146 238 __THS A STUDY OF THE EFFECT OF FLUORIDES ON THE DEVELOPMENT OF DENTAL CARIES IN RATS Thesis for the Degree of M. S. WCHIGAN STATE COLLEGE Kenneth jean Olson 1944 . LIBRARY , 3 Michigan State 1' E University F - - gala-g?- - ,- ii: -_ E .0 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJCIRC/DateDuest-p. 15 A STUDY OF THE EFFECT OF FLUORIDES ON THE DEVELOPMENT OF DENTAL CARIES IN RATS by Kenneth Jean Olson A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry l9hh ACKNOWLEDGEMENTS The author wishes to express his sin- cere appreciation and gratitude to Dr. Carl A. Hoppert, Professor of Ohemu istry, for his guidance, cooperation, and friendship., The writer also wishes to extend acknowledgement to hiw wife for her encouragement in the pursuit of higher learning. ,a 3".“- M q (.3 . A - J -' '4. 4&— (I. La‘SLO‘THA. ( TABLE OF CONTENTS TABLE or oonranrs INTRODUCTION HISTORICAL EXPERIMENTAL Group I Group II Group III Group IV Group V Group VI and VII TABLE OF CARIES DEVELOPMENT ANALYSIS OF DENTAL AND SKELETAL TISSUES Method Table of Analytical Data DISCUSSION CONCLUSIONS BIBLIOGRAPHY \OWNQUH 10 11 16 19 an 26 28 IN RODUC TI ON INTRODUCTION Why do teeth decay? A simple question indeed. Although our knowledge of the factors involved is considerable, the complete answer still lies within the realms of the unknown.‘ Since time immemorial, man has been confronted with the prob- lem of dental caries, yet today, in a world of scientific thought, we must still rely on the technique of repair. If teeth decay,-have them filled, if they ache,-pull them out, when they are gone,-do without, but see your dentist twice a year~~yet, why do teeth decay? A more ideal approach to the problem of dental caries is one of prevention. Obviously, however, any attempt to study human beings where diet and other factors must be controlled, presents difficulties and complications often times insur- mountable. In many cases where physiological reaction is comparable, experimental animals may be successfully substi- tuted for humans. In the study of tooth decay, it should be pointed out that rat caries and human caries are, perhaps, not entirely similar. For the most part, molar lesions in the rat resulting from a coarse grain diet are lesions of the occlusal surfaces (1). Smooth surface lesions such as proxi- mal, buccal, and lingual lesions (1) are not found in rat mo- lars. Consequently, these animals are of no help in studying inhibitory effects on smooth surface caries. From observa- tions made in humans, there is evidence that fluoride inhibi- tion is greater relative to the smooth surface caries than in pit and fissure lesions (1). In view of these tendencies, it -2- seems likely that by controlling the occlusal caries, the smooth surface caries would also respond. Because of its comparatively short life span, the al- bino rat is well suited for studying slowly developing phe- nomena such as dental caries. The period for producing se- vere caries with the common strain of albino rats varies considerably, two to four months being required as a rule. Fortunately, as a result of a genetic study of rat caries by Hunt and prpert, a susceptible strain of animals has been ’made available which appreciably shortens the eXperimental period (2). A fairly homozygous strain of susceptible rats has been developed in which cavities are produced within 20 to 30 days after the animals are pum on a caries producing diet. In view of the demonstrated influence of fluorides on tooth develoPment and decay and the availability of an un- usual caries susceptible type of rats, a study of the effect of t0pical applications of fluorides to the teeth of such rats as well as a dietary study of fluorides was undertaken. HI STORICAL -3- HISTORICAL A survey of dental history reveals that at some time or other, almost every element known to be present in dental tissue has been suspected.of bearing a relationship to dental caries. Some attributed the phenomenon to an excess of the element involved whereas others ascribed it to a deficiency. Relative to fluorine, some of the more pronounced ef- fects were observed and described before the etiological agent was known. In 1916, MdKay and Blaflk described the hy- poplastic nature of mottled teeth and studied degrees of its severity (5). Later, by feeding various levels of potassium fluoride, Cheyne produced graded mottling in rat molars (4). Previous to Cheyne, Smith, Lance and Smith, and Churchill, working independently, showed a similar effect caused during the calcification process of the tooth by the incorporation of excessive amounts