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The complex mechanical properties of biomaterials such as hair, horn, skin, or bone are determined by the architecture of the underlying fibrous bionetworks. Although much is known about the influence of the cytoskeleton on the mechanics of isolated cells, this has been less studied in tridimensional tissues. We used the hair follicle as a model to link changes in the keratin network composition and architecture to the mechanical properties of the nascent hair. We show using atomic force microscopy that the soft keratinocyte matrix at the base of the follicle stiffens by a factor of ∼360, from 30 kPa to 11 MPa along the first millimeter of the follicle. The early mechanical stiffening is concomitant to an increase in diameter of the keratin macrofibrils, their continuous compaction, and increasingly parallel orientation. The related stiffening of the material follows a power law, typical of the mechanics of nonthermal bending-dominated fiber networks. In addition, we used X-ray diffraction to monitor changes in the (supra)molecular organization within the keratin fibers. At later keratinization stages, the inner mechanical properties of the macrofibrils dominate the stiffening due to the progressive setting up of the cystine network. Our findings corroborate existing models on the sequence of biological and structural events during hair keratinization.

X-rays interact strongly with biological organisms. Synchrotron radiation sources deliver very intense X-ray photon fluxes within micro- or submicro cross-section beams, resulting in doses larger than the MGy. The relevance of synchrotron radiation analyses of biological materials is therefore questionable since such doses, million times higher than the ones used in radiotherapy, can cause huge damages in tissues, with regard to not only DNA, but also proteic and lipid organizations. Very few data concerning the effect of very high X-ray doses in tissues are available in the literature. We present here an analysis of the structural phenomena which occur when the model tissue of human hair is irradiated by a synchrotron X-ray micro-beam. The choice of hair is supported by its hierarchical and partially ordered keratin structure which can be analysed inside the tissue by X-ray diffraction. To assess the damages caused by hard X-ray micro-beams (1 microm(2) cross-section), short exposure time scattering SAXS/WAXS patterns have been recorded at beamline ID13 (ESRF) after various irradiation times. Various modifications of the scattering patterns are observed, they provide fine insight of the radiation damages at various hierarchical levels and also unexpectedly provide information about the stability of the various hierarchical structural levels. It appears that the molecular level, i.e. the alpha helices which are stabilized by hydrogen bonds and the alpha-helical coiled coils which are stabilized by hydrophobic interactions, is more sensitive to radiation than the supramolecular architecture of the keratin filament and the filament packing within the keratin associated proteins matrix, which is stabilized by disulphide bonds., ((c) 2009 Elsevier Inc. All rights reserved.)

Several aspects of the intermediate filaments' molecular architecture remain mysterious despite decades of study. The growth process and the final architecture may depend on the physical, chemical, and biochemical environment. Aiming at clarifying this issue, we have revisited the structure of the human hair follicle by means of X-ray microdiffraction. We conclude that the histology-based growth zones along the follicle are correlated to the fine architecture of the filaments deduced from X-ray microdiffraction. Our analysis reveals the existence of two major polymorph intermediate filament architectures. Just above the bulb, the filaments are characterized by a diameter of 100 Angstroms and a low-density core. The following zone upwards is characterized by the lateral aggregation of the filaments into a compact network of filaments, by a contraction of their diameter (to 75 Angstroms) and by the setting up of a long-range longitudinal ordering. In the upper zone, the small structural change associated with the tissue hardening likely concerns the terminal domains. The architecture of the intermediate filament in the upper zones could be specific to hard alpha-keratin whilst the other architecture found in the lower zone could be representative for intermediate filaments in a different environment.

