The histochemistry of dental decay

Arch . oral Biol . Vol .7, pp.207-219, 1962. Pergamon Press Ltd . Printed in Gt. Britain .

THE HISTOCHEMISTRY OF DENTAL DECAY* D . L . J . OPDYKE Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, Ohio, U .S .A .

Abstract-The application of histochemical methods to thin sawed sections of human carious teeth has enabled us to make the following observations : (1) The carious lesion is histochemically the most reactive site in teeth . (2) Reactions for calcium are intensely positive in the carious lesion, including small "white spots", indicating that the enamel mineral component in this site is more loosely bound than in the sound tooth . (3) Tests for protein give intense reactions at the same sites. An intense protein reaction can be demonstrated in the entire section by gentle demineralization . In the carious spot the protein, as well as the mineral, is more loosely combined . (4) Refluxing the sections in ethylenediamine eliminates the protein reaction and extends the mineral reaction to the entire section. The pH of the carious lesion is reported and a description given of the lipid component of enamel rods with histochemical evidence for its origin in enamel and spread into the dentine . By using the agar method of FRANCIS and MECKEL, modified by using lactic acid as the demineralizing agent instead of bacterially produced acid, it is possible to induce carious lesions in human teeth that are indistinguishable morphologically or histochemically from naturally occurring dental decay . In early dental decay the principal aetiologic agent is acid attack . A time relationship has been established for the order of appearance of the histochemical reactions in the decay process : first, unbound mineral ; second, the acidic reaction ; third, uncoupled protein ; and fourth, unmasked lipids . By treatment with enamel antisolubility agents before or concomitant with the exposure in the agar system, the in vitro carious process can be prevented . Several agents have been compared for protective action . In addition, after incipient carious processes have been induced, treatment with stannous fluoride solution causes the lesions to lose all of the distinguishing characteristic histochemical reactivity and the legions disappear . One method by which the former carious sites can be visualized is by specific staining for tin .

Tats research was designed to study the chemical and morphological nature of the decay lesion in human teeth by the application of histochemical methods to thin sawed sections and to compare naturally occurring dental decay with lesions artificially induced in human teeth in vitro . In addition, the effects of several anticaries agents on the histochemistry of these lesions are described . SOGNNAES and WISLOCKI (1950) have reported one of the first extensive studies on the histochemistry of teeth, but problems involved in making ground sections have tended to limit research in this field . The development of a dental saw in our

• This work was presented in part at the 38th and 39th annual meetings of the International Association for Dental Research . 207



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laboratories (GRAY and OPDYKE, 1960) has made it possible consistently to cut sections of intact molar teeth from 30 to 50 µ in thickness, thus providing reproducible uniform material for histochemical studies . METHODS

The teeth used in this study were extracted by dentists in the Cincinnati area and stored in buffered (pH 7) 10% formalin until use . Studies on naturally occurring dental decay were made on sawed sections of teeth stained by the procedures described in Table 3 . For investigations on induced carious lesions, molar teeth were split into quadrants along vertical lines with "tile-cut nippers" . The four segments could then be treated in different ways and cemented together with Eastman 910 cement . The teeth were sectioned on the dental saw and stained by appropriate methods . This procedure provides for three experimental variations and an adequate control in the same tooth . Since the sections were stained as one piece, it could be concluded with more certainty that differences in dye uptake were the result of differences in treatment . Artificial decay lesions were induced by placing quadrants of teeth, with the fractured surfaces protected by dental inlay wax, in tubes containing 15 ml of sterile dextrose agar and I Inl of N lactic acid, a modification of the FRANCIS and MECKEI method (1960) in that the acid was not derived by the action of micro-organisms . No growth of micro-organisms was observed . In order to produce lesions more similar to those seen in natural decay, molars were covered with dental inlay wax, small excavations (2-3 mm in diameter) were made in the wax, and the teeth were placed in the agar system for varying periods of time . The softened dental substance was gently flushed away at intervals, permitting the attack to extend deep into the dentine . Artificial "white spots" were induced in much the same manner . Numerous pinpoint holes were made in a wax covering applied to molar teeth, and the teeth were placed in the agar medium for 20 days . Teeth prepared in the above ways provided a tool for the investigation of anticaries agents . Comparisons were made between the effects of SnF Q (3120 p .p .m . Sn+-, 1000 p.p .m . F-), SnCI2 (3120 p,p .m . Sn' ), NaF (1000 p .p.m . F-), Na2 HPO4 (10%), and hexachlorophene (0 10%, solubilized with Triton X-100), all at unadjusted pH . Teeth were given ten 20 min treatments in the solutions before being placed in the agar system for 10 days, or were removed from the agar daily during the 10 day period for a 20 min treatment . Permeability of teeth was investigated by placing them for 2 weeks in I % solutions of toluidine blue, either at pH 4 . 5 in 0 . 5 % tartaric acid or at pH 9 in 1 % borax. The extent of penetration of the dye could he observed visually when these teeth were sectioned . To determine the importance of the organic matrix to histochemical reactions in enamel, sections were gently refluxed in ethylenediamine for 24 hr, removing essentially all of the organic components . Other sections were placed in 0-05 N HC1 for 10 or 30 min to observe the effect of partial demineralization on staining reactions .



