I
EXPERIMENTAL NUTRITION
1
MALNUTRITION AND TOOTH DEVELOPMENT IN THE RAT A low grade protein-calorie deficiency in lactating rats causes a major reduction in the rate of growth of the offspring and a lesser reduction in the development of the incisor and the first molar tooth. No evidence was observed of any interference in mineralization of these developing teeth or of their collagen formation.
Key Words: protein-calorie deficiency, growth, incisor, molar
A moderate level of protein deficiency throughout the reproductive cycle in rats resulted in offspring that grew very slowly and had smaller molars with an altered cuspal pattern, delayed eruption of the third molars and an increased susceptibility to dental caries.’ Later studies showed that the submandibular salivary glands of offspring of similarly malnourished female rats contained lower mean values of protein, ribonucleic acid and deoxyribonucleic acid.2 These changes were demonstrated to be attributable to protein deficiency at the tissue level rather than to a caloric insufficiency. The rate of saliva production was reduced and the overall amount of protein secreted in the saliva was reduced although the actual protein concentration of the saliva was higher than in the control rats3 studied the influence of a Nakamoto et low grade protein deficiency in lactating rats on the development of the incisor and first molars of their offspring in a similar experiment to those conducted earlier as a means to produce aform of protein-energy malnutrition. They studied 32 pregnant rats. The rats’ offspring were randomized at birth and eight were assigned to each dam. The control females were fed a diet containing 25 percent casein. The rats to be malnourished were fed an isoenergetic diet that provided only 6 percent protein with the remainder of the protein replaced by dextrose and dextrin. The pups were weighed at birth 356
NUTRllION REVIEWS VOL. 37. NO. 1 1 NOVEMBER 1979
(Day l),Day 5, Day 10,Day 15 and Day X), and a subgroup was injected with 45Ca at a dose of 100 pCi per 100 g body weight and sacrificed 11/2 hours later. In another subgroup, 45Ca was injected at the beginning of the experiment, followed by sacrifice on Day 5, 10, 15 or 20 to determine the half-life of 45Caas a measure of the rate of resorption. The developing incisor and first molar tooth were carefully dissected from the mandible, weighed and total calcium and 45Ca determined. Body weight differences were already evident by Day 5 and became increasingly striking throughout the experiment. By Day 20 the mean body weight of the offspring from the females fed 6 percent casein was about onethird that of the offspring of the control females fed the 25 percent casein diet (23g versus 60 g; p < 0.001). The authors did not mention any fatalities during this experiment. It is also not stated how many samples were represented by each point in the figures. With 32 females and eight offspring each, however, a maximum of 256 pups was available for the entire experiment. Likewise, it is not specified whether litter size was reduced during the experiment by the sacrifice of rats at various intervals or whether whole litters were sacrificed at each interval. In the former procedure, the stress of malnutrition on the females would be reduced and the effect on the offspring minimized as the number of suckling pups was reduced to fewer than eight. The mean weight of the incisor germs of the malnourished pups was slightly lower than
those of the adequately nourished pups as early as Day 5. The difference continued to widen with increasing age. By Day 20, the mean incisor weight of the malnourished pups was 25 percent less than that of the control group (p < .001). No difference in mean molar weight was observed until Day 10. By Day 20, the mean molar weight for the malnourished pups was 21 percent (p < .001) less than for the pups of the control females. The authors did not say whether the difference between development of the incisor and molar was statistically significant. Obviously both the incisor and first molar tooth were much less severely penalized in their development than the total body weight. The uptake of 45Ca was transiently lower for both the developing incisor and molar on Day 5, but no difference was observed at later intervals. The half-life of 45Ca was slightly longer in the incisors and molars of the malnourished than in the normal pups. The mean calcium contents of the incisors and molars began to be lower in the malnourished group by Day 10 with the depression in calcium content increasing as the experiment progressed. The final calcium contents in the malnourished