Relationship of Fluid Transport Through the Dentinto the Incidence of Dental Caries

RALPH R. STEINMAN and JOHN LEONORADepartment of Oral Medicine, School of Dentistry and Department of Physiology,School of Medicine, Loma Linda University, Loma Linda, California 92354, USA

The rate of fluid movement in the teeth of28-day-old rats is inversely related to theincidence of dental caries after 13 weeks ofthe same diets. The early hypomineralizationfound in the dentin at 28 days is a resultof altered metabolism imposed by a re-duced rate of fluid transport in the rats feda cariogenic diet.

It is generally accepted that three condi-tions are essential in the oral cavity fordental caries to occur: a suitable microbe,a substrate for the microbe, and a suscep-tible tooth. Susceptibility or resistance todental caries is usually thought of in termsof structure, which is determined, for themost part, during calcification. However,it is also plausible that susceptibility todental caries may be related to altered me-tabolism within the tooth.To maintain a rapid rate of metabolism

through the Embden-Meyerhof pathway inthe avascular dentin, as reported by Bosia,Arese, and Pezolli,1 a rapid fluid transportsystem is essential for nutrient uptake andwaste removal from the tooth structure.Any reduction in the rate of fluid transportthrough the dentin may have a deleteriouseffect on the health of the tooth. Underthese conditions, the rate of metabolismwould be altered and the end product ofmetabolism (lactic acid) might accumulatein the structure. The purpose of these ex-periments was to determine if the rate offluid transport is related to the incidenceof dental caries, to determine if the rateof fluid transport can be altered by sys-

This investigation was supported by a grant fromthe Don Baxter Foundation. Computer assistance wasobtained from the Loma Linda Scientific Computa-tion Facility, supported by Grant RR 00276 from theNational Institutes of Health, Bethesda, Md.

Received for publication April 17, 1970.

1536

temic means, and to find what effect thisalteration would have upon the incidenceof dental caries.

Materials and MethodsMale, 21-day-old Sprague-Dawley rats*

were fed either a high sucrose cariogenicdiet or Purina laboratory chow.t Citrul-line,t urea,§ and acriflavine hydrochloridewere obtained.

Pentobarbital sodium (Nembutal) wasthe anesthetic used.EXPERIMENT 1.-The rate of fluid move-

ment (FM) through the dentin beneath theocclusal grooves was determined on the28th day of life in rats fed either laboratorychow or a cariogenic diet, and in the anes-thetized and unanesthetized state. Groupsof ten or more rats were given an injectionof the fluorescent dye, acriflavine hydro-chloride (50 mg/kg) intraperitoneally. Therate at which the fluorescent dye movedin and disappeared from the odontoblasticprocesses was used as a criterion for theoccurrence of FM. The unanesthetized ratsfed laboratory chow and the rats fed thecariogenic diet were killed at 15, 30, 45,and 60 minutes, and at 45, 60, 70, 90, 135,and 180 minutes, respectively. The ratswere decapitated quickly and the upper andlower jaws were removed, frozen, andcoded. The frozen jaws were sectioned,2and viewed microscopically under ultra-violet light.3 Particular attention was paidto the dentin beneath each occlusal grooveto determine whether acriflavine hydro-chloride was present in the odontoblasticprocesses. When the dye was present, these

* Simonsen Laboratories Inc., Gilory, Calif.t Ralston-Purina Co., St. Louis, Mo.$ Nutritional Biochemical Corp., Cleveland, Ohio.§ J. T. Baker Chemical Co., Phillipsburg, N.J.11 Sigma Chemical Co., St. Louis, Mo.

FLUID TRANSPORT AND DENTAL CARIES

processes fluoresced a brilliant green. Toquantitate the results, a count of the flu-orescing and nonfluorescing areas wasmade and a ratio was expressed:

fluorescing occlusal grooves

(fluorescing + nonfluorescing grooves)FMR,

where FMR is fluid movement ratio.EXPERIMENT 2.-The systemic effects of

urea and citrulline on FM were studied inrats maintained on the cariogenic diet. Onthe 28th day, these compounds were in-fused through the subclavian vein or theinternal carotid artery. The chemicals weredissolved in saline in the concentrationsshown in Table 1. The dose administeredfor each solution was 0.5 ml/100 gm bodyweight. When the infusion was through thesubclavian vein, the vein was exposed andthe compounds were injected through ahalf inch (27G) needle attached to a 1 mldisposable tuberculin syringe. For infusionsthrough the arterial system, the left com-mon, external, and internal carotid arterieswere exposed. To infuse through the in-ternal carotid artery, the external carotidartery was ligated close to the bifurcationof the common carotid artery. The com-mon carotid artery was ligated just anteriorto the manubrium. Then polyethylene tub-ing (Clay Adams, PE 10) attached to a half

