BONE RESORPTION IN TISSUE CULTURE 613 



that both macrophages and osteoclasts are capable of resorbing bone 

 (Figs. 7 and 8), The formation of Howship's lacunae, however, is a 

 task restricted to the osteoclasts. It is of interest that large numbers 

 of macrophages appear relatively late in the culture period and that, 

 to date, we have not observed anv fusion of these mononucleated 

 cells to form large, multinucleated osteoclasts. Although we have 

 observed osteoclastic activity as early as 1 day in culture, it is prob- 

 able that these cells were present at the time of explantation and 

 were stimulated by the experimental conditions rather than being 

 formed de novo. With regard to the fate of the osteoclast, the longest 

 period that we have been able to follow one cell has been approxi- 

 mately 48 hours, during which time the cell appeared to enlarge 

 and become more granular. In several instances we have observed 

 the death of such granular osteoclasts, which was manifested by a 

 sudden gelation of the cytoplasm and cessation of cell movement. 

 Significantly, further bone resorption did not continue at these sites. 

 The aggressive action of these cells against the resorbing bone sur- 

 face supports the idea that osteoclasts actively participate in the 

 process of bone resorption. 



The hypothesis that osteoclasts actively secrete some product in- 

 volved in bone resorption is further supported by some of our time- 

 lapse studies, wherein it is possible to visualize the production of 

 large vacuoles at the border of the resorbing bone. Occasionally, in 

 cultures exhibiting extremely rapid and marked bone resorption, the 

 presence, growth, and fusion of huge bubblelike vacuoles has been 

 noted in resorbing areas at the height of resorptive activity. Time- 

 lapse films of such giant "bubbles" suggest that these unusual struc- 

 tures are intracytoplasmic. Their sudden, rapid contraction several 



Fig. 7. Four-day living culture, showing osteoclast (O), outlined by arrows, 

 in Howship's lacunae. BM, bone margin. (Enlargement of 16-mm frame, X 

 approx. 540. ) 



Fig. 8. Same field, 6* hours later, showing resorption of some of the bone 

 margin (BM) as the osteoclast (O) burrows deeper into the bone. (Enlarge- 

 ment of 16-mm frame, X approx. 540.) 



(Figures 7 and 8 were reproduced by permission from the author's paper in 

 Cinemicrographij in Cell Biology, edited by George G. Rose and published by 

 the Academic Press, Inc., New York, 1963.) 



