EFFECT OF STRONTIUM ON THE EPIPHYSEAL CARTILAGE PLATE OF RAT TIBIAE-HISTOLOGICAL

AND RADIOGRAPHIC STUDIES

Akira MATSUMOTO Department ()J'Pharmacology, .SchoolofDental.t fedicinc, Tsurnnn University, Tsurumi-ku, Yokohama 30, Japan

Accepted July 12, 1976

Abstract-Following dietary administration of strontium carbonate, histological and radiographic changes in the epiphyseal cartilage plate of the rat tibiae were examined in the present study. The weight gain of the rat fed a strontium diet was less than that of the control rats. Longitudinal growth of tibiae and endochondral ossification were inhibited by strontium administration. The widths of both proximal and distal cartilage plates increased enormously as has also been shown by other investigators. Sizes of chondroblasts in columns of proximal cartilage plate in rats fed a strontium diet were smaller than those of the control rats and were not different between upper and lower parts. It is suggested that strontium inhibits bone growth through the inhibitory action on the maturation process of chondroblasts and the succeeding endochondral ossification.

Oral administration of strontium salts to animals induces "strontium rickets" (1, 2, 3). There have been many reports on the calcification lesions in the epiphyseal cartilage plate induced by administration of dietary stable strontium to animals (1, 3, 4, 5, 6). These calcification lesions have been postulated to be due to the adverse effects of strontium on intestinal absorption mechanisms (7). In the present study, effect of dietary administration of strontium carbonate on the endochondral ossification in the epiphyseal cartilage plate of rat tibiae was examined by histological and radiographic methods to obtain basic data on the calcification lesions induced by this element. Cell size was also measured in order to determine the effect of strontium

on proliferating cartilage cells or flattened cells in columns of the epiphyseal cartilage plate.

MATERIALS AND METHODS Ninety male rats of Wistar strain, 4weeks old with an initial body weight of 50 to 60g
were divided into three equal groups of 30. Each group was provided anormal well balanced diet (control group), a low Ca diet (low Ca group) or astrontium diet (Sr group) respectively, and given deionized water ad libitunr for 4 weeks (8). Calcium content in the diets was 0.67 , in the control group and 0.04 in both low Ca and Sr groups; strontium content, 1.50°~ in the Sr group; phosphorus content, 0.64 in all groups. During the experimental period, the body weight of the animals was recorded in order to observe the effect of oral administration of strontium carbonate on the growth of the animals. The rats were decapitated under ether anaesthesia 4 weeks following the strontium

### administration. Both left and right tibiae were dissected out immediately and adherent

tissues were carefully removed. Tissues from 24 rats in each group were fixed in 10 neutral formalin and then radiographed using a Softex EMB type apparatus (Softex Co., Ltd., Tokyo). The length of the tibiae and the width of proximal and distal epiphyseal cartilage plates wererecorded on theradiographs. For histological examination, the right tibiae obtained from 24rats ineach group were decalcified in IV/ EDTA solution (pH 7.2) for 3 weeks, embedded in paraffin, sectioned midsagittally at 6,m1byusing aslidingmicrotome and stained with haematoxylin and eosin. The degree of endochondral ossification was examined microradiographically; here, the proximal heads oflefttibiae from 6rats ineach group wereused. After the fixation in 10% formalin, these tissues were dehydrated in ethanol, embedded in Rigolac (Riken Polyester Resin Co., Ltd., Tokyo) and polymerized (9). Midsagittal ground sections of 50heminthickness wereprepared. Contact microradiograins of these sections weretaken (75KV and 2mA ofcurrent intensity) byusing aSoftex CSM type apparatus (Softex Co., Ltd., Tokyo). Eastman Kodak 649-0 spectroscopic filmwas used and the exposure time
was 15min. Thefilmswere developed for 3min at 20°CinMicrofine (Fuji Photo Film Co., Ltd., Tokyo), fixedfor 10minat 20'C inFuji fix(Fuji Photo Film Co., Ltd., Tokyo), washed inrunning tap water for 30min, dried and mounted. Linear distance between the proximal and distal ends along the central axis ofthe sagittal plane ofa tibia was measured on the radiographs. Widths ofboth proximal and distal epiphyseal cartilage plates weremeasured along thecentral axis ofthe sagittal plane of the tibia. To estimate the size of the cells in the zone ofproliferating cartilage cells, one good section from each of4 rats in each group was selected. Photomicrographs ofthe central part of the epiphyseal cartilage plate were taken at an original magnification of 200. The
zone ofproliferating cartilage cells, that is,chondroblasts, wasdefined at the region where cartilage cells multiply and form columns offlattened cells (10). Since thenumber ofthese flattened cellsinacolumn wasfound to beapproximately 26inthepreliminary observations,
a column was arbitrarily divided into two parts, that is, upper (from the proximal end to the 13thcell)and lower (therest ofthecells)parts. Fifteen cellsineachpart ineach section
were randomly chosen on the printed pictures whose final magnification was 1700. The cellswere cut out and weighed. Assuming that the thickness and density ofthe paper were uniform and, therefore, the weight ofthe paper was proportional to the area, the weight of thecutout ofeach cellwasconverted to the area.

