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The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly., (Copyright 2000 Academic Press.)

Solutions of type I acid soluble collagen were studied in light and electron microscopy at concentrations over 40 mg/ml. Banded patterns spontaneously emerge in samples observed between crossed polars between slide and coverslip. The textures are interpreted as precholesteric, appearing at the transition between the isotropic phases, due to random molecular order, and the cholesteric phase corresponding to a highly organized three-dimensional structure. Type I collagen banded patterns correspond to regular undulations of the molecular directions with an observed periodicity in the range of 1 to 10 microm. This interpretation is verified by ultrastructural analysis of precholesteric samples gelled under ammonium vapors. Results are discussed in regard to banded patterns described either within synthetic polymer systems or within collagen extracellular matrices. Self-assembled liquid crystalline phases of collagen generate crimp morphologies. Their possible relationship with early secretion steps in the development of connective tissues is discussed.

Supramolecular assemblies following liquid crystalline cholesteric geometries have been described in biological systems from optical properties observed in polarized-light microscopy and structural data obtained in electron microscopy. Major biological macromolecules are discussed, including structural polymers of the extracellular matrix, genetic material in nuclei and chromosomes, and proteins of the cytoplasm. The liquid crystalline assembly properties of biological polymers have been demonstrated by experiments in vitro with molecules at basic structural levels, such as molecular chains of cellulose and chitin, triple helices of collagen, and double helices of DNA, and also with entities at higher states of organization as they appear in cells and tissues, such as cellulose and chitin crystallites, and collagen fibrils. It appears that the building of cellular and extracellular edifices implies self-ordering processes of the liquid crystalline type and that the study of these mesomorphic states will help resolve basic questions about the structure and morphogenesis of densely packed biological structures.

Liquid crystalline assemblies occur spontaneously in highly concentrated solutions of type I acid-soluble calf skin collagen. The degree of order, identified by optical microscopy in polarized light, varies from a random distribution of molecules at low concentrations to highly organized structures as the concentration increases up to 80 mg/ml. Ultrastructural studies using classical techniques of chemical fixation are inappropriate for liquid crystalline phases due to the absence of stable links maintaining their three-dimensional order. In order to analyse the collagen liquid crystalline phases by electron microscopy the viscous preparations were stabilized under ammonia vapour. Observations of the gels in polarized light indicated that the liquid crystalline order, established at acidic pH in a sol state, persists at neutral pH in a gel state. Transmission electron microscopic observations allow us to validate the geometrical model interpreted from observations in polarizing microscopy, that is continuously twisting orientations in cholesteric phases characterized by typical series of arced patterns when viewed in oblique sections. A significant result is that the ultrastructure of the stabilized liquid crystalline collagen faithfully mimics fibrillar patterns described in vivo in extracellular matrices. This strongly supports the hypothesis that liquid crystalline properties are involved in the morphogenesis of collagen matrices.

Microscopic studies at different scales have shown that in biological tissues the three-dimensional arrangement of chitin-protein or of collagen fibrillar networks can follow the same spatial distributions as those described in certain liquid crystals. The present work reviews the structural analogies established between the dense fibrillar organic matrix found in two materials: crab cuticles and compact bones. In both systems mobile fringes are described in polarizing microscopy, periodic cleavage aspects in scanning electron microscopy (SEM), and arched patterns in transmission electron microscopy (TEM). In parallel to these structural data, results obtained in vitro are recalled which corroborate the relationship established between ordered arrays of biopolymers and liquid crystalline assembly principles, highly concentrated solutions of purified collagen molecules spontaneously form ordered assemblies characterized in polarizing microscopy as cholesteric phases. This particular state of matter joins both fluidity and order and could correspond to a transient state of collagen or chitin secretion, before the stiffening of these skeletal structures, bone or cuticle, by molecular cross-links and crystalline deposition.

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.

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.

Twisted fibrous extracellular matrices observed in section often show alternating clear and dark bands. Three different methods of observation (high voltage electron microscopy, shadowing of thin sections and stereoscopic views) show the presence of ruffling effects and relief at the surface of crab cuticle sections. These effects appear uneven on both sides of the sections. As shown in a series of diagrams, the localization of the microtomy artefact is a function of the orientation of the cuticle laminae relative to the knife direction, and this creates variations in the position and the extent of the microtomy effect over each lamina. Confirmation of this analysis is obtained in a particular geometrical situation which appears in sections of tubercles in the crab cuticle where the twisted plywood stratification is deformed into a dome. By shadowing thin sections, perpendicular to the tubercle axis, nested crescents are visualized on the surface of the samples. All observations demonstrate that the clear and dark lamellae are due to a microtomy artefact which is a three-dimensional process, and not, as usually considered, due to chemical or physical variations in the structure.