20 9

THE HISTOCHEMISTRY OF DENTAL DECAY

The nature of the protein in teeth was studied by treating sections overnight with a number of enzymes, under the conditions listed in Table I . Sections were also placed in living cultures of Micro .sporon gypseum (a micro-organism that attacks keratin in skin infections) in Sabouraud's dextrose broth for 150 days at room temperature, and others were treated overnight with an aqueous suspension of an acetone powder of clothes moth larvae .

TABLE 1 . ENZYME TREATMENTS USED TO EXPLORE NATURE OF DENTAL PROTEINS

Sections of teeth were placed in 0-05 N HCI for 30 min at room temperature . rinsed, and treated overnight in the following solutions

Concentration (%)

Temp . (°C)

Pepsin - ~~ - ~ Trypsin Chymotrypsin Hyaluronidase Flastase

0^ 0-3 0. 1 0-1 0-025

37 37 37 37 37

Collagenase

1

37

I

Medium

Activating agent

0. 02 N HCI 0 . 5 % NaCl PH 7-4 0-5% NaCl PH 8-5 M/10 NaCl 0 . 1 M Borate-carbonate buffer pH 8 . 5 0 .1 M Phosphate buffer pH 7-5

None Ca+ Cap Na Co,

Some mineral stains were also carried out on samples of enamel ground to a fine powder in a mortar, and on several mineral salts (Table 2) .

FABLE 2 . REACTIONS OF SOLID COMPOUNDS WITH PI ALIZARIN RED S AT pH 8-4

Material Calcium chloride Calcium carbonate Sodium triphosphate Sodium pyrophosphate Dicalcium phosphate (anhydrous) Dicalcium phosphate (dihydrate) Hydroxylapatite Powdered enamel (60-mesh) Powdered enamel (200-mesh)

Result Red precipitate Coloured red No reaction No reaction Coloured red Coloured red Coloured red Coloured on some surfaces Coloured on some surfaces

The histochemical procedures applied to teeth were in general those described by PEARSE (1960) . The principal modifications, introduced to allow for the hardness of the tissue, was the use of very dilute staining solutions for longer periods of time than those employed for soft tissues . The tests used, with information as to details of procedure, are listed in Table 3 . Where no details are given, the procedures are given by PEARSE (1960) .

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D . L . J . OPancr TABLE 3 . 1-bSTOCHEMICAL PROCEDURES USED IN STUDY OF DENTAL CARIES

Kind of test Indicator (acid-base)

Mineral

Protein

Dye or method

Remarks

Methyl orange (pH 4 . 0-5 . 0) Methyl red (pH 4 .2-6 . 0) Bromcresol purple (pH 52-618) Bromcresol blue (pH 6 .0--7 . 6) Phenol red (pH 6 . 8-8 . 4) Toluidine blue

0 . 1 % solution, overnight

5 r 10- ' M in water, 20 hr at 30°C

Galloformic reaction (Cretin), staining by gallamine blue Silver replacement (Von Kossa) Alizarin red S Unmordanted haematoxylin Sodium rhodizonate Purpurins

McGEE-RUSSELL (1958)

Ninhydrin Ninhydrin Schiff Naphthol yellow S Chloramine Schiff Alloxan Schiff Mercury bromphenol blue Performic acid Schiff* Performic acid methylene blue*