group were about the same amount lower than in the controls as developing tooth weights were reduced. In other words, in all measures of mineral uptake and turnover in the developing teeth, no evidence of any clearcut influence on calcium metabolism and mineralization was observed. The authors point out that the calcium content of the incisor on Day 1 was ten times greater than that of the first molar. By Day 5, the calcium content of the molar had increased by 30-fold whereas the incisor’s calcium content increased only by a factor of ten. By Day 20, both developing teeth had approximately the same calcium content, indicating that more calcium deposition occurred in the molar postnatally in comparison with the incisor. By some measures, the developing incisor appeared to be penalized slightly more in the malnourished rats by Day 20 than the molar germs. Evidence is not provided to indicate whether this phenomenon was statistically significant. The different life histories and structures of incisors versus molars in rodents set the stage
whereby different influences of stress can be expected. Since the rat’s incisor continues to erupt throughout life to compensate for the loss of the incisal edges during mastication, it has an active germinative center that produces enamel and dentin sufficiently rapidly to cause its eruption at the rate of several millimeters per week. At the same time, the cross section of the incisor and the length above the gingiva increase until the rat reaches its adult weight. Thus throughout the 19-day experimental period here, the incisor is under the stress of malnutrition. In contrast, the rat molar crown is developmentally comparable to all teeth of primates and has a developmental period in the jaw followed by a brief period of eruption into the oral cavity. After eruption and root formation, no further development occurs. Indeed, the tooth decreases in vertical height throughout life as the cusps are worn away by mastication. In this experiment, the first molar crown would have attained its maximal size by about Day 15 and then erupted. Malnutrition causes retardation of eruption rather than any effect on the size of the crown.5 It would be interesting to know how the developing second and third molar germs were being affected by the experimental procedure. Each of the first, second and third molars in the rat develop successively later than the other. Whereas the first molar crown in both the control and malnourished rats likely had erupted or was in a late state of eruption into the oral cavity by Day 20,the third molar crown would still be developing quite actively and would not erupt for another two weeks. Presumably the severity of the deficiency increased as the experimental procedure progressed, if for no other reason than the increasing weight of the litter placed an increasingly greater stress on the dam. Thus it could be expected that the developing second molar and even more the third molar would be increasingly severely penalized than the first molar germ. Evidence that this is true has been published previously using differing criteria. In a similarly designed experiment, Holloway et a1.6 observed greater reductions in the bucco-lingual width and the mesio-distal length of the maxillary and mandibular third molars of the malnourished pups than in the first and second molars. NUJRlJlON REVIEWS VOL. 37. NO. 1 I NOVEMBER 1979
357
In a similar experiment, Nakamoto et al.’ studied 31 pregnant rats. After parturition, the rat pups were redistributed among the dams and the dams divided into two groups fed either the 25 percent or 6 percent protein diets. The pups were sacrificed on the day of birth or on Day 5, 10, 15 or 20. The developing incisors and first molars of the mandibles were collected. These tooth germs were incubated for six hours in an appropriate medium with 14C-prolineto determine the amount of collagen synthesis. Total hydroxyproline and I4C-hydroxyproline were determined after incubation and hydrolysis. Collagen synthesis in the molar germs was approximately 30 percent higher than in incisor germs. On Day 1, the incisor tooth germs contained about four times as much hydroxyproline as the molar tooth germs (p < 0.001). These differences agree with the differences in weights and calcium contents observed in the previous e ~ p e r i m e n tBy . ~ Day 10 up to Day 20,collagen synthesis in the developing teeth was approximately equal except on Day 15, when collagen synthesis was significantly lower in the incisors from the malnourished rats (p < 0.05). The total hydroxyproline contents of both the incisor and molar were lower for the rats from the malnourished females only on the fifth day. Addition of ascorbic acid to the medium did not alter the overall collagen synthesis by the tooth germs. As in the previous experiment with regard to calcium metabolism, synthesis and deposition of the organic matrix does not appear to be impaired sufficiently to interfere with the development of the tooth germs. The authors suggest that the developing molar should be affected more than the incisor since more of the molar is formed during the nursing period. While they state, “The present data indicate that the rate of collagen accumu-