inch (27G) needle was slipped into the com-mon carotid artery up to the internal carotidartery. The total dose of each compoundwas administered gradually over a 35 min-ute period. After ten minutes, the rats were

decapitated and the jaws processed as de-scribed in experiment 1.EXPERIMENT 3.-This study consisted of

two phases. In the first phase, the inci-dence of dental caries at 111 days of age in25 rats maintained on a cariogenic diet,was compared to the incidence in 23 ratsmaintained on laboratory chow. In the sec-

ond phase, the relationship between FMthrough the dentin to the incidence of den-tal caries was studied. By use of the FM-promoting capacity of urea and citrulline,various multiple doses dissolved in salinewere administered subcutaneously or intra-peritoneally for 91 days. Group 1, the con-

trol group of 20 rats, received 0.5 ml/100gm body weight of 0.9% saline subcuta-neously three times a day. Group 2, 17 rats,received 20 mg urea/ 100 gm body weightintrapertioneally twice a day. Group 3, 14rats, recevied 80 mg urea/ 100 gm bodyweight subcutaneously three times a day.Group 4, 14 rats, received 240 mg urea/100 gm body weight subcutaneously threetimes a day. The doses of urea and citrul-line were dissolved in 0.9% saline (0.5 ml/100 gm body weight). The rats were placed

TABLE 1COMPARISON OF THE EFFECTIVENESS OF UREA AND CITRULLINE TO

STIMULATE FLUID MOVEMENT BY INTRA-ARTERIAL ORINTRAVENOUS INFUSION

Dose Fluid MovementCompound (mM) Common Carotid Artery* Subclavian Vein SignificanceNoninfused ... 0.11 0.02 (14)t ...

Saline 155.0 0.14 0.02 (21) 0.14 ± 0.02 (18) NStUrea 6.25 0.25 ± 0.05 (12) ... <0.03 §

12.5 0.42 0.03 (12) ... <0.00125.0 0.66 0.04 (11) ... <0.00125.0 ... 0.22 ± 0.04 (5) 0.0550.0 ... 0.48 ± 0.07 (5) <0.001100.0 ... 0.76 + 0.03 (10) <0.001

Citrulline 6.25 0.31 ± 0.04 (7) ... <0.00112.5 0.46 0.04 (9) ... <0.00125.0 0.66 ±0.04 (9) ... <0.00112.5 ... 0.21 0.02 (13) 0.0225.0 ... 0.49 ± 0.03 (16) <0.00150.0 ... 0.76 ± 0.02 (8) <0.001

* Administered through the left common carotid artery with the external carotidartery ligated.

t Assay performed as described except that no infusion was done.i The change in FM because of saline infusion by either arterial or venous infusion

was not statistically significant compared with FM in the noninfused rats.§ Level of statistical significance compared with the appropriate saline infusion con-

trol.

Vol 50 No. 6 1537

1538 STEINMAN AND LEONORA

in raised cages and fed the high sucrosediet ad libitum during the experimentalperiod.The rats were weighed twice a week for

the first eight weeks, and once a weekthereafter. At the end of the experiment,an autopsy was performed and the jawswere removed. The teeth were coded, ex-amined, and scored for dental caries by themethod of Shaw et al.4 The adrenal glands,testes, ventral prostate, thymus, thyroid,submaxillary glands, spleen, kidneys, andpituitary gland were dissected free of ex-traneous tissue, and weighed on a torsionbalance.EXPERIMENT 4.-In this experiment an

attempt was made to ascertain the mech-anism whereby the daily systemic adminis-tration of urea resulted in a reduction inthe incidence of dental caries. Forty 21-day-old male rats were fed a cariogenicdiet for six weeks. Group 1, 20 rats, wasinjected subcutaneously three times a daywith urea (240 mg/100 gm body weight).Group 2, 20 rats, received saline inthe same volume three times a day. Theinjections were continued for three weekswhile the rats were fed the cariogenic diet.At the end of the third week, blood andsaliva samples were taken at 45, 120, and180 minutes after an injection of urea orsaline. The blood (0.5 ml) was drawn witha heparinized syringe from each rat througha heart puncture. The individual bloodsamples were centrifuged, and the plasmawas saved. Immediately after the blood col-

