DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 



Evans, 1912; Evans and Schulemann, 1914), and hence it may be concluded that 

 this diffusely stained material consists of devitalized protoplasm.* 



The sections also confirm the cleared preparations in revealing at the fracture- 

 site an increase in the vitally stained phagocytes as compared with corresponding 

 areas in control preparations. These blue cells are present among the degenerate 

 tissue-remnants, extravasated blood, and exudate. Often large macrophages 

 filled with blue granules appear in the blood-clot, and here the corpuscles have 

 almost disappeared, showing that there is a direct relationship between the pres- 

 ence of the macrophages and the disappearance of the blood-cells. The same 

 association is emphasized in the damaged muscle and other tissue. 



In addition to the large, irregularly shaped cells, which we may identify as 

 the clasmatocytes the normal mononuclear tissue-resident phagocytes which 

 show the most distinct blue granulation, there are great numbers of smaller dye- 

 containing cells. These are of various sizes, extending from a small, round, lym- 

 phocyte-like cell to the large polyblast. In the smaller cells the dyestuff is very 

 slight, even absent, and as a rule it is increased in amount progressively in the 

 larger sizes. It would seem that the larger cells are developed from the smaller 

 and that the cytoplasm not only hypertrophies, but with this acquires the power 

 of imbibing and storing the colloidal dyestuffs in granule form. The macrophages 

 are always larger and more numerous where muscle and other tissue is evidently 

 damaged. Later stages will show that there is always an excess of macrophage 

 tissue where protoplasm is degenerate, as evidenced by its diffuse staining with 

 trypan-blue and by its histological characters. 



Polymorphonuclear leucocytes are often seen in the tissues of the fracture- 

 area. No dye-granules were observed in them. Quite a number of fibroblasts 

 were found; occasionally a few small grains of blue were noted in them, but they 

 take the dyestuff very sparingly. There are, too, many tiny blood-vessels in 

 fact we have here to deal with granulation tissue. 



On the second day the sections show the beginning of the callus. This is a 

 fairly thick layer of basophilic cells lying along the original bone in the region of 

 the injury. These cells are evidently young osteoblasts, and it is plain that there 

 has been an extensive proliferation of them. Overlying this osteoblastic precallus 

 is a layer of fibrous connective tissue continuous on either side with the periosteum. 



In the precallus there are traces of new bone in the form of slender plates, 

 lying along the original bone, and, in places, short delicate trabecute arising from 



*The trypan-bluc staining which occurs in (a) devitalized tissue has several outstanding and distinctive characteristics. 

 The blue mass is made up of what appears to be degenerate tissue, in which the cells are often misshapen and broken up, 

 or it may be clumped together in a disorderly manner and mixed with exudate. The staining is dense and irregular and 

 involves the entire cell, including the nucleus. It is simply due to the absorption of the dyestuff by the necrotic protoplasm. 

 Its occurrence is thus good evidence of the presence of defunct tissue and exudate. Living protoplasm is able to protect 

 itself by excluding or segregating the dye, except as described brlmv in "c." 



This typical behavior of necrotic or moribund tissue toward trypan-blue is to be sharply distinguished from the (6) 

 true vital-staining, which is found in the macrophages. Here the dyestuff is housed within the cytoplasm in the form of 

 numerous discrete droplets or granules. Often the dye is found in the fluid of a vacuole. The nucleus does not take the 

 dye. Under low magnification, as with the binocular microscope, the entire cell is seen as a single blue granule, standing 

 out in sharp contrast to its unstained surroundings. Such an appearance is represented in the drawings of the cleared ribs 

 (figs. 1 to 5) and cleared skull (fig. 6). 



A third type of trypan-blue staining (c), distinct from both of the above, may be referred to here. It is found in certain 

 relatively inert living tissue (such as the elastic lamina? of blood-vessels), in fibrous connective tissue (as tendons and liga- 

 ments), and in scar tissue. It is a rather pale, even coloration and is easily distinguishable from the true vital staining in 

 the macrophages and from the staining of devitalized tissue. 



