The effect of vitamin D on the dentine of the incisor teeth and on the alveolar bone of young rats maintained on diets deficient in calcium or phosphorus

Arch.

oral Bid.

Vo1.9,

pp.447-460,

1964. Pergamon Press Ltd. Printed in Ot. Britain.

THE EFFECT OF VITAMIN D ON THE DENTINE OF THE INCISOR TEETH AND ON THE ALVEOLAR BONE OF YOUNG RATS MAINTAINED ON DIETS DEFICIENT IN CALCIUM OR PHOSPHORUS H. W. FERGUSONand R. L. HARTLES School of Dental Surgery, University of Liverpool, England Summary-Sixty month-old rats from our own colony were divided into six groups, one of which was a control group. For 10 weeks the remaining groups received diets deficient in vitamin D; calcium; calcium and vitamin D; phosphorus; and phosphorus and vitamin D. The animals were killed and their incisor teeth examined. Simple deficiency of vitamin D caused only very slight changes in the incisors. The greatest change was caused by diets deficient in calcium and vitamin D. Simple deficiency of calcium delayed mineralization of the apical portion of the dentine but the incisal part of the dentine was relatively little disturbed. Diets low in phosphorus even in the absence of vitamin D produced incisors which contained more mineral than the teeth formed on the diet deficient in calcium and vitamin D. This is in contrast to the situation found in bone. In general, deficiency of calcium or phosphorus had a lesser effect on the formation of the incisor tooth than it had on bone. The only exception was in the case of deficiency of calcium and vitamin D, when the apical portion of the tooth suffered a disturbance comparable with that of bone. Alveolar bone did not enjoy any priority for available mineral and reacted to deficiency states in a manner similar to that of the long bones. The implications of these results are discussed in relation to possible differences in the mineralization processes in teeth and bone. INTRODUCTION

IN A previous publication (FERGUSONand HARTLES,1963) it was shown that certain well defined changes occur in the long bones of young rats deprived of calcium or phosphorus in the presence and absence of vitamin D. It was found that a greater disturbance in the formation of the rat’s bone is caused by dietary deficiency of phosphorus than by lack of calcium. It seemed logical therefore to investigate the changes occurring in the forming incisor dentine and alveolar bone in animals maintained in similar experimental circumstances. MELLANBY(1923) showed that the changes in the teeth of dogs maintained on diets deficient in calcium were less severe than when “vitamin D” was excluded from the diet. In the rat, BECKYand RYDER(1931) reported that a rachitogenic diet with a Ca/P ratio of 4 causes histological changes in the predentine and dentine of the incisor teeth. On the other hand it has been claimed that a rachitogenic diet with a Ca/P ratio of 7.27 does not lower the ash content of the rat incisor, and that histologically the structure of the teeth is almost normal (KARSHAN, 1930, 1931, 1933; KARSHAN and ROSEBURY, 1932, 1933; ROSEBURY and KARSHAN,1931).

448

H. W. FERGUSON ANDR. L. HARTLES

GAUNT and IRVING (1940) in reassessing these somewhat conflicting results stressed the importance of the Ca/P ratio as well as the absolute mineral intake. They found in the rat, that when the intake of calcium and phosphorus is inadequate the consumption of a diet with a high Ca/P ratio has a greater effect on the bones than one with a low Ca/P ratio, whereas in the teeth the converse is the case. IRVING (1944) also showed that when vitamin D is given to rats with low-phosphorus rickets the newly formed pre-dentine becomes properly calcified whilst the existing dentine remains unchanged and poorly calcified. Some investigators, however, do not consider a lack of vitamin D to be essential in order to produce changes in the dentine. COLEMANet al. (1953) reported severe disturbances in the teeth and bones of rats maintained on diets deficient in phosphorus but containing adequate amounts of calcium and vitamin D. WEINMANNand SCHOUR(1945a, 1945b) and COLEMANet al. (1953) observed that the alveolar bone of rats receiving rachitogenic diets remains poorly calcified with the persistence of wide osteoid seams. On the other hand SOGNNAES(1961) in reviewing the dental aspects of mineral metabolism expresses the opinion that both the teeth and alveolar bone enjoy a certain priority in regard to the availability of minerals in various deficiency states. There is thus some conflict of evidence concerning the effects of differing deficiencies of calcium, phosphorus and vitamin D on the developing tooth and alveolar bone. We have maintained weanling rats on defined diets, low in calcium or phosphorus, both with and without added vitamin D. In all the experimental groups changes of varying degree and kind were produced in the incisor teeth and alveolar bone. These changes are described and discussed in relation to the changes in the long bones previously reported. EXPERIMENTAL Animals

Sixty weanling black and white hooded rats from our own colony were distributed between six groups. The animals were 21-24 days old and had been born to dams receiving a stock laboratory diet (Lever No. 4). Experimental diets

The several diets were those used by FERGUSONand HARTLES(1963). Full details of their preparation are given by HARTLESand LEAVER(1961a, b; 1962). The main components of all the diets were sucrose, egg albumin, and ground nut oil, with appropriate salt and vitamin supplements. The diets fed to the different groups were as follows. Group 1. Ten animals. Control diet HS7 ; contained O-56% Ca, 0.42 ‘J!,P, and 200 pg ergocalciferol per 960g diet. Group 2. Ten animals. Diet HS9; contained no added ergocalciferol, otherwise as HS7. Group 3. Ten animals. Diet HS8; contained O-026% Ca, 0.45 % P, otherwise as HS7.