of fluorides in drinking water (1). Cox, Matuschak, Dixon and Walker reported a milky appearance in rat molars fed varying levels of fluorine during the suck- ling period (5). These studies established a relationship between fluo- rine intake and the appearance of dental tissue in general and suggested a possible tiedup of fluorine to tooth decay. Commercial casein was found to contain 0.2% fluorine and when used in a diet for rats produced fluorosis (6). Inde- pendently of this work, commercial casein was shown to have protecting effects against exPerimental caries in rats (7). .4- Probably the first substantial evidence relating Optimal fluorine intake to caries resistance was furnished by Arm- strong and Armstrong and Breckus, who demonstrated the pres- ence of significantly more fluorine in the enamel of sound teeth than in the undamaged enamel of carious teeth. Subsequently, considerable research.has been done in an effort to relate fluorine to the prevention of dental caries. From.the standpoint of systemic and metabolic effects, mcLendon and Foster observed that the addition of small amounts of fluo- rine to the drinking water considerably delayed the onset of caries. The cracked corn diet used in this experiment, howe ever, contained .5 ppm fluorine (8). Miller showed a reduc- tion in the incidence of carious lesions in the molar teeth of rats by adding either NaF, CaFa or iodoacetic acid to the diet (9). Similar results were observed by McClure and Arnold who speculated that the mode of action might be due to anti- enzymatic action, antibacterial activity, or a modified saliva resulting from a fluoride diet. Prior to receiving a caries producing diet, McClure placed rats aged 40 days, 100 days, and 200 days on a fluoride sup- plemented ration. From this work, there appeared.favorable evidence that this pre-fluoride period affected a caries re- sistance in the rat teeth. Analyses of these teeth show an increase in both enamel and dentine fluoride. (McClure states that in light of these facts human teeth might be affected in a similar manner (10). A recent approach to the study of dental caries in rats -5- was made by Sognnaes in his work on the tOpical application of fluorides (1). Three groups of rats were placed on a caries producing diet, two of which received treatment and one served as control. Of the treated groups, one received tOpical applications beginning early in the eXperiment and continuing at two week intervals whereas the other group re- ceived treatment during the middle and latter parts of the experiment only. Extreme care was exercised to eliminate the possibility of effects due to ingested fluorine. In each treatment, the animal was anaesthetised, the lower mo- lars cleaned, dried, and swabbed with fluoride solution. In general, the control group showed twice the number of af- fected teeth per rat than the group receiving the treatment throughout the experiment. The contrast was less noticeable in the other grOUp, however. WOrk of the above nature supports the hypothesis that fluorides are effective in the prevention of dental caries and has established a basis for the subject of this thesis. EXPERIMENTAL -6- EXPERIMENTAL As previously mentioned, the susceptible strain of rats developed by Hunt and HOppert (a) was used throughout these eXperiments. Due to the varying degree of susceptibility of different families of these rats, it will be necessary to de- fine this factor relative to each individual eXperiment. It is of major importance that the relative susceptibility be born in mind when making comparisons of results between fami- lies. Because of this varying susceptibility, litter mates only were used as control animals. Besides stock animals, rats of varying degrees of sus- ceptibility were used. The more susceptible animals develOp definite caries after fifteen to twentybone days on the coarse rice caries producing diet (11) whereas the less sus- ceptible ones require 55 to 66 days under identical condi- tions to show comparable caries. It is well to mention at this point that after caries is initiated in the susceptible animal, large cavities appear much more rapidly than in the case of stock rats. Animals of these two types as well as stock animals will be referred to throughout the following groups of experiments. In several cases, t0pical application of fluoride solup tions has been the basis for dental treatment. In such cases, only the lower molars were treated as the uppers seldom de- cay (11). The solutions were applied a drOp to each side with a small round brush while an assistant eXposed the teeth