(Received February 6, 1935.)

Transcription

1 272 6 I2.3II.1:577. i6c THE EFFECT OF VITAMIN D ON THE CALCIUM CONTENT OF THE DENTINE. BY E. WILFRED FISH. (From the Hale Research Laboratory, Royal Dental Hospital, London.) (Received February 6, 1935.) INTRODUCTION. IT has been recognized for many years that rickets and the acute exanthemata in infancy may cause severe bypoplasia of the dental enamel [Bunon, 1746]. More recently Mellanby [1929] has shown that this condition and also hypoplasia of the dentine can be produced in laboratory dogs by depriving the animals of vitamin D and inducing experimental rickets at the time when the enamel is being laid down. Tbere is no experimental evidence that any effect is produced on either dentine or enamel, once it is formed, by exhibiting or withholding vitamin D, but it is claimed [McBeath, 1932; Mellanby, 1934]that the exhibition of therapeutic doses of the vitamin will botb prevent and arrest dental caries. This conclusion is based on a series of controlled clinical experiments, but does not coincide with general clinical experience. Apart from the fact that formed buman enamel is practically outside the pale of nutrition, it bas also been shown that dentine when exposed by caries is entirely shut off from the tissue fluids by calcific scar tissue [Tomes, 1848; Fish, 1932]. It would therefore appear theoretically that, once the teeth are formed, the incidence and progress of caries cannot be affected by any factor influencing the tissue fluids of the pulp and dentine. The suggestion that the exhibition of vitamin D can do so appears to rest on two assumptions: (i) That the exhibition of the vitamin will increase the calcium content of the dentine. (ii) That an increased calcium content of the dentine will prevent or arrest caries.

2 DENTINE CALCIUM AND VITAMIN D. 273 There is no experimental evidence that either of these assumptions is correct, indeed it has been found impossible to affect the calcium content of the dentine in any way whatever. There is a slight physiological increase in the calcium content during adolescence, but negative results have followed attempts to affect it by deprivation of calcium during pregnancy in dogs, parathyroidectomy in cats, or, in the other direction, by giving massive doses of vitamin D and calcium carbonate to dogs. Even in disease, when the calcium metabolism was profoundly disturbed, in certain dogs wbich became available, although the bones were pliable and radiolucent the calcium content of the dentine remained absolutely unaltered [Fish, 1932]. Since it has been found impossible to affect the calcium content of the dentine in any way, it is impossible to say whether the second assumption, that a higher calcium content would prevent caries, is correct or not, but it may be significant that the dog which has a calcium content several p.c. lower than that of man is absolutely immune to dental caries. The present report records a further unsuccessful attempt to alter the calcium content of the dentine by giving heavy doses of vitamin D and calcium over a longer period than in the previous experiment. METHOD. Two teeth were excised from each animal at the beginning of the feeding experiment, and the two symmetrically opposite teeth were removed at the end. A specimen of the dentine was prepared from eacb tooth and analysed. Each specimen was freed mechanically from pulp tissue and from enamel and cementum and was prepared from the cervical part of the tooth. The specimen thus consisted of the central mass of the dentine and did not include the odontogenetic zone. The pieces were dried at 1050 C. for 18 hours, when it was found that the weight remained constant since further drying for 3 days bad no effect on it. This dry weight was therefore taken as a basis for calculation and the calcium content was expressed as the proportion of calcium oxide to this weight. The specimen was next incinerated, and the total calcium content estimated by dissolving the ash in phosphoric acid, precipitating with oxalic acid and ammonia, redissolving the precipitate in sulphuric acid and titrating with potassium permanganate. The accuracy of the metbod was tested by cutting three specimens in halves and analysing each half separately. The results agreed to within ±002 p.c. 18-2

3 274 E. W. FISH. As a biological control two dogs are quoted from a previous experiment. Two teeth were excised, and the dogs were kept on a normal diet of meat and biscuits for 7 weeks wben they were killed and the two corresponding teeth were removed. One of these dogs was young, and the dentine was still being deposited on the pulp surface so that the recent deposit had to be removed from the later specimen before analysis. This is very important in every case with young animals, since it is desired to detect any change in the calcium content of the dentine which had already been deposited at the beginning of the experiment, and not merely to compare it with more recently formed dentine. The amount which must be cut away can easily be determined by comparing the size of the pulp chamber with that of the corresponding tooth extracted before the experiment began. The second dog was mature, and no appreciable amount of dentine had been added. Certain organs were removed, post-mortem, sectioned and stained with hiemotoxylin and eosin and with heemotoxylin and van Gieson. These included the thyroid and parathyroid glands, kidney, spleen, lung and ventricle of the heart, and a specimen of both jaw bone and long bone. Skiagrams of the head, limbs and lower jaw of each animal were taken under standardized conditions both before the experiment and after death. Blood calcium estimations were made from time to time during the experiment. Three dogs from the same litter were used. They had been bred on the experimental farm at Mill Hill and had lived and fed together. None of them had suffered from any apparent disease and the kennel history of each is available. One dog, DEL/4, was smaller than the other two and of somewhat different type. It happened to show extremely severe hypoplasia of the enamel, especially of the canines of the type usually associated in man with rickets or the acute exanthemata in infancy. This dog, however, had always been healthy, had a normal diet and had not had rickets or distemper. The basic diet of raw meat, hounds' meal and water on which they had always been fed was continued, but certain additions were made to the diet of each dog. DEL/2 received an addition of 4 g. of calcium carbonate for 60 days, followed by a normal diet for 38 days, then an addition of 4 g. of calcium carbonate and 50,000 units of vitamin D per day for 34 days. The vitamin was administered in the form of a solution of calciferol in arachis oil in a. gelatin capsule.

