Liquid crystallinity in condensed type I collagen solutions. A clue to the packing of collagen in extracellular matrices.

We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.     

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.     

Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.     

Liquid crystalline phases of sonicated type I collagen

The assembly properties of concentrated solutions of type I collagen molecules are compared before and after a 5-min sonication, breaking the 300-nm triple helices into short segments of about 20 nm, with a strong polydispersity. The collagen concentration of these solutions, sonicated or not, was increased up to 100 mg/ml by slow evaporation of the solvent. Whereas the non-sonicated solutions remain isotropic, the sonicated solutions transform after a few hours into a twisted liquid crystalline phase, well recognizable in polarizing microscopy. The evidence of a twisted assembly of collagen triple helices in vitro is new and relevant in a biological context since it was reported in various collagen matrices.     

Assembly of collagen fibril meshes using gold nanoparticles functionalized with tris(hydroxymethyl)phosphine-alanine as multivalent cross-linking agents

We report on the use of tris(hydroxymethyl)phosphine-alanine (THPAL) functionalized gold nanoparticles as a multivalent cross-linking agent to assemble collagen fibrils into a mesh-like structure. Atomic force microscopy (AFM) was used for characterization of the structure after adsorption onto an atomically flat mica substrate, revealing a mesh-like construct in which the collagen fibrils and the gold nanoparticles interact to form interconnected nodes measuring from 100 to 500 nm. As expected, the density of the collagen mesh can be increased with a higher initial concentration of gold nanoparticles. The maximum thickness of the meshes (～ 20 nm) obtained through cross-sectional height measurements confirms that the adsorbed structure consists of a single layer of collagen fibrils/gold nanoparticles assembled in two-dimensions. We propose that the capability of gold nanoparticles functionalized with the THPAL to bind to several collagen fibrils combined with the large persistence length of the fibrils, which was reported to be in the hundreds of nanometer range, are determinant factors for the preferential 2D growth of the mesh in solution.     

Twisted architectures in cell-free assembled collagen gels: study of collagen substrates used for cultures

Reprecipitated fibrils from collagen solutions assemble into aggregates often showing a remarkable twisted structure. We first observed these aggregates in collagen gels prepared to facilitate culture of epithelial cells. We verified that these structures form in the absence of cells and correspond to a process of self-assembly. Studies on reconstructed fibrils of collagen are generally based on the examination of thin specimens mounted onto coated grids prepared for electron microscopy. We rather applied the classical methods of fixation, embedding and ultramicrotomy, which allowed us to analyze the structure of these aggregates, several microns in diameter. Our gels were prepared from 2.5 mg/ml tropocollagen solutions usually chosen for cell and organ cultures. The time required to obtain twisted architectures, in these aggregates, depends on temperature and the presence of factors such as fetal calf serum proteins. Twist is observed at two different levels of organization. Microfibrils are gathered into twisted bundles which condense into cross-striated fibrils. These fibrils themselves aggregate and show a mutual twist whose orientation is left-handed as is the twist observed within each microfibril bundle. Several models of these architectures are presented. Planar twist, cylindrical twist and toroidal twist are described and their relation to the structure of certain liquid crystals is considered. Examples of orthogonal packing also have been observed. These structures obtained in vitro are very close to patterns already described in vivo in numerous collagen matrices.     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

Ultrastructural studies on the collagen of the marine sponge Chondrosia reniformis Nardo

The ultrastructure of isolated fibrils of Chondrosia reniformis sponge collagen was investigated by collecting characteristic data, such as fibril thickness, width, D-band periodicity, and height modulation, using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Therefore an adapted pre-processing of the insoluble collagen into homogeneous suspensions using neutral buffer solutions was essential, and several purification steps have been developed. Fourier transform infrared reflection-absorption spectroscopy (FT-IRAS) of the purified sponge collagen showed remarkable analogy of peak positions and intensities with the spectra of fibrillar calf skin type I collagen, despite the diverse phylogenetic and evolutionary origin. The sponge collagen's morphology is compared with that of other fibrillar collagens, and the typical banding of the separated single fibrils is discussed by comparison of topographical data obtained using AFM and corresponding TEM investigations using common staining methods. As the TEM images of the negatively stained fibrils showed alternating dark and light bands, AFM revealed a characteristic periodicity of protrusions (overlap zones) followed by two equal interband regions (gap zones). AFM and TEM results were correlated and multiperiodicity in Chondrosia collagen's banding is demonstrated. The periodic dark bands observed in TEM images correspond directly to the periodic protrusions seen by AFM. As a result, we provide an improved, updated model of the collagen's structure and organization.     

Collagen fibril structure is affected by collagen concentration and decorin

Collagen fibrils were obtained in vitro by aggregation from acid-soluble type I collagen at different initial concentrations and with the addition of decorin core or intact decorin. All specimens were observed by scanning electron microscopy and atomic force microscopy. In line with the findings of other authors, lacking decorin, collagen fibrils undergo an extensive lateral association leading to the formation of a continuous three-dimensional network. The addition of intact decorin or decorin core was equally effective in preventing lateral fusion and restoring the normal fibril appearance. In addition, the fibril diameter was clearly dependent on the initial collagen concentration but not on the presence/absence of proteoglycans. An unusual fibril structure was observed as a result of a very low initial collagen concentration, leading to the formation of huge, irregular superfibrils apparently formed by the lateral coalescence of lesser fibrils, and with a distinctive coil-structured surface. Spots of incomplete fibrillogenesis were occasionally found, where all fibrils appeared made of individual, interwined subfibrils, confirming the presence of a hierarchical association mechanism.     