.8 Unanestketized Rats

Purina Diet Cariog

4-

0 .6- ~~~~~Anesthetized Rats

5-Purina Diet Cariagf

election, the rats were given an injection ofpilocarpine (0.1 ml of 10%) subcutane-ously to induce salivation. Approximately0.5 to 1 ml of saliva was collected fromeach rat. The urea content of each plasmaand saliva sample was determined by themethod of Chaney and Marbach.5On the last day of the experiment, the

rats were bled to death through heart punc-tures two hours after the last injection ofurea or saline. The blood from each groupof rats was pooled and centrifuged. Theplasma from the saline and urea injectedrats was bioassayed for FM-promoting ac-tivity, as discussed under experiment 2, atdoses of 0.5, 1, and 2 ml plasma. Whennecessary, saline was added to the plasmato maintain the dose volume at 2 ml.

ResultsThe fluorescent dye appeared to move in

and disappear from the odontoblastic pro-cesses at a faster rate in the unanesthetizedrats fed the laboratory chow than in theunanesthetized rats fed the cariogenic diet(Fig 1). The peak response in the lattergroup was obtained at 45 minutes, and acomparable level of FM was obtained at15 minutes in the other rats. The responsein the rats fed the cariogenic diet appearedto be blunted. Pentobarbital sodium anes-thesia dramatically enhances the suppressiveeffect of the cariogenic diet on the rate ofFM.The relationship of the FM ratio to the

incidence of dental caries is shown in Figure2. The FM ratio in a 28-day-old rat demon-

Time-minutes

FIG 1.-Effect of diet, and diet with pentobarbital sodium anesthesia onfluid movement through odontoblastic processes of 28-day-old rats.

J Dent Res November-December 1971

FLUID TRANSPORT AND DENTAL CARIES

RATS 28 DAYSOF AGE

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strates an inverse relationship to the inci-dence of dental caries at 111 days of age.A high FM ratio is associated with a lowincidence of decay, and vice versa. Thesedifferences are statistically significant at the<0.001 level.The capacity for urea and citrulline to

stimulate FM when infused into the ratsfed the high sugar diet is shown in Table 1.The FM response is related to dose. Infu-sion through the internal carotid artery istwo to four times more effective thanthrough intravenous infusion. At higherdoses, the FM response obtained with ei-ther compound is equal to or greater thanthat found in rats fed the laboratory chow.The effect of long-term systemic admin-

istration of urea and citrulline on the inci-dence of dental caries is shown in Table 2.A highly significant reduction in dental

caries was obtained with the higher dosesof urea. The decrease in caries incidenceobtained with citrulline was equivalent tothat obtained with the lowest dose of urea,

but it was not statistically significant. Thedecrease in the number of carious lesionswas found to be a function of the totaldose of urea.

The effect of urea and of citrulline on

body weight at autopsy, and on the weightof various glands and organs, is summarizedin Table 3. With the larger doses of urea,

the body and thymus weights were reduced.Increased weight of the adrenal glands,testes, spleen, kidneys, and pituitary glandalso was found in rats receiving higherdoses of urea. The statistical significanceof the observed differences is given as com-

pared with the control rats.The concentrations of urea in the blood

plasma and saliva from rats receiving sub-cutaneous injections of urea three times a

day for three weeks, are shown in Table 4.Forty-five minutes after an injection of urea,

there was a sharp rise in the blood plasmaconcentration of urea, from 0.185 to 0.296mg/ml. There was a corresponding rise inurea concentration in the saliva, from 0.121to 0.253 mg/ml. The concentration of ureain the saliva was always less than that inthe blood. Two hours after an injection,the blood urea dropped down to almost thecontrol level. After three hours, the ureaconcentration both in the blood and salivawas within the control range. Table 5 showsthe FM-promoting capacity of plasma ob-tained one hour after the last injection ofurea in 28-day-old rats that were main-tained on a high sugar diet for one week.The data show that as little as 0.5 ml ofthis plasma was capable of stimulating a

significant rise in FM, and 2 ml of plasmafrom the saline injected rats was ineffective.

