12] tinuoug | iterative activity of chondrocytes (percent labeled proliferative zone cells)—as well as to the inability Studies fradiat, § 1., and blood- ot the tissue to clacify and undergo bony replacement. However, the autoradiographic studies with 7HTdR -.;] to delineate any specific cellular mechanism TABLE 56. Group Division or Rats py Group “ich, in the absence of deficient calcification, might . plain why rickets occurs. With time, the proliferative 1 Purina Chow, ad lub. Purina Chow, pair-fed with cer 40, # the competent cells to synthesize DNA seems normal. 3 Rachitogenic diet fortified with phosphate and vitamin D: ad lib. Rachitogenic diet, 21 days, ad lib. Rachitogenic diet (14 days) and phosphate (7-10 days) Rachitogenic diet (14 days) and vitamin D,» (7-10 days) Rachitogenic diet (14 days) and phosphate plus vitamin D, (7-10 days) posure, fF vupacity of chondrocytes decreases, but the ability of ogy of ntane. § 340), Rachitogenic diets enriched with phosphate were more effective than vitamin D alonein reversing the even- ). Thee tual decline in proliferative potential of rachitic carti- iduced E 7, 269-5 ity of & ! jae. The present autoradiographic investigation was wanlertaken to determine whether the abnormalities vtserved during the development of rickets in rats reflected an altered ability of cartilage and bone cells 4 and§ tu produce the extracellular collagenous components itional F vision F 4 5 6 7 | of their respective matrices. We have used *H-pro- 2 heta . line as a tracer based upon evidence that it is a selec- tive marker for collagen formation.{-) The inBone triecllular pathway involving the Golgi apparatus F ty which protocollagen is synthesized in chondrocytes '4< been established by electron microscopic auto- idiography.4% 1 The export of radioproline from ‘hondroeytes and osteoblasts and its incorporation into collagen with time has been effectively demon- ‘ular I lism § gly- § ritic fk ysis f die- F ~trated by light microscopic autoradiography ,“@! and by experiments conducted in vitro.¢% This study Iso permitted a further investigation of the capac- itv of dietary supplements of phosphate and/or vitanun Des to heal rickets, and the opportunity to ‘valuate the results from several other labora- ones 17 which suggest that collagen formation is accelerated in rickets. tter § MATERIALS AND METHODS ita- § tal to that employed previously by Kunin and his coworkers. 7 8) Rickets was produced in 6 male weanling Sprague-Dawley rats (40-50 g) utilizing a high caleium (1.2%), low phosphate (0.1%), vitamin ')-free diet™®) for 21-23 days. For comparison, litterinates were fed either Purina Laboratory Chow (cal‘ium, 1.42%; phosphorus, 0.96%) ad kbitum, or pair- In ; rity F age FE ely ate arral § The experimental protocol was essentially identi- ied with this commercial preparation in amounts consumed daily by the rats on the rachitogenic diet. ‘\ third group was sustained on the basal rachitogenic ‘het which was supplemented with NaH»PQ, (calcium to phosphorus ratio = 1.4:1), and vitamin De ‘10 IU. per gram diet) ad libitum. Free access to de- “nized distilled water was permitted, and the colony «a8 maintained under shielded incandescent lighting ‘l constant temperature rooms at 22°C. In order to Numberof rats . . Dietary regimen group 4 sacrificed at 21 days 23 days 3 3 3 3 3 3 3 3 3 3 3 3 3 3 - study the ability of inorganic phosphate and vitamin D. to heal rickets, 3 groups of 6 rats each were raised on the basal rachitogenic ration for 14 days. Subsequently, these animals were maintained for an additional 7-10 days on diets which were selectively enriched with either NaHsPO,, vitamin De, or both, as outlined above. The division of rats by group is listed in Table 56. On the 21st day of the experiment, all the animals were injected intraperitoneally with 2.0 »Ci *H-pro- line* per gram body weight in 0.1-0.3 ml 0.01 N HCl. Three rats from each group were sacrificed by cervical dislocation at 4 hr and 3 days after isotope injection. These time periods were chosen so that both the initial pattern of isotope uptake by chondrocytes and bone cells could be followed, as well as the subsequent rates of endochondral ossification and for- mation of new bone by osteoblasts. The tibias re- moved from the rats at autopsy were fixed in 10% neutral formalin, decalcified in 10% EDTA (pH 7.47.6), embedded in paraffin and sectioned with the bones oriented in their long axes on a rotary microtome at 4 «. Slides bearing deparaffinized sections were autoradiographed by dipping in liquid Kodak NTB-2 emulsion. After a 9-week exposure period in a freezer, the slides were developed for 5 min in Kodak D-19 {20° C), fixed in acid fixer, washed thoroughly, and stained through the emulsion with hematoxylin and eosin. Autoradiographic Studies The pattern of silver grains developed in the emul- sion was examined to determine which cell types in * L-proline-3,4-"H, New England Nuclear Corporation, Lot No. 343-207, Specific Activity 5.86 Ci/mM.