6 DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 



At the time selected the animals were anesthetized and bled, the required 

 tissues being at once dissected out and fixed in 10 per cent formalin neutralized 

 with magnesium carbonate. Care was taken to leave the soft parts surrounding 

 the bones undisturbed. After fixing for 24 hours and washing in running tap-water 

 for the same length of time the tissues were passed through a graded series of alco- 

 hols ending with two changes of absolute. They were then cleared in benzine and 

 oil of wintergreen, after the method of Spalteholz (1914), and examined. Of the 

 cleared specimens the ribs and skulls were most satisfactory, for it was impossible to 

 make the larger bones transparent because of their greater density and thickness. 



Certain of the skulls and long bones were then decalcified, embedded, and 

 sectioned (table 1), so that the various stages could be studied microscopically. 

 A few were cleared after having been decalcified ; some of these also were afterwards 

 sectioned. The most valuable sections were those simply cleared and mounted 

 with no stain other than the try pan-blue and those lightly counterstained with 

 carmine, which afforded a satisfactory contrast with the blue. Hematoxylin and 

 eosin for cellular detail were used, and also (in special cases) methyl-green and 

 safranin. 



OBSERVATIONS. 



In the description of the gross and microscopic findings in the vitally stained 

 healing wounds of bone, the different stages, as shown by a study of cleared gross 

 specimens of ribs and skulls and of cleared and stained sections, will be taken up 

 in order. Later on, the various aspects of vital staining will be analyzed, con- 

 solidated, and finally reduced to a summary. But before proceeding to a portrayal 

 of the changes in the vital staining of wounded bone and the tissues immediately 

 surrounding the site of the wound it will be well to describe briefly the appearance 

 of the vitally stained normal bone and its tissue environment. 



A good example of this is seen in figure 1, drawn with the aid of the binocular 

 microscope from a cleared rib taken from a rat vitally stained for 2 days with 

 trypan-blue. It is intended as a control of the stages afterward to be described. 



The clear shaft of the bone (B) is easily seen surrounded by the periosteum (p) 

 and inclosing a well-marked medullary canal (MC). To the left the intercostal 

 vessels and nerve (N) are faintly sketched in. The fasciculi of intercostal muscle 

 (MUS) are suggested by the intercrossing lines. 



It will be noted at once that black dots appear throughout the drawing. These 

 are seen as blue granules in the original specimen and represent individual cells 

 which have phagocytized and stored the dyestuff. These cells are more concen- 

 trated in the medullary cavity, the periosteum, and in the connective tissue around 

 the bone. In the intermuscular septa they are conspicuous as granular streaks. 

 These cells are macrophages and their presence in the regions described, as is well 

 known, is normal. Goldmann (1909), for instance, has recorded similar findings, 

 as in his Taf. xi, No. 2, where he shows the histological appearance of the vitally 

 stained interfibrillar cells in the muscle of the tongue. 



