BONE RESORPTION IN TISSUE CULTURE 615 



study liy Goldhaber and Barrnett (1960), however, was carried out 

 on resorl)ing bone tissue cultures under time-lapse microcinematog- 

 raphy. This procedure permitted visualization of active osteoclastic 

 bone resorption prior to the histochemical staining of the very same 

 osteoclasts (Figs. 9 to 14). In view of the intimate association of 

 this enzyme with the mitochondria, the likelihood appears that 

 osteoclasts have a high metabolic activit}', supporting the interpreta- 

 tion of previous cinematographic evidence concerning the active role 

 of osteoclasts in the process of bone resorption. 



Some Chemical Inhibitors of Resorption 



The high energy requirement of bone-resorbing cells is suggested 

 by the finding that addition to the culture medium of 2,4-dinitro- 

 phenol (5 X 10 "' m) inhibits resorption during the 2-week culture 

 period, presumably by inhibiting svnthesis of adenosine triphosphate 

 (Figs. 15 and 16). Under these conditions, bone formation was not 

 totally inhibited (Figs. 17 and 18). Of further interest was the find- 

 ing that addition to the medium of sodium malonate (5 X 10~'' m), 

 a powerful inhibitor of succinic dehvdrogenase, inhibits bone resorp- 



FiG. 9. Two-dav resorbing culture showing indistinct osteoclast (O) against 

 hollowed-out bone margin (BM). (Portion from enlargement of 16-mm frame, 

 X approx. 400.) 



Fig. 10. Continuation from Fig. 9. Active resorption proceeding, as may 

 be seen from new position of osteoclast (O) and the resorbing bone margin 

 (BM). (X approx. 400.) 



Fig. 11. Continuation from Fig. 10. Resorption still progressing. Note new 

 position of osteoclast ( O ) and adjacent bone margin ( BM ) . Total time interval 

 between Fig. 9 and Fig. 11 was IGJ hours. ( X approx. 400.) 



Fig. 12. Continuation from Fig. 1 1 after reaction mixture for histochemical 

 disclosure of succinic dehydrogenase was added to tissue culture medium. 

 Note that outline of osteoclast (O) is becoming more distinct as a result of 

 the initiation of the color reaction. BM, bone margin. ( X approx. 400.) 



Fig. 13. Continuation from Fig. 12. Development of color reaction pro- 

 ceeding in osteoclast (O). No further bone resorption occurring owing to 

 toxicity of the histochemical procedure. BM, bone margin. (X approx. 400.) 



Fig. 14. Continuation of histochemical reaction for succinic dehydrogenase. 

 Note intensity of color reaction in osteoclast (O). Time interval between Fig. 

 12 and Fig. 14 was 8 hours. BM, bone margin. (X approx. 400.) 



(Figures 9 to 14 were reproduced by permission from the author's paper in 

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

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