358
NUTRlT/ON REVIEWSIVOL. 37. NO. 1lINOVEMBER 1979
lation based on total hydroxyproline content per day was always less in the molar than in the incisor tooth germs,” this is not evident from their figures and no further evaluation was provided to support this statement. In any case, this would be a reversal from the previous experiment where the developing incisor was possibly more severely affected by the nutritional stress than the molar. These studies represent a refinement to those conducted earlier when the gross observations of smaller molars, later eruption and higher caries susceptibility were reported. The current studies suggest that these results are not attributable to alterations in calcium metabolism, mineralization or collagen formation in the dentinal matrix. 0
1. Nutrition, Tooth Size and Caries Susceptibility. Nutrition Reviews 19: 311-314, 1961 2. Protein Deficiency and Tooth and Salivary Gland Development. Nutrition Reviews 32: 24-27, 1974
3. Malnutrition, Salivary Volume and Protein Concentration. Nutrition Reviews 33: 178-180, 1975 4. T. Nakamoto. H.M. Mallek and S.A. Miller: The Effect of Protein-Energy Malnutrition on the Growth of Tooth Germs in Newborn Rats. J. Dent. Res. 58: 1115-1122, 1979 5. J.H. Shaw and D. Griffiths: Dental Abnormalities in Rats Attributable to Protein Deficiency during Reproduction. J . Nutr. 80: 123141, 1963 6. P.J. Holloway, J.H. Shaw and E.A. Sweeney: Effects of Various Sucrose: Casein Ratios in Purified Diets on the Teeth and Supporting Structures of Rats. Arch. Oral Biol. 3: 185-200, 1961 7. T. Nakamoto, H.M. Mallek and S.A. Miller: In Vitro Collagen Synthesis of Tooth Germs from Newborn Rats with Protein-Energy Malnutrition. J. Dent. Res. 58: 1717-1721, 1979
EXPERIMENTAL NUTRITION
1
MALNUTRITION AND TOOTH DEVELOPMENT IN THE RAT A low grade protein-calorie deficiency in lactating rats causes a major reduction in the rate of growth of the offspring and a lesser reduction in the development of the incisor and the first molar tooth. No evidence was observed of any interference in mineralization of these developing teeth or of their collagen formation.
Key Words: protein-calorie deficiency, growth, incisor, molar
A moderate level of protein deficiency throughout the reproductive cycle in rats resulted in offspring that grew very slowly and had smaller molars with an altered cuspal pattern, delayed eruption of the third molars and an increased susceptibility to dental caries.’ Later studies showed that the submandibular salivary glands of offspring of similarly malnourished female rats contained lower mean values of protein, ribonucleic acid and deoxyribonucleic acid.2 These changes were demonstrated to be attributable to protein deficiency at the tissue level rather than to a caloric insufficiency. The rate of saliva production was reduced and the overall amount of protein secreted in the saliva was reduced although the actual protein concentration of the saliva was higher than in the control rats3 studied the influence of a Nakamoto et low grade protein deficiency in lactating rats on the development of the incisor and first molars of their offspring in a similar experiment to those conducted earlier as a means to produce aform of protein-energy malnutrition. They studied 32 pregnant rats. The rats’ offspring were randomized at birth and eight were assigned to each dam. The control females were fed a diet containing 25 percent casein. The rats to be malnourished were fed an isoenergetic diet that provided only 6 percent protein with the remainder of the protein replaced by dextrose and dextrin. The pups were weighed at birth 356
NUTRllION REVIEWS VOL. 37. NO. 1 1 NOVEMBER 1979
