JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ment of skin structures in mice have been carried out by removing portions of the skin from the trunk of embryos and cultivating these upon clots prepared from equal parts of fowl plasma and chicken embryo extract (Hardy, 1949 and 1951). Some of the embryonic skin was removed before hair follicle development had apparently begun. The cultures were continued until past the time when the mice would have been born and a comparison was made of the structures which developed in the cultures with those appearing in normal young mice of the same age. The cultivated material showed normally developed epidermis, hair follicles and some differentiation of cells in sebaceous glands. Close similarity with the same structures in normal young mice was observed in the hair papillae, root-sheaths and the growing hairs, the last consisting of a cortex and cuticular scales which developed with normal changes in the constituent cells. The rate of cell division was high and even after 25 days' cultivation, dividing cells were present in the epidermis, dermis and hair follicles. When the cultures were prepared from the embryos of coloured mice, pigment-producing cells and pigmented hairs were easily distinguished. Pigment cells were active in some cases where hair follicles failed to form keratinising fibres, and these cells produced pigment as if a hair were present to receive it. Hardy concluded that the dermis and epidermis together were sufficient for forming and maintaining the hair follicles, no other cellular structures being required. Neither circulating blood nor nervous stimulation appeared necessary for follicle formation and the absence of blood circulation affected only the quantity of keratin produced by the hair follicles and the epidermis. The effect of blood supply on the quantity of keratin formed by the skin of the adult mouse is seen when the wave of activity passes through the coat during the cycle of hair growth, the blood supply increasing markedly with active growth in a certain area. The increased blood supply is required for maximum production of keratin only. The potentialities for diverse development of embryonic tissues prevent the results obtained with them being necessarily applicable to adult tissues. However, the fact that the development of embryonic skin tissues can be influenced by external conditions is shown by the growth of skin from the chick embryo (Fell, Mellanby & Pelc, 1954). When comparable fragments from the trunk and limbs were cultivated in media with and without the addition of excess vitamin A alcohol then marked differences were observed in the development of the skin. The tissue expected to form a keratinising epidermis did so in the absence of excess vitamin whereas when excess vitamin A was present a mucus-secreting membrane resulted. A mucous membrane readily takes up sulphate to form polysaccharide sulphates, but the animal uses very little to form sulphur amino acids such as the cystine in keratin. Addition to the media of sulphate containing a radio-active isotope of sulphur revealed that the keratinising epithelium took up very little sulphate whereas 120

THE SKIN AS A COMMUNITY OF STRUCTURES the tissue cultivated in the presence of excess vitamin A utilised sulphate like a normal mucous membrane. Thus, the development of the tissue was not uniquely determined and could be influenced in the absence of the intact animal. No experiments on uptake of labelled cystine or methionine in such cultures have been reported, but would be of interest in making clear-cut the difference in metabolism of two types of epithelium. The effect of vitamin A and the relation to the action of oestrogens have been investigated using older tissues. Hardy, Biggers and Claringbold (1953) cultivated portions of the vagina of a prepuberal mouse and showed that this tissue retained its non-keratinising epithelium. However, when oestrogens such as oestradiol, oestrone and diethylstilboestrol were added to the medium then a typical squamous, keratinising epithelium was produced. This work was extended by the cultivation of similar tissue from prepuberal rats (Kahn, 1954), and in this case it was noted that the epithelium did change to a keratinising type if cultivation in the standard medium was continued for a sufficient length of time. Such a change was accelerated by the addition of oestrone to the medium, but was prevented if excess vitamin A was present. Tissue cultivated in the presence of excess vitamin A and then transferred to the standard medium underwent normal conversion to a keratinising condition. ADULT MAMMALIAN SKIN The ability of adult rabbit skin to survive in a stirred fluid mhdium at body temperature was investigated by Medawar (1947, 1948), using a grafting technique to determine whether survival had occurred. Small squares of skin consisting of epidermis and part of the dermis were removed from an animal, cultured under desired conditions and then transplanted to the animal from which they had been removed. In the presence of air or an air-oxygen mixture, cultivation for as long as eight days was possible during which time migration of epithelial cells from the epidermis and the hair follicles occurred to provide a layer enclosing the tissue specimen completely. The cells of this layer underwent normal division and gave rise to a true keratinised epidermis. The continued formation of this tissue was taken as evidence that the characteristic functional activity of the skin epidermis had not been impaired. When the enclosing epithelium was removed from the dermal tissue then successful grafting could be carried out. The skin was able to survive for a week at body temperature in the medium in the complete absence of atmospheric or dissolved oxygen, but under these conditions no movement nor division of the epithelial cells took place. In the presence of air, tissues cultured in media containing more than 10 -• M iodoacetate did not survive. Iodoacetate is a powerful inhibitor of certain enzymes involved in the production of energy from sugars, i.e., involved in glycolysis, and 121