4 DENTINE CALCIUM AND VITAMIN D. At the end of the experiment the dog was refusing food and losing condition; it was therefore killed. The blood calcium was: At the beginning of the experiment 11.1 mg./100 c.c. of serum On the 19th day 12-4 On the 60th day 12-3 At death Not available'. DEL/3 received an addition of 4 g. of calcium carbonate and 50,000 units of vitamin D per day for 25 days, then 4 g. of calcium carbonate and 75,000 units of vitamin D for 35 days, normal diet for 38 days, and finally 4 g. of calcium carbonate and 50,000 units of vitamin D for 34 days. On the 60th day this dog was drinking prodigiously, refusing food and became comatose, but entirely recovered in about a week on normal diet. At the end of the experiment (135 days) it was again refusing food and losing condition and was killed. The blood calcium was: At the beginning of the experiment 11*8 mg./100 c.c. of serum On the 19th day 13-9 On the 60th day 19-6 At death Not available'. DEL/4 received an addition of 4 g. of calcium carbonate and 50,000 units of vitamin D per day for 60 days; it was then drinking continuously, refusing food and becoming comatose. The calcium carbonate and the vitamin were discontinued, and the dog recovered somewhat but relapsed and had to be killed on the 65th day. At the postmortem there were marked hamorrhages of the intestine. The blood calcium was: At the beginning of the experiment 11.1 mg./100 c.c. of serum On the 19th day 15X2 On the 60th day (killed 65th day) 21X1,. 275 RESULT. It was found that these heavy doses of vitamin D almost doubled the normal calcium content of the blood; the slight rise in the case of DEL/2 when on calcium carbonate alone may not be significant. 1 Previous experience would suggest that from the clinical symptoms the blood calcium must have been of the order of 20 mg./100 c.c. of serum.

5 276 B. W. FISH. The skiagrams showed a very marked increase in the opacity of the bones to X-rays, but no apparent change in the opacity of the teeth. There was no histological change or abnormal deposition of calcium observed in any of the tissues examined, with the possible exception of bone. There appeared to be some increased deposition of calcium salts, as indicated by staining reactions, in some new bone which was forming, especially at the points of attachment of muscles and ligaments. The analyses showed that there was no significant change in the calcium content of the dentine. The results may be tabulated: TABLE I. Calcium oxide percentages Description A AVerage of dog Before After Difference difference DEL/2 L! 33*46 ±j E *87 DEL/ L[ j DEL/4 L! 34* *28 L Young v ] 35* (control) Mature T F (control) F DIscussIoN. The difference in the calcium content of the dentine of the teeth excised before and after the experiment falls well within the normal range of individual tooth variation. In this respect the results correspond with those of the earlier experiments in which two dogs on toxic doses of calciferol ranging from 50,000 to 400,000 units a day for 6 weeks showed an average difference of p.c. of calcium oxide after the experiment. In these cases the blood calcium rose to 22-5 and 22.3 mg./100 c.c. of serum respectively. Two other animals investigated at that time were put on therapeutic doses of calciferol (2000 units a day) for 7 weeks. One of them showed an average difference of -0 3 p.c. and the other an average difference of p.c. of calcium oxide in the dentine of the teeth removed before and after the experiment. Taking the ten pairs of teeth extracted from the five dogs on very high doses of vitamin D together, there was an average difference of

6 DENTINE CALCIUM AND VITAMIN D p.c. of calcium oxide in the teeth excised before and after the experiments. It is therefore reasonable to assume that high doses of vitamin D and calcium carbonate do not affect the calcium content of the dentine. At the same time the increase in opacity of the bones to X-rays, in conjunction with their histological appearance and the high blood calcium, suggests that there was a considerable deposition of calcium salts in such tissues as were able to avail themselves of it. The dentine, unlike bone, consists almost entirely of matrix and has no cells in it except for the fibrillar processes of the odontoblasts. It would also appear from this and earlier investigations [Fish, 1932] that, unlike bone, dentine is devoid of any mechanism by which calcium salts can be deposited in it or withdrawn from it to any appreciable extent. When calcium salts are deposited in the dentine in the form of a translucent zone in response to local injury, they occupy the lumen of the tubules and block them up; they are not incorporated in the matrix. Similarly the slight increase in calcium content observed in human teeth in the first few years after eruption is associated with an anatomical reduction in the diameter of the tubules. It would therefore appear that, in the only two conditions under which any increase in the calcium content of dentine has been observed, it is due to new deposits in the tubules and not to an increase in the calcium content of the collagen matrix already present. SUMMARY. 1. It has been reported that therapeutic doses of vitamin D have brought about prevention and arrest of dental caries. There is no experimental evidence, bowever, that the exhibition of the vitamin produces any effect on formed dentine or enamel. Earlier attempts to modify the calcium content of the dentine have failed. 2. Continuing these earlier experiments three dogs were given maximal doses of vitamin D together with calcium carbonate over extended periods. 3. Analvses of the dentine showed no change in its calcium content. Skiagrams of the bones showed an increase in their opacity to X-rays, and histologically there appeared to be new deposits of highly calcified bone matrix, but there were no apparent changes in the other tissues examined. The blood calcium was markedly increased.

7 278 E. W. FISH. REFERENCES. Bunon (1746). Experience8 et Demonstrations Faites c l'h6pital de La Salpgtriere pour servir de suite et de preuves a PEssai sur lew Maladies des Dents. Fish, E. W. (1932). An Experimental Investigation of Enamel, Dentine and the Dental Pulp. McBeath, E. C. (1932). J. den. Res. 12, 723. Mellanby, M. (1929). Sp. Rep. Ser. Med. Res. Coun. No Mellanby, M. (1934). Ibid. No Tomes, J. (1848). Dental Physiology and Surgery, p. 203.