TABLE 2EFFECT OF SUBCUTANEOUS INJECTION OF UREA AND CITRULLINE ON

INCIDENCE OF DENTAL CARIES IN MALE RATS

Dose Injec- Statis-mg/100 gm tions tical

Body Per Caries Percent Signifi- No.Chemical Weight Day Score Reduction cance Animals

Saline 3 33.3 4.2* ... ... 20Urea 20t 2 22.5 ± 2.0 33 0.02 17

80 3 9.0 ± 2.3 73 0.002 13240 3 3.5 ±0.7 90 0.001 12

Citrulline 60 2 23.9 ±4.9 29 0.16 14* Standard error of the mean.t Administered intraperitoneally.

Vol 50 No. 6 1539

1540 STEINMAN AND LEONORA

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DiscussionThe data show that the rate of FM

through the odontoblastic processes is sig-nificantly effected by the dietary regime(Fig 1). In contrast to the rate observed inrats fed Purina laboratory chow, a cario-genic diet significantly suppresses the rateof FM. Pentobarbital sodium anesthesiapotentiates the suppressive effect of the highsugar diet. Correlation of the FM ratio tothe incidence of dental caries demonstratesthat a high incidence of caries is associatedwith a suppressed FM. The common de-nominator between these two parameters isthe cariogenic diet. A high FM ratio is cor-related with an exceptionally low incidenceof decay (Fig 2).

Previous studies have shown that thesuppressive effect of a cariogenic diet onFM is discernible as early as two days afterthe rats have been placed on this diet. How-ever, in acute experiments that last only 45minutes, the suppressive effect can be over-come by the systemic administration ofurea or citrulline through intravenous orintra-arterial infusion (Table 1). With thesechemicals, it is possible to establish FM ata level equal to or greater than that ob-served in rats fed laboratory chow. Fromthe data, it is apparent that the intra-arterial infusion is significantly more effec-tive in re-establishing FM than the intra-venous infusion. This suggests that thesecompounds exercise their effect on FM byactivating a mechanism in the central ner-vous system.

Application of the beneficial systemiceffect of urea and citrulline as observed inthe acute experiments to a chronic experi-ment that required daily administrationover a period of 13 to 16 weeks resultedin a significant reduction in the incidenceof dental caries (Table 2). Urea was sig-nificantly more effective than citrulline,because urea is a terminal metabolic prod-uct that is not metabolized further, butcitrulline can be diverted into other meta-bolic pathways. With urea, the decrase incaries was a function of the dose. The cor-relation between suppressed FM and in-creased dental decay suggests that the rateof fluid transport is important for resistanceto dental caries.

The beneficial systemic effect of urea isin agreement with the findings of McClure,6who reported a significant decrease in the

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I Dent Res November-December 1971

FLUID TRANSPORT AND DENTAL CARIES

TABLE 4COMPARISON OF UREA CONCENTRATION IN THE PLASMA AND SALIVA AT

VARIOUS TIMES IN RATS RECEIVING EITHER SALINE OR UREA BYINJECTION THREE TIMES PER DAY

Bleeding Time Urea ConcentrationMaterials Injected Postinjection (mg%)Subcutaneously (minutes) Plasma Saliva

Saline control 45 18.5 + 0.5 (20) 12.1 ± 0.4 (20)120 15*

Ureat 45 29.6 ± 1.5 (14) 25.3 ± 1.4 (14)120 20.Ot180 17.3 + 0.5 (20) 11.7 0.3 (20)

* Urea concentration in the pooled plasma at the time the rats were bled.t Dose of urea 240 mg/kg body weight, three times per day.