by manipulating a nasal speculum in the oral cavity. Rats treated -7- daily become very gentle as a result of thoughtful handling. At two week intervals by a similar technique, the teeth were inspected and carious lesions were recorded after the follow- ing manner: fi/‘j l’ // H/ ,‘5/ //’/(/~k ”2/, J R L Such data gives information relative to the initiation time of caries which has proved to be significant Upon several oc- casions. ‘BEPERIMENTAL GROUP I Four cages of three animals each were started shortly after weaning on the coarse rice caries producing diet and maintained on it until sacrificed. Each cage contained two animals of the more susceptible and of the less susceptible type. Rats l, 2 and 3 served as controls. Rats 4, 5 and 6 received t0pical application daily of a 1% solution of NaF at a neutral pH. Rats 10, 11 and 12 received the same treat- ment with the fluoride solution maintained at a pH of a with 301. Animals 15, 14 and 15 received the solution established at a pH of 9 with NaOH. The relative time of induced caries along with cavity deveIOpment are indicated in the accompany- ing table. It is significant to note that no perceptable de- lay in caries initiation and develOpment was imparted to the more susceptible animals by tOpical treatment with fluoride Experimental Group I. Photograph showing caries after 170 days of daily treatment. (3) control, (5) tOpical NaF soln., pH 7, (l2) tOpical NaF soln., pH 6, (15) topical NaF soln., pH 9. These are less susceptible animals. EXperimental Group II. Photograph _ showing caries after 150 days of topi— cal daily treatment with NaF soln., pH 7. (55 & 34) controls, (55) treat- ed. These are less susceptible ani- mals. -9- solutions. Although little difference can be seen in the photograph between numbers 5, 5 and 12, the table shows con- siderable delay in caries initiation and development. No decay appeared in animal number 15 as a result of the basic (fluoride treatment. However, it must be mentioned that upon sacrifice of the animal the upper incisors had grown somewhat askew. This malformation may have impaired the normal functioning of the molars although the animal appeared normal in every other respect. I EXPERIMENTAL GROUP II Four animals of the less susceptible series were placed on the caries producing diet shortly after weaning. Two of these four litter mates were treated daily with a 1% NaF so- lution. The other two served as controls. The caries de- welOpment of the control animals compares very favorably with that of the control animal of the same type in Group I. Animal 56 was discarded after 85 days due to apparent res- piratory ailment. At this time, however, no decay was notice- able in the treated lower molars. After 115 days the teeth of animal 55 were caries free. These marked differences can be observed in the accompanying photograph. EXPERIMENTAL GROUP III In this eXperiment, an attempt was made to study the ef- fects of basic NaF solution applied t0pica11y to the lower mo- lars when the animals were given a period of one week treat- ment before being subjected to the caries producing diet. The treatments were made daily and other general procedure was the -10- same as in experimental Group I. It was thought that per- haps resistance could be enhanced by starting the fluoride applications seven days prior to feeding the coarse rice diet. The data in the table for Group II, however, shows no appreciable difference from that in Group I. Six ani- mals of the less susceptible strain were chosen for the experiment, being started shortly after weaning. The three control animals compare favorably with the corresponding animals in Groups I and II and animals 16 and 18 ShOW’COD- siderable delay in caries formation. more will be said about animal 17 in the later discussion of results. EXPERIMENTAL GROUP IV This eXperiment comprised a study of the effect of t0pical fluoride applications to the lower molars of stock albino rats. The table for this group shows that although caries initiation occurred at approximately the same time as it did in the less susceptible series, the development of moderate and severe cavities was considerably slower. Four cages of these animals were placed on the coarse rice diet and treated as follows: Cage 1, animals number 43, 45, 46 and 47 (44 escaped), controls; Cage 2, animals number 48, 49, 50, 51 and 52, basic solution of 1% NaF topical treatment weekly; Cage 3, animals number 53, 54, 55, 56 and 57, neutral solution of 1% NaF topical treat- ment daily; Cage 4, animals number 58, 59, 60, 61 and 62, basic solution of 1% NaF tOpical treatment