(Day l),Day 5, Day 10,Day 15 and Day X), and a subgroup was injected with 45Ca at a dose of 100 pCi per 100 g body weight and sacrificed 11/2 hours later. In another subgroup, 45Ca was injected at the beginning of the experiment, followed by sacrifice on Day 5, 10, 15 or 20 to determine the half-life of 45Caas a measure of the rate of resorption. The developing incisor and first molar tooth were carefully dissected from the mandible, weighed and total calcium and 45Ca determined. Body weight differences were already evident by Day 5 and became increasingly striking throughout the experiment. By Day 20 the mean body weight of the offspring from the females fed 6 percent casein was about onethird that of the offspring of the control females fed the 25 percent casein diet (23g versus 60 g; p < 0.001). The authors did not mention any fatalities during this experiment. It is also not stated how many samples were represented by each point in the figures. With 32 females and eight offspring each, however, a maximum of 256 pups was available for the entire experiment. Likewise, it is not specified whether litter size was reduced during the experiment by the sacrifice of rats at various intervals or whether whole litters were sacrificed at each interval. In the former procedure, the stress of malnutrition on the females would be reduced and the effect on the offspring minimized as the number of suckling pups was reduced to fewer than eight. The mean weight of the incisor germs of the malnourished pups was slightly lower than
those of the adequately nourished pups as early as Day 5. The difference continued to widen with increasing age. By Day 20, the mean incisor weight of the malnourished pups was 25 percent less than that of the control group (p < .001). No difference in mean molar weight was observed until Day 10. By Day 20, the mean molar weight for the malnourished pups was 21 percent (p < .001) less than for the pups of the control females. The authors did not say whether the difference between development of the incisor and molar was statistically significant. Obviously both the incisor and first molar tooth were much less severely penalized in their development than the total body weight. The uptake of 45Ca was transiently lower for both the developing incisor and molar on Day 5, but no difference was observed at later intervals. The half-life of 45Ca was slightly longer in the incisors and molars of the malnourished than in the normal pups. The mean calcium contents of the incisors and molars began to be lower in the malnourished group by Day 10 with the depression in calcium content increasing as the experiment progressed. The final calcium contents in the malnourished group were about the same amount lower than in the controls as developing tooth weights were reduced. In other words, in all measures of mineral uptake and turnover in the developing teeth, no evidence of any clearcut influence on calcium metabolism and mineralization was observed. The authors point out that the calcium content of the incisor on Day 1 was ten times greater than that of the first molar. By Day 5, the calcium content of the molar had increased by 30-fold whereas the incisor’s calcium content increased only by a factor of ten. By Day 20, both developing teeth had approximately the same calcium content, indicating that more calcium deposition occurred in the molar postnatally in comparison with the incisor. By some measures, the developing incisor appeared to be penalized slightly more in the malnourished rats by Day 20 than the molar germs. Evidence is not provided to indicate whether this phenomenon was statistically significant. The different life histories and structures of incisors versus molars in rodents set the stage
whereby different influences of stress can be expected. Since the rat’s incisor continues to erupt throughout life to compensate for the loss of the incisal edges during mastication, it has an active germinative center that produces enamel and dentin sufficiently rapidly to cause its eruption at the rate of several millimeters per week. At the same time, the cross section of the incisor and the length above the gingiva increase until the rat reaches its adult weight. Thus throughout the 19-day experimental period here, the incisor is under the stress of malnutrition. In contrast, the rat molar crown is developmentally comparable to all teeth of primates and has a developmental period in the jaw followed by a brief period of eruption into the oral cavity. After eruption and root formation, no further development occurs. Indeed, the tooth decreases in vertical height throughout life as the cusps are worn away by mastication. In this experiment, the first molar crown would have attained its maximal size by about Day 15 and then erupted. Malnutrition causes retardation of eruption rather than any effect on the size of the crown.5 It would be interesting to know how the developing second and third molar germs were being affected by the experimental procedure. Each of the