DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. 51 



of undifferentiated mesenchyme, which is what would be expected in any tissue that 

 proliferates as rapidly as this tissue necessarily must. It would be interesting to see 

 whether, if one of the cultures were kept alive without further explanation, it would 

 again differentiate after a certain equilibrium of proliferation had been reached. 



Some workers have claimed that certain substances present in the medium 

 of tissue cultures prevent the growth of the connective tissues. For example, 

 "Walton (1914) states that liver extract inhibits the growth of adult mammalian 

 connective tissue in plasma cultures, and Russel (1914) claims that gentian violet, 

 in solution of 1/20,000 in the medium of tissue culture, prevents the growth of 

 connective tissue but not of endothelium. The reason for this neither writer 

 explains, nor does either state what structure of the cell is affected by the sub- 

 stance so that the cells do not grow out, or whether the medium may simply not 

 attract the cells to migrate and that the cells themselves are uninjured. 



Thus a review of the literature on the living connective-tissue cells shows 

 that the study of the living tissue has not presented decided proof as to whether 

 the connective-tissue fibers arise from the cells or are formed from an intercellular 

 substance. Evidence is presented on both sides, and the question remains as 

 completely at a dead-lock as when the observations were confined to fixed and 

 stained preparations. 



OBSERVATIONS IN GENERAL. 



A few general observations as to what takes place in the tissue cultures of con- 

 nective tissue are given here in order to show what factors influence the growth 

 of the fibers. 



Cultures from the subcutaneous tissue of chick embryos of various ages were 

 made in the usual manner (W. H. and M. R. Lewis, 1915; M. R. Lewis, 1916). 

 Lewis and Lewis have shown that while the cells in the new growth in tissue 

 cultures are under somewhat abnormal conditions as regards environment and 

 nourishment, nevertheless they are actively growing cells which undergo normal 

 division and which grow out as definite types of cells that is, nerve-cells, muscle- 

 cells, heart-muscle cells, endoderm of the intestine, epithelial cells of the skin, 

 and connective-tissue cells. 



As has been stated, no fibrin is present in the medium, and no substance 

 which coagulates. 



The subcutaneous tissue can be removed as a thin, transparent sheath from 

 the skin of chick embryos of 10 days or older. It proved difficult to isolate the 

 subcutaneous tissue from embryos younger than 8 days; and in these embryos a 

 piece of skin or one of the deeper skin fascias or the arachnoid tissue was used 

 for explanation. The connective tissue of embryos less than 8 days old is com- 

 posed of cells without definite fibrils; that of embryos of 11 days and over contains 

 definite bundles of fibers. The new growth from an explanted piece of subcuta- 

 neous tissue of embryos of 8, 9, and 10 days proves very satisfactory for the study 

 of the development of the fibrils. The growth can be kept alive and healthy by 

 frequent baths of fresh Locke's solution, plus 10 per cent bouillon, plus 0.25 per 

 cent dextrose; and fibrils begin to develop in the new growth in from 48 to 72 