daily. Perhaps the most significant observation made in this experiment -11- is that caries in the treated animal deve10ps somewhat more slowly after initiation. The weekly application apparently helped very little in caries prevention although in spite of the limited number of animals involved the ratio to the daily treated animals is surprisingly proportional. EXPERIMENTAL GROUP V Because of the recognized importance of prenatal care, this eXperiment was set up to determine whether the addition of fluoride to the diet of the maternal organism might in- fluence the deve10pment of caries in the offspring. In view of the fact that apposition of primary dentin and enamel be- gins after 20 days in utero (12), it seemed reasonable to expect that rats might benefit by the use of a fluoride supplement during their prenatal deve10pment. Accordingly, a mother of the more susceptible strain was placed on the fine rice diet (11), containing 250 ppm NaF, at first signs of pregnancy and maintained on it until the young animals were weaned. The offspring were then transferred to the coarse diet, maintaining the same level of NaF, and were also given daily t0pica1 applications with.neutra1 1% NaF solution. Unfortunately, due to the small litter, only two animals received treatment, the experiment having but one control which received the fluoride*diet but not the tOpi- cal treatment. With reference to the table and photograph, the treated animals bear the numbers 25 and 26 and the con- trol animal number 27. It is significant to note that caries deve10pment was practically negligible in all of the .25. 26 , 27 ” imperiousntal Group V. The mother of these animals received 250 ppm.NaF in her diet from early pregnancy and the young were con- tinued on the same level until sacrificed. 25 and 26 also received daily topical ap- plications of NaF soln., pH 9. The picture below shows the ingrowing upper incisors of these animals due to the grind- ing off of the opposing lowers. Note the caries free molars after 120 days. These animals were of the more susceptible series. -13- rats. Birth, lactation and care of the young were normal on this level of fluorine. The treated animals showed normal growth and physical deve10pment throughout the ex- periment except for extreme fluorosis of the incisors. The lowers, apparently, became soft, the tips fractured and, as a result, stOpped the normal attrition and grinding with the uppers. The photograph of numbers 25 and 26 reveals a complete circle formation of the upper incisors, which in certain cases resulted in abcessed gums. All teeth were extremely white from the earliest stages of visible de- velopment. The results are impressive because these ani- mals were from a highly susceptible strain (ls-21 days). Obviously, the curved upper incisors became functionally abnormal but this point was not reached until sometime past the period for severe caries formation. EXPERIMENTAL GROUPS VI Am) VII. It was thought better to discuss these two groups to- gether in view of the fact that the main difference between them lies in the prenatal administration of fluoride. The mother of the animals in Group VI received 125 ppm.NaF in her diet and the young were maintained on the same level until weaned. The rats used in Group VII received fluoride only through treatment. Animals 67-69 served as controls in Group VI while, shortly after weaning, numbers 63-66 received daily topical application of 1% NaF solution made basic with 1% Naficoa. From the photographs, very little difference can be observed 67 68 50 EXperimental Group VI. (above) controls, (below) molars receiving tOpical application daily of NaF solution made basic with NaHCOa. The mother received 125 ppm.NaF in her diet and the young were maintained on the same level until weaned. These more susceptible animals were sacrificed after 90 days of treat- ment. EXperimental Group VII. (72 & 73) controls. (70 & 71)“ topically treated animals daily with.NaF solution made basic with.NaHCOa. The eXperimental period of these animals was 90 days. Note no appreciable difference in controls and treated animals. These are more suscepti- ble teeth and may be compared with numbers 63-69 above to show effects of prenatal fluoride feeding at the spe- cified level. -15- between the treated and control animals, although the caries deve10pment table shows some delay in cavity initiation and enlargement. In Group VII rats 72 and 73 represent the controls and numbers 70 and 71 received parallel treatment with those in Group VI. Here again neither the picture nor the table re- veals any prevention