EFFECT OF VITAMIN D, Ca AND P ON RAT TEETH AND BONE

Group 4. Ten animals.

449

Diet HSlO; contained no added ergocalciferol, otherwise

as HS8. Group 5. Ten animals.

Diet HS13; contained 0.6% Ca, 0.06 % P, otherwise as

HS7. Group 6. Ten animals.

Diet HS14; contained no added ergocalciferol, otherwise

as HS13. The main features of diets were as follows: Group 1 2 3 4 5 6

Diet HS7 HS9 HS8 HSlO HS13 HS14

Ca Normal Normal Deficient Deficient Normal Normal

P Normal Normal Normal Normal Deficient Deficient

Vitamin D Normal Deficient Normal Deficient Normal Deficient

Housing

The animals were housed in screen-bottomed wire cages without access to direct sunlight and at a temperature of 21-22°C. The rats were given the diets and deionized water in unrestricted amounts. Duration of experiments

Eight animals from each group were killed by ether inhalation after 10 weeks, the remaining two after 14 weeks on the diet. Preparation of material

Immediately after killing the mandibles and maxillae were carefully removed from the skulls. One half of the material was used for the preparation of decalcified sections, the other half for the examination of undecalcified sections. Decalcified specimens

After dissection the material was placed immediately in 10% form01 saline for a minimum period of 3 weeks. Decalcification was then carried out in 5 % (v/v) aqueous formic acid and the progress of demineralization checked radiographically. Subsequent preparation was by a double-embedding technique. Serial sections were cut on a Spencer rotary microtome and stained with Ehrlich’s haematoxylin and eosin. UndecalciJied specimens

The incisor teeth with the alveolar bone attached were removed from the jaws. The tissues were placed in 80% ethanol for a minimum period of 24 hr and then dehydrated with acetone. They were then placed in a polymerizing polyester resin (CEMAR;Cromwell & Co.). When the blocks were hard, 100 TVsections were cut on a machine incorporating a 4 in. rotating diamond-impregnated steel cutting disc. The sections were placed in close contact with Kodak MR plates and radiographed with a Raymax 60 X-ray machine, using 30kV, 15 mA for 17 min at a target distance of 20 cm. It should be emphasized that in order to obtain adequate differentiation in

450

H. w.

hXCHJSON

AND

R. L.HARTLFS

those samples containing enamel and dentine, the photographic negatives were often over exposed for one tissue or the other. If equivalent exposures had been given the demarcation between enamel and dentine would have been obliterated. This is a justifiable procedure in this instance since a quantitative radiographic assessment was not intended. Determination of ash content of incisor teeth

A further thirty weanling rats were distributed between six groups as before and received the respective diets for 10 weeks. They were killed and the incisors carefully removed from the jaws. A preliminary attempt to separate the enamel and dentine by flotation in a bromoform acetone mixture of defined specific gravity was unsuccessful. A fluid of a specific gravity adequate for separation of the two hard tissues in the normal incisor as used by HARTLES (1951) proved useless in separating the tissues of the incisor formed on deficient diets. It was felt that much work would be necessary to determine the exact nature of the fractions being separated before this technique could be applied. In the circumstances it was considered better to obtain ash values from portions of the intact tooth. The teeth were cut transversely into two equal portions, and the apical and incisal halves so obtained were treated independently. The tooth portions were extracted with boiling ethanol in a soxhlet continuous extraction apparatus for 16 hr and dried at 105°C for 1 hr. The dry fat-free tooth samples were weighed and ashed at 800°C to constant weight. RESULTS Growth and general condition of animals

Observations on the growth and condition of the animals on the different diets have already been reported (FERGUSON and HARTLES 1963). Histology and microradiography Teeth. The material illustrated was taken from animals which were 14 weeks old

and had been maintained on their respective diets for 10 weeks. The rats which had received the diets for 14 weeks exhibited similar and only slightly more severe signs; they are not illustrated. The rat incisor is generally believed to be completely replaced in 6-7 weeks. We have as yet no information concerning the effect of the various diets on the rate of eruption of the incisor. Figure 1 shows a general low-power view of a longitudinal section of a decalcified lower incisor from a Group 1 animal. It is deemed to be normal and indicates the position of two labial areas, one apical (A) the other more incisal (B) which are illustrated at higher magnifications in subsequent figures. The photomicrographs of the decalcified sections from region A in all groups are shown in Fig. 2. The corresponding microradiographs are illustrated in Fig. 3. Figure 4 shows photomicrographs of the more incisally situated region B for all groups, and the corresponding microradiographs are shown in Fig. 5. Figures 6 and 7 illustrate photomicrographs and microradiographs respectiveiy for samples of alveolar bone from all groups.

