J. periodont. Res. 5: 191-195. 1970 Dietary influences on the formation of dental calculus in rats TRA L. SHANNON, EDWARD C. CARROLL AND KENNETH O. MADSEN Oral Physiology Research Laboratory, Veterans Administration Hospital, and Department of Biochemistry, University of Texas Dental Branch, Houston, Texas Eleven diets were evaluated for their ability to produce dental calculus in albino rats. A di containing 50 % cornstarch, 32 % nonfat dry milk, 3 % liver powder, 5 % celluflour, 1 % cottonseed oil, $% powdered sucrose, 1 % calcium chloride dihydrate, 2.7% sodium dihydrogen phosphate monohydrate, and 0.3 % magnesium sulfate (Francis and Briner 1969) was remarkably effective. On the basis of a 0-3 calculus scoring system, an average score of 2.36 (S. D. = 0.65) resulted after 4 weeks’ exposure to the above diet and, after 8 weeks, the average score was 2.59 G. D. = 0.56). While it was found that, in general, high cornstarch and milk powder dicts were associated with higher calculus scores, no other diet remotely approached the above diet in calculogenicity. Introduction A reliable method for the production of reproducible amounts of dental calculus in animals has not been available, While sev- eral studies (Baer, Stephan and White 1961, Baer and White 1966a, Baer and White 1966b, Baer ef al. 1967, Baer 1968, Baer, Kyes and White 1969, Baumhammers, Lan- day and Pinkus 1968, Kakahashi, Baer and White 1964, Smith er al. 1963, and Zipkin and McClure 1952) have dealt with dietary induction of calculus in rodents, a singularly superior diet has not been identified. ‘At a meeting of the International Associa- tion for Dental Research a diet was de- scribed that produced a measurable, re- producible amount of calculus in rats (Fran- cis and Briner 1969). Since this diet was superior to 24 other diets tested, it was suggested that other investigators would find it suitable for experimental calculus studies, The present study explores the effect of this diet on the formation of calculus in albino rats. It is compared to other diets, and modifications thereof, that have been offered for this same purpose. Material and Methods In the first experiment 11 different diets were ‘ested over an 8-week period in Sprague-Dawley albino rats, Twelve rats were placed on each diet (ad libitum) at approximately 30 days of age with sexes evenly distributed within each diet group. Initial body weights for the 11 groups ran- ged from 94.2 (SD. = 5.45) to 114.8 (SD. = 10,08). Animals were weighed at weekly intervals and weight gains during the experi- mental period were calculated in grams per week, Supported in part by a grant from the Warner-Lambert Research Institute, Morris Plains, New Jersey.192 Table I shows the composition of the diets. Diet No. 1 (control) was Purina Rat Chow. Diet No. 2 was this control chow made into a mush consistency with tap water and kept damp at all times. This procedure was employed since Baer (1968) had reviewed data from several investigators indicating that the physical consistency of the diet and its effects on the local en- vironment were important. Likewise, Diets No. 3 and 4, each containing 15 % Lagos cocoa hulls, were of interest because of previous studies in cotton rats (unpublished) which suggested that these diets would leave residues in the mouth, Diet No. 3 was a modification of Diet No. 9. The calculogenic diet of primary interest was Diet No. 5 (Francis and Briner 1969). It is high in carbohydrate. Diet No. 6, a high protein diet, has been found to be calculogenic by investigators at SHANNON, CARROLL AND MADSEN the NIDR (Baer and White 1966b, Baer et al, 1967, and Baer ef al. 1969). Diet No. 7, a high fat diet, has been employed by Smith e¢ al. (1963), Kakahashi et al, (1964), Baer and White (1966a), Baer (1968), and Baer et al. (1967). Diet No. 8 is a high carbohydrate diet (Baer er al, 1961, and Baumhammers et al. 1968). Diets No. 9 and 10 are modifications of Diet No. 8 in which different amounts of corn oil were incorporated at the expense of cornstarch and nonfat dry milk. Diet No. 11, also high in carbohydrate, was described by Zipkin and McClure (1952). The salt mixture contained sodium chloride, ferric citrate and copper sulfate. In the second experiment, Diet No. 5 was fed to a group of 12 albino rats for only four weeks to determine the effect of a shorter feeding period on calculus deposi- tion. Table | Experimental diets" Percent Composition by Weight Constituent Diet Number 5 6 Cornstarch Nonfat Dry Milk ‘Whole Milk Powder sucrose** Casein Ground Oats Crisco Cottonseed Oi! Com Oil Celluflour (alphacel)| Lagos Cocoa Hulls Liver Powder (1 :20) Brewer's Yeast Whole Dried Yeast Cod Liver Oil Salts Mb CaCl,» 2420 NataPO, « Hi MgSO. Perr ee Purina rat chow Purina rat chow as a mush pad tft hr BB 188 Phtatteatattea!Barit Phte@tinwatrrrGrBrras brent bight rrr rr Bele Petrrtty tr sere er eye i 5 4 1 2 8. 