagen's in vivo functions. Collagen's molecular packing arrangement helps control cellular communication, attachment and movement, and conveys its tissue-specific biomechanical properties. Recent progress in understanding collagen's molecular packing, fibrillar structure, domain organization, and extracellular matrix (ECM) interactions in light of X-ray fiber diffraction data provides significant new insights into how the ECM is organized and functions. In this review, the hierarchy of fibrillar collagen structure is discussed in the context of how this organization affects ECM–“ligand” interactions, with specific attention to collagenolysis, integrins, fibronection, glycoprotein VI receptor (GPVI), and proteoglycans (PG). Understanding the complex structure of collagen and its attached ligands should provide new insights into tissue growth, development, regeneration, and disease.

Morphological analysis of the acute effects of overdistension on the extracellular matrix of the rat urinary bladder wall

To investigate the morphological effects of acute overdistension in the structure of the extracellular matrix of the bladder wall in rats. The bladders of a group of 6 male Wistar rats were transurethrally overdistended for 3 hours. Another identical group (the control group) was only submitted to a sham operation. Specimens from the bladder dome were analyzed with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). LM--The control group bladders had a 4 to 5 layer urothelium, a lamina propria, and a smooth muscle layer with longitudinal and transversal fibers. The overdistended bladders presented an intense interstitial infiltrate in the lamina propria, and a less intense infiltrate among the smooth muscle fibers. TEM--The cells of the overdistended bladders had a significant amount of vacuoles, unlike the control bladders, where such vacuoles were scarce or absent. SEM--A delicate three-dimensional mesh of collagen fibrils was observed in the lamina propria of the bladder walls from the control group. Whilst for the control group this mesh consisted of distinct geometric structures, with mostly circular cellular spaces surrounded by the fibrils, the overdistended group showed evidence of distortion of the mesh, with flattened and elongated cellular spaces. Acute bladder overdistension induces structural modifications, altering the arrangement and interaction of collagen fibrils, as well as incipient tissue damage as edema in the lamina propria and smooth muscle layers.

The pentapeptide NKISK affects collagen fibril interactions in a vertebrate tissue.

The pentapeptide NKISK has been reported to inhibit the binding of decorin, a proteoglycan on the surface of collagen fibrils, to fibronectin, a tissue adhesion molecule. Because of our interest in fibril-fibril binding as it relates to changes in length of ligament or tendon (during growth or contracture), we investigated the potential of this peptide to dissociate fibrils. The peptide permitted the release of intact fibrils into suspension for examination under the electron microscope (which has not previously been possible in mature vertebrate tissues).

Mineral Content and Salt-dependent Viscosity in the Dermis of the Sea Cucumber Cucumaria frondosa

Samples from the deep dermis of the sea cucumber Cucumaria frondosa were analyzed to determine the concentrations of Na, K, Ca, and Mg in fresh tissues as well as in those that had been incubated in filtered seawater or extracted extensively in deionized water. Fresh tissues were in equilibrium with seawater with respect to the above minerals, and showed a relative concentration of K, Mg, and Ca. Water-extracted tissues were enriched in Mg and Ca and depleted in K and Na. The Ca and Mg could be removed from the tissues by extraction in a divalent cation chelator or in 0.1 M HCl. Biomechanical (creep) tests showed that dermal specimens in which the resident cells had been lysed by freezing and thawing had very low viscosities in buffered solutions of a divalent cation chelator containing either 0.03 M or 1.0 M NaCl. In contrast, their viscosities were quite high in similar solutions containing 0.3 M NaCl. The aggregation of isolated collagen fibrils in the presence of the dermal glycoprotein stiparin showed a similar dependence on the concentration of NaCl: the fibrils aggregated in 0.3 M NaCl, but not in 0.03 or 1.0 M NaCl. The above results are inconsistent with the hypothesis that collagen fibril interactions in C. frondosa dermis are regulated by cellular control of the extracellular [Ca 2+]. The results are consistent with the hypothesis that stiparin mediates mechanical interactions between collagen fibrils in dermal tissues as well as in isolated fibrils.