EFFECTOF

VITAMIN

~,ca

AND

P 0N

RATTEETH

AND

BONE

451

Group 1. A typical decalcified section deemed to represent a sample of normal rat incisor dentine is shown in Fig. 2a. The pre-dentine was narrow and remained so in the more incisal region (Fig. 4a). The microradiograph (Fig. 3a) shows that the dentine was evenly calcified with a sharply defined margin. In the apical region the dentine was more highly mineralized than the overlying enamel. In the incisal region (Fig. 5a) the enamel was relatively more radiopaque than the underlying dentine. Group 2. Normal calcium and phosphorus, low-vitamin-D. A simple deficiency of vitamin D appeared to result in the formation of dentine almost indistinguishable from the control dentine. There seemed to be a very slight increase in the width of the apical pre-dentine (Fig. 2b) and in a few isolated instances there was evidence of incomplete fusion of calcifying areas (Fig. 4b) in the incisal region. The microradiographs were similar to those of the control animals (Fig. 3b, 5b). Group 3. Low calcium, normal phosphorus, normal vitamin D. The effect of a simple deficiency of calcium in the apical region is shown in Figs 2c, 3c. The predentine was wider than normal and there was an increased irregularity of the calcification front. The odontoblasts showed some signs of disorganization. Figure 4c and 5c show examples of more incisal dentine. The pre-dentine was of normal width. The mineralization of the dentine was normal in its outer portion, but the radiograph revealed some undermineralization of the inner dentine. Group 4. Low calcium, normal phosphorus, low vitamin D. In the apical region the lack of calcium and vitamin D resulted in a great increase in the relative width of the pre-dentine (Fig. 2d). The microradiograph revealed a very poorly mineralized narrow hand of dentine (Fig. 3d). In contrast with Group 3 animals the widened pre-dentine persisted into the incisal region which remained poorly mineralized (Figs 4d, 5d). Group 5. Normal calcium, low phosphorus, normal vitamin D. In the apical region simple deficiency of phosphate resulted in the formation of a widened band of pre-dentine (Fig. 2e), with a narrow band of mineralized dentine (Fig. 3e). In the more incisal region the width of the pre-dentine was much reduced but the margin was irregular and the dentine was laid down in longitudinal undulating incremental striations (Fig. 4e). The differential pattern of mineralization corresponding to these lines was revealed in the microradiograph (Fig. 5e). Group 6. Normal calcium, low phosphorus, low vitamin D. The apical predentine was again very wide, the dentine narrow and poorly mineralized (Figs. 2f, 3f). In the incisal region the incremental striations were not so well marked as in Group 5 animals, but there appeared to be an incompleteness in the coalescence of the calcifying areas (Fig. 4f). This effect was clearly shown in the microradiograph (Fig. Sf). Despite these disturbances the pre-dentine layer was narrow. Alveolar bone The photomicrographs illustrated in Fig. 6 are of samples of decalcified alveolar bone taken from an area adjacent to the lingual surface of the incisor tooth. Figure 7 shows microradiographs of a similar area but at a lower magnification. The animals had received the diets for 14 weeks after weaning.

H. W. F~RGUSON

452

AND R.L.HARTLE.S

Group 1. Control. Figures 6a and 7a show bone from an animal deemed to represent a normal sample of rat alveolar bone. It is a simple type of bone with poorly developed Haversian systems. Group 2. Normal calcium, normal phosphorus, low vitamin D. The bone formed on the diet simply lacking added vitamin D appeared indistinguishable from the bone of the control animals (Figs 6b, 7b). Group 3. Low calcium, normal phosphorus, normal vitamin D. A simple calcium deficiency produced a grossly porotic appearance with many large cavities (Fig. 6~). There was no demonstrable osteoid tissue in the decalcified sections. The microradiograph emphasized the porotic nature of the bone (Fig. 7~). Group 4. Low calcium, normal phosphorus, low vitamin D. The double deficiency of calcium and vitamin D produced a marked difference in the bone compared with that from Group 3 animals. The porosis, whilst still evident, was much reduced and in some cases had almost disappeared. There were wide seams of lighter staining osteoid (Fig. 6a) and the microradiograph showed many poorly mineralized areas (Fig. 7a). The condition resembled osteomalacia rather than osteoporosis. Group 5. Normal calcium, low phosphorus, normal vitamin D. The low phosphorus diet even in the presence of adequate vitamin D produced alveolar bone showing clear signs of osteomalacia, there was a large amount of lighter staining osteoid tissue which was poorly mineralized (Figs 6e, 7e). Group 6. Normal calcium, low phosphorus, low vitamin D. The alveolar bone in this group of animals had very wide osteoid seams (Fig. 6f). The microradiograph again showed the paucity of mineral, and the margins were ill-defined. The differences between the bone of animals in Groups 5 and 6 were of degree rather than of kind. Ash content of incisor teeth

The percentage ash content of the teeth of the different groups is shown in Table 1. There were insufficient samples available for statistical evaluation ; nevertheless, certain distinct trends were apparent. TABLE 1. PERCENTAGE ASH CONTENT OFTHE APICAL ANDINCISALHALVES UPPER INCISORS OF RATS MAINTAINED ON THE SIX DIETS FOR 10 WEEKS

Group

1 2 3 4 5 6

Diet

Control Low vitamin D Low calcium Low calcium Low vitamin D Low phosphorus Low phosphorus Low vitamin D

OF THE DRY FAT-FREE AFTER WEANING*

wt. of teeth (mg)

Apical wt. of ash (mg)

Ash %

wt. of teeth (mg)

Incisal wt. of ash (mg)

Ash %

30.0 28.5 20.9

19.5 17.9 12.6

65.1 62.7 60.0

53.8 52.5 51.3

38.1 36.4 35.5

70.8 69.5 69.3

14.6 17.7

6.3 10.6

43.1 59.9

34.3 38.6

18.1 27.2

52.4 7n.c

16.5

9.4

53.1

36.0

25.2

69.9

* Data in this and subsequent

tables are the average of five animals.