7 3 reresererrnvaee CIT heer Ie * Fat soluble vitamins; 2 drops weekly per rat. ** Granulated in Diets 4 and 6; powdered in Diet . *** HMW salts mixture in Diets 6 and 7.DIET AND CALCULUS 193 Table Il Weight gain and calculus score after 8 weeks’ exposure to the test diets | - ; Weight Gain Calculus Score wae Prelees oe (gmk) (per tooth surface) Moan 8.0. Mean 8D. 1 | Controt Chow (Purina) 268 ar 0.00 0.00 2. | Control Chow (as mush) 254 70 ou7 039 3 | Cocoa Hulls (high carbohydrate) 73 33 0.62 0.70 4 | Cocoa Hulls (ground cats) 255 6s 0.10 0.30 5 | Francis-Briner (8) (high carbohydrate) 79 4g 259 0.56 68 | High Protein 209 67 86 056 7 | High Fat 28 105 075 0.66 8 | High Carbohydrate (0 % corn oil) 193 54 131 0.63 9 | High Carbonydrate (10 % corn cil) 166 33 oz 0.58 10 igh Carbohydrate (20 % corn cil) a7 24 082 0.60 11 | High Carbohydrate (with Yeast) 135 27 1.19 os At the end of the test period the animals were sacrificed by decapitation, the heads were autoclaved and freed of soft tissue, and calculus deposition was scored using a binocular microscope at 20X. All scoring was done by the same observer (E.C.C.). A buccal surface score and a lingual surface score were recorded for each of the 12 molars in each animal, Thus 24 values were available for each animal. A calculus rating was assigned to each tooth surface as 0, 1, , or 3. A zero score indicated no detectable calculus. If calculus was present but in- volved less than one-fourth of the tooth surface, a value of 1 was assigned. Grade 2 was given when calculus covered one-fourth to one-half of the surface, and a value of 3 was recorded if more than one-half of the surface was involved, The 24 values for each animal were summed and divided by the number of tooth surfaces studied. The final figures were thus based upon the aver- age expression for a single tooth surface. Results Means for weekly weight gain after 8 weeks on the different diets are presented in Table I], With the 24.6 gm/wk gain for the control group taken as 100 %, the range of response was from 113 % for the high-fat diet (No. 7) to 35% for the high-carbohydrate diet with 20 % corn oil (No. 10). Animals on Diet No. 5 over the 8-week period gained weight at a level 73 % of that for the controls. This difference in mean weekly weight gain was significant at the 0.05 level. The initial weight mean for the animals on Diet No. 5 (114.8 grams) was significantly more than that for controls (106.1 grams) but this difference was barely significant at the 0.05 level. Table IH Effect of time of exposure to diet No. 5 on caleulus deposition Calculus Score (per tooth surface) Dental Area 4 Weeks on Diet Weeks on Diet 8.0. 2.45 2.38 059 065 252 © 059 056 Lingual All Areas.194 Statistics derived from calculus scores are also presented in Table II. The five dicts with greatest calculogenicity, Diets No. 5, 8, 11, 9, and 10, were all high in carbohydrate. Of these diets, however, only Diet No. 5 ‘was outstanding in its ability to produce calculus. The mean surface score of 2.59 (SD. = 0.56) resulting from 8 weeks on this diet far exceeded that produced by any other regimen in the study. Table III outlines the production of cal- culus when Diet No. 5 was fed over 4- and 8-week test periods. The calculus score after 8 weeks was only about 10 % greater than after 4 weeks, Discussion All of the high carbohydrate (cornstarch) diets produced smaller weight gains than did the control diets. These diets were all dry, light powders that were readily scat- tered during eating so that food intake rather than nutritional adequacy may have accounted for the poor weight gains. Diet No. 10 was not powdery, however, and was clearly inadequate. In general, better dietary adequacy, as indicated by higher weight gains, was asso- ciated with the lower calculus scores. This indicated that systemic factors, related to dietary inadequacy, could have influenced some of the calculus scores in these studies. On the other hand, high cornstarch diets produced the lowest weight gains and, in general, the highest calculus scores. The high cornstarch diets also contained some- what similar levels of milk powder (22- 32%), so the combination of high corn- starch and milk powder levels was associated with higher calculus production. This was not invariable since the second most cal- culogenic diet (Diet No. 8), while similar in adequacy to Diet No. 5 and also of high comstarch and milk powder content, pro- duced significantly less calculus than did SHANNON, CARROLL AND MADSEN Diet No, 5. Thus, while cornstarch and milk powder at high levels may promote calculus, other features of Diet No. 5 must contribute strongly to its calculogenicity. The addition of 10% (Diet No. 9) or 20 % (Diet No. 10) of oil to the high corn- starch-milk powder diet did not have a significant effect on calculus production. Likewise, in a different basal diet, high