incidence of dental caries in rats fed a dietcontaining 1 to 2% urea. However, certaindifferences in experimental design shouldbe noted. With urea in the diet, adminis-tration was correlated with food consump-

tion, and in our experiment urea was ad-ministered three times a day when foodconsumption was low. No urea was admin-istered during the night when food con-sumption is high.The beneficial systemic effect of urea is

emphasized by examination of the bloodand saliva urea levels in the rats that re-

ceived the highest dose of urea 45 minutesto three hours after injection (Table 4).There was an initial sharp rise in urea con-

centration in both fluids, and in 2 to 3hours the level returned to the control levelfound in saline-injected rats. These datashow that during a 24 hour period therewere three relatively brief periods when theurea concentration in the biologic fluidswas elevated, and it remained low duringthe night when the rats ate the major por-tion of their food. The intermittent rise inurea level in the oral cavity is considered

to have little or no effect on conditions atthe bottom of the food plug where decayoccurs, because the time for urea to pene-

trate to the bottom of the plug is too short.The rats were housed in raised cages tofurther reduce the availability of extraneousurea in the oral cavity.The idea that urea must exercise its ef-

fect through a systemic mechanism ratherthan through a surface reaction is con-firmed with the following observations.Others have shown that a reduction in foodconsumption is often associated with a de-crease in dental caries. However, rats un-der stress may have a reduction in foodconsumption but have an increase in dentaldecay.8 A reduction in growth rate also isassociated with an increase in dental decay.9In our experiment, the rats receiving thehighest dose of urea were under stress, as

indicated by the increased adrenal andpituitary weights and the reduced thymusand body weights (Table 3). The kidneyswere hypertrophied because of the increasedstress from rapidly eliminating the ureaload from the body fluids. In spite of stress,

TABLE 5PLASMA LEVEL OF FLUID MOVEMENT-PROMOTING PRINCIPLE AFTER

SUBCUTANEOUS INJECTION OF SALINE OR UREADose(ml)

Materials Plasma Level ofInjected + Fluid Signifi-

Intravenously Saline Movement cance

Saline 0 + 2 0.14 0.02 (18) ...

ControlPlasma* 2 +0 0.16 0.02 (16) NStExperimentalPlasmat 0.5 + 1.5 0.23 ± 0.03 (10) 0.01§

1.0 + 1.0 0.29 ± 0.03 (10) 0.0022.0 + 0.0 0.30 ± 0.03 (10) 0.002

* Plasma obtained from saline injected rats by heart puncture.t Level of significance compared with saline infused rats.$ Plasma obtained from urea injected rats by heart puncture.§ Experimental plasma compared with control plasma.

Vol. 50 No. 6 1541

1542 STEINMAN AND LEONORA

a significant decrease in the incidence ofdental caries was achieved.More evidence for the systemic effect of

urea becomes apparent when the FM re-sponse of 28-day-old rats to plasma ob-tained from the urea- and saline-injectedrats is compared (Table 5). The intravenousinfusion of 2 ml of control plasma fromthe saline-injected rats had no significanteffect on the suppressed FM ratio. In con-trast, as little as 0.5 ml of plasma from theurea-injected rats caused a significant risein the FM ratio. The urea concentration in0.5 ml of plasma was 0.1 mg. This concen-tration of urea is seven times less than theminimum effective dose required to stimu-late FM when infused intravenously intothe assay rat. The urea concentration in thehighest dose (2 ml) used is only 54% ofthe minimum effective dose of urea. Theinevitable conclusion from these data is thata hormonal factor, not urea, was the re-sponsible factor in the plasma that stimu-lated FM. Evidence submitted elsewherefor publication shows that urea and othercompounds of the ornithine cycle, and car-bamyl aspartate of the pyrimidine cycle,exercise their effect on FM through thehypothalamus. Under the influence of urea,the hypothalamus secretes the hypothalamic,parotid hormone-releasing factor, whichstimulates the parotid gland. The lattergland secretes a parotid hormone that stim-ulates FM by acting on the odontoblasts.Surgical interruption of this endocrine axis,such as parotidectomy, renders these com-pounds ineffective in stimulating FM. Theireffectiveness is dependent on an intact en-docrine axis. Evidence for the existence ofthe hypothalamic-parotid gland endocrineaxis has been published previously.'0The tooth is a unique structure, even

when compared with bone, to which itshows certain structural similarities. Boneis a relatively vascularized structure, where-as dentin is avascular, and is dependent ona rapid fluid transport system for nutrientuptake and waste removal. Bone is sur-rounded by body fluid, and teeth are ex-posed to the atmosphere. The aerobicenvironment may be of importance ininfluencing metabolic pathways in theteeth.""12 Beneath the occlusal food plug,a more anaerobic environment can existthat could adversely affect the metabolicpathways. In germfree animals'3 on a high

