570 G. NICHOLS, JR. 



three-phase model appeared to be vahd, and further evidence in 

 favor of an acid mechanism for the mobihzation of Ca from the 

 skeleton was provided. 



The foregoing review of our experiments with regard to bone 

 mineral resorption has perhaps indicated the lines which our think- 

 ing has followed and some of the difficulties we have encountered. 

 Obviously more questions have been raised than answered, and 

 there is patently much more to be done. It would seem, however, 

 that such in vitro model systems not only are feasible but are prob- 

 ably sufficiently good prototypes to offer by analogy possible ex- 

 planations of in vivo phenomena. Especially encouraging is the 

 evidence suggesting that the bone fragment in vitro functions as a 

 three-phase system, indicating that it may be an even better model 

 of the situation in vivo than we had hoped. 



Though the majority of our work so far has been concerned with 

 the resorption of inorganic material, experiments have been begun 

 to study resorption of the organic phase. 



The first of these grew from evidence recently obtained by Flana- 

 gan and others that C^^-labeled proline from the incubation medium 

 is incorporated into the stable collagen of fresh bone fragments dur- 

 ing incubation (Flanagan and Nichols, 1962; Deiss et ah, 1962). 

 This observation suggested a way of examining at a chemical level 

 the notion advanced from histologic and physiologic observations 

 (Lacroix, 1956; Jowsev, I960; Talmage et al., 1959) that bone re- 

 sorption occurs at sites microscopically remote from areas of new 

 bone formation, and at the same time possibly developing a method 

 for studying collagen turnover time in bone. Since proline label in- 

 troduced during in vitro incubation in labeled medium would be 

 incorporated into the stable collagen only at sites of new bone for- 

 mation, further incubation of such samples in medium containing 

 only unlabeled proline, with examination of the collagen labeling 

 of samples removed at various times thereafter, should indicate the 

 biological life of the collagen deposited during the initial labeling. 



Woods, who is carrying on these studies, has obtained the results 

 shown in Fig. 9, in two t\'pical experiments using metaphyseal bone 

 from 50-day-old rats. The closed circles show the counts found in 