of caries formation by treatment. It must be remembered that in cases involving more susceptible animals, topical applications of fluoride have proved of no avail. However, when fluoride supplementation of the diet was imposed during prenatal life, carious le- sions were inhibited to a marked extent. For general com- parison, animals 70-73 of Group VII with animals 1, 2, 4, 6, 10, ll, 13 and 14 of Group I may be contrasted with numbers 25, 26 and 27 of Group V and numbers 63 through 69 of Group VI. These are all essentially homozygous sus- ceptible rats and not in a single case has an inconsistency occurred to cast doubt on the tenet that prenatal fluoride feeding affords marked protection against the deve10pment of tooth decay in rats. -l6- TABLE OF CARIES DEVELOPMENT IN DAYS Emperimgntal Group I Animal Initial moderate Severe Number Animal Treatment R L R L R L Killed 1 ms. control”) 17 21 27 2'7 54 54 54 2 m;s. ' 21 21 27 27 54 54 54 3 L.S. ' 55 55 75 75 100 100 170 4 (151.3.) tOpical NaF, pH 7,daily 15 15 27 27 54 54 54 6 (M.S.) tOpical NaF, pH 7,daily' 21 21. 27 27 54 54 54 5 (L.s.) tOpical NaF, pH 7,daily 150 60 170 150 -— 170 170 10 (M.S.) topical NaF, pH 6,daily 21 25 27 3O 54 54 54 11 (M.S.) topical NaF, pH 6 ,daily 19 19 27 27 54 54 54 12 (L.S.) topical NaF, pH 6,daily 7O 70 150 150 -- 170 170 l5 (M.S.) tOpical NaF, pH 9,daily 17 21 27 27 54 54 54 14 (M.S.) tOpical NaF, pH 9,daily 14 21 27 27 54 54 54 15 (L.S.) tepical NaF, pH 9,daily none none none 170 _¢f Egperimental Group II __#: 33 L.S. controls 65 6O 90 80 106 95 120 34 L.S. ' 54 7O 80 90 106 -» 120 35 (L.S.) tOpical NaF, pH 7,daily 120 120 -- -- ~-- —~ 120 36 (L.S.) topical NaF, pH 7,daily - --- -~ ~- --- -- 85 (l) M.S. and L.S. mean more susceptible reapectively. and less susceptible -17- Experimental Group III J Animal Initial Mbderate Severe Number Animalfigreatment R L R A, L, R L Killed 19 L.S. controls 60' 52 72 60 9O 72 150 20 L.S. ' 52 52 72 72 9O 90 150 21 L.S. ' 85 110 110 140 140 -- 150 16 (L.S.) topical NaF, pH 9,daily -- 140 --~ 150 --- -- 150 17 (L.S.) topical NaF, pH 9,daily 60 60 8O 80 100 100 150 18 (L.S.) tOpical NaF, pH 9,daily -- -— ~~ -- ------ ~150 Experimental Group IV 43 (Stock)control 6O 60 74 74 none 110 45 ' V 90 90 106 106 ' 110 46 " ' 90 90 106 106 ' 110 47 ' 7 60 9O 90 106 ' 110 48 tOpical NaF, pH 9, weekly 60 none none none ' 110 49 topical NaF, pH 9, weekly 75 90 90 106 V 110 50 t0pical NaF, pH 9, weekly 90 90 none 106 3 110 51 topical NaF, pH 9, weekly none none none none ” 110 52 tOpical NaF, pH 9, weekly 90 90 106 none ” 110 53 topical NaF, pH 7, daily 106 106 none none " 110 54 topical NaF, pH 7, daily none none ' 7 ' 110 55 topical NaF, pH 7, daily ' ' 9 ' V 110 56 topical NaF, pH 7, daily 106 none ' fl " 110 57 tOpical NaF, pH 7, daily 90 V 106 ' ' 110 58 tepical NaF, pH 9, daily 60 60 none none ' 110 59 tOpical NaF, pH 9, daily none none ' fl 7 110 60 tOpical NaF, pH 9, daily 9 ' ' T 3 110 -13- Experimental Group IV (cont'd) Animal Initial Mbderate Severe Number gAnimal Treatment R L R ‘L R L Killed_ 61 tOpical NaF, pH 9, daily 106 106 none none none none 110 62 tOpical NaF, pH 9, daily 106 none I ' ' ” 110 27 (11.3.) 250 ppm, NaF in diet pre- natal through eXperiment none none none 120 25 (M.S.) same as 27 plus tOpical NaF, pH 9, daily 3 fl 1 120 26 (M.S.) same as 25 fl ' ' 120 EXperimental Group VI 67 (M.S.) 125 ppm Naf 60 60 90 75 .- 90 90 in diet pre- 68 (M.S.) natal until 45 45 6O 60 so so so beginning of 69 (M.S.) treatment 60 none none none none none 90 63 §M.S.§ same as 75 '75 90 90 none none 90 64 M.S. control plus75 75 9O 9O ' ' 90 65 II.S. topical NaF:none 75 none 90 9 9 90 basic with 1% NaHCOa 66 (M.S.) daily 75 75 90 90 3 3 90 Experimental Group VII 72 n;s. control 30 3O 45 45 9O 73 11.8. " 3O 3O 45 45 90 7O M.S. topical NaF 45 45 60 90 basic with 1% NaHCOs 71 (11.5.) daily so 50 45 45 90 ANALYSIS OF DENTAL AND SKELETAL TISSUES -19- ANADYSIS OF DENTAL AND SKELETAL TISSUES In order to gain information relative to fluorine dis- tribution in bone and dental tissue, analyses were made wherever possible. The recording and explanation of this data appears at a later point in the thesis. For the benefit of future workers, it was thought worthwhile to discuss somewhat in detail a few of the prob~ lems involved in fluorine determination. Since the original method of Willard and Winter (13) several modifications have appeared in the literature. Several weeks were spent with little success in trying to obtain acceptable results by the method of Armstrong (1“). This procedure was chosen for initial investigation because no ashing appeared to be neces- sary, thus eliminating a somewhat variable step. The Arn- strong method was carried out as follows: A weighed sample was introduced into a distillation flask and the fluorine distilled over as hydrofluosilic acid, dilute NaOH being ad- ded to keep the distillate alkaline. 