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The Effects of Nutritional Disturbances Upon Calcification and Decalcification of Teeth in Experimental Animals. A Progress Report*
THE EFFECTS OF NUTRITIONAL DISTURBANCES UPON CALCIFICATION AND DECALCIFICATION OF TEETH IN EXPERIMENTAL ANIMALS. A PROGRESS REPORT* By JOHN ALBERT MARSHALL, D.D.S., Ph.D., San Francisco, California (Read before the American Dental Association, Cleveland, Ohio, September 10-14, 1923)
A
T T H E last meeting of the Ameri ample, the deciduous cuspids and molars can Dental Association in Los were, in some instances, retained for a Angeles, I read a preliminary re much longer period of time than was the port on a group of nutrition experiments case in the control animals, and the re conducted under the supervision of W. P. sorption of their roots was very much Lucas, head of the Department of delayed. On examination of the perma Pediatrics, University of California, and nent set, it was observed that some of Dr. M artha Jones, of the George W il them exhibited a marked hypoplasia. One liams Hooper Foundation for Medical lower cuspid, in particular, showed strik Research. At necropsy, the heads of the ing symptoms of arrested growth. D ur animals were given to me for examina ing the earlier stages of calcification of tion and identification of dental lesions. this tooth, the tip of the crown was ap Although complete pathologic data parently normal in development. About have been compiled, these are not ap one-third of the distance from the tip pended here, but will be reported else toward the gingival line, there was a where by Dr. Jones. My interest in definite constriction produced by cessa the heads has been, as I have stated, only tion of function of the ameloblasts, so in so far as the teeth and supporting that the crown presented a definite nar structures are concerned. rowing toward the line of hypoplasia. The data formerly presented dealt Other teeth exhibited a sim ilar stunted mainly with four abnormalities : those re growth. In addition to these evidences lating, first, to the shedding of decidu of improper nutrition, there was a well ous teeth of dogs; second, to the forma defined irregularity in the position of the tion of the permanent teeth; third, to the teeth, particularly the lower incisors. position of the permanent teeth, and In one case, the teeth were clumped to fourth, to the relation in the arch be gether in a bunch, one or two standing tween the deciduous teeth and perma directly lingual to the others in the arch. nent ones. By imposing a restricted diet, Finally, some of the deciduous teeth we were able to produce lesions resem were so firmly attached that even the bling rickets, some of the outstanding eruption of their successors did not force symptoms of which were those referred to the first teeth from their sockets. above, namely, the effects on the position The diet, which was responsible for and the eruption of the teeth. For ex these changes, was not described at that * Aided by grants from the American Dental time, nor will it be now. However, I Association and from the Board of Research, am permitted to state that all the food elements were present in what we believe University of California. Jour. A. D. A., January, 1924
6
Marshall— The Effects of Nutritional Disturbances to be sufficient amounts. The only alteration was concerned with the pro portions of calcium and phosphorus. These two elements exist as calcium
7
phosphate in both bones and teeth, and the quantities fed the animals were ap parently so at variance with the needs of nature that faulty metabolism re sulted. In one set of experiments, the phosphorus was given in excess, and in another, too much lime was administered. Between these two extremes there is an optimum quantity of each element, a quantity which will supply fully all physiologic requirements. Such a
Fig. 1.— Upper and lower jaws of normal animal, which are partially disarticulated to show the osseous development.
Fig. 3.— Opposite view of same side of jaw as shown in Figure 2.
quantity will not be detrimental to the organism as a whole. These elements were, so far as we know, the only variable factors in the whole dietary regimen. Utilizable proteids, as well as vitamins, were given in the form of meat and other fresh foods. The inorganic elements were supplied in the form of the McCol lum salt mixture, except the phosphorus and calcium, which were given in varying quantities as mentioned above. It is hardly necessary for me to call attention to the fact, now well recog Fig. 2.-—Hypoplasia of permanent lower nized, that diet exerts a profound influ cuspid; malposition of permanent lower in ence on the growth and function of bones cisors. and teeth. Although there are many
8
The Journal of the American Dental Association
uncorrelated observations reported which are being compiled which will be re need confirmation, the experiments of ported at a later date. Although the macroscopic abnormali Shipley1, Howe2, McCollum3, Grieves4, ties which have been enumerated are dis tinct evidence of irregularities in calci-
Fig. 6.—Note the hollow enamel caps of first and second maxillary molars, in which normal dentin is entirely absent. Fig. 4.— Abnormal dentition and bone, the result of diet. Note particularly the reten tion of deciduous teeth and the porosity of cortical bone.
