Hair grown in vitro is used by L'Oréal as a model for the study of the influence of nutrients or cosmetics on hair fibre production and the keratinisation process. In the field of cosmetics, X-ray microdiffraction could be useful to evaluate and compare the efficacy of different cosmetic treatments particularly in the field of hair growth treatment and for the development of products for the prevention of hair loss.

Human hair fibre is made of keratin, a protein synthesised and progressively organised into filaments by the cortical cells of the keratogenous zone of the bulb. The hair fibre keratin contains two structures: -keratin and amorphous keratin. In the -structure, molecules of keratin are organised into tetramers (or protofilaments) and form hexagonal superstructures: the microfilaments. These structures give the typical X-ray SAXS and WAXS diffraction patterns which have been studied for many years using X-ray diffraction [1]. However, as yet, the corresponding molecular structure is not very clear [2] and the keratinisation process, during which keratin is formed and its structure becomes organised and stabilised, has not been completely elucidated.

The microfocus beamline, ID13, provides intense synchrotron radiation microbeams. A monochromatic (=0.948 Å) microbeam of 10 µm was selected. This makes it possible to obtain diffraction data for hair, from molecular to supramolecular structures, in less than 30 s exposure time. Synchrotron X-ray micro-diffraction studies along the bulb and the hair fibre allowed us to follow the keratinisation process and the progressive organisation of the keratin.

In vivo grown hairs were dissected out from scalp biopsies with their intact bulb. Some of them were grown in vitro in a nutritive solution for four days.

Scanning along the follicle (Figure 120) made it possible to follow the keratinisation process and the progressive organisation of the keratin:
- molecular organisation appears progressively in the bulb. Keratin is gradually organised from amorphous to structure. The formation of -helices is completed inside the bulb.
- supramolecular organisation appears only outside of the bulb. Filament structure is observed far from the bulb.
- The medium axis of -helices tilts randomly around the main axis of the hair in the bulb: the organisation of the keratin is not stabilised. Changes in oscillation vanish when distance from the bulb increases, and disappear in the fibre.

Our microdiffraction experiments can be compared to experiments done by Mercer [3] with human plucked hairs, using a 100 µm collimated X-ray beam (copper anode filament) and an exposure time of 24 hours. The five zones, designated by Mercer from A to D, are described by diffraction patterns, birefringence, and thermal stability. The birefringence showed that the amount of oriented structures increases progressively between about 300 and 700 µm along the fibre. This corresponds with our observations with the WAXS patterns about the formation of the -helices and the simultaneous formation of the dimers.

Our SAXS observations show that the supramolecular structure of microfibrils is achieved at over 1000 µm. This seems to correspond to the consolidation zone, as defined by Mercer in terms of thermostability of the keratin structure. However, Mercer proposed that the supramolecular structures could be explained by a progressive aggregation of smaller organised structures, and caused with less importance, by an extrusion-like phenomenon. SAXS observations made by microdiffraction indicate that supramolecular organisation exists early on in the time frame of the keratinisation process. However, the progressive evolution of the diffraction patterns to the fibre SAXS pattern could indicate that a phenomenon of densification occurs simultaneously to the keratinisation process.

Comparisons between structures observed for in vitro and in vivo grown hair, whether in the bulb or in the fibre, indicate there is no evidence of any structural difference. Moreover, no variation in the transition in vivo/in vitro zone has been observed. In vitro and in vivo fibres exhibited the same structure, which is encouraging for in vitro growth technique development.

References
[1] R.B. Corey, R.W.G. Wyckoff, The Journal of Biological Chemistry, 114, 407-416 (1936).
[2] B. Busson, F. Briki, J. Doucet, Journal of Structural Biology, 125, 1-10 (1999).
[3] E.H. Mercer, In: Keratin and Keratinisation ­ An Essay in Molecular Biology, Pergamon Press New York (1961).

Principal Publication and Authors
F. Baltenneck (a), B.A. Bernard (b), J.-C. Garson (a), P. Engström (c), C. Riekel (d), F. Leroy (a), A. Franbourg (a), J. Doucet (e), Cellular and Molecular Biology, In press.

(a) L'OREAL Recherche, Aulnay-sous-Bois (France)
(b) L'OREAL Recherche, Clichy (France)
(c) KCK, Chalmers University of Technology, Göteborg (Sweden)
(d) ESRF
(e) LURE, Université Paris Sud, Orsay (France)