60$ perchloric acid di- luted to the boiling point of 140 degrees 0. acted as an oxi- dining medium in the distilland. Acid washed and ignited quarts was introduced to furnish the silica and a small amount of sodium perchlorate inhibited the distillation of perchlor- ates which, when in excess, interfere with the titration. The temperature of 140 degrees 0. was maintained throughout the 150 ml. distillation by the introduction of water by means of a capillary tube sealed to a separatory funnel. This Opera- tion proved to be tedious and difficult. The 150 ml. distillate -20 .. was then concentrated to a point where it could be quanti— tatively diluted to 10 ml. One milliliter aliquots were then used for titration. The aliquots were introduced into a small vial (about 14.5 by l.’+ cm.) buffered with a drop of solution and titrated to the pink end point of sodium ali~ sarin sulphonate with 0.000“ N thorium nitrate. A micro burst was used for this purpose and read to the nearest 0.005 ml. A very fine titration curve bearing a straight line re- lationship between gammas of fluoride and mls. of titer was obtained when known amounts of NaF were used, but actual sam- ples of bone tissue presented almost insurmountable difficul~ ties. The distillate contained a portion of lipid material which when concentrated to the necessary point made quantita» tive transfer to the 10 ml. volumetric flask quite impractical. The titration vessels were tiny and color matching was diffi- cult, which all resulted in variable results. It is quite obs vious that titer additions to the transferred aliquot of such small magnitude would make a large percentage difference in the total volume. Ordinarily, this factor would probably be neg» ligible but where an adsorption indicator is involved, it seems reasonable that drastic changes in the ratio of constituents involved in the solution might have an appreciable affect upon the end point. In view of the favorable results reported by Armstrong such difficulties were, apparently, overcome by the author. At any rate, this method had to be abandoned, although the experience gained proved valuable. Further work, with the method of Hoskins and Ferris (15), -21- proved satisfactory so that this method was adopted. The de- tailed method follows: Upon sacrifice of animals, the femurs and teeth were re— moved and.placed in 95% alcohol for a day. The teeth were then carefully removed from the mandibles and supporting tisa sues and pooled. In all cases, the upper molars, lower men lars, upper incisors, lower incisors and femurs were analysed separately. Great care was taken at this point to save all parts of the teeth in view of Perry and Armstrong's observa— tion of differences in fluorine content between various parts of the tooth (16). It was observed by McClure that the flucr~ ine content in enamel and dentine increases and decreases pro- portionally which is the reason for analysing whole teeth in an effort to establish general relative distribution (17). The pooled samples were ground.in an agate mortar, passed through a 50 mesh sieve and remixed in the mortar. This methp od removes tendon and fleshy tissue from femurs and yields a fine homogenous sample for analysis. The material was then ether extracted for 16-20 hours, dried for an hour at 110 de- grees 0., and weighed to the third significant figure (also third decimal place) in a silica crucible. The samples were then ashed in a muffle furnace at 660 degrees for forty min- utes which allows for about 96% recovery of the element. Fluoride recovery ranges from 86% to 97% between the tempera» tures 520 and 720 degrees centigrade (15) which necessitates accurate control of the temperature during aching. The ashed tissue was then transferred to a 250 m1. Olaissen type dis- tillation flask and washed down with 110 ml. 50% sulphuric -22- acid which had been previously boiled in the concentrated state. This eliminates an unexplained factor which makes for high results in the titration. To prevent excessive etching of the flask, readily accessible silica was fur- nished by a small amount of powdered porous plate. In order to maintain the most efficient temperature of 140 degrees 0., modified steam distillation was used. A steam inlet was in- troduced into the distalland and by controlling two