IN VITRO STUDIES OF BONE RESORPTIVE MECHANISMS 567 



that it is, in fact, at least a three-phase system consisting of mineral- 

 ized fibers covered by a very thin layer of fluid which is separated 

 from the incubation medium by a layer of metabolicalh' active liv- 

 ing cells embedded in a stroma of collagen and polysaccharide 

 ground substance, as illustrated in Fig. 7. 



This view, which is consistent with the histology of the tissue, 

 points out possible ways to explain such observations as the dis- 

 crepanc)' between the accumulation of H+ ion and the accumulation 

 of organic anions in the medium. As previously noted (Fig. 1), only 

 1 equivalent of H+ ion (measured by the manometric technique) 

 was released into the medium for every 3 equivalents of lactate plus 

 citrate. In the three-phase model the retained protons could be 

 either retained in the layer of fluid bathing the mineralized fibers, 

 thereby maintaining the pH of the layer at a lower level than the 

 inculcation medium, or taken up by the hydroxyapatite crystals. 

 Moreover, the possibility was suggested that the ratio of protons 

 and other ions retained in the sample to those released into the 

 medium might l)e modified by changes in cell metabolism or the per- 

 meability of the cell layer, which might change under the influence 

 of some of the factors studied ( Borle, Nichols, and Karnovsky, 1960 ) , 

 Thvis the three-phase model suggested possible mechanisms for the 

 observed modification of l)one mineral solubility, on the one hand, 

 and for the influence of cell metabolism on the steady-state medium 

 Ca concentration on the other, while pointing to possible reasons 

 for the failure of organic anion concentration in the medium to cor- 

 relate with medium Ca and P concentrations. 



With these views in mind, Schartum determined to seek evidence 

 that the pH at the mineral surface was kept at a lower level than 

 the inculcation medium bv living cells and that the presence of this 

 H+ ion gradient was responsible for the cell-dependent fraction of 

 the total Ca concentration in the incubation medium (Schartum 

 and Nichols, 1962). His argument was simple: If inactiyating the 

 cells of a bone sample abolished such a H+ ion gradient and thus 

 lowered the steady-state Ca concentration in the medium, a similar 

 effect should be observed if the gradient were abolished by another 

 means in incubations using samples with actively metabolizing cells. 

 Previous experiments ( Borle et al., unpublished data ) had indicated 