first, second and third molars in the rat develop successively later than the other. Whereas the first molar crown in both the control and malnourished rats likely had erupted or was in a late state of eruption into the oral cavity by Day 20,the third molar crown would still be developing quite actively and would not erupt for another two weeks. Presumably the severity of the deficiency increased as the experimental procedure progressed, if for no other reason than the increasing weight of the litter placed an increasingly greater stress on the dam. Thus it could be expected that the developing second molar and even more the third molar would be increasingly severely penalized than the first molar germ. Evidence that this is true has been published previously using differing criteria. In a similarly designed experiment, Holloway et a1.6 observed greater reductions in the bucco-lingual width and the mesio-distal length of the maxillary and mandibular third molars of the malnourished pups than in the first and second molars. NUJRlJlON REVIEWS VOL. 37. NO. 1 I NOVEMBER 1979
357
In a similar experiment, Nakamoto et al.’ studied 31 pregnant rats. After parturition, the rat pups were redistributed among the dams and the dams divided into two groups fed either the 25 percent or 6 percent protein diets. The pups were sacrificed on the day of birth or on Day 5, 10, 15 or 20. The developing incisors and first molars of the mandibles were collected. These tooth germs were incubated for six hours in an appropriate medium with 14C-prolineto determine the amount of collagen synthesis. Total hydroxyproline and I4C-hydroxyproline were determined after incubation and hydrolysis. Collagen synthesis in the molar germs was approximately 30 percent higher than in incisor germs. On Day 1, the incisor tooth germs contained about four times as much hydroxyproline as the molar tooth germs (p < 0.001). These differences agree with the differences in weights and calcium contents observed in the previous e ~ p e r i m e n tBy . ~ Day 10 up to Day 20,collagen synthesis in the developing teeth was approximately equal except on Day 15, when collagen synthesis was significantly lower in the incisors from the malnourished rats (p < 0.05). The total hydroxyproline contents of both the incisor and molar were lower for the rats from the malnourished females only on the fifth day. Addition of ascorbic acid to the medium did not alter the overall collagen synthesis by the tooth germs. As in the previous experiment with regard to calcium metabolism, synthesis and deposition of the organic matrix does not appear to be impaired sufficiently to interfere with the development of the tooth germs. The authors suggest that the developing molar should be affected more than the incisor since more of the molar is formed during the nursing period. While they state, “The present data indicate that the rate of collagen accumu-
358
NUTRlT/ON REVIEWSIVOL. 37. NO. 1lINOVEMBER 1979
lation based on total hydroxyproline content per day was always less in the molar than in the incisor tooth germs,” this is not evident from their figures and no further evaluation was provided to support this statement. In any case, this would be a reversal from the previous experiment where the developing incisor was possibly more severely affected by the nutritional stress than the molar. These studies represent a refinement to those conducted earlier when the gross observations of smaller molars, later eruption and higher caries susceptibility were reported. The current studies suggest that these results are not attributable to alterations in calcium metabolism, mineralization or collagen formation in the dentinal matrix. 0
1. Nutrition, Tooth Size and Caries Susceptibility. Nutrition Reviews 19: 311-314, 1961 2. Protein Deficiency and Tooth and Salivary Gland Development. Nutrition Reviews 32: 24-27, 1974
3. Malnutrition, Salivary Volume and Protein Concentration. Nutrition Reviews 33: 178-180, 1975 4. T. Nakamoto. H.M. Mallek and S.A. Miller: The Effect of Protein-Energy Malnutrition on the Growth of Tooth Germs in Newborn Rats. J. Dent. Res. 58: 1115-1122, 1979 5. J.H. Shaw and D. Griffiths: Dental Abnormalities in Rats Attributable to Protein Deficiency during Reproduction. J . Nutr. 80: 123141, 1963 6. P.J. Holloway, J.H. Shaw and E.A. Sweeney: Effects of Various Sucrose: Casein Ratios in Purified Diets on the Teeth and Supporting Structures of Rats. Arch. Oral Biol. 3: 185-200, 1961 7. T. Nakamoto, H.M. Mallek and S.A. Miller: In Vitro Collagen Synthesis of Tooth Germs from Newborn Rats with Protein-Energy Malnutrition. J. Dent. Res. 58: 1717-1721, 1979
