Pure cultures of fibroblasts displayed marked differences in their activity in the plasma of young, middle aged, and old chickens. The rate of cell multiplication varied in inverse ratio to the age of the animal from which the plasma was taken. There was a definite relation between the age of the animal and the amount of new tissue produced in its plasma in a given time (Text-figs. 1 to 10). The chart obtained by plotting the rate of cell proliferation in ordinates, and the age of the animal in abscissæ, showed that the rate of growth decreased more quickly than the age increased (Text-fig. 12). The decrease in the rate of growth was 50 per cent during the first 3 years of life, while in the following 6 years it was only 30 per cent. When the duration of the life of the cultures in the four plasmas was compared, a curve was obtained which showed about the same characteristics (Text-fig. 11). The duration of life of the fibroblasts in vitro varied in inverse ratio to the age of the animal, and decreased more quickly than the age increased.

As the differences in the amount of new tissue produced in the plasma of young, middle aged, and old chickens were large, the growth of a pure culture of fibroblasts could be employed as a reagent for detecting certain changes occurring in the plasma under the influence of age. But the method possesses the necessary accuracy only when it is used as has already been described, and by technicians thoroughly trained in the details of its application.

A comparative study of the growth of fibroblasts in media containing no serum, and serum under low and high concentrations, was made in order to ascertain whether the decreasing rate of cell multiplication was due to the loss of an accelerating factor, or to the increase

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of an inhibiting one. In high and low concentrations of the serum of young animals, no difference in the rate of multiplication of fibroblasts was observed. This showed that the serum of an actively growing animal did not contain any accelerating agent. The same experiments were repeated with the serum of a 3 year old and a 9 year old chicken. The medium made of a high concentration of serum had a markedly depressing effect on the growth, and this effect was greater in the serum of the older animal (Text-fig. 13).

The results of the experiments showed in a very definite manner that certain changes occurring in the serum during the course of life can be detected by modifications in the rate of growth of pure cultures of fibroblasts, and that these changes are characterized by the increase of an inhibiting factor, and not by the loss of an accelerating one. It appeared, therefore,that the substances which greatly accelerate the multiplication of fibroblasts and are found in the tissues do not exist in the blood serum, or are constantly shielded by more active inhibiting factors. The curve which expresses the variations of the inhibiting factor in function of the age was compared with that showing the variations of the rate of healing of a wound according to the age of the subject. For wounds of equal size, the index of cicatrization, which expresses the rate of healing, varies in inverse ratio to the age. The different values of the index of cicatrization of a wound 40 sq. cm. in area, taken from measurements made by du Noüy, were plotted in ordinates, and the age of the subject in abscissæ (Text-fig. 14). The curve showed a decrease in the activity of cicatrization which resembled the decrease in the rate of growth of fibroblasts in function of the age of the animal. This suggested the existence of a relation between the factors determining both phenomena.

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