Collagenous network in cartilage of human femoral condyles. A light microscopic and scanning electron microscopic study

To determine the spatial arrangement of collagen fibrils in articular cartilage of the human femoral head, three healthy femoral heads, obtained at necropsy, were examined by light microscopy and scanning electron microscopy. Light microscopic observations revealed no collagen fibril organization. Scanning electron microscopic observations showed a fine fibrillar texture throughout the articular cartilage. At the articular surface, smooth and fibrillated areas were detectable. Underneath the articular surface, the collagen network in the superficial zone showed a tighter appearance when compared with the homogeneous collagen network of the matrix in the deeper zones. The calcified cartilage zone was well demarcated from the uncalcified cartilage. The arcade model of Benninghoff [Z. Zellforsch. Mikrosk. Anat. 2: 783-862 (1925)] could not be confirmed. It was concluded that the organization of collagen fibrils in hyaline cartilage shows a three-dimensional network of randomly oriented fibrils.     

Strain-controlled enzymatic cleavage of collagen in loaded matrix

The purpose of this investigation is to support the novel hypothesis that collagenous matrices are intrinsically "smart" load-adapting biomaterials. This hypothesis is based fundamentally on the postulate that tensile strain directly modulates the susceptibility of collagen molecules to enzymatic degradation (i.e., protects molecules which are under load from cleavage). To test this postulate, collagenase (Clostridiopeptidase A) was applied to a uniaxially loaded, anisotropic, devitalized, collagenous matrix in which a subset of fibrils was loaded in tension while the remaining fibrils carried little or no load. The collagen degradation pattern (as assessed by polarization and transmission electron microscopy) was found to correspond inversely to the tensile stress field such that fibrils under lower tensile load were preferentially cleaved. These results have immediate implications for tissue engineering of load-bearing collagenous matrices in vitro and may contribute significantly to our understanding of synthesis, remodelling, and pathogenesis of collagen matrices in vivo.     

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment

Fibrillar collagen–integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril–integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.     

New Fibrous Long Spacing Form of Collagen

PRECIPITATION of collagen from solution in the presence of chondroitin sulphates (or certain other glycosaminoglycans), followed by dialysis against water, leads to the formation of the fibrous long spacing (FLS) polymorphic form of collagen. Electron microscopy shows this to consist of banded fibrils with a periodicity in the range 1200–2400 Å and a symmetric intraperiod structure, in contrast to the 600–700 Å periodicity and polarized fine structure characteristic of native collagen fibrils. FLS was originally reported by Highberger et al.^1–3 and has been observed in three principal modifications, FLS I, FLS II and FLS III, differing in periodicity and arrangement of dense bands^4–6. This letter reports a new form of FLS, denoted FLS IV.     

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

The self-assembly in films dried from aqueous solutions of a modified amyloid beta peptide fragment is studied. We focus on sequence Abeta(16-20), KLVFF, extended by two alanines at the N-terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days. The secondary structure of the fibrils is studied by FT-IR, circular dichroism and X-ray diffraction (XRD), which provide evidence for beta-sheet structure in the fibril. From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63+/-18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have b and c dimensions 1.9 and 4.4 nm with an a axis 0.96 nm, corresponding to twice the peptide backbone spacing in the antiparallel beta-sheet.     

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.     

Mechanism of in vitro collagen fibril assembly. Kinetic and morphological studies

The kinetics of in vitro fibril assembly of Type I collagen preparations that contain different amounts of covalently cross-linked oligomers was studied with turbidimetry. Fibril formation showed a lag phase with no solution turbidity and a growth phase with a sigmoidal increase in the solution turbidity. The length of the lag phase was inversely related to both the total collagen concentration and the amount of covalently cross-linked oligomers in the solution. Double logarithmic plots of t1/4, the amount of time it takes for 1/4 of the collagen to assemble into fibrils, versus the total collagen concentration were linear but the slope decreased from -0.84 to -2.3 with decreasing amounts of covalently cross-linked oligomers in the samples. Electron microscopy showed the formation of unbanded microfibrils with diameters in the range of 3-15 nm early in the lag phase and larger diameter banded fibrils coexisting with the microfibrils near the end of the lag phase. Centrifugation of the solution at the lag phase prolonged the lag time, presumably by removal of microfibrils, but subsequent growth of the fibrils was unaffected. The results suggest a cooperative nucleation-growth mechanism for the in vitro assembly of collagen fibrils which is consistent with the results of an equilibrium study of the fibril assembly reaction we reported earlier (Na, G. C., Butz, L. J., Bailey, D. G., and Carroll, R. J. (1986) Biochemistry 25, 958-966).     

An electrostatic model for collagen fibrils. The interaction of reconstituted collagen with Ca++, Na+, and Cl−

A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å. The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α-amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect. The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3–5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M^?1, respectively. The model also predicts that an average 4-mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties. The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility.     

Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels

Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering.