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Effects of chronic protein deficiency on the formation of the rat incisor teeth
Archs oral Bid. Vol 26. pp. Printed in Great Bntam
459 lo 466,
1981
OOO3-9969.81;060459-08$02.00/O Pergamon
Press Ltd
EFFECTS OF CHRONIC PROTEIN DEFICIENCY ON THE FORMATION OF THE RAT INCISOR TEETH P. L. GLICK and DOROTHYJ. ROWE Veterans’ Administration Medical Center, Iowa City, IA 52240, U.S.A. and Dows Institute for Dental Research, The University of Iowa, College of Dentistry, Iowa City, IA 52242, U.S.A.
Summary-Chronic protein deficiency was induced by a 1 per cent lactalbumin diet for 10 weeks. The incisors were smaller than the control animals fed an 18 per cent lactalbumin diet. Likewise the dentine appositional rate, determined by tetracycline labeling, was reduced. The width of the predentine increased, suggesting impairment of mineralization. However, no disturbances in dentine mineralization were observed by microradiography or electron microscopy. The ultrastructure of odontoblasts, predentine and enamel appeared normal. Electron probe microanalysis or mineralizing dentine and enamel revealed no differences between the mineral composition of protein-deficient and control animals. Thus, the effect of protein deficiency on dentine formation seems to be more related to the rate at which predentine mineralizes than to the degree of it; formation and mineralization of enamel and dentine proceeded normally although at a reduced rate. The increased caries susceptibility associated with chronic protein deficiency does not appear to be due to abnormal tooth formation.
INTRODUCTION Protein calorie malnutrition (PCM) is one of the major nutritional problems in the world today. The term PCM encompass#es a wide variety of clinical conditions, ranging from the moderate states of generalized malnutrition to the severe syndromes of marasmus, resulting from both protein and caloric deprivation, and kwashiorkor, resulting from long-term protein restriction (Jelliffe, 1966). Oral examinations of individuals experiencing PCM have shown alterations affecting tooth structure, which is often related to rapid carious destruction (Baume and Meyer, 1966; Sweeney, Saffin and deleon, 1971). On the other hand, laboratory stud& of experimental animals subjected to protein and calorie deficiencies have not revealed any alterations in mineral composition (Navia et al., 1970; Di Orio, Miller and Navia, 1973) or histological appearance of dental tissues (Hunter, 1950), although reductions in tooth size and eruption rates have been consistently reported (Shaw and Griffiths, 1963; Navia et al., 1970, Di Orio et al., 1973). The variety of dietary regimens used to produce PCM in experimental animals has made it difficult to cortelate the observed physiological responses with the distinct features of the specific type of PCM, as observed in man. However, Edozien (1968) described a diet which when fed ad libitum to young growing rats was capable of producing most of the clinical and biochemical features of the human condition of kwashiorkor. Subsequent experiments using this model allowed for con trolled studies of the metabolic effects of experimental protein deficiency in laboratory animals (Enwonwu and Sreebny, 1971; Anthony, 1973; Anthony and Edozien, 1975; Glick and Rowe, 1981). Our objective was to investigate the alterations 459
in tooth formation which result from chronic protein deficiency, using the experimental model simulating human kwashiorkor, and to compare these effects with the changes which have been observed in bone development in rats subjected to the same dietary regimen.
MATERIALS AND METHODS A total of 276 Sprague-Dawley rats 43 days of age were placed either on a protein-deficient (1 per cent lactalbumin) or on an 18 per cent lactalbumin control diet for 10 weeks. Weight gain, food and calorie intake were recorded throughout the experimental period. Determinations were made of serum protein, albumin, calcium and phosphate, as reported previously (Glick and Rowe, 1981). In both the 9th and 10th weeks of the experimental period, 5 protein-deficient and 5 control animals were injected with oxytetracycline (20 mg/kg) and killed 3 days later. Maxillary and mandibular incisors from these animals were removed and prepared for direct radiography, microradiography and fluorescence microscopy. Additional incisors from animals not previously injected with tetracycline were prepared for electron microscopy and electron probe microanalysis. Direct radiography Following the removal of the surrounding tissues, incisors were radiographed using a fine-grain radiograph film (Kodak Industrial Type R.) with a Picker industrial portable X-ray unit at 90 kV and a target distance of 100 cm.
P. L. Glick and Dorothy J. Rowe
460 Histological
procedures
Tissues were fixed in 75 per cent methanol, dehydrated in alcohols, defatted in ether-acetone and embedded in a partially polymerized methyl-methacrylate. Following polymerization, 80 pm thickly ground cross sections of maxillary and mandibular incisors were prepared in a para-sagittal plane. Additional mandibular incisors, 5 each, from proteindeficient and control animals were prepared in a para-sagittal plane extending to the base of the tooth. The ground sections were stained with toluidine blue and basic fuchsin. Microradiographr
Ground sections were radiographed using a Siemens X-ray diffraction generator employing nickelfiltered copper radiation, in a specially designed microradiographic camera allowing intimate contact of the sections with the radiographic emulsion (Kodak 649-O).The microradiographs were developed in Kodak-HRP developer. Fluorescent
microscopy
Cross sections of maxillary and mandibular incisors were used to determine the dentine appositional rate. Such sections were mounted using a non-fluorescent glycerin and observed with a Leitz incident-light fluorescent microscope. From a total of 40 teeth: direct measurements of the distance between the two tetracycline labels on the labial aspect of the teeth were made using an eyepiece reticule. Calibration of the magnification factors allowed for the appositional rate of dentine to be expressed as km dentine formed per day. The actual thickness of the predentine layer was then measured in transmitted light. Electron microscopy
An additional group of 5 control and 5 proteindeficient animals were anaesthetized with Nembutal and perfused through the ascending aorta with a 2.0 per cent solution of glutaraldehyde in 0.1 M sodium cacodylate (pH 7.3) for 15 min. Maxillary incisors were quickly dissected and split in a sagittal plane using a revolving diamond blade cooled with normal saline (Walton and Eisenmann, 1974). The incisors were returned to glutaraldehyde for an additional 1 h period, then washed in 0.1 M cacodylate buffer and post-fixed in 1 per cent osmium tetroxide. Following routine procedures, the undemineralized tissue was embedded in Epon 812. Semi-thin (0.5 pm) survey sections were prepared from the labial aspect of the tooth in a plane parallel to the axis of the odontoblasts and were then stained with toluidine blue. Ultra-thin sections were prepared with a Reichert OMU-2 microtome using a diamond knife. Sections were double-stained with 1 per cent uranyl acetate in pure methanol, followed by lead citrate, and observed in a Siemens 101 electron microscope. Electron probe microanalysis
Twenty mandibular incisors from 5 control and 5 protein-deficient rats were dissected from animals not previously injected with tetracycline. The surrounding soft tissue and alveolar bone were removed and the tissue fixed in 95 per cent methanol. Following dehy-
dration, the incisors were sectioned in a transverse plane in an anatomical location similar to that chosen for fluorescence microscopy (Figs 1 and 2). The incisors were embedded in Buehler epoxide and subsequently polished, using successive grits of silicon-carbide abrasive to a 5 pm diamond abrasive. The polished flat surfaces were then coated with a heavy conductive layer of carbon and examined in an ARLEMX electron probe. Two crystal spectrometers, both using ammonium dihydrogen phosphate crystals, were optimized for analysis of calcium and phosphate. The operating conditions for analysis were 12 kV and 80nA current as established on a Fluorapatite (FAP) reference standard; the beam was defocused to approximately 50 pm. The regions analyzed were the newly formed dentine on the labial surface of the tooth adjacent to the predentine-dentine junction and a region of enamel intermediate between the surface enamel and the dentine-enamel junction. Estimations of calcium and phosphate concentration were made based on the net X-ray counts relative to the FAP reference standards. As the objective of this part of the experiment was to determine relative changes in mineral concentration between control and proteindeficient tissue, the data were not corrected for either mass loss, atomic number, absorption or fluorescent effects (Edie and Glick, 1979, 1980).
RESULTS
At the termination of the experimental period, rats fed the 1 per cent lactalbumin diet developed the clinical features of experimental kwashiokor. Body weight decreased from an initial value of 114 to 69 g, whereas weight of the control animals increased to 381 g. Serum protein and albumin were depressed to approx. 40-50 per cent of that of the control animals and total serum calcium was depressed by 10 per cent (Glick and Rowe, 1981). Gross evaluation of teeth from protein-deficient animals indicated a reduction in size; however, the form of the tooth as well as the characteristic orange iron pigmentation of the enamel were normal. Radiographic examination of the teeth revealed normal features of development, and no alterations of either the form or mineralization pattern were apparent (Figs l-4). Microradiographs of the mandibular incisors revealed no hypoplastic alterations in developing enamel or any abnormal features in the calcification of the dentine. The ultrastructure of odontoblasts from protein-deficient animals did not differ from that of the control group (Fig. 5) and both the development and mineralization of the predentine appeared normal (Fig. 6). The daily appositional rates of the dentine in both mandibular and maxillary incisors were significantly reduced to approximately 32 per cent of that of control animals; however, the thickness of the predentine layer was increased to 135 per cent of that of the control group (Table 1; Figs 7 and 8). Electron probe microanalysis of dentine adjacent to the predentine junction from protein-deficient animals revealed no change in either calcium or phosphorus from that of the control group. Likewise, pre-eruptive
Etrect of low-protein diet on rat incisor
461
Table 1. Effects of protein deficiency on the appositional rate of dentine and the thickness of the predentine Control Appositional rate (pm/day): Mandibular incisors Maxillary incisors Predentine thickness (pm): Mandibular incisors Maxillary incisors
Protein-deficient
t
d,
PG
17.6 + 0.6 23.7 + 1.7
13.1 * 2.2 14.7 + 1.7
3.8 8.0
8 8
0.01 0.001
11.2 + 1.1 13.3 _t 1.7
16.3 + 2.1 16.9 i 1.2
4.8 3.9
8 8
0.01 0.01
enamel from protein-deficient animals was similar to that of the control group (Table 2). DIXUSSION
Rats fed a 1 per cent ‘lactalbumin diet ad libitum for 10 weeks experienced a progressive loss of weight (Glick and Rowe, 1981). The reduced size of the incisors in these animals appeared to be the result of a decreased rate of dentine apposition. This reduction was similar to that observed on the endosteal surfaces of tibiae from rats placed on the same diet (Glick and Rowe, 1981). Although a decreased appositional rate in bone is a common feature of protein deficiency (Himes, 1978), the one study which measured the appositional rate of dentine did not observe a reduction (Pack, 1978). As the rats in that study were fed a 5 per cent protein diet for approx. 1 yr, the degree of dietary restriction was not as severe as our study in which the animals were subject to a 1 per cent lactalbumin diet, consisting of approx. 0.8 per cent protein. Thus, protein deficiency may need to be severe before the dentine appositional rate is affected. The increased width of the predentine layer in the protein-deficient animals may be considered to be an impairment of the normal mineralization process (Schour and Massler, 1949). However, as no disturbances in the mineralization pattern were observed either by microradiography or electron microscopy, the impairment may be more related to the rate at which predentine mineralizes rather than to the completeness of the mineralization process. Although the animals did exhibit a significant (10 per cent) reduction in serum calcium (Glick and Rowe, 1981), the degree of this alteration did not appear to be sufficiently great as to affect mineralization. When either young or adult rats are fed a low-calcium diet, the
only effect on dentine formation is an increase in the predentine width (Ferguson and Hartles, 1964; Rasmussen, 1977). The additional stress of pregnancy of lactation is needed to create hypomineralization (Rasmussen, 1977). The normality of the mineralization process is also indicated by the unaltered mineral content of both the enamel and dentine. Other investigators using chemical analysis found no specific alterations in mineral composition attributable to protein deficiency (Shaw and Griffiths, 1963; Navia et al., 1970; Di Orio et a[., 1973). As chemical analysis reflects the mean mineral composition of dentine and enamel formed both before and during the period of protein deficiency, we used topical electron-probe microanalysis to determine the concentration of calcium and phosphate on the tissue actually formed during the period of protein deficiency and no changes were observed. Incisors from protein-deficient rats exhibited no macroscopic or microscopic abnormalities other than a reduction in size. The odontoblasts showed no changes suggestive of impairment of either collagen synthesis or mineralization. Hunter (1950), investigating the effects of diets deficient in protein and amino acids, also found no microscopic alterations of the adult rat incisors. Other organs, i.e. muscle, spleen, ovary and intestines, on the other hand, showed marked atrophy. He concluded that the dental tissues possess a high priority for the limited supply of amino acids. The relationship between protein deficiency and dental caries remains unclear. In man, Sweeney et al. (1971) described hypoplasia in deciduous incisors which they attributed to chronic neonatal PCM. Baume and Meyer (1966) found an association between severe dietary imbalance during early childhood and structural alterations of both deciduous and
Table 2. Electron probe microanalysis of enamel and dentine from control and protein-deficient animals Control Calcium, wt% Den tine Enamel Phosphate, wt% Dentine Enamel
Protein-deficient
26.4 & 5.5 37.1 + 0.5
26.9 k 1.3 37.2 + 0.8
15.4 + 0.5 18.7 + 0.1
15.4 _t 0.5 18.9 f 0.2
Results based on 20 sections from 5 protein-deficient and 5 control animals. No significant difference between control and experimental teeth.
P. L. Glick and Dorothy J. Rowe
462
permanent teeth. Studies have shown an association between protein deficiency and caries. Protein deficiency in rats, either pregnant or lactating, produces reduced tooth size, delayed tooth eruption and high caries incidence in the offspring (Shaw and Griffiths, 1963; Navia et al., 1970; Di Oro et al.. 1973). Rat molars normally exhibit hypomineralized regions which undergo further mineralization or maturation after the teeth erupt. When salivary function is impaired, the maturation of these regions is disturbed, predisposing the tooth to caries (Francis and Briner, 1966; Speirs, 1967). Protein deficiency adversely affects the development and function of submandibular salivary glands (Menaker and Navia, 1973). Thus, it is possible that the increased caries susceptibility associated with protein deficiency results from reduced salivary function, rather than from abnormal tooth development. Acknowledgements-We gratefully acknowledge the technical assistance of Mr Lee Wicks and Dr Richard Harris. This research was supported by Veterans Administration Medical Research Funds.
REFERENCES Anthony L. E. 1973. Effects of protein calorie malnutrition on drug metabolism in rat liver microsomes. J. Nutr. 103, 81 l-820. Anthony L. E. and Edozien J. C. 1975. Experimental protein and energy deficiencies in the rat. J. Nutr. 105, 63 l-648. Baume L. J. and Meyer J. 1966. Dental dysplasia related to malnutrition, with special references to melanodontia and odontoclasia. J. dent. Res. 45, 726-741. Di Orio L. P., Miller S. A. and Navia J. M. 1973. The separate effects of protein and calorie malnutrition on the development and growth of rat bones and teeth. J. Nutr. 103, 856865. Edie J. W. and Glick P. L. 1979. Irradiation effects in the electron microprobe quantitation of mineralized tissues. J. Microsc. 117, 285-296. Edie J. and Glick P. 1980. Electron irradiation effects in the EPMA quantitation of organic specimens. In: Scanning Electron Microscope 1980/11, pp. 271-284. Scanning Electron Microscope, Inc., AMF O’Hare, II. Edozien J. C. 1968. Experimental kwashiokor and marasmus. Nature 220, 917-919.
Enwonwu C. 0. and Sreebny L. M. 1971. Studies of hepatic lesions of experimental protein calorie malnutrition in rats and immediate effects on refeeding on adequate protein diet, J. Nutr. 101, 501-514. Fereuson H. W. and Hartles R. L. 1964. The effect of vsamin D on the dentine of the incisor teeth and on the alveolar bone on young rats maintained on diets deficient in calcium or phosphorus. Archs. oral Biol. 9, 447460.
Francis M. D. and Briner W. W. 1966. The development and regression of hypomineralized areas of rat molars. Arc/Is orul Biol. 11, 349-354. Click P. L. and Rowe D. J. 1981 Effects of chronic protein deficiency on skeletal development of young rats. C&if: Tissue lnt. 33, 223-231. Himes J. H. 1978. Bone growth and development in protein calorie malnutrition. W/d Rev. Nutr. Diet. 28, 143-187. Hunter H. A. 1950. Hypoproteinemia in relation to the dental tissues. J. dent. Rex 29, 73-86. Jelliffe D. B. 1966. The Assessment of the Nutritional Status of the Community. Monograph Series 53, pp. 179-193. World Health Organization, Geneva. Menaker L. and Navia J. M. 1973. Effect of undernutrition during the perinatal period on caries development in the rat. III. Effects of undernutrition on biochemical parameters in the developing submandibular salivary gland. J. dent. Res. 52, 688-691. Navia J. M., Di Orio L. P., Menaker L. and Miller S. A. 1970. Effects of undernutrition during the perinatal period on caries development in the rat. J. dent. Res. 49, 1091-1098. Pack A. R. C. 1978. Lower incisor tooth development studied in protein-deficient rats with intravital labelling techniques. Archs oral Biol. 23, 1145-l 149. Rasmussen P. 1977. Histological and microradiographic observations on teeth during calcium deprivation in rats. Stand. J. dent. Res. 85, 549-556.
Schour I. and Massler M. 1949. The teeth. In: The Rat in Laboratory Inorstigations (Edited by Farris E. J. and Griffiths J. Q.) Chap. 6, pp. 104-166. Hafner, New York. Shaw J. H. and Griffiths D. 1963. Dental abnormalities in rats attributable to protein deficiency during reproduction. J. Nutr. 80, 123-141. Speirs R. L. 1967. Factors influencing ‘maturation’ of developmental hypomineralized areas in the enamel of rat molars. Curies Res. 1, 15-31. Sweeney E. A., Saffin A. J. and R. de Leon 1971. Linear hypoplasia of deciduous incisor teeth in malnourished children. Am. J. clin. Nutr. 24, 29-31. Walton R. E. and Eisenmann D. R. 1974. Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration. Archs oral Biol. 19, 171-182.
Effect of low-protein
Plates
diet on rat incisor
1 and 2 overleaf
463
464
P. L. Glick and Dorothy J. Rowe
Plate 1. Fig. 1. Radiograph of a maxillary incisor from a control animal. The vertical line indicates the region from which sections were prepared for microscopy. x 2.8 Fig. 2. Radiograph of a maxillary incisor from a protein-deficient animal. The form and mineralization pattern of the tooth appears normal. x 2.8 Fig. 3. Radiograph of a mandibular incisor from a control animal. The vertical line mesial to the first molars indicates the region from which sections were prepared for microscopy. x 2.8 Fig. 4. Radiographs
of a mandibular incisor from a protein-deficient animal. The form and mineralization pattern of the tooth appear normal. x 2.8
Fig. 5. Electron micrograph of an odontoblast from a protein-deficient animal. The organizational pattern of both the endoplasmic reticulum and Golgi apparatus is similar to that of control animals, x 8800 Fig. 6. Electron micrograph of the dentine-predentine junction from a protein-deficient animal, illustrating the pattern of dentine mineralization. The region of initial mineralization, as well as subsequent dentine mineralization, corresponds to that of control animals. x 8800 Plate 2. Fig. 7. Fluorescent micrographs of maxillary incisors of rats fed a protein-deficient (a) or control (b) diet. The decreased distance between the two tetracycline labels, which is observed in the experimental animals, reflects the reduced rate of dentine formation. x 270 Fig. 8. Lightmicrographs of mandibular incisors of rats fed a protein-deficient (a) or control (b) diet. The predentine in the experimental animal is wider than that of the control; no differences are evident in the morphology of the odontoblasts or in mineralization pattern of the predentine. Toluidine blue. x 1600
Effect of low-protein
diet on rat incisor
Plate 1.
465
466
P. L. Glick and Dorothy J. Rowe
Plate 2.
459 lo 466,
1981
OOO3-9969.81;060459-08$02.00/O Pergamon
Press Ltd
EFFECTS OF CHRONIC PROTEIN DEFICIENCY ON THE FORMATION OF THE RAT INCISOR TEETH P. L. GLICK and DOROTHYJ. ROWE Veterans’ Administration Medical Center, Iowa City, IA 52240, U.S.A. and Dows Institute for Dental Research, The University of Iowa, College of Dentistry, Iowa City, IA 52242, U.S.A.
Summary-Chronic protein deficiency was induced by a 1 per cent lactalbumin diet for 10 weeks. The incisors were smaller than the control animals fed an 18 per cent lactalbumin diet. Likewise the dentine appositional rate, determined by tetracycline labeling, was reduced. The width of the predentine increased, suggesting impairment of mineralization. However, no disturbances in dentine mineralization were observed by microradiography or electron microscopy. The ultrastructure of odontoblasts, predentine and enamel appeared normal. Electron probe microanalysis or mineralizing dentine and enamel revealed no differences between the mineral composition of protein-deficient and control animals. Thus, the effect of protein deficiency on dentine formation seems to be more related to the rate at which predentine mineralizes than to the degree of it; formation and mineralization of enamel and dentine proceeded normally although at a reduced rate. The increased caries susceptibility associated with chronic protein deficiency does not appear to be due to abnormal tooth formation.
INTRODUCTION Protein calorie malnutrition (PCM) is one of the major nutritional problems in the world today. The term PCM encompass#es a wide variety of clinical conditions, ranging from the moderate states of generalized malnutrition to the severe syndromes of marasmus, resulting from both protein and caloric deprivation, and kwashiorkor, resulting from long-term protein restriction (Jelliffe, 1966). Oral examinations of individuals experiencing PCM have shown alterations affecting tooth structure, which is often related to rapid carious destruction (Baume and Meyer, 1966; Sweeney, Saffin and deleon, 1971). On the other hand, laboratory stud& of experimental animals subjected to protein and calorie deficiencies have not revealed any alterations in mineral composition (Navia et al., 1970; Di Orio, Miller and Navia, 1973) or histological appearance of dental tissues (Hunter, 1950), although reductions in tooth size and eruption rates have been consistently reported (Shaw and Griffiths, 1963; Navia et al., 1970, Di Orio et al., 1973). The variety of dietary regimens used to produce PCM in experimental animals has made it difficult to cortelate the observed physiological responses with the distinct features of the specific type of PCM, as observed in man. However, Edozien (1968) described a diet which when fed ad libitum to young growing rats was capable of producing most of the clinical and biochemical features of the human condition of kwashiorkor. Subsequent experiments using this model allowed for con trolled studies of the metabolic effects of experimental protein deficiency in laboratory animals (Enwonwu and Sreebny, 1971; Anthony, 1973; Anthony and Edozien, 1975; Glick and Rowe, 1981). Our objective was to investigate the alterations 459
in tooth formation which result from chronic protein deficiency, using the experimental model simulating human kwashiorkor, and to compare these effects with the changes which have been observed in bone development in rats subjected to the same dietary regimen.
MATERIALS AND METHODS A total of 276 Sprague-Dawley rats 43 days of age were placed either on a protein-deficient (1 per cent lactalbumin) or on an 18 per cent lactalbumin control diet for 10 weeks. Weight gain, food and calorie intake were recorded throughout the experimental period. Determinations were made of serum protein, albumin, calcium and phosphate, as reported previously (Glick and Rowe, 1981). In both the 9th and 10th weeks of the experimental period, 5 protein-deficient and 5 control animals were injected with oxytetracycline (20 mg/kg) and killed 3 days later. Maxillary and mandibular incisors from these animals were removed and prepared for direct radiography, microradiography and fluorescence microscopy. Additional incisors from animals not previously injected with tetracycline were prepared for electron microscopy and electron probe microanalysis. Direct radiography Following the removal of the surrounding tissues, incisors were radiographed using a fine-grain radiograph film (Kodak Industrial Type R.) with a Picker industrial portable X-ray unit at 90 kV and a target distance of 100 cm.
P. L. Glick and Dorothy J. Rowe
460 Histological
procedures
Tissues were fixed in 75 per cent methanol, dehydrated in alcohols, defatted in ether-acetone and embedded in a partially polymerized methyl-methacrylate. Following polymerization, 80 pm thickly ground cross sections of maxillary and mandibular incisors were prepared in a para-sagittal plane. Additional mandibular incisors, 5 each, from proteindeficient and control animals were prepared in a para-sagittal plane extending to the base of the tooth. The ground sections were stained with toluidine blue and basic fuchsin. Microradiographr
Ground sections were radiographed using a Siemens X-ray diffraction generator employing nickelfiltered copper radiation, in a specially designed microradiographic camera allowing intimate contact of the sections with the radiographic emulsion (Kodak 649-O).The microradiographs were developed in Kodak-HRP developer. Fluorescent
microscopy
Cross sections of maxillary and mandibular incisors were used to determine the dentine appositional rate. Such sections were mounted using a non-fluorescent glycerin and observed with a Leitz incident-light fluorescent microscope. From a total of 40 teeth: direct measurements of the distance between the two tetracycline labels on the labial aspect of the teeth were made using an eyepiece reticule. Calibration of the magnification factors allowed for the appositional rate of dentine to be expressed as km dentine formed per day. The actual thickness of the predentine layer was then measured in transmitted light. Electron microscopy
An additional group of 5 control and 5 proteindeficient animals were anaesthetized with Nembutal and perfused through the ascending aorta with a 2.0 per cent solution of glutaraldehyde in 0.1 M sodium cacodylate (pH 7.3) for 15 min. Maxillary incisors were quickly dissected and split in a sagittal plane using a revolving diamond blade cooled with normal saline (Walton and Eisenmann, 1974). The incisors were returned to glutaraldehyde for an additional 1 h period, then washed in 0.1 M cacodylate buffer and post-fixed in 1 per cent osmium tetroxide. Following routine procedures, the undemineralized tissue was embedded in Epon 812. Semi-thin (0.5 pm) survey sections were prepared from the labial aspect of the tooth in a plane parallel to the axis of the odontoblasts and were then stained with toluidine blue. Ultra-thin sections were prepared with a Reichert OMU-2 microtome using a diamond knife. Sections were double-stained with 1 per cent uranyl acetate in pure methanol, followed by lead citrate, and observed in a Siemens 101 electron microscope. Electron probe microanalysis
Twenty mandibular incisors from 5 control and 5 protein-deficient rats were dissected from animals not previously injected with tetracycline. The surrounding soft tissue and alveolar bone were removed and the tissue fixed in 95 per cent methanol. Following dehy-
dration, the incisors were sectioned in a transverse plane in an anatomical location similar to that chosen for fluorescence microscopy (Figs 1 and 2). The incisors were embedded in Buehler epoxide and subsequently polished, using successive grits of silicon-carbide abrasive to a 5 pm diamond abrasive. The polished flat surfaces were then coated with a heavy conductive layer of carbon and examined in an ARLEMX electron probe. Two crystal spectrometers, both using ammonium dihydrogen phosphate crystals, were optimized for analysis of calcium and phosphate. The operating conditions for analysis were 12 kV and 80nA current as established on a Fluorapatite (FAP) reference standard; the beam was defocused to approximately 50 pm. The regions analyzed were the newly formed dentine on the labial surface of the tooth adjacent to the predentine-dentine junction and a region of enamel intermediate between the surface enamel and the dentine-enamel junction. Estimations of calcium and phosphate concentration were made based on the net X-ray counts relative to the FAP reference standards. As the objective of this part of the experiment was to determine relative changes in mineral concentration between control and proteindeficient tissue, the data were not corrected for either mass loss, atomic number, absorption or fluorescent effects (Edie and Glick, 1979, 1980).
RESULTS
At the termination of the experimental period, rats fed the 1 per cent lactalbumin diet developed the clinical features of experimental kwashiokor. Body weight decreased from an initial value of 114 to 69 g, whereas weight of the control animals increased to 381 g. Serum protein and albumin were depressed to approx. 40-50 per cent of that of the control animals and total serum calcium was depressed by 10 per cent (Glick and Rowe, 1981). Gross evaluation of teeth from protein-deficient animals indicated a reduction in size; however, the form of the tooth as well as the characteristic orange iron pigmentation of the enamel were normal. Radiographic examination of the teeth revealed normal features of development, and no alterations of either the form or mineralization pattern were apparent (Figs l-4). Microradiographs of the mandibular incisors revealed no hypoplastic alterations in developing enamel or any abnormal features in the calcification of the dentine. The ultrastructure of odontoblasts from protein-deficient animals did not differ from that of the control group (Fig. 5) and both the development and mineralization of the predentine appeared normal (Fig. 6). The daily appositional rates of the dentine in both mandibular and maxillary incisors were significantly reduced to approximately 32 per cent of that of control animals; however, the thickness of the predentine layer was increased to 135 per cent of that of the control group (Table 1; Figs 7 and 8). Electron probe microanalysis of dentine adjacent to the predentine junction from protein-deficient animals revealed no change in either calcium or phosphorus from that of the control group. Likewise, pre-eruptive
Etrect of low-protein diet on rat incisor
461
Table 1. Effects of protein deficiency on the appositional rate of dentine and the thickness of the predentine Control Appositional rate (pm/day): Mandibular incisors Maxillary incisors Predentine thickness (pm): Mandibular incisors Maxillary incisors
Protein-deficient
t
d,
PG
17.6 + 0.6 23.7 + 1.7
13.1 * 2.2 14.7 + 1.7
3.8 8.0
8 8
0.01 0.001
11.2 + 1.1 13.3 _t 1.7
16.3 + 2.1 16.9 i 1.2
4.8 3.9
8 8
0.01 0.01
enamel from protein-deficient animals was similar to that of the control group (Table 2). DIXUSSION
Rats fed a 1 per cent ‘lactalbumin diet ad libitum for 10 weeks experienced a progressive loss of weight (Glick and Rowe, 1981). The reduced size of the incisors in these animals appeared to be the result of a decreased rate of dentine apposition. This reduction was similar to that observed on the endosteal surfaces of tibiae from rats placed on the same diet (Glick and Rowe, 1981). Although a decreased appositional rate in bone is a common feature of protein deficiency (Himes, 1978), the one study which measured the appositional rate of dentine did not observe a reduction (Pack, 1978). As the rats in that study were fed a 5 per cent protein diet for approx. 1 yr, the degree of dietary restriction was not as severe as our study in which the animals were subject to a 1 per cent lactalbumin diet, consisting of approx. 0.8 per cent protein. Thus, protein deficiency may need to be severe before the dentine appositional rate is affected. The increased width of the predentine layer in the protein-deficient animals may be considered to be an impairment of the normal mineralization process (Schour and Massler, 1949). However, as no disturbances in the mineralization pattern were observed either by microradiography or electron microscopy, the impairment may be more related to the rate at which predentine mineralizes rather than to the completeness of the mineralization process. Although the animals did exhibit a significant (10 per cent) reduction in serum calcium (Glick and Rowe, 1981), the degree of this alteration did not appear to be sufficiently great as to affect mineralization. When either young or adult rats are fed a low-calcium diet, the
only effect on dentine formation is an increase in the predentine width (Ferguson and Hartles, 1964; Rasmussen, 1977). The additional stress of pregnancy of lactation is needed to create hypomineralization (Rasmussen, 1977). The normality of the mineralization process is also indicated by the unaltered mineral content of both the enamel and dentine. Other investigators using chemical analysis found no specific alterations in mineral composition attributable to protein deficiency (Shaw and Griffiths, 1963; Navia et al., 1970; Di Orio et a[., 1973). As chemical analysis reflects the mean mineral composition of dentine and enamel formed both before and during the period of protein deficiency, we used topical electron-probe microanalysis to determine the concentration of calcium and phosphate on the tissue actually formed during the period of protein deficiency and no changes were observed. Incisors from protein-deficient rats exhibited no macroscopic or microscopic abnormalities other than a reduction in size. The odontoblasts showed no changes suggestive of impairment of either collagen synthesis or mineralization. Hunter (1950), investigating the effects of diets deficient in protein and amino acids, also found no microscopic alterations of the adult rat incisors. Other organs, i.e. muscle, spleen, ovary and intestines, on the other hand, showed marked atrophy. He concluded that the dental tissues possess a high priority for the limited supply of amino acids. The relationship between protein deficiency and dental caries remains unclear. In man, Sweeney et al. (1971) described hypoplasia in deciduous incisors which they attributed to chronic neonatal PCM. Baume and Meyer (1966) found an association between severe dietary imbalance during early childhood and structural alterations of both deciduous and
Table 2. Electron probe microanalysis of enamel and dentine from control and protein-deficient animals Control Calcium, wt% Den tine Enamel Phosphate, wt% Dentine Enamel
Protein-deficient
26.4 & 5.5 37.1 + 0.5
26.9 k 1.3 37.2 + 0.8
15.4 + 0.5 18.7 + 0.1
15.4 _t 0.5 18.9 f 0.2
Results based on 20 sections from 5 protein-deficient and 5 control animals. No significant difference between control and experimental teeth.
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permanent teeth. Studies have shown an association between protein deficiency and caries. Protein deficiency in rats, either pregnant or lactating, produces reduced tooth size, delayed tooth eruption and high caries incidence in the offspring (Shaw and Griffiths, 1963; Navia et al., 1970; Di Oro et al.. 1973). Rat molars normally exhibit hypomineralized regions which undergo further mineralization or maturation after the teeth erupt. When salivary function is impaired, the maturation of these regions is disturbed, predisposing the tooth to caries (Francis and Briner, 1966; Speirs, 1967). Protein deficiency adversely affects the development and function of submandibular salivary glands (Menaker and Navia, 1973). Thus, it is possible that the increased caries susceptibility associated with protein deficiency results from reduced salivary function, rather than from abnormal tooth development. Acknowledgements-We gratefully acknowledge the technical assistance of Mr Lee Wicks and Dr Richard Harris. This research was supported by Veterans Administration Medical Research Funds.
REFERENCES Anthony L. E. 1973. Effects of protein calorie malnutrition on drug metabolism in rat liver microsomes. J. Nutr. 103, 81 l-820. Anthony L. E. and Edozien J. C. 1975. Experimental protein and energy deficiencies in the rat. J. Nutr. 105, 63 l-648. Baume L. J. and Meyer J. 1966. Dental dysplasia related to malnutrition, with special references to melanodontia and odontoclasia. J. dent. Res. 45, 726-741. Di Orio L. P., Miller S. A. and Navia J. M. 1973. The separate effects of protein and calorie malnutrition on the development and growth of rat bones and teeth. J. Nutr. 103, 856865. Edie J. W. and Glick P. L. 1979. Irradiation effects in the electron microprobe quantitation of mineralized tissues. J. Microsc. 117, 285-296. Edie J. and Glick P. 1980. Electron irradiation effects in the EPMA quantitation of organic specimens. In: Scanning Electron Microscope 1980/11, pp. 271-284. Scanning Electron Microscope, Inc., AMF O’Hare, II. Edozien J. C. 1968. Experimental kwashiokor and marasmus. Nature 220, 917-919.
Enwonwu C. 0. and Sreebny L. M. 1971. Studies of hepatic lesions of experimental protein calorie malnutrition in rats and immediate effects on refeeding on adequate protein diet, J. Nutr. 101, 501-514. Fereuson H. W. and Hartles R. L. 1964. The effect of vsamin D on the dentine of the incisor teeth and on the alveolar bone on young rats maintained on diets deficient in calcium or phosphorus. Archs. oral Biol. 9, 447460.
Francis M. D. and Briner W. W. 1966. The development and regression of hypomineralized areas of rat molars. Arc/Is orul Biol. 11, 349-354. Click P. L. and Rowe D. J. 1981 Effects of chronic protein deficiency on skeletal development of young rats. C&if: Tissue lnt. 33, 223-231. Himes J. H. 1978. Bone growth and development in protein calorie malnutrition. W/d Rev. Nutr. Diet. 28, 143-187. Hunter H. A. 1950. Hypoproteinemia in relation to the dental tissues. J. dent. Rex 29, 73-86. Jelliffe D. B. 1966. The Assessment of the Nutritional Status of the Community. Monograph Series 53, pp. 179-193. World Health Organization, Geneva. Menaker L. and Navia J. M. 1973. Effect of undernutrition during the perinatal period on caries development in the rat. III. Effects of undernutrition on biochemical parameters in the developing submandibular salivary gland. J. dent. Res. 52, 688-691. Navia J. M., Di Orio L. P., Menaker L. and Miller S. A. 1970. Effects of undernutrition during the perinatal period on caries development in the rat. J. dent. Res. 49, 1091-1098. Pack A. R. C. 1978. Lower incisor tooth development studied in protein-deficient rats with intravital labelling techniques. Archs oral Biol. 23, 1145-l 149. Rasmussen P. 1977. Histological and microradiographic observations on teeth during calcium deprivation in rats. Stand. J. dent. Res. 85, 549-556.
Schour I. and Massler M. 1949. The teeth. In: The Rat in Laboratory Inorstigations (Edited by Farris E. J. and Griffiths J. Q.) Chap. 6, pp. 104-166. Hafner, New York. Shaw J. H. and Griffiths D. 1963. Dental abnormalities in rats attributable to protein deficiency during reproduction. J. Nutr. 80, 123-141. Speirs R. L. 1967. Factors influencing ‘maturation’ of developmental hypomineralized areas in the enamel of rat molars. Curies Res. 1, 15-31. Sweeney E. A., Saffin A. J. and R. de Leon 1971. Linear hypoplasia of deciduous incisor teeth in malnourished children. Am. J. clin. Nutr. 24, 29-31. Walton R. E. and Eisenmann D. R. 1974. Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration. Archs oral Biol. 19, 171-182.
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Plate 1. Fig. 1. Radiograph of a maxillary incisor from a control animal. The vertical line indicates the region from which sections were prepared for microscopy. x 2.8 Fig. 2. Radiograph of a maxillary incisor from a protein-deficient animal. The form and mineralization pattern of the tooth appears normal. x 2.8 Fig. 3. Radiograph of a mandibular incisor from a control animal. The vertical line mesial to the first molars indicates the region from which sections were prepared for microscopy. x 2.8 Fig. 4. Radiographs
of a mandibular incisor from a protein-deficient animal. The form and mineralization pattern of the tooth appear normal. x 2.8
Fig. 5. Electron micrograph of an odontoblast from a protein-deficient animal. The organizational pattern of both the endoplasmic reticulum and Golgi apparatus is similar to that of control animals, x 8800 Fig. 6. Electron micrograph of the dentine-predentine junction from a protein-deficient animal, illustrating the pattern of dentine mineralization. The region of initial mineralization, as well as subsequent dentine mineralization, corresponds to that of control animals. x 8800 Plate 2. Fig. 7. Fluorescent micrographs of maxillary incisors of rats fed a protein-deficient (a) or control (b) diet. The decreased distance between the two tetracycline labels, which is observed in the experimental animals, reflects the reduced rate of dentine formation. x 270 Fig. 8. Lightmicrographs of mandibular incisors of rats fed a protein-deficient (a) or control (b) diet. The predentine in the experimental animal is wider than that of the control; no differences are evident in the morphology of the odontoblasts or in mineralization pattern of the predentine. Toluidine blue. x 1600
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