EFFECTOFVITAMIN

~,ca

453

AND P 0~ RAT TEETH AND BONE

It can be seen that when the diet was deficient in calcium, the presence of vitamin D caused a great increase in the percentage ash content of the teeth. When the diet was deficient in phosphorus, the percentage of ash in the teeth was less affected by the presence or absence of vitamin D. Table 2 shows the data rearranged as a percentage of the control value and also includes comparable data for bone derived from an earlier study (FERGUSONand HARTLES,1963).

TABLE

2. PERCENTAGE ASH

AND

CONTENTOFINCIS~RT~~TH

EXPRESSED AS A PERCENTAGEOF

THE CONTROL

HUMERUS

VALUE Tooth

Diet

Group

Control Low vitamin D Low calcium Low calcium Low vitamin D Low phosphorus Low phosphorus Low vitamin D * Values derived from

TABLE 3. ABSOLUTE CORRESPONDING

Group 1 2 3 4 5 6

WEIGHTS

Bone*

Apical

Incisal

100.0 100.5 69.3

100.0 96 1 92.2

100.0 98.2 97.9

68.0 60.0

66.2 90.5

74.0 99.6

52.1

81.6

98.7

FERGUSON

and HARTLES

(1963).

OF DRY FAT-FREE INCISOR TOOTH AND HUMERUS

WEIGHTS OF ASH EXPRESSED AS A PERCENTAGEOF

Diet Control Low vitamin D Low calcium Low calcium Low vitamin D Low phosphorus Low phosphorus Low vitamin D

* Values derived from

AND

THE

VALUES

Incisor tooth Incisal Tooth Ash

Whole tooth Tooth Ash

100.0 91.8 64.6

100.0 97.6 95.4

100.0 95.5 93.2

100.0 96.6 86.2

100.0 944 83.6

48.7 59.0

32.3 54.4

63.8 71.7

47.5 71.4

58.4 67.2

42.3 65.7

55.0

48.2

66.7

66.1

62.7

60.0

Humerus* Bone Ash

Apical Ash Tooth

100.0 120.6 49.5

100.0 121.2 34.4

100.0 95.0 69.7

47.1 54.9

32.4 33.2

55.8

29.1

FERGUSON

THE CONTROL

and HARTLES(~~~~)

Table 3 gives the amounts of tooth formed and mineral deposited expressed as percentage of the control value, and again includes comparable data for bone.

a

DISCUSSION

The notion that the forming tooth might react differently from bone when confronted with deficiencies of calcium or phosphorus has been put forward by GAUNT and IRVING(1940) and further elaborated by IRVING(1957). It is claimed that in the

454

H. W. FERGU~~N ANDR. L. HARTLES

rat when the intake of both calcium and phosphorus is inadequate, the calcification of the teeth is more disturbed by diets with a Ca/P ratio of 05 than by diets with a ratio of 1.0 or 4.0. Conversely bone formation is more disturbed by diets with a high Ca/P ratio. It seemed appropriate therefore to investigate the effects on the incisor dentine of the consumption of defined diets deficient in either calcium or phosphorus in the presence and absence of vitamin D. The dietary deficiencies were as severe as we could achieve; the low calcium diets (0.026 % Ca, w/w dry basis) had a Ca/P ratio of 0.058; that of the low phosphorus diets (0.06% P) was ten; and that of the control diet 1.33. The deficient diets contained less calcium or phosphorus than those used by GAUNT and IRVING(1940) but in contrast were never simultaneously inadequate in both minerals. The experiments were therefore not an attempt at confirmation of their findings, but an elaboration. Reaction of teeth to low calcium diets When the diet was lacking both calcium and vitamin D (Group 4), the width of pre-dentine was greatly increased in the apical region (Fig. 2d) and the mineralized zone was narrow (Fig. 3d). The ash content of the apical half of the whole tooth was only 43.1 per cent (Table 1). The formation of the dentine was thus greatly disturbed and the disturbance was only slightly ameliorated in the more incisal portion of the tooth (Figs 4d and 5d). The ash content of the incisal half of the tooth had risen to 52.4 per cent. When the diet was low in calcium but adequate with respect to vitamin D (Group 3) the situation was very different. In the apical region the pre-dentine was less wide, although wider than normal, and better mineralized (Figs 2c, 3c) with an ash content of 60 per cent. In the incisal region the tooth approached normalcy (Fig. 4c) although the mineralization appeared incomplete near to the pre-dentine margin (Fig. 5~). The ash content of the incisal half of the tooth had risen to 69.3 per cent, which is close to the control value of 70.8 per cent. Thus the presence of vitamin D resulted in a great improvement in the formation of the dentine even when the intake of calcium was very low. It is most interesting to compare the effects of low calcium diets on the incisor tooth with that on the bone. When the diet is low in calcium, the presence of vitamin D prevents the accumulation of any demonstrable osteoid in rat bone; instead, a porotic condition develops (FERGUSONand HARTLES,1963). This observation suggests that in the presence of the vitamin the rate of mineralization of bone keeps pace with the rate of matrix formation even when there is a severe deficiency of calcium. In the case of the incisor tooth this is not so. The animals of Group 3 showed an increased width of pre-dentine in the apical region of the incisor (Fig. 2~). Thus the rate of mineralization was less than that of matrix formation. In the more incisal part of the tooth the pre-dentine was of normal width although the most recently formed dentine was not completely mineralized (Figs 4c, 5~). This suggests that the pre-dentine formed in these circumstances was a calcifiable matrix but that the rate of mineralization had been slowed down.

