BONE DESTRUCTION BY MULTINUCLEATED GIANT CELLS 517 



salt as the coupling agent; for succinic dehydrogenase, the method 

 of Sehgman and Rutenburg (1951); the method of Nachlas et al. 

 (1958a) for diphosphopyridine nucleotide-linked dehydrogenases, 

 malic, lactic, and glutamic; for DPN diaphorase, DPNH (0.5 mg/ 

 ml) was substituted as substrate in the procedure of Nachlas et al. 

 (1958a); for the triphosphopyridine nucleotide-linked isocitric de- 

 hydrogenase, that of Nachlas et al. ( 1958Z? ) ; and for TPN diaphorase 

 demonstration, TPNH (0.5 mg/ml) was substituted as substrate in 

 the last-mentioned procedure. In the demonstration of the above 

 diaphorases and dehydrogenases, tetranitro blue tetrazolium was 

 substituted for nitro-BT to minimize artifactitious staining on lipid- 

 aqueous interfaces (Rosa and Tsou, 1961). Control sections were 

 incubated in the absence of substrate, and in the demonstration of 

 cytochrome oxidase were treated with 10 ~^ mole of NaCN prior 

 to incubation. All incubations were for ^ o hour except for acid phos- 

 phatase and isocitric deh\xlrogenase, which were for 1 hour. Maxil- 

 lae and tibiae of 3-day-old rats were processed to compare the en- 

 zyme activities of skeletal osteoclasts with those of the giant cells 

 about the implants. 



Details of the Experimental Groups 



Group 1. The scapula sample was removed, decalcified, and 

 implanted 14 days later. Groups of 3 to 7 animals were killed at 

 weekly intervals after implantation up to 8 weeks. In some groups, 

 prior to implantation, the decalcified scapulae were blotted on filter 

 paper and weighed to the nearest 0.1 mg, and after removal were 

 similarly reweighed. 



Group 2. Rats were put on the rachitogenic diet of Jephcott and 

 Bacharach ( 1926) for 28 days. Portions of the scapula were removed 

 and devitalized by repeated freezing and thawing but were not 

 decalcified. The animals were in the meantime returned to a normal 

 diet and 2 weeks later the bone was implanted. Groups of 4 to 17 

 animals were killed at weekly intervals up to 4 weeks later, when 

 the implant was removed and sectioned. 