BURSTONE (1955) MAZIA

et at. (1953)

PEARSE (1951) BARRNETT and SELIGMAN (1952)

Sulphydryl Lipid

Oil red 0 Sudan black Oil blue N Sudan IV

Enzymes

Alkaline phosphatase Acid phosphatase Succinic dehydrogenase

14ucopolysaccharide

Alcian blue

Pigment

Argentafin Bleaching with H,O,

j

Satd. solution in an aqueous solution saturated with caffeine overnight BURSTONE (1958)

' Methods reported to be specific for the keratins . RESULTS

A . Naturally Occurring Decay Lesions Reaction with indicators Sound enamel and dentine did not stain with any of the acid-base indicators tested . However, carious areas, including the early "white spot" stage, coloured brilliantly with indicators having specific colour reactions in the pH range 4 .2-46 (methyl orange, methyl red) (Fig . 1) . When tests were carried out in solutions of controlled pH values from 3 to 5, maximum staining occurred between pH 4 . 2 and 4 . 6 with both methyl red (an acid dye) and toluidine blue 0 (a basic dye) .



THE HtFTOCHEMMMY OF DENTAL DECAY

21 t

It was observed in the staining with toluidine blue that the interprismatic substance in normal enamel was intensely metachromatic at pH 4, an indication of acid mucopolysaccharide content . In the carious lesion, however, the acid pH and consequent intense basophilia of the decaying tooth masked this metachromasia so that it could not be seen . The carious spot appeared as a dense blue-black spot against a background of the lavender of the normal enamel metachromasia (Fig . 2) . The mineral reaction

The procedures applied are usually considered to be tests for calcium, but such other elements as aluminum, iron, barium, strontium and magnesium also give positive reactions . In dental tissues, calcium must be largely responsible for the reactions observed . With all of the methods employed, the grossly decayed areas as well as the tiniest of white spots stained intensely, whereas sound enamel gave no reaction . The staining response could be duplicated in sound enamel by exposing sections of teeth to 0 . 05 N HCI at 37°C for 10 min, or by refluxing overnight in ethylenediamine ; after this gentle demineralization or deproteination, the entire section stained with the mineral reagents . In a further test of the mineral reaction to determine the physical state of reacting materials, the solid compounds listed in Table 2 were stained overnight with 0 . 1 °o aqueous alizarin red S solution brought to pH 8 .4 with NH4 OH . The reactions with the powdered enamel were most interesting in that the size of the particle was not the determining factor (60-200 mesh) . Particles ranging from 3 to 400 µ showed staining on some surfaces . While several of the particles were smaller than 10 µ in thickness, fragments from sawed sections of teeth of the same thickness did not show a mineral reaction in the sound enamel . The possibility that carious or pre-carious enamel was involved was ruled out by grinding a 1 mm square piece of sound enamel which was stained with results as described above . The protein reaction

Intact healthy enamel did not stain with any of the protein reagents listed in Table 3 . All of the methods gave strongly positive reactions in the decayed areas : the most intense staining was seen with the mercury bromphenol blue reagent as used by MAZIA, BREWER and ALFERT (1953) (Fig . 4) . Protein reactions in white spots depended on the size of the lesion . Those less than 1 mm in diameter failed to react, whereas those of larger dimensions coloured an intense blue with the mercury bromphenol blue reagent . The protein reaction in decayed areas was eliminated by gently refiuxing the sections in ethylenediamine for 24 hr, a treatment which is capable of removing all of the organic components . (Following this treatment the entire section gave an intense positive reaction for minerals) . Likewise, placing the sections in 0 .05 N HCI for 30 min made the entire section protein reactive, or "exposed" the protein . The observation of positive reactions in methods otherwise specific for keratins (PEARSE, 1951) suggested the application of a sulphydryl test . Consequently, reactions for -SH and -SS- were carried out by the BARRNErT and SELIGMAN method (1952)



