MEASUREMENT OF THE INHERENT GROWTH ENERGY OF TISSUES

1. The residual growth energy of fibroblasts is expressed by the extent of their migration and multiplication in a non-nutrient medium. 2. The residual energy of fibroblasts is related to their inherent energy and the variations of the inherent energy can be ascertained by the measurement of the residual energy

ARTIFICIAL ACTIVATION OF THE GROWTH IN VITRO OF CONNECTIVE TISSUE

The experiments have shown that extracts of tissues and tissue juices, under certain conditions, accelerate the growth in intro of the connective tissue from about three to forty times. This activating power was found in many tissues. It was much more marked, however, with the extracts of embryos, of adult spleen, and of the Rous sarcoma. The power diminished directly with the dilution of the extracts, and appeared not to apply to the tissues of a heterologous animal. The power was reduced when heated at 56° C., and removed when heated at 70° C. It was diminished markedly by filtration through a Berkefield filter and was completely suppressed by filtration through a Chamberland filter. Possibly the finding of the activating power of tissue extracts will have no immediate practical application. Nevertheless, it may be indirectly useful by leading to the discovery of some of the factors determining the growth of tissues and of the unknown laws of cell dynamics, and may ultimately throw light on the mechanism of the cicatrization of wounds

GROWTH-PROMOTING FUNCTION OF LEUCOCYTES

Two main facts were brought to light by the preceding experiments: first, the presence of growth-activating substances in the leucocytes; second, the setting free of these substances in tissues and fluids where leucocytes accumulate. The existence of growth-promoting substances within the body of the leucocytes was to be expected. Leucocytes are embryonic cells and it is well known that embryonic tissues contain substances which stimulate cell proliferation. But the experiments gave a direct experimental proof of this fact. Then, during the whole life and even in extreme old age, there is a supply of growth-promoting substances within the organism which is potentially capable of restoring the activity of the resting cells. Embryonic tissue juice, as is known, can rejuvenate cells which have ceased to multiply in vitro and show evidences of degeneration. If the growth-activating substances of leucocytes can be transferred in vivo to tissue cells, they may play a similar rôle. Therefore, it was important to find out whether the growth-activating substances were set free either by the secretions of the living leucocytes or by diffusion from the body of the cells after they were injured or dead, and whether this phenomenon occurred actually in vivo. Indeed, the idea that leucocytes secrete substances necessary to normal physiological processes is by no means new. Long ago, Ranvier described the lymph cells as mobile unicellular glands, and Renaut thought that their function was to bring to fixed cells the necessary food material. This rôle of the leucocytes was considered by him as absolutely essential. According to his theory, differentiated cells could not live in the absence of the lymph cells, which supply them with the substances required for their activity. The presence of such substances in the leucocytes was shown by our experiments and the growth-activating power acquired by inflamed connective tissue demonstrated that the leucocytes could actually bring these substances to the fixed cells. Under certain conditions, this property of the white cells of the blood may cause the resumption of the activity of tissues which are in a resting state. In the adult organism, the tissues have ceased to grow and the blood plasma has acquired marked inhibiting properties. But growth-promoting substances are still stored in leucocytes, glands, and muscle tissue. The leucocytes could supply fibroblasts or epithelial cells with the necessary food material if they were present where cell proliferation is needed. The existence of mechanisms causing leucocytes to invade tissues in need of repair is certain. The initiation of healing seems to depend on the coming of the leucocytes to the wounded tissue. When they are missing, as happens when the wound is protected from all external irritation, cicatrization is greatly delayed. On the contrary, when staphylococci, turpentine, and other irritants are applied at the surface of the wound, granulations appear after less than 48 hours. These irritants, although different in nature, have the common characteristic of determining an inflammation of the tissues and the migration of leucocytes from the vessels to the surface of the wound. Possibly the white cells bring the substances which adult tissues require in order to cicatrize or regenerate. They would have the function of storing away the growth-promoting substances characteristic of embryonic tissues, and bringing them to the regions of the organism where they are needed