EFFECT

OF VITAMIN

D, Ca

AND

P ON RAT TEETH

AND

BONE

455

In the absence of vitamin D, the low calcium diet results in the formation of an osteomalacic bone. In the incisor, the corresponding diet produced a severe disturbance of the dentine (Group 4, Fig. 2d). The pre-dentine was very wide in the apical region and remained much wider than normal in the more incisal portion (Fig. 4d), there is therefore a very marked delay in the mineralization process. Thus, in these circumstances of combined calcium and vitamin D deficiency the bone and tooth react in a not dissimilar manner producing a poorly mineralized tissue with excessive amounts of either osteoid or pre-dentine. Whereas, when the diet simply lacks calcium, the tooth and bone react differently. These findings may be explained in part by the fact that the bone formed in the doubly-deficient state is resorbed with difficulty, whilst in the presence of the vitamin the bone is readily resorbable; in neither case is dentine resorbed. Therefore bone and dentine approximate closely in their behaviour in the absence of adequate calcium and vitamin D because in these circumstances both tissues are virtually non-resorbable. The question remains as to why more mineral is deposited in the incisor on low calcium diets when the vitamin is present. We have already observed that vitamin D raises the serum calcium when the diet is low in calcium from about 67 pg/ml to about 85 pg/ml, compared with a normal value of about 120 pg/ml (FERGUSONand HARTLES, 1963). We are not entirely convinced that the great differences in the formation of bone and dentine brought about by the presence of the vitamin can be explained by this comparatively small difference in serum calcium concentration Consideration must be given to the possibility that vitamin D is also contributing to the preparation of a matrix which is calcifiable even when calcium is in short supply. Reaction of teeth to low-phosphorus diets In the apical region of the incisors there was a great increase in the width of the pre-dentine irrespective of the presence or absence of vitamin D (Figs 2e, 2f) and the mineralized dentine was narrow (Figs 3e, 3f). In the incisal regions of the teeth, in both Groups 5 and 6 the pre-dentine was narrow, but the quality of the dentine formed was changed. In the presence of the vitamin (Group 5) an incremental pattern was observed (Fig. 4e) which was also apparent in the microradiograph (Fig. 5e). In the absence of vitamin D (Group 6) the incremental pattern was less clear and there was lack of fusion of calcifying areas (Figs. 4f and 5f). Despite these changes more and better mineralized dentine appeared to be formed on the low-phosphate diets than on the low-calcium, low-vitamin D diets (Group 4). Reaction qf alveolar bone to the de$cient diets It has been stated (SOGNNAES,1961) that alveolar bone enjoys some priority with regard to available minerals when compared with the extra-oral skeletal structures. Our results do not support this view; the alveolar bone reacted in a manner similar to the long bones (FERGUSONand HARTLES,1963). Simple deficiency of vitamin D had no demonstrable effect. Simple calcium deficiency produced a predominantly porotic condition with no evidence of osteoid formation (Figs 6c, 7~). Deficiency of calcium and vitamin produced osteomalacia (Figs 6d, 7d). The low phosphate diets produced

456

severe osteomalacia 7e, f).

H. W. FERGUSONAND

which was exacerbated

R.

L. HARTLES

by the absence

of vitamin

D (Figs 6e, f;

The ash content of the incisor teeth formed on the different diets The ash determinations have been carried out on portions of whole teeth because of the difficulty of separating the enamel, with its varying density, from the dentine. It is necessary to be cautious in drawing conclusions from such data, but with this proviso in these circumstances it is a justifiable procedure. The tooth with the lowest percentage ash content was that formed on a diet low in calcium and vitamin D (Group 4, Tables 1 and 2). The bone with the lowest ash content is that formed on a diet low in phosphorus and vitamin D (Table 2, Group 6). A simple dietary deficiency of calcium permitted the formation of a tooth with an ash content, even in the apical half, of more than 90 per cent of the control value; in similar circumstances the ash content of the bone is about 70 per cent of the control value (Table 2). When the diets were deficient in phosphorus, either in the presence or absence of vitamin D, the ash content of the incisor was much greater than that of the bone (Table 2).