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D . L . J . OPDYKE

before and after "exposing" the protein by treating the sections with 0 .05 N HCI . The sections untreated with acid gave a very faint reaction for sulphydryl in the pulp cavity, loose tissue attached to the root, and in the carious spot . The reactions for disulphide linkages were slightly more intense. However, following the "exposure", the reaction for sulphydryl was marked, and the reaction for disulphide was very intense . This was true for both enamel and dentine . Following these observations, sections whose proteins were "exposed" by acid attack were treated overnight with several enzymes as shown in Table I . There was no loss of reactivity to protein reagents as a result of these treatments . In addition, sections were boiled in acidified water overnight without detectably removing any of the protein . Additional sections were placed in living cultures of Microsporon gypseum in Sabouraud's dextrose broth . This is a micro-organism that attacks keratin in skin infections . The sections were left in the culture for as long as 150 days with frequent examination . Other sections were treated with an aqueous suspension of an acetone powder of clothes moth larvae . None of these treatments affected the staining reaction of the protein . The lipid reaction When sections of teeth containing decayed areas were stained with the lipid stains, the decayed areas stained brilliantly but there was no dye uptake by normal enamel . This staining could subsequently be extracted with a mixture of chloroform and methanol or of ethanol and diethyl ether . If the sections were extracted prior to staining, no colouring occurred . On microscopic evaluation, it was observed that in incipient caries none of the fatty material was interprismatic but was distributed on the enamel rods in a linear fashion, periodically arranged in bands perpendicular to the axis of the enamel rods (Figs. 5 and 6) . This periodicity is roughly 4 µ from band to band . In areas where the decay was more advanced, no enamel rods are even discernible . The whole area is deeply stained and no morphology is distinguishable . Deep to these severe lesions, fat droplets were readily seen in the dentinal tubules extending toward the pulp cavity (Fig . 7) . Some of the tubules were filled, whereas others contained only a few droplets . Apparently, these fat droplets came from the decay lesion and spread through the proximal dentine, radiating out from the carious spot . Enzyme reacriom With unfixed teeth, only a few hours after extraction, there was no alkaline phcsphatase (BURSTONE, 1958) except for that in the blood vessels of the pulp cavity . There was also no alkaline phosphatase activity in the decay lesion . Tests for acid phosphatase were also negative . In these same unfixed sections, an intense reaction for succinic dehydrogenase was found at the base of plaque masses on the surface of the enamel but not in the thin threadlike filaments . In addition, there was a strong reaction in the dentinal tubules surrounding the carious lesion . Aside from these sites there was no detectable enzymatic activity by any of the methods used except for that in the pulp .



THE HISTOCHEMISTRY OF DENTAL DECAY

21 1

41ucopolysaccharide reaction In addition to the stain uptake for toluidine blue at controlled pH values, stains with alcian blue showed the normal enamel and the dentinal tubules to be richly reactive for acid mucopolysaccharide (AMPS) substances . In the enamel immediately under the plaque mass, the AMPS was depleted . i n dentinal areas surrounding the carious lesion, there was a depletion of AMPS from the dentinal tubules . Pigment materials in enamel Enamel was found to contain a pigment material which had the following characteristics : It was granular, argyrophil (deposited metallic silver from solutions of the ammoniacal silver complex), and argentaffin (deposited metallic silver from solutions of silver nitrate), and was readily bleached with peroxide . In these respects, it is indistinguishable from the naturally occurring melanins of skin or retina . Surrounding carious lesions, this pigment material migrates to form a dense line of granules around the decayed area . B . Artificially Induced Lesions When quartered molars were placed in the agar system containing lactic acid for 20 days, lesions grossly resembling those of natural caries were seen . The histochemical changes were also comparable to those seen in the natural decay process ; the intensity of the reactions depended on the length of time in the agar tube . In the earliest lesions, after 1-4 days, it was possible to show a mineral reaction with alizarin red S (Fig . 8) . The acid reaction, demonstrated with the indicator methyl red, appeared after 4-6 days in the system . Next, in 6-10 days, the enamel showed an intense protein reaction (Fig . 9) and, finally, after 10-20 days, fatty material could be demonstrated (Fig . 10) . Interprismatic mucopolysaccharide could be demonstrated in the enamel (Fig . 11) in increased concentrations with areas of depleted AMPS at the limits of the lesions . Similar results were obtained with teeth that had been protected with dental inlay wax in which small excavations or pin-point lesions had been made . The only difference was that the reaction for protein was seen only in lesions more than I mm in diameter, although the pin-point spots gave the acid response with indicator . the mineral reaction (Fig . 12), and the fat reaction as anticipated . In all of these incipient lesions, the surface of the enamel appeared to be intact (Figs . 12 and 13)_ C . Evaluation of Anticaries Agents When teeth were placed in solutions of SnF2 , SnCl 2 , NaF, Na2 HPO 4, or hexachlorophene for ten 20 min treatments prior to or during the exposure to the acidagar system, only the fluoride-containing agents altered the rate of development of caries-like lesions . As measured by the mineral reaction . SnF 2 gave complete protection and NaF gave partial protection (Fig . 14) . In one study, numerous small lesions were developed in teeth by the methods described . One half of each tooth was then covered with dental wax, and the entire tooth was placed in the SnF 2 solution for ten 20 min periods . Sections prepared