RESULTS Changes inthebodyweight Asseen in Fig. 1,average body weights in allgroups ofrats showed gradual increases during the experimental period but the weight gain was somewhat different among the groups. None of the rats died during the experimental period. In the group ofrats fed
alow Ca diet (low Cagroup), the average body weights were not significantly different from those of the control group until the 5th day ofthe experiment. However, they were sigFIG. 1. Daily changes in the body weights in groups of rats

. • ® control,

,. x low Ca,

_ Sr groups. Each point represents the average value in each group. Vertical bars represent the standard deviations

.

Fio. 2. Upper, middle and lower pictures represent the left tibiae from control, low Ca and Sr rats respectively. Note the differences in the lengths of the tibiae, and in the degrees of calcification among the groups. Arrows indicate the proximal cartilage plates. Magnification x 1.3.nificantly greater from the 6th to the 22nd day (p<0.05-0.01). The differences were not

significant again from the 23rd to the 28th day. The weight gain in the group of rats fed a strontium diet (Sr group) was least among the three groups of animals throughout the ex perimental period. From the 3rd day of the experiment, the average body weight began to show significant decreases from the control values (p<0.05-0.001). Radiographic findings As seen in Fig. 2, calcification of the rat tibiae was poor in the group of rats fed a low calcium diet. In the group of rats fed a strontium diet, it was found that both the longi tudinal growth ofthe tibiae and the calcification were inhibited. One of the most remarkable changes due to strontium administration was the marked increase of the width of the epi

TABLE 1. Changes in the length of the tibiae and in the width of proximal and distal epiphyseal cartilage plates

Mean-istandard deviation of six left tibiae in each group are shown. * Significant difference from the control value (p<0 .001).

FIG. 3. Microradiographs of the proximal heads of rat tibiae.

a: Control group. Magnification x 18. b: Low Ca group. Trabecular bones are thinner as compared with the control specimen. Magnification x 18.

c: Sr group. Mineralization was not observed at the site of the wide epiphyseal cartilage plate. Magnification x 18.physeal cartilage plate.

Table 1 shows that tibial length and the widths of proximal and distal cartilage plates

were significantly different between the control and Sr groups (p<0.001). But no significant differences of the lengths of the tibiae and the widths ofcartilage plates were found between low Ca and the control groups. The differences were significant between low Ca and Sr groups (p<0.001). Microraddlograp/i cfindings Fig. 3a-c shows microradiographs at the proximal heads of rat tibiae in the control, low

FIG. 4. Photomicrographs of the proximal heads of rat tibiae.

a: Chondroblasts in control group. Haematoxylin and eosin. Magnification 400. b: Chondroblasts in low Ca group. No appreciable histological changes were found. Haematoxylin and eosin. Magnification x400.

c: Chondroblasts in Sr group. Note the irregular arrangement of cells in the columns and small size of the cells as compared with the control specimen. Haematoxylin and eosin. Magnification 400. d: The zone of provisional calcification in control group. Lacunae of the hyper trophic cartilage cells are opened at the end of the columns. Haematoxylin and eosin. Magnification x 400.

e: The zone of provisional calcification in low Ca group. Opening of the lacunae of the hypertrophic cartilage cells are also seen at the end of the columns. A number of hypertrophied endosteal cells are visible around the trabecular bones. Haematoxylin and eosin. Magnification x400. f: The zone of provisional calcification in Sr group. Note that lacunae of the hypertrophic cartilage cells are not opened, that some cells are extremely flattened at the end of the columns and that osteoid tissue rather than tra becular bone is evident. Haematoxylin and eosin. Magnification x400.Ca and Sr groups, respectively. In the low Ca group, the trabecular bones were thinner

than those in the control group. In the Sr group, mineralization was not observed at the site of the wide epiphyseal cartilage plate (Fig. 3c). Histological findings Fig. 4a-f shows histological appearances of the flattened cells inthe columns at the proxi mal head ofthe rat tibiae inthe control, low Ca and Srgroups, respectively. No remarkable histological changes were found in the low Ca group as compared with the control group. In the Sr group, the arrangement of the columns seemed to be in disorder and the size of the cells appeared to be small. At the zones of provisional calcification, lacunae of the hypertrophic cartilage cells were opened at the end of the columns in the control specimen (Fig. 4d). Opening of the lacunae of the hypertrophic cartilage cells was also seen at the end of the column in the low Ca group, and a number ofhypertrophied endosteal cells which may remove the trabecular bone as has been reported by other investigators (11, 12) could be seen around the trabecular bones (Fig. 4e). In the Sr group, no such opening of the lacunae was observed; some of the hypertrophic cartilage cells are extremely flattened at the end of the columns and osteoid tissue was observed instead of trabecular bone (Fig. 4f).
Cell size Table 2 shows the average areas of the flattened cells in columns of the proximal epi physeal cartilage plate in the rat tibiae. In both upper and lower parts of the columns, the average sizes ofthe cells in the low Ca group were not significantly diffrent from those of the control group, but those inSr group were only inthe lower part significantly smaller than those in the control group (p<0.001). The average sizes ofthe cells in the lower part were signifi cantly larger than those in the upper parts in both control and low Ca group (p<0.01), but

no significant difference was found between lower and upper parts in the Sr group.