The absolute weights of dry fat-jiee incisor teeth formed on the diflerent diets The tooth with the smallest mass and the smallest amount of ash was that formed on the diet low in calcium and vitamin D (Table 3, Group 4). These were the conditions in which the reaction of the tooth, especially in the apical portion, corresponded most closely to that of bone. When the diet was deficient in calcium alone, the mass of tooth formed and the amount of mineral deposited were over 80 per cent of the control value (Table 3, Group 3) a much higher figure than for the corresponding bone. When the diet was low in phosphorus, with or without vitamin D, the mass of tooth formed and mineral deposited was reduced to about two-thirds of the control value (Table 5, Groups 5 and 6). In similar circumstances the bone mass and particularly the amount of mineral deposited was reduced more than in the teeth. The histological and analytical evidence show that the incisor teeth and bone of the rat react differently when formed in conditions of calcium or phosphorus deficiency The relative amount of mineral deposited in the incisors was always greater than in the bone. The percentage of ash in the tooth was greater than in the bone except when a deficiency of calcium was accompanied by lack of vitamin D. It would appear therefore that in relation to the actual deposition of mineral, the incisor tooth was most susceptible to lack of vitamin D when the diet was deficient in calcium. In contrast the quantity of mineral deposited in the teeth on diets low in phosphorus was less affected by the presence or absence of the vitamin. Nevertheless a deficiency of phosphorus per se had a.much greater effect than a deficiency of calcium in reducing the quantity of tooth formed. Even so the reduction in mass was not so great as in the case of bone. It has been argued above that the differences between the reactions of bone and teeth to a simple lack of calcium and a concomitant deficiency of vitamin D may be

EFFECT

OF VITAMIN

D, CB AND P ON RAT

TEETH

AND

BONE

451

explained in part by the resorbable nature of bone. Such an explanation is unlikely to account for the different reaction of the bone and teeth to the rachitogenic lowphosphorus diets. The fact that mineral is preferentially deposited in the teeth in these circumstances suggests that, since the humoral factors are the same, the origin of the differences must lie in the process concerned locally with the actual deposition of mineral or with the preparation of the matrix for mineralization. In the circumstances of our experiments a simple deficiency of vitamin D appeared to have little effect on the development of the incisor tooth. We are satisfied that the diets to which ergocalciferol has not been added are deficient in vitamin D. This view is supported by the observation that the addition of a physiological amount of the vitamin to a diet deficient in calcium and the vitamin produces a marked response in the teeth and bone (Groups 3 and 4). The conclusion can be drawn, therefore, that when the dietary content of calcium and phosphorus is adequate the requirement of the rat for vitamin D is very low. Nevertheless the small departures from normal which were occasionally observed underlined the necessity for further study. No comments have been made regarding the condition of the enamel in the different groups because we are not yet in a position adequately to do so. It is however interesting to note that there were signs of disturbed mineralization of the enamel of animals in Group 4 (Fig. 5d) which were not apparent in other groups. Acknowledgements-The authors wish to thank Mr. L. F. GORE and Miss ANNE HARRISONfor their skilled assistance in the preparation of material, Mr. R. P. WILLIAMSfor his care and maintenance of the animals, Mr. J. S. BAILLIEfor the photography, and Automatic Telephone Co Ltd., for the use of their microradiographic equipment. Gifts of icing sugar and groundnut oil are gratefully acknowledged from Messrs. TATEand LYLELtd., and Messrs. J. BIBBYand Sons Ltd., respectively. R&sum&Des rats, &g&s de 60 mois, provenant de notre klevage, sont divisks en 6 groupes, dont l’un constitue le groupe tkmoin. Les autres groupes sont soumis pendant 10 semaines ?I une nourriture dkficiente en vitamine D; calcium; calcium et vitamine D; phosphore; phosphore et vitamine D. Les animaux sont sacrifiks et leurs incisives sont examinkes. Une simple dtficience en vitamine D provoque que de trks ltgbres modifications des incisives. Les altkrations les plus prononckes sont provoqutes par des rtgimes dtficients en calcium et vitamine D. Une dkficience simple en calcium retarde la mixkalisation de la portion apicale de la dentine, mais la dentine coronaire est relativement peu atteinte. Avec des rkgimes pauvres en phosphore, m&me en l’absence de vitamine D, les incisives contiennent plus de sels minkraux que les dents form&es pendant l’utilisation d’un rkgime dkficient en calcium et en vitamine D. Ce fait contraste avec la situation observke au niveau de 1’0s. En gtSra1, une dkficience en calcium et en phosphore a un effet moindre sur le dkveloppement de l’incisive que sur celui de 1’0s. Le seule exception est observke dans le cas d’une dkficience en calcium et en vitamine D, lorsque la partie apicale de la dent subit une altkration comparable & celle de 1’0s. L’os alvkolaire ne b&kficie d’aucune prtfkrence pour les sels minkraux disponibles et rkagit vis-&vis de conditions de d&icience d’une man&e identique %celle des OSlongs. Les conskquences de ces rksultats sur des diffkrences Bventuelles dans le mkanisme de minkralisation des dents et de 1’0s sont envisagkes.