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D . L. i . OPDYKE

from these teeth showed the expected staining for minerals and proteins in the enamel that had been covered with wax, but there was no evidence of these reactions in the half that had been treated with SnF2 solution . It was also possible to demonstrate a marked effect of SnF2 on the permeability of enamel . Two teeth were half coated with inlay wax and were then given ten 20 min treatments with SnF2 solution . After removal of the wax, the teeth were kept for 2 weeks in 1 % toluidine blue solution, one at pH 4-5 in 0 .5 % tartaric acid and the other at pH 9 in 1 % borax . The untreated half of the tooth from the acid dye bath showed penetration of the dye halfway through the enamel, while the SnFQ treated segment showed no penetration even through tiny cracks . There was no dye penetration in the tooth that had been in the basic dye bath . There was only one observable effect of SnF2 on intact enamel . This was a movement of pigment granules from the enamel surface toward the dentino-enamel junction . In some instances this resulted in a complete clearing of the yellow colour from the superficial half of the enamel, with the development of a dense brown zone halfway through the enamel thickness . DISCUSSION The nature of the major changes in naturally occurring dental decay is clearly demonstrated by the use of appropriate histochemical procedures . Carious lesions are regularly acidic in nature, showing pH values in the range 4 .2-4-6 ; this is somewhat lower than the figure of 5 .5 reported by MACGRECOR (1959) . The intense staining with mineral reagents in decayed areas does not indicate that there is more calcium present in the regions visualized by the stain, but it can and probably does mean that the mineral is in a more loosely bound state and is accessible for reaction with the specific dyes . These observations confirm and extend those of SULLIVAN (1954), who used ground sections and alizarin red S, This conclusion is further supported by the fact that otherwise normal enamel reacts with calcium stains after gentle demineralization in HCI or after removing organic components with ethylenediamine . It was interesting that both soluble and insoluble calcium salts showed red coloration with alizarin red S, while powdered enamel demonstrated this reaction only on some surfaces . Since the same result was obtained with a small piece of sound enamel that was reduced to a powder, the reaction cannot be attributed to the presence of carious surfaces in the ground material . Changes must occur in the crushing process, perhaps resulting from the uneven production of intense heat, that are similar to the chemical disruption that takes place in decay . Results with the protein stains suggest that, in normal enamel, mineral and protein are very intimately and tightly associated, and that both become "exposed" as a result of the decay process . Staining with the performic acid Schiff and performic acid methylene blue reagents, strongly positive reactions in the tests for sulphydryl, marked resistance to digestion by proteolytic enzymes, and failure to dissolve by prolonged boiling in acidified water, would all suggest that the organic component of enamel is, at least in part, a keratin. Resistance to attack by Microsporon gypseum