fication, it was not realized what pro found changes had occurred in the struc ture of the teeth until a microscopic ex amination of the sections had been made. Sherman5 and many others all point to In one of the animals, the dentin under neath a thin enamel cap had become com this developmental phenomenon. decalcified, and had lost all the This paper is essentially a progress re pletely characteristic structural elements of the port in which certain histologic character-
Fig. 5.— Abnormal dentition and bone, showing the effects produced by dietary in fluences in an upper jaw.
istics are presented. The previous ex Fig. 7.—Maxillary cuspid showing enamel periments are being repeated and ex cap (right) and a fibrous vascular mass tended, and further data on this problem which was found in place of normal dentin.
Marshall— The Effects of Nutritional Disturbances
9
tissue. The separation between the gone profound changes. In its place enamel cap and the underlying dentin there was loose areolar connective tis was easily affected at necropsy, merely sue, richly supplied with fine capillaries by exerting a slight pulling pressure with the fingers on the enamel. This resulted in the complete removal of the enamel in one piece. By this operation, the soft, decalci fied dentin was exposed. There was no normal dentin in the tooth, although the •enamel was intact. The soft tissue underneath the enamel was prepared for
Fig. 8.—Looking down into the enamel cap from the gingival toward the tip of the crown.
sectioning in the usual manner, namely, by immersing in a 10 per cent solution of formaldehyde, then carrying it through various strengths of alcohol up to abso lute, and finally in xylol. N o decalci fying fluid was used at any time. From xylol it was transferred to melted paraf fin, and, when cold, cut into sections for staining and study. On examination, it was found that both the inorganic and organic portions of the dentin had been altered to such an extent as to render their identification impossible. N either dentin tubules nor tubular contents remained. Even the organic matrix, which is very character istic in the decalcified section, had under
Fig. 9.— Photomicrograph of fibrous mass showing, even at a comparatively low magni fication, the inflammatory reactions occurring just before necropsy.
which freely anastomosed throughout the structure. At various places in the sec tion there were inflammatory tissue ele
10
The Journal of the American Dental Association
ments which indicated that active proc dentin, but, from its histologic structure, esses of tissue changes were occurring it resembles an inflamed pulp. The illustrations presented, as well as just before necropsy. These facts throw the specimen which I have demon strated, indicate better than a description 'm .
*
v
jfr
-rs‘Av. - í ■.?Hv ..
.A
'fc
j
'
Fig. 10.—High power photomicrograph of tissue. Note enlarged and engorged capil laries at A. Throughout the area are evi dences of round cell infiltration and of other inflammatory reactions.
*: w f w
* * ' f t **' *
Fig. 12.—Mallory connective tissue stain of tip of specimen, showing odontoclastic layer on both sides, at A ; enlarged capillary at B.
the extent of these lesions. It is discon certing to examine a skull with teeth Fig. 11.— Same as Figure 10. intact, and to discover that under an apparently normal enamel, the dentin, doubt on our classification of this tissue. which we expect to find normal as well, Is it dentin or pulp? From its inti has either undergone processes of ex mate relation to enamel, it is obviously tensive décalcification, or else has not
11
Marshall— The Effects of Nutritional Disturbances been formed at all. I am led to the former conclusion, however, especially when the developmental stages of tooth growth are recalled. This development of enamel from the enamel organ, and dentin from the odontoblastic layer, is a simultaneous occurrence in so far as cal cification of each tissue is concerned. A normal enamel has never been observed to have been formed without an under lying normal dentin. If there had been no dentin formed at any time in these teeth, it is inconceivable that the size and shape of the enamel cap would be normal, or that it would be without some evidence of lack of a supporting calcified structure. I am referring at this moment not to the cases of hypoplasia, but what I believe to be the more advanced con dition, namely, that in which a normal enamel cap overlies a decalcified tissue, rather than an uncalcified one. There remains, then, this other hypoth esis which may serve to explain this process of internal décalcification of den tin. The administration of a diet de ficient or improperly balanced in so far as calcium and phosphorus is concerned, resulted in a retardation of the deposi tion of these elements. Owing to the requirements of the growing animal for increasing amounts of calcium and phos phorus, the organism endeavored to meet this demand by robbing its own tissues of previously calcified material. The bones were first affected and were perhaps robbed of as much lime as possible. This source of supply was quickly exhausted, however, and the other tissue in the body capable of supplying the deficiency more readily was the formed dentin. The odontoblasts became odontoclasts and slowly removed the inorganic salts from the walls of the pulp chamber and canal. As this occurred, the cavity became wider and wider, until finally the whole area of dentin was invaded. There are in sup port of this hypothesis only these two facts: first, animals at necropsy ex hibit normal enamel, but decalcified den
tin; second, this decalcified dentin is without the normal structural elements, but contains, instead, abundant evidence of a definite inflammatory reaction. Throughout the tissue there is round cell infiltration, proliferation and engorge ment of blood capillaries. T his inflam mation gives evidence of profound changes occurring in the involved tissue, such as might accompany processes of décalcification. I advance this explanation of these facts as one which more nearly correlates the various observations of the experi ment. It will be extremely interesting to follow this research farther, and to note the effects of such deficient dietaries on pregnant animals, and on the young. Such a program, however, demands more funds than are at my disposal, but it is hoped that additional support will be made immediately available. The direct application of this research to the health of children from birth to maturity is at once apparent, for, in the task of seeking to determine those environmental and nutritional factors which are prejudicial to animals, a wider concept of the prob lems in infant mortality, child welfare, public health, and social service will be attained. B
ib l io g r a p h y
1. S h i p l e y , P. G.: Faulty D iet and Its Relation to the Structure of the Bone T A M. A. 79: 1563 (Nov. 4) 1922. 2. H o w e , P e r c y R.: Décalcification of Teeth and Bones, and Regeneration of Bone Through Diet, J. A. M. A. 79: 1565 (Nov. 4) 1922. 3.
S h ip l e y , P a r k , M c C o l l u m
and
Sim -
4. M c C o l l u m , Sim m o n d s , K in n e y G r ie v e s : Relation of Nutrition to
and
: Studiesin Experimental Rickets, VI, Am. J. Hygiene 1: 512 (July) 1921.
m onds
Tooth Development and Tooth Preservation. I, A Preliminary Study of Gross Maxillary and Dental Defects in Two Hundred and Twenty Rats on Defective and Deficient Diets, Bull. Johns Hopkins Hosp. 33: 216 (June) 1922. 5. S h e r m a n , L. C., a n d S m i t h , S . L. : The Vitamins, Chem. Catalog Co., N Y 1922.
A
T T H E last meeting of the Ameri ample, the deciduous cuspids and molars can Dental Association in Los were, in some instances, retained for a Angeles, I read a preliminary re much longer period of time than was the port on a group of nutrition experiments case in the control animals, and the re conducted under the supervision of W. P. sorption of their roots was very much Lucas, head of the Department of delayed. On examination of the perma Pediatrics, University of California, and nent set, it was observed that some of Dr. M artha Jones, of the George W il them exhibited a marked hypoplasia. One liams Hooper Foundation for Medical lower cuspid, in particular, showed strik Research. At necropsy, the heads of the ing symptoms of arrested growth. D ur animals were given to me for examina ing the earlier stages of calcification of tion and identification of dental lesions. this tooth, the tip of the crown was ap Although complete pathologic data parently normal in development. About have been compiled, these are not ap one-third of the distance from the tip pended here, but will be reported else toward the gingival line, there was a where by Dr. Jones. My interest in definite constriction produced by cessa the heads has been, as I have stated, only tion of function of the ameloblasts, so in so far as the teeth and supporting that the crown presented a definite nar structures are concerned. rowing toward the line of hypoplasia. The data formerly presented dealt Other teeth exhibited a sim ilar stunted mainly with four abnormalities : those re growth. In addition to these evidences lating, first, to the shedding of decidu of improper nutrition, there was a well ous teeth of dogs; second, to the forma defined irregularity in the position of the tion of the permanent teeth; third, to the teeth, particularly the lower incisors. position of the permanent teeth, and In one case, the teeth were clumped to fourth, to the relation in the arch be gether in a bunch, one or two standing tween the deciduous teeth and perma directly lingual to the others in the arch. nent ones. By imposing a restricted diet, Finally, some of the deciduous teeth we were able to produce lesions resem were so firmly attached that even the bling rickets, some of the outstanding eruption of their successors did not force symptoms of which were those referred to the first teeth from their sockets. above, namely, the effects on the position The diet, which was responsible for and the eruption of the teeth. For ex these changes, was not described at that * Aided by grants from the American Dental time, nor will it be now. However, I Association and from the Board of Research, am permitted to state that all the food elements were present in what we believe University of California. Jour. A. D. A., January, 1924
6
Marshall— The Effects of Nutritional Disturbances to be sufficient amounts. The only alteration was concerned with the pro portions of calcium and phosphorus. These two elements exist as calcium
7
phosphate in both bones and teeth, and the quantities fed the animals were ap parently so at variance with the needs of nature that faulty metabolism re sulted. In one set of experiments, the phosphorus was given in excess, and in another, too much lime was administered. Between these two extremes there is an optimum quantity of each element, a quantity which will supply fully all physiologic requirements. Such a
Fig. 1.— Upper and lower jaws of normal animal, which are partially disarticulated to show the osseous development.
Fig. 3.— Opposite view of same side of jaw as shown in Figure 2.
quantity will not be detrimental to the organism as a whole. These elements were, so far as we know, the only variable factors in the whole dietary regimen. Utilizable proteids, as well as vitamins, were given in the form of meat and other fresh foods. The inorganic elements were supplied in the form of the McCol lum salt mixture, except the phosphorus and calcium, which were given in varying quantities as mentioned above. It is hardly necessary for me to call attention to the fact, now well recog Fig. 2.-—Hypoplasia of permanent lower nized, that diet exerts a profound influ cuspid; malposition of permanent lower in ence on the growth and function of bones cisors. and teeth. Although there are many
8
The Journal of the American Dental Association
uncorrelated observations reported which are being compiled which will be re need confirmation, the experiments of ported at a later date. Although the macroscopic abnormali Shipley1, Howe2, McCollum3, Grieves4, ties which have been enumerated are dis tinct evidence of irregularities in calci-
Fig. 6.—Note the hollow enamel caps of first and second maxillary molars, in which normal dentin is entirely absent. Fig. 4.— Abnormal dentition and bone, the result of diet. Note particularly the reten tion of deciduous teeth and the porosity of cortical bone.
fication, it was not realized what pro found changes had occurred in the struc ture of the teeth until a microscopic ex amination of the sections had been made. Sherman5 and many others all point to In one of the animals, the dentin under neath a thin enamel cap had become com this developmental phenomenon. decalcified, and had lost all the This paper is essentially a progress re pletely characteristic structural elements of the port in which certain histologic character-
Fig. 5.— Abnormal dentition and bone, showing the effects produced by dietary in fluences in an upper jaw.
istics are presented. The previous ex Fig. 7.—Maxillary cuspid showing enamel periments are being repeated and ex cap (right) and a fibrous vascular mass tended, and further data on this problem which was found in place of normal dentin.
Marshall— The Effects of Nutritional Disturbances
9
tissue. The separation between the gone profound changes. In its place enamel cap and the underlying dentin there was loose areolar connective tis was easily affected at necropsy, merely sue, richly supplied with fine capillaries by exerting a slight pulling pressure with the fingers on the enamel. This resulted in the complete removal of the enamel in one piece. By this operation, the soft, decalci fied dentin was exposed. There was no normal dentin in the tooth, although the •enamel was intact. The soft tissue underneath the enamel was prepared for
Fig. 8.—Looking down into the enamel cap from the gingival toward the tip of the crown.
sectioning in the usual manner, namely, by immersing in a 10 per cent solution of formaldehyde, then carrying it through various strengths of alcohol up to abso lute, and finally in xylol. N o decalci fying fluid was used at any time. From xylol it was transferred to melted paraf fin, and, when cold, cut into sections for staining and study. On examination, it was found that both the inorganic and organic portions of the dentin had been altered to such an extent as to render their identification impossible. N either dentin tubules nor tubular contents remained. Even the organic matrix, which is very character istic in the decalcified section, had under
Fig. 9.— Photomicrograph of fibrous mass showing, even at a comparatively low magni fication, the inflammatory reactions occurring just before necropsy.
which freely anastomosed throughout the structure. At various places in the sec tion there were inflammatory tissue ele
10
The Journal of the American Dental Association
ments which indicated that active proc dentin, but, from its histologic structure, esses of tissue changes were occurring it resembles an inflamed pulp. The illustrations presented, as well as just before necropsy. These facts throw the specimen which I have demon strated, indicate better than a description 'm .
*
v
jfr
-rs‘Av. - í ■.?Hv ..
.A
'fc
j
'
Fig. 10.—High power photomicrograph of tissue. Note enlarged and engorged capil laries at A. Throughout the area are evi dences of round cell infiltration and of other inflammatory reactions.
*: w f w
* * ' f t **' *
Fig. 12.—Mallory connective tissue stain of tip of specimen, showing odontoclastic layer on both sides, at A ; enlarged capillary at B.
the extent of these lesions. It is discon certing to examine a skull with teeth Fig. 11.— Same as Figure 10. intact, and to discover that under an apparently normal enamel, the dentin, doubt on our classification of this tissue. which we expect to find normal as well, Is it dentin or pulp? From its inti has either undergone processes of ex mate relation to enamel, it is obviously tensive décalcification, or else has not
11
Marshall— The Effects of Nutritional Disturbances been formed at all. I am led to the former conclusion, however, especially when the developmental stages of tooth growth are recalled. This development of enamel from the enamel organ, and dentin from the odontoblastic layer, is a simultaneous occurrence in so far as cal cification of each tissue is concerned. A normal enamel has never been observed to have been formed without an under lying normal dentin. If there had been no dentin formed at any time in these teeth, it is inconceivable that the size and shape of the enamel cap would be normal, or that it would be without some evidence of lack of a supporting calcified structure. I am referring at this moment not to the cases of hypoplasia, but what I believe to be the more advanced con dition, namely, that in which a normal enamel cap overlies a decalcified tissue, rather than an uncalcified one. There remains, then, this other hypoth esis which may serve to explain this process of internal décalcification of den tin. The administration of a diet de ficient or improperly balanced in so far as calcium and phosphorus is concerned, resulted in a retardation of the deposi tion of these elements. Owing to the requirements of the growing animal for increasing amounts of calcium and phos phorus, the organism endeavored to meet this demand by robbing its own tissues of previously calcified material. The bones were first affected and were perhaps robbed of as much lime as possible. This source of supply was quickly exhausted, however, and the other tissue in the body capable of supplying the deficiency more readily was the formed dentin. The odontoblasts became odontoclasts and slowly removed the inorganic salts from the walls of the pulp chamber and canal. As this occurred, the cavity became wider and wider, until finally the whole area of dentin was invaded. There are in sup port of this hypothesis only these two facts: first, animals at necropsy ex hibit normal enamel, but decalcified den
tin; second, this decalcified dentin is without the normal structural elements, but contains, instead, abundant evidence of a definite inflammatory reaction. Throughout the tissue there is round cell infiltration, proliferation and engorge ment of blood capillaries. T his inflam mation gives evidence of profound changes occurring in the involved tissue, such as might accompany processes of décalcification. I advance this explanation of these facts as one which more nearly correlates the various observations of the experi ment. It will be extremely interesting to follow this research farther, and to note the effects of such deficient dietaries on pregnant animals, and on the young. Such a program, however, demands more funds than are at my disposal, but it is hoped that additional support will be made immediately available. The direct application of this research to the health of children from birth to maturity is at once apparent, for, in the task of seeking to determine those environmental and nutritional factors which are prejudicial to animals, a wider concept of the prob lems in infant mortality, child welfare, public health, and social service will be attained. B
ib l io g r a p h y
1. S h i p l e y , P. G.: Faulty D iet and Its Relation to the Structure of the Bone T A M. A. 79: 1563 (Nov. 4) 1922. 2. H o w e , P e r c y R.: Décalcification of Teeth and Bones, and Regeneration of Bone Through Diet, J. A. M. A. 79: 1565 (Nov. 4) 1922. 3.
S h ip l e y , P a r k , M c C o l l u m
and
Sim -
4. M c C o l l u m , Sim m o n d s , K in n e y G r ie v e s : Relation of Nutrition to
and
: Studiesin Experimental Rickets, VI, Am. J. Hygiene 1: 512 (July) 1921.
m onds
Tooth Development and Tooth Preservation. I, A Preliminary Study of Gross Maxillary and Dental Defects in Two Hundred and Twenty Rats on Defective and Deficient Diets, Bull. Johns Hopkins Hosp. 33: 216 (June) 1922. 5. S h e r m a n , L. C., a n d S m i t h , S . L. : The Vitamins, Chem. Catalog Co., N Y 1922.