H. W. FERGUSONAND R. L. HARTLES

458

Zusanunenfassnng-60-Monate alte Ratten eigener Zucht wurden in 6 Gruppen eingeteilt, von denen eine als Kontrollgruppe diente. IO-Wochen lang erhielten die iibrigen Gruppen Diaten, die sich durch Mange1 an Vitamin D, Calcium, Calcium und Vitamin D. Phosphor, und Phosphor und Vitamin D auszeichneten. Die Tiere wurden get&et und ihre Schneidezahne untersucht. Einfacher Mange1 an Vitamin D verursachte lediglich sehr leichte Vednderungen in den Schneidezahnen. Die starkste Veranderung wurde durch Diaten mit Mange1 an Calcium und Vitamin D verursacht. Einfacher Mange1 an Calcium hemmte die Mineralisation des Dentins im apikalen Bereich; dagegen war der inzisale Teil des Dentins relativ wenig gestiirt. Kostformen mit niedrigem Phosphorgehalt bewirkten selbst bei Fehlen von Vitamin D Schneidezlhne, welche mehr Mineralien als diejenigen ZPhne enthielten, die unter der Calcium- und Vitamin-D-Mangeldilt gebildet wurden. Dies steht im Gegensatz zu den im Knochen beobachteten Verhaltnissen. Im allgemeinen zeigte ein Mange1 an Calcium oder Phosphor weniger Wirkung auf die Entwicklung der Schneidezlhne als auf den Knochen. Die einzige Ausnahme war im Falle des Calcium-und Vitamin-D-Mangels zu beobachten, bei dem der apikale Anteil des Zahnes eine mit dem Knochen vergleichbare Storung erlitt. Der Alveolarkamm wurde fiir verfiigbare Mineralien nicht bevorzugt und reagierte auf Mangelzustlnde in einer den Extremitatenknochen lhnlichen Weise. Die Folgerungen aus diesen Ergebnissen werden in Beziehung zu moglichen Unterschieden der Mineralisation von Zlhnen und Knochen diskutiert.

REFERENCES BECKS, H. and RYDER, W. B. 1931. Experimental

rickets and calcification

of dentine.

Arch. Path.

(Lab. Med.) 12, 358-386.

COLEMAN,R. D., BECKS, H.. COPP, D. A. and FRANDSEN,A. M. 1953. Skeletal changes of severe phosphorus deficiency in the rat. II. Skull, teeth and mandibular joint. Oral. Surg. 6, 756-764. FERGUSON,H. W. and HARTLES,R. L. 1963. The effect of vitamin D on the bones of young rats receiving diets low in calcium or phosphorus. Arch. oral. Biol. 8,407418. GAUNT, W. E. and IRVING, J. T. 1940. The influence of dietary calcium and phosphorus upon tooth formation. J. Physiol. 99, 18-29. HARTLES,R. L. 1951. The effect of a high-sucrose diet on the calcium and phosphorus content of the enamel and dentine of rat incisors. Biochem. J. 48,245-249. HARTLES,R. L. and LEAVER,A. G. 1961a. Citrate in mineralized tissues-III. The effect of purified diets low in calcium and vitamin D on the citrate content of the rat femur. Arch. owl. Biol. 5, 38-44.

HARTLES,R. L. and LEAVER,A. G. 1961b. Citrate in mineralized tissues-IV. The relation of vitamin D intake and calcium nutrition to the citrate content of the rat femur. Arch. oral. Biol. 5,274-283.

HARTLES,R. L. and LEAVER,A. G. 1962. Citrate in mineralized tissues-V. The effect of purified diets low in phosphates, adequate in calcium and containing varying amounts of vitamin D, on the citrate content of the rat femur. Arch. oral. Biol. 7, 557-562. IRVING, J. T. 1944. Action of vitamin D upon incisor teeth of rats consuming diets with high or low Ca/P ratio. J. Physiol. 103, 9-26. IRVING, J. T. 1957. Calcium Metabolism. Chap. 13. p. 141. Methuen, London. KARSHAN, M. 1930. III. Calcification of teeth and bones on rachitic and non-rachitic diets. J. dent. Res.

10,267.

KARSHAN, M. 1930. Calcification Res.

KARSHAN, M. 1931. Calcification Res.

of teeth and bones on rachitic and non-rachitic

diets. J. dent.

of teeth and bones on rachitic and non-rachitic

diets. J. dent.

10,267. 11,64.

KARSHAN,M. and ROSEBURY,T. 1932. Correlation of chemical and pathological of rats on rachitic and non-rachitic diets. J. dent. Res. 12, 437439.

changes in teeth

EFFECT OF VITAMIND, Ca AND P ON RAT TEETH AND BONE KARSHAN, M. and ROSEBURY, T. 1933. Correlation of chemical and pathological and bones on rachitic and non-rachitic diets. J. dent. Res. 13, 305-310. MELLANBY, M. 1923.

The effect of diet on the structure

ROSEBURY, T. and KARSHAN, M. 1931. Pathological synthetic diets. J. dent. Res. 11, 137-148. R. F. London.

SOGNNAES,

1961. Mineral

Metabolism,

of teeth. changes

Vol. 1, Part B.

459 changes in teeth

Brit. dent. J. 44, 1031-1049. in the teeth of rats Chap.

produced

15, pp. 713-714.

by

Academic,

WEINMANN, J. P. and SCHOUR, 1. 1945a. Experimental studies in calcification. 1. The effect of a rachitogenic diet on the dental tissues of the white rat. Amer. J. Path. 21,821-831. WEINMANN, J. P. and SCHOUR, 1. 1945b. Experimental studies in calcification. II. The effect of a rachitogenic diet on the alveolar bone of the white rat. Amer. J. Path. 21, 833-856.

PI.ATES 1 -- 7 OVERLEAF

EFFECT OF VITAMIN

D.

ca

AND

P ON RAT TEETH AND

BONE

FIG. I. Longitudinal decalcified section of lower incisor of a normal rat, 14 weeks old, showing gross histological features. E.S., enamel space; E.M., enamel matrix; 0, odontoblasts; O.E.,odontogenicepithelium; Lab. D.,Iabial dentine; Lin. D.,iingual dentine; P.D., pre-dentine; Ar., artifact. Haematoxylin and eosin. x 13.

