in Bone Produced GIRAUD
by Thyrocalcitonin” PH.D.,
M.D. and S. TUN-CHOT
has a direct action on bone. This conclusion is well supported by kinetic data [Y-4], in vitro bone culture studies [5-71, changes in urinary hydroxyproline excretion [S] and experimental evidence from perfusion of the isolated cat’s tibia . To determine the morphologic changes induced by the hormone, we have studied the bones of treated animals by both quantitative radiology and quantitative histology. We have found that in parathyroidectomized animals thyrocalcitonin (1) increases metaphyseal bone, (2) reduces the number of osteoclasts in the affected areas and (3) increases the proportion of partially mineralized and unmineralized osteoid to total trabecular bone volume.
[ 17 ] have reported that diaphyseal cortical bone is increased by thyrocalcitonin. The dose of hormone administered, however, was considerably in excess of that used in our investigations. With the dose of hormone we used, probable compensatory hypersecretion of parathyroid hormone induced by thyrocalcitonin prevented observable bone changes. For this reason subsequent investigations were carried out on parathyroidectomized animals.
STUDIES IN INTACT
STUDIES IN PARATHYROIDECTOMIZED
Male hooded rats weighing between 40 and 60 gm. were used. Parathyroidectomy was performed by hot wire cautery. Only animals with a plasma calcium level of 3.2 mEq. per L. or lower were selected for study. Two experiments were performed. In each, twelve rats were given thyrocalcitonin (partially purified by salt precipitation) and twelve rats were given solvent buffer. In experiment I the animals were given 40 MRC mu. four times daily, in experiment II 80 MRC mu. once daily. All animals were fed a standard cube diet. During the study, ten of the twenty-four animals treated and five of the twenty-four animals that were not treated died. On the twenty-eighth day the surviving rats were killed and their skeletons preserved. Prior to killing the animals, survey roentgenograms were taken. Marked differences were apparent between the caudal vertebrae of the animals that were treated and those that were not. Metaphyseal bone was markedly increased in the vertebrae of the rats that received thyrocalcitonin (Fig. 1). The changes produced by
The effect of thyrocalcitonin was first investigated in intact rats. Twenty-four male hooded rats, weighing between 40-60 gm., were used. Half the animals were treated with hormone purified by salt precipitation and dissolved in 0.1 M acetate buffer [IO]. A dose of 80 MRC milliunits (mu.) was administered subcutaneously four times daily. The remaining animals received a similar volume of buffer alone. On the twenty-eighth day the animals were killed, their skeletons dissected, roentgenograms obtained and scrutinized bone by bone. The features of the vertebrae, the long bones and small bones of the paws were studied in detail. No obvious gross differences were detected between control and treated animals. Since our original studies, other investigators
* From the Departments of Chemical Pathology and Diagnostic Radiology, Royal Postgraduate Medical School and Hammersmith Hospital, London, W.12., England and from the Centre du Mttabolisme Phosphocalcique (D. Hioco, Chef du Laboratoire), HBpital Lariboisitre, Paris X(e), France. This work was performed during the tenure of an Established Investigatorship of the American Heart Association (G.V.F.) and was supported in part by grants from the American Heart Association and the Medical Research Council. VOL.
Bone Changes Produced by Thyrocalcitonin-Foster
vertebrae were then scanned with a microdensitometer. This technic allowed expression of bone mineral in terms of millimeters of aluminium. In both experiments I and II the bone mineral in the proximal metaphysis of the treated animals was approximately 50 per cent greater than that in the control animals. No significant difference in mid-diaphyseal bone was found (Fig. 2). In addition to roentgenologic assessment, the increase in trabecular bone was confirmed by quantitative histologic technics. Longitudinal undecalcified sections were cut from the tenth caudal vertebrae of the animals in experiment II and stained with toluidine blue. Trabecular bone in the vertebrae of the treated animals
tenth and of the ninth, FIG. 1. Roentgenograms eleventh caudal vertebrae of a control rat from experiment I (lefl) and a treated rat (Gghl). A marked increase in trabecular bone adjacent to the growth cartilage is apparent in the vertebrae of the treated animal.
the hormone were similar to those observed in patients with osteopetrosis and occasionally in patients with primary or secondary hyperparathyroidism. To place these observations on a more quantitative basis the rat tails were excised and roentgenograms of them were obtained. An aluminium step-wedge served as a standard of radioopacity /72]. The films of the tenth caudal EXPERIMENT
LO MC. milliunils lhyracalcitonin four limes daily .7 _
Bone Mineral Expressed As mm.Al.
fi0 MRC. milliunils lhyrocalcitonin once daily
.5 0 10 Proximal Me;qs\y$s
FIG. 2. Bone mineral in the tenth of control and thyrocalcitonin-treated in millimeters of aluminium. In the ditions used, 1 mm. of aluminium is mg. per cmz. of bone ash.