burners, one under the steam flask and one under the Olaissen flask, the desired temperature of the distilland was easily main~ taineeathroughout the 200 m1. distillation. The distillate was then diluted to 250 ml. in a volumetric flask prior to titration. Twenty-five milliliter aliquots were introduced into a 125 ml. Erlenmeyer along with an equal amount of 95% ethyl alcohol, .2 m1. of 0.05% sodium alisarin sulfonate in— dicator and 5 ml. of buffer. The indicator is of adsorp- tive nature and according to Hoskins and Ferris, its consis- [tent behavior is a function of the concentration of all con- stituents present in the solution including the relative pro- portions of the buffer. The buffer is made up of monochlor- acetic acid and NaOH which must bear a molecular ratio of 0.5 and be present in the titration sample at about 0.2 total molarity. In view of the relatively large volume used for analysis, the addition of 0.3 to 2 mls. of titer does not efe fect an appreciable volume change. This larger volume seemed to be an advantage over the method of Armstrong. The titration is based on the following reaction: Th plus 4?" yields ThFu. 0.001 n thorium nitrate was used for -25- the titration and the end point was indicated by a color change from yellow to a faint pink. A standard comparison color was used in all cases. The final answer in filfluoride is significant in the fourth decimal place and seldom varied in triplicate ali~ quote in the third. It will be noticed in the table of analytical data that in many cases, fluoride differences ap— pear in the first significant figure. Because of apparent physiological insignificance, no effort was made to estab- lish the third. It should be born in mind, therefore, that all data herein is of a relative significance only. -24- TABLE OF ANALYTICAL DATA Experimental Group 1 (less susceptible series) Animal Upper Upper Lower Number Animal Treatment Femurs' Molars Incisors Incisors W 763‘ if 37‘— 3 control 0.034 0.066 0.026 5 topical NaF, neu- tral, daily (L.S.) 0.114 0.151 0.065 12 topical NaF, acid, daily (L.S.) 0.127 0.134 0.123 15 topical NaF, basic, d‘ily (L.S.) 0e169 0e162 ‘”“” Experimental Group 11 (less susceptible series) 33 a 31 controls 0.082 0.0u5 '0.029 35 topical NaF, neu- tral, daily 0.127 0.110 0.370 Experimental Group III (less susceptible series) 19, 20 a 21 controls 0.116 0.171 0.07s 16 d 18 topical NaF, basic - daily 0.139 0.113 0.073 17 same as 16 a 18 (more caries) 0.130 0.147 0.066 Experimental Group IV (stock animals) 43-47 controls 0.056 0.023 0.01M 0.015 48-52 topical NaF, basic, weekly 0. 0.033 0.033 0.019 53-57 topical NaF, noun tral, daily 0.103 0.059 0.035 0.03? 58-62 topical NaF, basic, daily 0.093 0.064 0.047 0.052 Experimental Group V (more susceptible series) 2? 25-26 controls, dietary prenatal NaF (250 ppm) con't. through experiment 0.142 same diet as con- trols plus tepical Na! neutral,daily 0.333 0.12“ 0.169 mount .25- Experimental Group VI (more susceptible series) InimaI’ *Upper ‘Upper Lower Number Animal Treatment Femurs Molars Incisors Inctsors W W..— 67-69 controls, 125 ppm ' NaF dietary pre- natal only, no treatment 0.063 0.089 0.042 63~66 same as controls plus topical NaF basic with 1% Na3003, daily 0.112 0.128 0.073 Experimental Group VII (more susceptible series) 72 a 73 controls 0.040 0.033 0.046 70 d 71 topical NaF basic with 1% romeo} daily 0.074 0.127 0.043 DISCUSSION -26~ DISCUSSION Rate of varying degrees of susceptibility were used to study oral and systemic effects of fluorides on dental caries. Tepical applications of r% NaF solutions when administered daily resulted in appreciable delay of caries initiation and development in the lower molars of less susceptible and stock animals. Identical treatment of more susceptible rats proved to be no help in the prevention of tooth decay. The method used in applying the fluoride solutions to the teeth resulted in the intake of approximately 50 mgm. of the element over a period of 100 days. Consequently, any tendency toward caries prevention may be due to systemic or local oral effects or both. Animals maintained on this level of fluoride appeared normal and no noticeable fluorosis of the teeth resulted. Sognnaes, by eliminating effects due to ingested fluoride, demonstrated that topical applications of fluoride solutions were beneficial in preventing dental caries in white rats (1). In the case of the more susceptible animals, striking evidence of caries prevention resulted from systemic effects of fluoride on prenatal and early postnatal organisms. These experiments involved feeding high levels of fluoride to moth» are at first signs of pregnancy and maintaining the young on the same diet through varying experimental periods. 