PIATF

i

H. W. FERGUSONAND R. L. HARTLES

FIG. 2. Longitudinal decalcified sections corresponding to the apical region A. in Fig. 1. Haematoxylin and eosin. x 112. (a) Group 1, normal 14 weeks old. (b) Group 2, low-vitamin D, normal Ca and P. Very slight increase in width of predentine, with slight irregularity of pre-dentine/dentine margin. (c) Group 3, low-Ca, normal vitamin D and P. Increased width of pre-dentine with irregularity of margin, and some degeneration of odontoblasts. (d) Group 4, low-Ca, low-vitamin D, normal P. Very marked increase in width of pre-dentine with irregular calcification and disturbance of odontoblasts. (e) Group 5, normal Ca and vitamin D, low P. Very wide pre-dentine with a reasonably regular margin but some disorganization of odontoblasts. (f) Group 6, normal Ca, low vitamin D and P. Very wide pre-dentine with an Irregular margin, odontoblasts disturbed. PLATE 2

FIG. 3. Microradiographs of undecalcified sections from apical region A. Corresponding with Fig. 1. x44. (a) Group 1, normal 14 weeks old. At this stage of development the dentine is more highly mineralized than the overlying enamel. (b) Group 2, low vitamin D, normal Ca and P. Similar to (a) with well-defined margin. (c) Group 3, low Ca, normal vitamin D and P. Note irregular mineralization of dentine margin. (d) Group 4. low Ca, low vitamin D, normal P. Dentine has broken away from enamel. ‘Considerable reduction in width of mineralized dentine with marked irregularity of calcification front. (e) Group 5, normal Ca and vitamin D, low P. Reduction in width of dentine but the margin less irregular than in (c) or (d). (0 Group 6, normal Ca, low P and vitamin D. Reduced width of dentine, with slight irregularity of margin. PLATE 3

H. W. FERGUSON AND R. L. HARTLES

FIG. 4. Longitudinal decalcified sections corresponding to the region B in Fig. 1. Haematoxylin and eosin. x 112. (a) Group 1, normal 14 weeks old. Pre-dentine narrow. (b) Group 2, low vitamin D, normal Ca and P. Very slight increase in width of predentine with a suggestion of incomplete fusion of calcifying areas. (c) Group 3. low Ca, normal vitamin D and P. Pre-dentine only slightly wider than normal with some irregularity of the calcification pattern. with irregular (d) Group 4, low Ca and vitamin D, normal P. Wide pre-dentine margins. Odontoblast layer lost in preparation. (e) Group 5, low P, normal vitamin D and Ca. Incremental zones of hypo and hypermineralization are apparent. The pre-dentine although irregular is only slightly wider than normal. (f) Group 6, low P and vitamin D, normal Ca. Considerable disturbance of formed dentine but width of pre-dentine almost normal. PLATE 4

EFFECT OF VITAMIN D,Ca

AND

P ON RAT TEETH AND BONE

FIG. 5. Microradiographs of undecalcified sections from region B corresponding with Fig. 4. x 44. (a) Group 1, normal 14 weeks old. In this more incisal region the enamel is more highly mineralized than the dentine. tb) Group 2, low vitamin D, normal Ca and P. Width of dentine similar to normal with a well defined margin. (c) Group 3, low Ca, normal vitamin D and P. Layer of dentine adjacent to enamel well mineralized but more recently formed dentine shows signs of disturbed mineralization. td) Group 4, low Ca, low vitamin D, normal P. Very narrow band of mineralized dentine with irregular margin. Some disturbance of enamel mineralization. te) Group 5, low P, normal Ca and vitamin D. Incremental pattern of hypo and hypermineralization. (f) Group 6, low P and vitamin D, normal Ca. Dentine narrow with areas of disturbed mineralization. PLATE

5

H. W.

FERGUSON AND

R. L.

HARTLES

FIG. 6. Longitudinal sections of decalcified lingual alveolar bone adjacent to the incisor. Haematoxylin and eosin. x 112. (a) Group 1, normal 18 weeks old. (b) Group 2, low vitamin D, normal Ca and P. No convincing difference in general appearance from (a). (c) Group 3, low Ca, normal vitamin D and P. Extremely porotic bone but an absence of osteoid seams. (d) Group 4, low Ca and vitamin D, normal P. A reduction in the number and size of porotic cavities with the presence of osteoid seams. (e) Group 5, low P, normal vitamin D and Ca. Wide osteoid seams. (f) Group 6, low P and vitamin D, normal Ca. Very wide osteoid seams.

PLATE 6

EFFECT0~ VITAMIN

0,

ca

ANO

P ON RAT TEETH ANO BONE

FIG. 7. Microradiographs of part of the lingual alveolar bone. x 44. (a) Group 1, normal eighteen weeks old. (b) Group 2, low vitamin D, normal Ca and P. Similar to (a) but with slight irregularity of margins. (c) Group 3, low Ca, normal vitamin D and P. Reduction in bone mass with very large porotic cavities. (d) Group 4, low Ca and vitamin D, normal P. Reduction in bone mass, many radiolucent areas with variation in the radiodensity of the margins. (e) Group 5, low P, normal vitamin D and Ca. Reduction in bone mass but margins of radiolucent zones are regular. (0 Group 6, low P and vitamin D, normal Ca. Pattern similar to (e) but margins of bone are irregular.

PLATE 7