LATENT LIFE OF ARTERIES

When a segment of artery, killed by heat, formalin or glycerin is transplanted, it undergoes a rapid degeneration. Its muscle fibers disappear while the tissue of the host reacts by building a new wall of connective tissue. When the transplanted vessel has been preserved in a condition of latent life, no degeneration of the wall occurs, or the wall undergoes only partial degeneration. The muscle fibers can keep their normal appearance, even for a long time after the operation. It is, therefore, demonstrated that arteries can be preserved outside of the body in a condition of unmanifested actual life. The best method of preservation consists of placing the vessels, immersed in vaselin, in an ice box, the temperature of which is slightly above the freezing point. From a surgical standpoint, the transplantation of preserved vessels can be used with some safety. When the arteries were kept in defibrinated blood or vaselin and in cold storage, the proportion of positive results was 75 and 80 per cent., and this can probably be increased

HETEROTRANSPLANTATION OF BLOOD VESSELS PRESERVED IN COLD STORAGE

The experiments show merely that blood vessels transplanted from dog to cat can act as arteries for seventy-seven days at least; and that having spent several days in cold storage does not interfere with their ordinary functions. The animals operated upon must, however, be kept under observation for several months, or, indeed, for several years, before any conclusion can be drawn concerning the practicability of this method of preservation and heterotransplantation of blood vessels

TRANSPLANTATION IN MASS OF THE KIDNEYS

Among so many etiological factors, it is impossible to discriminate which are responsible for the complications which took place in our experiments. An attempt to explain the occurrence of nephritis, cedema or calcification of the arterial system, for instance, will not be made, but the technique of the operations will be modified in order to suppress as much as possible the causes which may originate these secondary changes. The purpose of this article was not to analyze minutely the physiological or pathological character of the functions of transplanted kidneys, but merely to ascertain whether these functions are efficiently reëstablished. It is to be concluded that an animal which has undergone a double nephrectomy and the grafting of both kidneys from another animal can secrete almost normal urine with his new organs, and live in good health at least for a few weeks. This demonstrates that it is possible to reëstablish efficiently the functions of transplanted kidneys

CONTRIBUTIONS TO THE STUDY OF THE MECHANISM OF THE GROWTH OF CONNECTIVE TISSUE

When connective tissue cells have been cultivated for a certain length of time in a medium which has been repeatedly changed, a definite relation arises between the rate of growth of the cells and the composition of the medium. It is possible, by adding to the culture medium a given quantity of certain substances, such as embryonic juices, to foresee the extent to which a fragment of tissue composed of a given strain of cells will increase in a given time. The rate of growth of a strain of cells can be accelerated or retarded by the addition to the medium of activating or retarding substances. The dynamic condition of a strain of connective tissue cells, which have been living in a given medium for some time, is not a definitely acquired characteristic, but a temporary state, and is the product or function of the medium in which the cells are living, and is readily modified merely by altering the composition of the medium. A knowledge of the characteristics of the growth of connective tissue described has led to a new result,—the indefinite proliferation of a strain of connective tissue cells outside of the organism. The strain of connective tissue originally obtained from a fragment of chick embryo heart, which had been pulsating in vitro for 104 days, was still actively alive after sixteen months of independent life and more than 190 passages. The rate of proliferation of the connective tissue sixteen months old equalled and even exceeded that of fresh connective tissue taken from an eight day old embryo. It appears, therefore, that time has no effect on the tissues isolated from the organism and preserved by means of the technique described above. During the sixteenth month of life in vitro the cells increased rapidly in number and were able in a short time to produce a large quantity of new tissue. This fact, therefore, definitely demonstrates that the tissues were not in a state of survival, as was the case in certain earlier experiments, but in a condition of real life, since the cells of which they were composed, like microorganisms, multiplied indefinitely in the culture medium

ON THE PERMANENT LIFE OF TISSUES OUTSIDE OF THE ORGANISM

In two series of experiments made at the end of 1911 and at the beginning of 1912, new techniques were developed with the view of investigating the problem of prolonging indefinitely the life of tissues isolated from the organism. These techniques are far from perfect and will doubtless be modified in the future. But they have already permitted the establishment of new facts. Fragments of connective tissue have been kept in vitro in a condition of active life for more than two months. As a few cultures are now eighty-five days old and are growing very actively, it is probable that, if no accident occurs, the life of these cultures will continue for a long time. In some cases the rate of growth of the tissues increased in direct ratio to the age of the culture. Fragments of heart pulsated rhythmically at the beginning of the third month of their life in vitro. These facts show that experiments made with these or with more perfect techniques and followed over long periods of time may lead to the solution of the problem of permanent life of tissues in vitro, and give important information on the characters acquired by tissues liberated from the control of the organism from which they were derived