olg j. t. irving and c. s. iiandelman 



Methods 



Rats. Young male animals of the Holtzman strain weighing 80 

 to 100 gm were used. 



Operative technique. Under ether anesthesia, a piece of the 

 scapula was removed aseptically. Part was preserved for histological 

 examination (called the reference section) and the other part was 

 treated in various ways and ultimately implanted into the same ani- 

 mal under the skin of the back, the implants thus being autogenous. 

 At stated intervals the animals were sacrificed and the bone and 

 surrounding tissue removed for examination. Some animals in each 

 group were injected with trypan blue before the implant was re- 

 moved, to detect the presence of phagocytic cells around the implant. 



Treatment of bone implants. Decalcification was carried out 

 with 0.5 M ethylenediamine tetraacetic acid at pH 7.0 for 7 to 10 

 days. The bone was well washed before implantation. Some of the 

 reference bones that had been decalcified were ashed at 900° C to 

 determine if decalcification had been complete. Devitalization was 

 carried out by repeated freezing to — 40°C and thawing. The 

 presence of empty osteocyte lacunae was taken as evidence of de- 

 vitalization. In no case was any fixative employed. 



Histological methods. The following were used: hematoxylin 

 and eosin; the von Kossa method for phosphate in calcified tissues, 

 the sections being treated with thiosulfate before mounting; the 

 Gomori (1933) method for calcified tissues. 



Histoenzymologic methods. For the demonstration of en- 

 zymes, decalcified scapulae which had been implanted for 2 weeks 

 were frozen in isopentane, cooled by a dry ice-acetone mixture. 

 Sections 10 microns thick were cut on a cryostat, mounted on cover- 

 slips, and incubated at room temperature in the various substrates. 

 The following procedures were used: for cytochrome oxidase, that 

 of Burstone (1961a), using 8-hydroxy-l:4-naphthoquinone as a 

 coupling agent; Burstone's method (1961^) for acid phosphatase, 

 using naphthol AS-BI phosphate as substrate, and fast red violet LB 