Alternative mRNA Processing Occurs in the Variable Region of the Pro-α1(XI) and Pro-α2(XI) Collagen Chains

An analysis was performed of differential splicing of primary transcripts in the noncollagenous variable region located in the amino terminus of the pro-alpha 1(XI) and pro-alpha 2(XI) collagen chains. The results for the pro-alpha 2(XI) chain showed that human cartilage or fibroblasts in culture contain transcripts in which a single highly acidic exon encoding for 21 amino acids is present or absent. For the chicken pro-alpha 1(XI) chain a more complex pattern of alternative splicing was detected with six possible variants. Of special interest was the alternative use of two exons (called IIA and IIB) in which IIA encodes for 39 amino acids and is highly acidic (estimated pI = 3.2), whereas IIB encodes for 49 amino acids and is highly basic (estimated pI = 10.6). A similar alternative use of exon IIA or exon IIB was also observed for human chondrocytes. Northern blotting with probes specific for IIA or IIB showed that both exons are present in transcripts from cartilage but exon IIB is preferentially utilized in transcripts from tendon. Present results suggest that both the pro-alpha 1(XI) and pro-alpha 2(XI) chains of type XI collagen undergo limited processing in vivo and that the noncollagenous variable region is initially retained on the surface of the fibrils. Differential splicing in the variable region may potentially influence the interaction of collagen fibrils with other molecules of the extracellular matrix and more specifically with sulfated glycosaminoglycan chains or with hyaluronan. Such interactions may play a key role in establishing both the organization of the collagen fibrils within the extracellular matrix and in limiting the diameter of collagen fibrils.

Autoantibodies to cartilage collagens in relapsing polychondritis.

Relapsing polychondritis is a systemic disease associated with a destruction of cartilage in various parts of the body. Sera from six patients with relapsing polychondritis and one patient with microscopic polyarteritis nodosa as well as from six controls were analyzed by immunoblotting and ELISA. All patients had autoantibodies against native collagens II and IX. The serum from one patient showed a strong reaction with all three collagen chains of the high molecular weight fraction of collagen IX after denaturation; sera from four patients showed autoantibodies against alpha 2 (XI) and sera from three patients showed autoantibodies against the covalently cross-linked gamma component of collagen XI. The presence of autoantibodies against collagens II, IX, and XI, which form the major fibrillar scaffold in cartilage and mediate the interaction of collagen fibrils and proteoglycan, suggests that autoantibodies against cartilaginous collagen may play a crucial role in the pathogenesis of relapsing polychondritis and microscopic polyarteritis nodosa.

Response to growth factors of human dermal fibroblasts in a quiescent state owing to cell-matrix contact inhibition

Mitogenic responses to various growth factors were compared for quiescent human dermal fibroblasts cultured under three different conditions; serum depletion, cell-cell contact inhibition and cell-matrix contact inhibition. The non-dividing fibroblasts cultured under a low serum condition (0.2% fetal bovine serum, FBS) or in a confluent culture with 10% FBS resumed multiplying upon exposure to any one of or any combination of the growth factors examined; epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF) and transforming growth factor (β (TGF-β). The only exception was the lack of effect of TGF-β on the cells under a low serum condition. In contrast, the proliferation of fibroblasts which were growth-arrested in contracted collagen gel by cell-matrix contact inhibition was not stimulated by any of the growth factors examined except for PDGF. It is currently accepted that the mechanism of growth stimulation or signal transduction after binding of each growth factor to the specific receptor depends on the kind of growth factor. The results suggest that the signal transductions delivered by EGF, b-FGF or TGF-β are inactivated by a high level of interaction of collagen fibrils with the cell membrane (under the condition of cell-matrix contact inhibition); whereas the signal transduction by PDGF is unaffected. The finding supports the existence of a specific growth stimulation pathway for PDGF.

Two affinity immunoelectrophoretic methods for studying collagen interaction with von Willebrand factor antigen.

Two new immunoelectrophoretic methods are described for studying the interaction of collagen fibrils with von Willebrand factor antigen (vWF: Ag). In the first, the sample was electrophoresed through a collagen-agarose wedge into an antibody-agarose area, and immunoprecipitin lines was detected by staining. Different immunoprecipitin patterns were obtained with the vWF: Ag of normal plasma, commercial FVIII preparations, and von Willebrand disease (vWD) type IIa plasma as the result of collagen binding of vWF: Ag. In the other method, the sample was electrophoresed into agarose for preliminary separation of forms, followed by migration in the second dimension through a collagen spacer gel into an antibody-agarose area. This method demonstrated preferential binding of high molecular weight forms of vWF: Ag in normal plasma and slight binding of the lower molecular weight forms of antigen found in vWD type IIa plasma. The affinity wedge method is a convenient general method for finding quickly a useful concentration of affinity reagent.