caudal vertebrae rates is expressed experimental conequivalent to 155
FIG. 3. Longitudinal sections of the tenth caudal vertebra of a control rat (top) and a thyrocalcitonintreated animal ((lottom). ‘Irabecular bone central to the growth cartilages in the treated animal is markedly increased relative to the control.
was increased compared to that in the control animals (Fig. 3). By using an integrating eyepiece the percentage of trabecular bone relative to total bone volume was measured. In the areas assessed, trabecular bone was approximately twofold greater in the vertebrae of treated animals (Fig. 4). From these investigations we conclude that thyrocalcitonin affects bone in the absence of parathyroid hormone and may sustain its effect for prolonged periods. Thyrocalcitonin acts on bone by inhibiting resorption [7,2,4-61. To ascertain if the effect of the hormone was mediated by affecting boneresorbing cells, the osteoclasts and chondroAMERICAN
clasts were counted in histologic sections. Cell counts were confined to the region of the primary spongiosa. In both the proximal and distal metaphyses the number of resorbing cells in the vertebrae was less in treated animals than in control animals (Table I). Although no conclusions can be drawn about cellular activity, the fiindings indicate that thyrocalcitonin reduces the number of bone-resorbing cells. The work of other investigators supports this contention . The hormone has been shown to suppress the increase in osteoclasts observed in in vitro cultured bone stimulated by either vitamin A or parathyroid hormone. Reduction PROXIMAL METAPHVSIS
TABLE RESORBING l’ENI‘I1
Metaphysis Proximal Distal
Treated Rats 10.0 7.1
1 .l 1.0
Control Rats 27.0 20.0
f 3.1 ItI 0.9
* Cells per longitudinal section. .4n area distal to the growth cartilage was counted.
can be differentiated (Fig. 5). We have employed toluidine blue and Solachrome cyanine R for this purpose. The results of our findings in the primary spongiosa of the caudal vertebrae II are summarized in of rats in experiment Table II. In treated animals the total trabecular bone was 38.5 per cent of total bone volume compared to 26.2 per cent in control animals.
FIG. 4. Volume of trabecular bone expressed as a percentage of the total volume in longitudinal sections of the tenth caudal vertebrae of control and treated rats in experiment II. The end zones measure 500 p and comprise mainly primary spongiosa including some calcified cartilage. The intermediate zones each measure 1,000 p and comprise secondary spongiosa. Little or no trabecular bone is found in the intermediate zones. Values for control animals are indicated by clear bars, treated animals by solid bars.
in number of osteoclasts can therefore account for the increased trabecular bone found in the caudal vertebrae of treated animals. In our parathyroidectomized rats the number of osteoclasts was too low to be counted accurately. For this reason we could not conclude that there was an effect on bone formation rate. In further studies the changes in the components of trabecular bone induced by thyrocalcitonin were assessed. In the growing animal trabecular bone is composed of calcified cartilage, fully mineralized bone, partially mineralized osteoid, and unmineralized osteoid. Using special staining technics these components VOL.
FIG. 5. Histologic section of a single trabecula stained with toluidine blue to differentiate calcified cartilage (C.C.), fully mineralized osteoid (F.M.O.), partially mineralized osteoid (P.M.O.), and unmineralized osteoid (U.O.)
TABLE II COMPONENTS OF TRABECULAR BONE IN THE PRIMARY SPONGIOSAOF THE PROXIMAL METAPHYSISOF THE SEVENTH
of Total Bone
Components Total trabecular bone volume Mineralized cartilage Fully mineralized bone Partially mineralized and unmineralized osteoid
26.2 + 1.5 38.5 13.5 f 1.0 15.3 11 .O f 1 .O 18.0 1.7
i i f
2.2 1.5 1.2
Of the components of trabecular bone in treated animals, only mineralized cartilage was not significantly increased. The greatest increase was found in fully mineralized bone. A marked increase, however, was also found in partially mineralized and unmineralized osteoid. This latter finding is compatible with thyrocalcitonin having an effect on bone formation. COMMENTS
Two theoretical possibilities can explain how thyrocalcitonin increases trabecular bone. These are schematically represented in Figure 6. The components of trabecular bone are indicated by roman numerals. Mineralized cartilage and mineralized bone, which are affected by cellular resorbing activity, are indicated by cross hatching. Compared to control animals, there is an absolute increase in trabecular bone in thyrocalcitonin-treated animals. The first explanation for the increase is that the hormone exerts its effect solely by diminishing the rate of resorption of mineralized tissue. As this process appears to be mediated by the chondroclasts and KEY: 1.
Partiallv mineraked osteoid. Unmineralized osttoid
... lll. IV.