fat alone was not associated with calculus production (Diet No. 7). High protein alone (Diet No. 6) was also ineffective. If the 5 % of sucrose in Diet No. 5 were important to its calculogenicity, it would seem to have been so either because of interaction with the rest of the diet (none of the other high cornstarch-milk powder diets contained sucrose) or because it was fed as a powder. Granulated sucrose at higher levels, 13 and 18 % in Diets No. 4 and 6, was not associated with calculus production. No additional information was obtained from the longer test period since the small increment in calculus score provided by the additional 4 weeks was uniformly distributed over each dental area. This indicated that later studies need not involve more than a 4-week experimental period. The calculus scores were high and of similar magnitude for buccal, lingual, maxillary, mandibular, left and right dental areas. This was true when Diet No. 5 was fed for either 4 weeks or 8 weeks (Table III). Rats have a highly alkaline saliva and, if the diet were acting locally, this characteri- stic could be related to the generalized calculus response found with Diet No. 5. As already noted, the major components of Diet No. 5 do not appear to be sufficiently distinctive, when compared to the other diets studied, to account, by themselves, for the high calculus scores associated with this diet. Since Diet No. 5 is distinguished mainly by its content of 4% of a simple, but somewhat unusual, soluble and acidic,DIET AND CALCULUS salts mixture (Table 1), it may be speculated that these salts under alkaline oral condi- tions may be the factor which causes all surfaces to readily accumulate calculus de- posits. Four and 6 % of HMW* salt mix- ture, albeit in the absence of cornstarch and milk powder (Diets No. 6 and 7), were not associated with the higher calculus scores. Interaction of the salt mixture with other components of Diet No. 5 must also be considered important to the calculus pro- duction with this diet and the possibility of systemic effects cannot be discounted. These results confirmed the high calculus response reported by Francis and Briner (1969) for Diet No. 5. It appears desirable, however, to improve on its acceptance by the rats so that the question of dietary adequacy will not complicate its use in the search for anticalculus factors. Our pre- liminary suggestions to account for the cal- culogenicity of Diet No. 5 do not appear to be at variance with the written and oral reports by Francis and Briner (1969). References Baer, P. N., R. M. Stephan and C. L. White. 1961. Studies on experimental calculus forma- tion in the rat. IT. Effect of age, sex, strain, high carbohydrate, high protein diets. J. Periodont. 32: 190-196. Buer, P.N. and C. L. White. 1966a. Studies on experimental calculus formation in the rat. VIII. Effect of function. J. Periodont. 37: 34-35. Baer, P. N. and C. L. White. 1966b. Studies on experimental calculus formation in the rat. IX. The effect of varying the protein and fat content of the diet on calculus deposition and alveolar bone loss. J. Periodont. 37: 113-116. * Hubbel, Mendel, and Wakeman. In Hawk, P. logical Chemistry. 12th Editon, Biakiston, Philadelphia, 1947, p. 1273. Phy 195 Baer, P. N., G. R. Hawkins, H. Wells, N. Mantel, and I. Zipkin. 1967. Studies onex- perimental calculus formation in the rat. XI. Relation to diet and selected salivary con- stituents. J. Periodont. 38: 323-329. Baer, P. N. 1968. Use of laboratory animals for calculus studies. Ann. N.Y. Acad. Sci, 153: 230-239, Baer, P. N., P. H. Keyes, and C. L. White. 1969. Studies on experimental calculus forma- tion in the rat. XIIT. Effect of strain, anti- biotics and foster feeding. J. Periodont. 40: 20-21. Baumhammers, A., M. A. Landay, and D. T. Pinkus. 1968. Dental calculus inhibition in rats through pancreatin applications. Arch. oral, Biol. 13: 353-356. Francis, M. D. and W. W. Briner. 1969. Animal calculus Methods of dietary production and control. Abstracts of Papers, International Association for Dental Research. Abstract No. 193, Kakahashi, S., P. N. Baer and C. White, 1964. Studies on experimental calculus formation in the rat. VIL. Effect of selective desaliva- tion of the major salivary glands. J. Peri- odont. 35: 467-469. Smith, L. W., P. N. Baer, C. T. G. King and C. L. White. 1963. Studies on experimental calculus formation in the rat. TI, Calculus formation as influenced by high fat, high carbohydrate diets, and sucrose in the drink- ing water. J. Periodont. 34: 327-329. Zipkin, I. and F. J. McClure. 1952. Deposition of fluorine in bones and teeth of the growing rat. J. Nutr. 47: 611-62 Address: Oral Physiology Research Laboratory Veterans Administration Hospital 2002 Holcombe Boulevard Houston, Texas 77031 USA Oser, B. L., and Summerson, W. H. PracticalThis document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.