THE HISTOCHEMISTRY OF DENTAL DECAY

215

and by a suspension of clothes moth larvae may have been due to insufficient activity of the preparations or to inability to penetrate to the protein surface . An unanticipated observation was that the staining characteristics of the organic material in the dentine, as in the case of the enamel, were much like those of the keratins . The existence of collagen in the dentine has been clearly established by electron microscopy ; however, much of the organic component in the dentine is definitely not collagen as we know it in the rest of the animal organism . The fatty material observed in carious enamel and dentine is clearly not exogenous . Its presence in the very early lesions and in such a distribution suggests that it is an integral part of the organic component of the enamel rod, laid down when the structure was forming (hence its 4 µ increment) . It is hypothesized that, just as the mineral and protein are "unbound" by the decay process, the lipid moiety is released from its binding materials by action of the carious process on the lipid derivatives . It is further considered a good possibility that this lipid material, once set free, accompanies or even precedes the spread of the decay process into the dentine via the tubules . A most interesting observation was that the dentinal tubule, normally filled with AMPS substances, had no enzyme activity by the methods employed . In areas adjacent to decay areas, however, the AMPS disappeared, fat droplets appeared (Fig . 7) and the enzymatic activity by succinic dehydrogenase was marked . The changes seen in artificially induced lesions were in all respects similar to those observed in natural dental decay . In addition, it was possible to show that, in vitro, uncoupling of mineral was the first change that could be demonstrated histochemically, followed by development of the acid reaction, the protein reaction, and the lipid reaction, in that order, This would indicate that a certain amount of demineralization must occur before protein becomes accessible to react with the staining solutions . Of the several potential anticaries agents tested, only SnF 2 showed marked effectiveness . Not only did this agent protect enamel from attack in the dextrose-agar system, but it abolished reactions for mineral and proteins in already induced smoothsurface lesions . The previously carious enamel appeared normal histologically and histochemically . The migration of pigment from the surface of enamel toward the dentino-enamel junction in teeth treated with SnF 2 is a surprising and unexplained phenomenon . Since enamel treated in this way shows no uptake of basic dye, it does not appear to have been made acidic : the movement of pigment inward would not seem to be the result of acid attack .

SUMMARY AND CONCLUSIONS A.

Naturally occurring dental decay

In the examination of decay lesions found in extracted human teeth, the following conclusions may be made based on histological and histochemical observations of thin, sawed, cross-sections of the teeth,



216

D. L. J .

OFDYxe

I . The carious lesion is characterized by the presence of loosely bound mineral, protein and fat, and by its pH of 4-2-4-6 . 2 . The minerals, proteins and fat are intrinsic components of the sound enamel . These are uncoupled or unbound in the decay state and made reactive to histochemical reagents . 3 . The minerals of sound enamel can be removed by acids which "expose" proteins . 4 . The organic component of sound enamel can be removed by ethylenediarnine which "exposes" minerals . 5 . The protein component of enamel has solubility, enzyme indigestibility and tinctorial characteristics like the keratins . The dentine has a scleroprotein histochemically similar to that of the enamel in addition to its collagen . 6 . The fatty materials in a carious lesion can he extracted with fat solvents before or after staining . 7 . Lipids demonstrate the progress of decay into the dentine . 8 . Small "white spots" give positive mineral reactions and reactions with indicators but only the larger ones (greater than I mm in diameter) give positive reactions for proteins and fats . B . Artificially induced decor

Similar histological and histochemical studies made on teeth in which decay lesions had been induced by exposure to lactic acid in agar tubes enable us to conclude as follows about artificially induced decay . l . Artificial dental decay, that is indistinguishable in staining characteristics from the naturally occurring lesions, can be induced by simple acid attack . The reactions for minerals, proteins, fats and pHH are identical . 2 . There is a time relationship in the order of appearance of the histochemically demonstrable components visualized in dental decay, viz . minerals, pH, proteins and fats . 3 . The protein reactivity in the artificial lesion is a function of the surface area of the tooth involved . C . Effects produced by anticaries agents

Observations on teeth treated ten times with 20 min treatments in solutions of several anticaries agents and subsequently subjected either after or during the treatments to the agar tube acid attack method lead to the following conclusions . 1 . Stannous fluoride offers complete protection against artificially induced enamel caries . Sodium fluoride offers minimal protection . 2 . Stannous fluoride abolishes reactions for minerals and for proteins in already induced lesions . The previously carious enamel appeared normal histologically and histochemically . 3 . Stannous fluoride produces a characteristic effect halfway through the enamel thickness. This is manifest by a migration of superficial pigment to a zone midway in the enamel .

THE HISTOCHEMISTRY OE DENTAL DECAY

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Acknowledgements-This work would not have been possible without the generous co-operation of a number of Cincinnati dentists . Particular thanks are due Dr . ROBERT E. APPLEGATE, whose encouragement and advice have been especially appreciated . REFERENCES BARRNETT, R . J . and SetIGMAN, A . M . 1952 . sulfhydryl groups . Science 116, 323-327 .