Inhibition of resorption alone
increased bone formation
100 Y Z 3 i
2 * 3*
0 CONlROL ANIMALS
FIG. 6. The two theoretical thyrocalcitonin could increase text for explanation.)
osteoclasts, one might expect the increase in trabecular bone to be due to an increase in mineralized cartilage and mineralized bone, the relative amounts of partially and unmineralized osteoid remaining unchanged. The is that thyrocalcitonin second explanation exerts its effect by both reducing the rate of resorption and increasing the rate of bone formation. Under these circumstances an increase in collagen deposition would also be found. The increase in partially and unmineralized osteoid, however, can be explained in one of three ways: (1) thyrocalcitonin decreases the rate of bone mineralization; (2) thyrocalcitonin increases the rate of bone formation; and (3) thyrocalcitonin interferes with the resorptive processes involved in unmineralized collagen turnover. At this time we have no evidence to indicate which of these three ways the hormone may act. In man an increase in unmineralized osteoid is found in osteomalacia and rickets, primary and secondary hyperparathyroidism, and thyrotoxicosis. Of these, rickets, osteomalacia and hyperparathyroidism are associated primary with low blood phosphate levels. Since thyrocalcitonin lowers plasma phosphate in parathyroidectomized animals  it is tempting to attribute failure to mineralize collagen tissue to insufficient available phosphate. Such a conclusion is tenuous since normal or high phosphate levels are usually found in hyperthyroidism and secondary hyperparathyroidism. It is equally possible to attribute the increase in unmineralized osteoid in primary and secondary hyperparathyroidism to a direct effect of thyrocalcitonin. These conditions are probably associated with a compensatory hypersecretion of the hormone. In short, no conclusion may be reached from correlations with clinical conditions associated with similar chemical and histologic findings. Recently, thyrocalcitonin has been reported to increase the number of osteoblasts in in u&o bone cultures . This finding suggests that the hormone stimulates bone formation. Such an increase would provide a reasonable explanation of our findings. SUMMARY
possibilities by which trabecular bone. (See
Our investigations indicate that thyrocalcitonin increases metaphyseal bone mineral and reduces the number of osteoclasts in the affected area. The increase in fully mineralized metaAMERICAN
physeal bone is associated with an increase in partially mineralized and unmineralized osteoid. Thyrocalcitonin unquestionably inhibits bone resorption. In addition it either increases the rate of bone formation, reduces the rate of bone mineralization or interferes with the resorptive phase of unmineralized collagen turnover. Our conclusions are based on studies in parathyroidectomized animals. Presumably similar changes could be induced in intact animals given larger and more frequent doses of hormone for longer periods. In the dosage used in intact animals, the effects of thyrocalcitonin were most likely obscured by a compensatory increase in parathyroid hormone secretion. REFERENCES
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5. FRIEDMAN,J. and RAISZ, L. G. Thyrocalcitonin: inhibitor of bone resorption in tissue culture. S&me, 150: 1465, 1965. 6. ALIAPOULIOS,M. A., GOLDHABER,P. and MUNSON, P. L. Thyrocalcitonin inhibition of bone resorption induced by parathyroid hormone in tissue culture. Science, 151: 330, 1966. 7. GAILLARD, P. J. Bone culture studies. J. Bone & Joint Surg., 48B: 386, 1966. 8. MARTIN, T. J., ROBINSON,C. J. and MACINTYRE, I. The mode of action of thyrocalcitonin. Latmt, 1: 900, 1966. 9. MACINTYRE, I. and PARSONS, J. A. Blood-bone calcium equilibrium in the perfused cat tibia and the effect of thyroid calcitonin. J. Physiol., 183: 3IP, 1965. IO. BAGDIANTZ, A., FOSTER, G. V., EDWARDS, A., KUMAR, M. A., SLACK, E., SOLIMAN, H. A. and MACINTYRE, I. Extraction and purification of calcitonin. Nature,203: 1027, 1964. II. WASE, A. W., SOLEWSKI,J., RICKES, E. and SEIDENBERG, J. Action of thyrocalcitonin on bone. Nature, 214: 388, 1967. 12. FOSTER, G. V., DOYLE, F. H., BORDIER, P. and MATRAJT, H. Effect of thyrocalcitonin on bone. Lancet, 2: 1428, 1966. 13. REYNOLDS, J. J. Inhibition by thyrocalcitonin of bone resorption induced in vitro by vitamin A. Proc. Roy. Sot. London s.B., in press. 14. GUDMUNDSSON, T. V., MACINTYRE, I. and SOLIMAN, H. A. Isolation of thyrocalcitonin and a study of its effects in the rat. Proc. Roy. Sot. London s.B., 164: 460, 1966. 15. GAILLARD, P. J. Bone culture studies with thyrocalcitonin. Koninkl. Nederl. Akad. Wetenschap. (s.C), 70: 309, 1967.