RHEOLOGY OF STRATUM CORNEUM--II 19 treatment the elastic modulus increases significantly at a given relative humidity. The behaviour of stratum corneum described above contrasts with that observed if only lipid materials are removed, in which case there is no loss in the material's water-retaining capacity and no change in its elastic properties. A similar result, i.e. no change in elastic modulus, is obtained if the corneum is extracted only with water (lipid left intact). Based on findings of this type, Middleton (7), in particular, has proposed that water-soluble hygroscopic materials (e.g. urea, free amino-acids and carboxylic acid salts) are retained within the corneum by a semi-permeable membrane system which breaks down when lipids are removed. Water can pass freely through the membrane when it is intact but the water binding materials cannot be removed. This hygroscopically bound water is believed to act as corneum plasticizer. However, it is not at all dear how this hygro- scopically bound water plasticizes the corneum proteins which are respon- sible for its mechanical integrity. One possible mechanism will now be suggested. Due to their ionic or dipolar nature, the hygroscopic substances will be bound to the proteinaceous components of the stratum corneum. Location of these bulky molecules between protein chain segments will create a more open structure than would exist in their absence leading to enhanced protein hydration. Upon removal of the hygroscopic molecules the protein matrix collapses with the resultant formation of more protein-protein bonds (mainly hydrogen bonds and ionic interactions). An analogy can be drawn with, for example, the action of dilute phenol solutions on wool (15). When a wool fibre is stressed in such solutions it swells considerably and becomes much weaker as compared with its strength in water. This is believed to be due to the fact that phenol is much more effective in disrupting certain hydrogen bonds in the wool structure. In the case of the stratum corneum the naturally occurring hygroscopic substances act in a similar manner to phenol on wool. The higher modulus values obtained for the hygroscopic- free corneum samples (Fig. 1) result from the rigidity that the additional bonds formed (after removal of the hygroscopic substances) impart to the structure. Due to the reduction in swelling capacity of the corneum imposed by this structural collapse, water uptake will also be less as indicated by the TGA results presented here (Table II) and water-binding isotherms obtained by other authors (2, 4). The essential feature of this model is that aqueous plasticization of the corneum is due to direct protein hydration both in the presence or absence of hygroscopic substances. This is in accord with the DTA data of Bulgin et al (10) which indicates that bound water

20 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS in the stratum corneum is associated with proteins rather than with hygro- scopic substances (or lipids). Infra-red spectra of untreated and extracted stratum corneum were recorded to determine whether the molecular changes outlined above could be confirmed by this technique. Normal and polarized spectra failed to detect any change in the absorption band frequencies or intensities of the principal protein component (keratin). Consequently, any explanation that the differences in mechanical properties observed are due to a conforma- tional change in the keratin or an alignment of the protein filaments can be ruled out. Attempts to force alignment or conformer change of the keratin by large extensions of the corneum ( 50•o) also failed. This is not too surprising since the keratin filaments are completely contained within the corneum cells and the fibrils in contiguous cells are mechanically indepen- dent. The cell membrane/desmosomal system is, therefore, the only con- tinuous phase pervading the entire cell structure of the corneum and, as such, is likely to be the load bearing component. Indirect evidence for the cell membrane system rather than the keratin being the rheologically active material in the stratum corneum, is provided by the fact that isolated keratin films swell considerably in water, almost to the point of dissolution, and display no detectable elasticity. This behaviour contrasts with that of untreated and extracted corneum (the extracted material should contain only keratin and cell membrane components) both of which have finite elastic modulus values in water (see 100•o rh data in Fig. 1). The concept that the mechanical strength of the corneum resides in the cell membrane system is supported by the fact that the cell membrane protein (16) has a much higher cystine content (x 3-4) than the keratin filaments (17) leading to more permanent (with regard to disruption by water) disulphide cross-links in the membrane. Unfortunately, no independent experimental data can be obtained for the cell membranes in their native state since the ir spectrum of the whole corneum is principally that of the keratin filaments, owing to their preponderance in the structure (• 60•o of the dry weight), and isolation of the cell membranes always involves their partial destruction. CONCLUSIONS (1) Elastic moduli can be used to detect changes in the stratum corneum. (2) Extraction of the corneum with organic solvents or surface active agents followed by aqueous extraction reduces the water-retaining power of this substrate.