Morphology of the interaction of collagen fibrils with normal human platelets and thrombasthenic platelets

The ultrastructure of the platelet contacts with collagen fibrils (CF) as well as the course taken by CF on the platelet surface were studied on ultrathin sections of platelets and CF. Platelets from normal donors and from a patient with thrombasthenia were incubated in citrated plasma with collagen. For electron microscopy a protein-stabilizing fixation procedure was applied. Platelet-collagen contacts (PCC) are characterized by a distance of 7±3 nm between the platelet membrane and the CF; the gap contains electron-dense bridges. The PCC of normal and thrombasthenic platelets are morphologically identical. Hence, it is unlikely that the glycoproteins IIb/IIIa-complex, which is absent in thrombasthenic platelets, plays a significant role in the platelet collagen interaction. The CF induce random movements of the platelets and their pseudopods, whereby the fibrils, which often show multisite attachment, coil up around the platelet surface; they become bent and often display drastic directional changes. CF are found inside invaginations of the platelet membrane as well as in depressions of the platelet surface. These processes require an involvement of the platelet's contractile system, which appears to interact reversibly with the platelet plasma membrane.

Proteoglycan-type 1 collagen fibril interactions in calcifying and non-calcifying connective tissues

Proteoglycan-type 1 collagen fibril interactions in calcifying and non-calcifying connective tissues

Collagen polymorphism: Its origins in the amino acid sequence

The polymorphic forms of ordered collagen aggregation in vitro and in vivo are reviewed. The axially projected structures of a class of fibrils known as fibrous long spacing (FLS) collagen are solved using simulated positively stained banding patterns based on the amino acid sequence. This method is also used to solve the axial projection of a 670 Å ( D) periodic structure with a symmetrical banding pattern (DPS) re-precipitated from skin collagen. The relation between the obliquely striated and 110 Å periodic forms of collagen is discussed. The specificity for the formation of FLS, DPS and segment long spacing (SLS) collagen is shown to be in the distributions of various amino acids in the sequence. Different residues are important for each type of structure, their importance being dependent on the chemical conditions and the presence of other macromolecules. The interaction of collagen fibrils with proteoglycans in vivo is discussed in terms of the amino acid sequence. Also the factors which affect collagen morphology in the presence of mucopolysaccharides and proteoglycans in vitro and in vivo are discussed. Some insight is gamed into the principles which govern the self-assembly of molecules into ordered fibrous aggregates.

The effect of chelating agents on collagen interfibrillar matrix interactions in connective tissue

1. 1.The interaction of collagen fibrils and the interfibrillar matrix of tendon has been studied. 2. 2.Treatment of tendon with chelating agents at pH 7.5 results in the subsequent dispersion of collagen fibrils in dilute acetic acid. 3. 3.The dispersed collagen fibrils have an amino acid composition which is almost identical with the best preparations of tendon gelatin, indicating the chemical purity of the collagen. 4. 4.The action of chelating agents can be reversed by equilibration of the treated tendon with calcium acetate prior to acetic acid extraction. 5. 5.The interfibrillar matrix can be isolated in a purified form virtually free of collagen. 6. 6.It is suggested that metal ions play a part in the interaction of collagen fibrils and the interfibrillar matrix in tendon.

The interaction of collagen and acid mucopolysaccharides. A model for connective tissue.

1. The interaction of acid mucopolysaccharides of connective tissue with solubilized collagen of native, or near-native, structure was investigated by free solution electrophoresis at pH7.0. 2. Complex-formation was detected by the appearance of a third peak in the ascending limb only, indicating reversible association. 3. Complex-formation was destroyed by prior heating of solubilized collagen, indicating a probable requirement for high molecular weight or internal structure of the protein. 4. Hyaluronate and chondroitin sulphate of mol.wt. 50000 gave complexes with soluble collagen at I 0.4, whereas heparin and chondroitin sulphate of mol.wt. 15000-18000 did not. All mucopolysaccharides yielded complexes at I 0.1. The stability of the complex appears mainly dependent on electrostatic forces and is increased with increase in chain length of the polysaccharide. 5. Solubilized collagen interacted to yield gels with the ;native' chondroitin sulphate-protein macro-molecule from cartilage. 6. A schematic model for the interaction of collagen and chondroitin sulphate-protein macromolecules shows parallel-ordered interaction of collagen fibrils with chondroitin sulphate side chains of the chondroitin sulphate-protein macromolecule. The biological implications of this model are discussed, particularly in relation to the ordered structures and the ionic-network properties of the intercellular components of connective tissue.