0n high levels of fluoride, extreme fluorosis resulted in ingrowing upper incisors, but the molars remained practically caries free. Lower levels of similar fluoride administration reduced fluorosis considerably and likewise prevented caries develop- 'ment significantly. Animals appeared physiologically normal -27- after 100 days on a diet containing 250 ppm NaF. In general, the results are in agreement with those of Finn and Hodge (18), Killer (9), and Cox and co~workers (19), who report reductions in caries incidence resulting from the incorporation of fluorides in the diet. Although the t0pioa1 application of fluorides to the teeth of the more susceptible animals proved ineffective, fluoride administered prenatally and continued postnatally, imparted noticeable resistance. ' Femur bones, incisors, and molars of the experimental animals were analyzed for fluoride content. With but few ex— ceptions, large tissue increases in the element resulted from fluoride intake. Not in all cases, however, was this increase accompanied by a pr0portional decrease in caries. This ob- servation was also reported by McClure who suggested that there might be some effects from local reactions within the oral cavity (17). Results of this work support the view that fluoride en~ richment of the diet of the maternal organism plays a role in the caries resistance of the offspring. However, the analysis of the skeletal and dental tissues indicate there is no con- sistent relationship between the fluoride content of the tis— sues and the incidence of caries in a given animal. This points to the existence of other factors, systemic and local. 1. 2. 3. 125- CONCLUSIONS Topical application of fluoride solutions to the lower molars of stock white rats and to rats of moderate susceptibility to dental caries resulted in considerable delay of caries initiation and development. The same treatment had no perceptible effect on rate of a more susceptible type. Administration of fluoride to the maternal organism proved very effective in the pregention of dental caries in the offspring of highly susceptible animals. Topically treated animals which involved an intake of about 50 mgm. of fluoride over a hundred day period revealed no physiological abnormalities, whereas rats maintained on a diet containing 250 ppm NaF showed fluorosed incisors. The topical application of fluoride solutions to the teeth of rats or the enrichment of the diet with sodium fluoride resulted usually in marked increases in the fluoride content of the bones and teeth. However, there was no consistent relationship between the fluoride con- tent and the incidence of dental caries. BIBLI OGRAPHY 1. 2. 3. 6. 7. 8. 9. 10. 11. 12. .29- BIBLIOGRAPHY Bognnaes, R. F., Effect of Topical Fluorine Application on Experimental Rat Caries. Brit. Den. J., 70:“33-437 (l9hla). Hunt, R. 3., and Hoppert, C. A., Inheritance of Bus- ceptibility and.Resistance to Caries in Albino Rats. J. Am. Col. Dent., p 33. March, 194“. McKay, F. 8., and Black, G. V., An Investigation of Mottled Teeth. o. Cosmos, 58:u77, 731 and 39n (1916). Cheyne, V. D., Production of Graded nottling in Molar Teeth of Rats by Feeding Potassium Fluoride. , J. Den. Res., 21:1u5 (19u2). Cox, G. J., Hatuschak, n. 0., Dixon, 8. F., and.Wa1ker, W. E., Mottled Enamel in Rat Molars. Science, 90:83 (1939). Hodge, H. 0., LuceeClausen, E.‘M., and Brown, E. F., Fluoroeie in Rats Due to Contamination with Fluorine of Commercial Casein. The Effects of Darkness and of Controlled Radiation upon the Pathology of the Teeth. J. Nutrition, 17:33} (1939). Lilly, C. A., Lessened Incidence of Caries When Casein Re— places Milk in the Coarse Corn Meal Diet. Proc. Soc. Exp. Biol. Med., 39:389 (1938). McClendon, J. F., Foster, I. 0., Effect of Dietary Fluorine in Delaying Dental Caries. J. Den. Res., 21:139 (19MB). Miller, B. F., Inhibition of Experimental Dental Caries in the Rat by Fluoride and Iodoacetic Acid. Proc. Soc. Exper. Biol. Med., 39:389 (1938). McClure, F. J., Observations on Induced Caries in Rats. IV. Inhibiting Effect of Fluoride Ingested Post Eruptively and Prior to the Caries Pro- ducing Diet. J. Den. Res., 22:37 (19h3). Hoppert, C. A., Webber, P. A., and Canniff, T. L., The Production of Dental Caries in Rats Fed an Adequate Diet. J. Den. Res.. 12:161 (1932). Hoffman, M. M., Schaue, 1., Rate of Gradients of Growth in Rat nolars as Demonstrated by Injections of Alizarin Bed 8. J. Den. Res., 17:30? 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