sucrose diet, the hypomineralization foundin the enamel and dentin beneath the foodplug might be explained as a consequenceof reduced fluid transport, along with ashift to a more anaerobic metabolism andthe accumulation of lactic acid in the den-tin. These changes prepare the tooth for asuccessful attack by the unfavorable ex-ternal environment.The authors suggest that the early changes

in teeth which eventually lead to cariouslesions are the result of altered metabolismthat includes a reduced rate of fluid trans-port and external conditions imposed bythe food plug and its microbial inhabitants.These conclusions do not minimize the im-portance of the external conditions, butrather give added importance to them byshowing how external conditions may in-fluence metabolism within the teeth. Teethwill not decay in the absence of the mi-crobe and its products on the external sur-face. The external factors cannot be ignored,but for dental caries to be controlled, itmight be well to consider how the alteredinternal metabolic processes contribute tothe problem of dental caries. Considerationof both the internal and external factors isa more complete approach to the preven-tion of dental caries. Teeth decay becauseof an initial alteration in the internal metab-olism. Reduction in the rate of fluid trans-port, as found in the teeth of animals main-tained on the high sucrose diet, may be ofprimary importance in understanding thealterations in metabolism and pathologicsequelae.

ConclusionsThe rate of fluid transport was suppressed

significantly in rats maintained on a highsucrose diet as compared with rats fedPurina laboratory chow. Fluid transportmay be stimulated by the systemic admin-istration of urea or citrulline.The systemic administration of urea to

rats on a high sucrose diet significantly re-duces the incidence of dental caries. Thisreduction in dental caries may be the resultof stimulation of the dentin fluid transportsystem produced by the hypothalamic-parotid endocrine axis.

References1. BoslA, A.; ARESE, P.; and PEZZOLI, M.:

Glycolytic Enzymes in Bovine Hard Dental

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FLUID TRANSPORT AND DENTAL CARIES

Tissues at Different Ages, Calcif TissueRes 6:93-102, 1970.

2. STEINMAN, R.R.; HEWES, C.G.; and WOODS,R.W.: Histochemical Analysis of Lesionsin Incipient Dental Caries, J Dent Res 38:592-605, 1959.

3. STEINMAN, R.R.: The Movement of Acri-flavine Hydrochloride Through Molars ofRats on a Cariogenic and Non-CariogenicDiet, J South Calif Dent Assn 35:151-157,1967.

4. SHAW, J.H.; SCHWEIGERT, J.M.; MCINTIRE,J.M.; ELVEHJEM, C.A.; and PHILLIPS,P.H.: Dental Caries in the Cotton Rat,J Nutr 28:333-345, 1945.

5. CHANEY, A.L., and MARBACH, E.P.: Modi-fied Reagents for Determination of Ureaand Ammonia, Clin Chem 8:130-132, 1962.

6. MCCLURE, F.J.: Observations on InducedCaries in Rats. VI Summary Results ofVarious Modifications of Food and Drink-ing Water, J Dent Res 27:34-40, 1948.

7. MUNTZ, J.A., and MILLER, B.F.: FactorsInfluencing Penetration of Synthetic De-tergents and Certain Other Compounds

Into Dental Plaque Material, J Dent Res22:73, 1943.

8. STEINMAN, R.R.: The Effect of StressUpon the Incidence of Dental Caries, JSouth Calif Dent Assn 28:367-369, 1960.

9. STEINMAN, R.R.; HARDINGE, M.G.; andWOODS, R.W.: Experimental Caries WithHuman Foods, J Dent Res 37:865-873,1958.

10. LEONORA, J., and STEINMAN, R.R.: Evi-dence Suggesting the Existence of a Hypo-thalamic-Parotid Gland Endocrine Axis,Endocrinology 83:807-815, 1968.

11. BORLE, A.B.; NICHOLS, N.; and NICHOLS,G., JR.: Metabolic Studies of Bone InVitro. II. The Metabolic Patterns of Accre-

12. BORLE, A.B.; NICHOLS, N., and NICHOLS,G., JR.: Metabolic Studies of Bone InVitro. II. The Metabolic Patterns of Accre-tion and Resorption, J Biol Chem 235:1211-1214, 1960.

13. BRINER, W.W.; and ROSEN, S.: Effect ofFluoride on Hypomineralized Areas in theRats fed a Cariogenic Diet, Arch OralBiol 12:1077-1084, 1967.

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