The putative transformation of alpha-helices into beta-sheets has been studied for more than 50 years in the case of hard alpha-keratin. In a previous study of stretched keratin fibers, we specified the conditions for beta-sheet appearance within horsehair: the formation of beta-sheets requires at least 30% relative humidity. However, this phenomenon was observed in the whole tissue. Then there was no clear chemical identification of the beta-sheets (keratin or matrix proteins) and the exact location of the beta-sheets across the fiber could not be specified. In this study, using wide-angle x-ray scattering and high spatial resolution infrared microspectroscopy, we could determine and characterize the structural elements across hair sections stretched in water, which provides new information about the aforementioned transition. Our results show that the process can be split into three steps: 1), unraveling of the alpha-helical coiled-coil domains, which starts at roughly 5% macroscopic strain; 2), further transformation of the unraveled coiled-coils into beta-sheet structures, which occurs above roughly 20% macroscopic strain; and 3), spatial expanding of the beta-structured zones from the sample center to its periphery.

Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard alpha-keratin intermediate filaments under in vivo conditions. In short, the alpha-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.

The mechanical behavior of human hair fibers is determined by the interactions between keratin proteins structured into microfibrils (hard alpha-keratin intermediate filaments), a protein sulfur-rich matrix (intermediate filaments associated proteins), and water molecules. The structure of the microfibril-matrix assembly has already been fully characterized using electron microscopy and small-angle x-ray scattering on unstressed fibers. However, these results give only a static image of this assembly. To observe and characterize the deformation of the microfibrils and of the matrix, we have carried out time-resolved small-angle x-ray microdiffraction experiments on human hair fibers stretched at 45% relative humidity and in water. Three structural parameters were monitored and quantified: the 6.7-nm meridian arc, which is related to an axial separation between groups of molecules along the microfibrils, the microfibril's radius, and the packing distance between microfibrils. Using a surface lattice model of the microfibril, we have described its deformation as a combination of a sliding process and a molecular stretching process. The radial contraction of the matrix is also emphasized, reinforcing the hydrophilic gel nature hypothesis.

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.

Synchrotron X-ray micro-diffraction studies along the follicle and the hair fibre allowed us to follow the keratinization process and the progressive organization of the keratin: i) molecular organization appeared progressively in the follicle; the formation of alpha-helices was completed inside the follicle. ii) supramolecular organization appeared only outside the follicle, far from the bulb; filament structure was observed far from the follicle. Comparisons between structures observed for in vitro and in vivo grown hair, whatever in the bulb or in the fibre, indicate there is no evidence of any structural difference. More, no variation in the transition in vivo/in vitro zone has been observed. In vitro and in vivo fibres exhibited the same structure.

The origin of the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns is discussed in terms of side-chains conformations. We show it to reveal specific configurations of the side chains which are common to all two-stranded alpha-helical coiled coils. Combining literature data on crystallised coiled coil pieces and molecular dynamics results with our X-ray diffraction pattern simulations, we propose rules for the attribution of chi1 torsion angles for coiled coils involved in fibres whose structure cannot be resolved at atomic resolution: in a (a b c d e f g) heptad repeat, a and d residues, respectively, adopt mean t and g+ configurations, whereas statistical rules are given for the other residues., (Copyright 1999 Academic Press.)

Low-angle X-ray scattering patterns of hard alpha-keratin fibers have been studied for more than 50 years but a completely convincing modelling has never been presented. The models which have been proposed so far are specific to the sample and cannot be adapted to others, mainly because they do not use a parametric analytical expression of the distribution function describing the relative positions of the microfibrils. Our new approach is based on a paracrystal distribution function. In addition, a huge background originating from a non-ordered matrix is taken into account. Various hard alpha-keratins from different origins have been studied using our approach. From the rather good modellings obtained, it appears that the diameter of the microfibril is not origin dependent (7.4 nm) whereas the distances between microfibrils and their electron density profiles are. Hair microfibrils can be reasonably approximated by a solid cylinder but a core and an outer ring are necessary for porcupine. Our method is of course not limited to keratin microfibrils; it can be used for modelling equatorial X-ray scattering profiles of all types of hexagonal fibrillar assemblies, which are in fact widely found in biological tissues.