Histochemical demonstration of protein-hound

BDRSTONE, M . S . 1955 . An evaluation of histochemical methods for protein groups . J . Histochem. Cytochem . 3, 1-18 . BURSTONE, M. S. 1958 . Histochemical comparison of naphthol AS-phosphates for the demonstration of phosphatases . J. nat . Cancer Inst . 20, 601-615 . FRANCIS, M . D . and MECKEL, A . H . 1960 . An in vitro method for producing carious lesions . J. dent . Res . 39, 697-698 (Abstract No . 127) . GRAY, J. A . and OPOYKE, D . L . 1960 . A device for thin-sectioning of hard tissues . J . dent . Res. 39, 698 (Abstract No . 128) . MACGREGOR, A . B . 1959 . Acid production and the carious process . J . dent. Res . 38, 1055 . MAZIA, D ., BREWER, P. A. and ALFERT, M . 1953 . The cytochemical staining and measurement of protein with mercuric bromphenol blue . Biol . Bull ., Woods Hole 104, 57-67 . McGEE-RUSSELL, S. M . 1958 . Histochemical methods for calcium . J . Histochem . Cytochem . 6, 22-42 . PEARSE, A . G . E . 1951 . The histochemical demonstration of keratin by methods involving selective oxidation . Quart . J. micr . Sci. 92, 393-402 . PEARSE, A . G . E . 1960 . Histochemistry, Theoretical and Applied (2nd Ed .) . Little-Brown, Boston . SOGNNAES, R . F . and WisLocKi, G . B . 1950 . Histochemical observations on enamel and dentine undergoing carious destruction . Oral Surg . 3, 1283-1296. SULLIVAN, H . R . 1954 . The formation of early carious lesions in dental enamel--L 33, 218-230 .

PLATES I AND 2 OVERLEAF

J. dent . Res .



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OPPYKE

PLATE I FIG . 1 . Molar tooth stained with methyl red showing characteristic colour at pH 42. Observe absence of staining in the sound enamel and dentine whereas the carious lesion stains brilliantly. x 20 . FIG . 2 . Section stained with toluidine blue at pH 4 . Observe the intense uptake of dye in the very acidic carious region which appears dark blue and the lavender metachromasia of the interprismatic material in the sound enamel . x 250 .

Pro . 3 . Section stained with alizarin red S. Carious region is intense red . Less distinct red areas are regions where the section has included some predentine . x5 . FIG . 4. Mercury bromphenol blue stain for protein . The intense blue marks the unbound protein of the carious area as well as the tip of the pulp cavity . x 5 . FIG . 5. Oil red 0 stain for lipids . Early carious lesion . Only the carious area is stained . x 250 . FIG . 6 . Oil red 0 stain of incipient lesion . Observe the regular 4 µ periodicity of the staining on the individual enamel rods . x 1000. FIG . 7 . Oil red 0 stain for lipids. Observe the fat droplets in the dentinal tubules . This always occurs in dentine deep to the enamel lesion and is detectable when no other signs of dentinal involvement are apparent . x 1000 .

THE HMTOCHEMLSTRY OF DENIAL DECAY

PLAIT

I

D . L . J . OPDYKE

PLATE 2

THE HISTOCHEMISTRY OF DENTAL DECAY

PLATE 2 FIG . 8 . Mineral reaction as shown by alizarin red S stain . Artificial decay produced by acid attack in agar system in 10 days . x200 .

Fic . 9 . Same as above but stained for protein . Observe the brown line "front" of pigment materials . x 200 . Fm . 10 . Similar to above but stained for fats . Observe pigment "front" and a healed crack or lamella . x 120 . Fm . 11 . Similar to above but stained with alcian blue for acid mucopolysaccharides . x 200 . Flc . 12 . Cross section of a tooth showing small induced lesions . Stained for minerals . x 8 . Ftc. 13 . Two small spots from above tooth. Compare with natural lesions, x 250 . Fio . 14 . Mineral reactions on tooth quadrants treated in agar system for 20 days . From left to right : (a) control quadrant, (b) SnF,-protected quadrant, (c) NaF-treated quadrant, and (d) quadrant unprotected by any anticaries agent . x200 .

219