DISCUSSION Storey reported that the weight gain in Sr group was less than that in the control animals (6). Similar results were obtained in the present experiment. It is apparent that

a dietary administration of strontium carbonate results in a retardation of growth in young animals. TABLE 2. Areas of the flattened cells in columns of the proximal epiphyseal cartilage plate in the rat tibia

Mean standard deviation of the means in each group are shown. * Significant difference from the control value (p<'0 .001). + Significant difference between the upper and lower parts (p<0.01).

### Harrison and Fraser (13) reported that rats on a low Ca diet with complete vitamin

supplements failed to grow. In the present experiment, however, the weight gain of the rats in the low Ca group was similar to, or greater than, that in the control group. This discrepancy would bedue to the difference ofthecompositions ofthe diets; milk casein was not included in their diet. In the present experiment, the rats in the low Ca group ap parently ingested more food totake up thesmall amount ofcalcium contained inmilk casein in the diet and thus attained such a growth curve (Fig. 1). Longitudinal growth of tibiae and endochondral ossification are apparently inhibited by strontium administration (7, 14). However, the zone ofhypertrophic cartilage plate in Srgroup became wider (Figs. 3c and 4c) as has already been shown byother investigators (1,4,5,6,15, 16). Theaverage sizeofcellsincolumns ofproximal cartilage plate inSrgroup was smallerthan seenin lowCa and control groups. Inaddition, cellsizesinSrgroup were not different between upper and lowerparts (Table 2). These results suggest that inhibition ofthe maturation process occurred in the chondroblasts inthe Sr group. It is also possible that strontium interfered with calcium metabolism locally or sys temically, and such metabolism isconsidered toplay an important roleincellfunction (2, 17, 18, 19). Further studies are under way to determine the process(es) initially inhibited after ingestion ofstrontium carbonate. Acknowledgements: Thanks are due to Prof. M. Chiba for pertinent suggestions and help in preparing the manuscript and alsoto Mr. K. Ogiwara for technical assistance.

REFERENCES 1) LEHNERDT,

F.: Beitr. path. Anat. 46,468 (1909) 2) SOBEL,

A. E., GOLDFARB,

A.R. AND

KRAMMER,

B.: Proc. Soc. exp. Biol. Med. 31, 869 (1933) 3) FOLLIS,R.H.: FednProc. 14,403 (1955) 4) LEHNERDT,

F.: Beitr. path. Anat. 47,215 (1910) 5) SHIPLEY,P.G., PARK,E.A., MCCOLLUM, E.V., SIMMONDS,

N. ANDKINNEY,E.M.: Johns Hopkins Hosp. Bull. 33,216 (1922) 6) STOREY,

E.: Aust. Ann. Med. 10,213 (1961) 7) CORRADINO,

R.A., EBEL,.1.G., CRAIG,P.H., TAYLOR, A.N. ANDWASSERMAN, R.H.: Calcif. Tiss.Res. 7, 81 (1971) 8) MATSUMOTO,

A.: Tsurumi Univ.dent. J. 1,21 (1975) 9) SUGA,S.: Shikai Tenbo27, 495 (1966) (inJapanese) 10) MAXIMOW,

A.A. AND

BLOOM, W.: A Text Book ofHistology, 7th ed., p. 126, Saunders Co
., Philadelphia and London (1957) 11) OKUDOH,

M.: Bull. TokyoMed. Dent. Univ.20, 131 (1973) 12) SALOMON,

C.D.: Calcif. Tiss. Res. 8, 320 (1972) 13) HARRISON,

M. AND FRASER,

R.: J. Endocr. 21, 197 (1960) 14) CORRADINO,

R.A., EBEL,J.G., CRAIG,P.H., TAYLOR, A.N. ANDWASSERMAN, R.H .: Calcif Tiss.Res. 7, 93(1971) 15) STOREY,

E.: J. BoneJt. Surg. 47B, 145 (1965) 16) ENGFELDT,

B., HJERTQUIST,

S. AND LARGERGREN,

C.: Acta Soc. Med. Uppsal. 67, 239 (1962) 17) SOBEL,

A.E.: Ann. N. Y.Acad. Sci. 60, 713 (1954) 18) DRINKER,

N. AND ZINSSER,

H.H.: J. biol. Chem. 148, 181 (1943) 19) DEAMER,

D.W. ANDCORNWELL, D.G.: Biochim. Biophys. Acta 116, 555 (1966)