57^2 G. NICHOLS, JR. 



and ability to incorporate further label if transferred back to radio- 

 acti\e niedimn. There are, howe\'er, pitfalls in this approach. In 

 another experiment antibiotic was omitted by error from the me- 

 diinn, which soon developed a healthy growth of bacteria — ap- 

 parently an organism capable of breaking down collagen, since 

 radioactivity was rapidly lost and was almost totally gone by 48 

 hours. 



Although no true bone resorption has been demonstrated in these 

 preliminar)- experiments, the results offer strong corroboration to 

 the view that bone resorption occurs at sites remote from bone for- 

 mation, and that most bone collagen once laid down has a biological 

 life of considerable duration. Moreo\ er, it would appear likelv that 

 the cells at sites of new collagen formation are not displaced and 

 continue synthetic acti\it\ during prolonged experiments in vitro; 

 certainh' the\' do not begin to resorb the newlv formed bone during 

 this time. The contaminated experiment further supports this ^•iew 

 of the integrit) of the synthetic sites, since when collagenoK tic bac- 

 teria are present, the newly incorporated label is rapidly removed. 



Finally, Woods and I have recentlv begun to look for exidence 

 of the collagenoK tic acti\'ity which must be present somewhere in 

 bone. For these experiments we ha\'e used a system of macerated 

 fresh bone incubated at 37°C under N- in phosphate buffers ranging 

 in pll from 5.5 to 8. Tn this SNstem the cells are intact but free swim- 

 ming rather than bound in their normal anatomical sites. Since 

 known collagenases break up collagen into small peptides rather 

 than individual amino acids (Gallop ct al., 1957), we have used an 

 increase in total ultrafiltrable hvdrox\proline following incubation 

 as an index of "collagenase-like" acti\it\'. So far only a few qualita- 

 tive results ha\e been obtained. Howe\er, ultrafiltrable peptide 

 hydroxyproline can be shown to increase after 4 hours of incuba- 

 tion in this SN'stem, a phenomenon which is completelv abolished 

 by previous heating of the s\stem to 70 °C for 5 minutes. The pH 

 optimum of this acti\'itv is still in doubt, although it seems at present 

 to lie between pH 6 and 7. Though onlv of the most preliminar\- 

 sort, these few obserxations and Gross's elegant experiments ( Gross 

 and Lapiere, 1962), which he will describe later in this s\niposium 

 ( chapter 26 ) , encourage us to believe that soon much more evidence 



