The decalcification and discoloration of intact noncarious human tooth crowns by an oral strain of lactobacillus acidophilus

Research THE

DECALCIFICATION AND DISCOLORATION OF INTACT NONCARIOUS HUMAN TOOTH CROWNS BY AN ORAL STRAIN OF LACTOBACILLUS ACIDOPHILUS+

I’ELLO11V discoloration of the involved enamel and dentin<: is characteristic of hlmlan dental caries. This pigment has been isolated and idcntificd as a melanoidin.l As such Leicester” stated that it can be formed only as a result of Ihe breakdown of the protein matrix of the enamel and dcntinc involving thr c;sidation of tyrosine by an enzyme such as tlol~ ositlase, and that “prOtcolytic” bacteria are required for this process. In a series of studies performed in this laboratory, it has been shown, howcvcr, that a pigment similar to, if not identical with, that present in the natural lesion CiIrl be prodnced chcmicall~- I,- the reaction between some of the carbonylcontaining derivatives of glucose and the amino acids or intact proteins of tooth structure which contain functional amino groups not joined in a peptide linkage (Fig. 1) .8, 4 It has been demonstrated that this pigment can be formed in the protein matrices of decalcified noncarious human tooth crowns by substances elaborated by oral lactobacilli in a synthetic medium containing g1ucose.j Since many investigators have implicated the oral La~ctobacillus acidophilus in the initiation of the carious lesion in human teeth, this study was undertaken to determine whether a pure culture of a strain of this organism could decalcify as well as discolor intact noncarious human tooth crowns.

A

Materials

and Methods

Seven newly extracted human permanent teeth free from any evidence of dent,al caries mere cleaned by scrubbing with pumice and washing with distilled water. The teeth were sectioned at the necks, and the crowns covered with pink baseplate wax. A window, approximately 1 cm. in diameter, was carved in the wax so as to expose a part of the labial surface of each crown. Each wax-invested crown was incubated at 37O C. in a tube containing 10 C.C. sterile yeast extract dextrose broth and 0.5 cc. 95 per cent ethyl alcohol. The tubes were examined at the end of twenty-four hours for evidence of bacterial growth. In the presence of turbidity the crown was removed, washed thoroughly with distilled water, and placed in a tube containin, 0‘ 10 C.C. sterile yeast extract dextrose broth and 0.5 (a.~. 95 1)~ cent ethyl alcohol. In the absence of turbidity the broth was replaced by an equal amount of test medium minus the alcohol. The t,ubes were reincnl)ated at 37’ C. This procedure was repeated until no growth was observed in alcohol-free test broth for a period of three consecutive days.

1kLEIZEX ASD SPIES : THE DECALCIFICATION AND DISCOLORATION

ORAL STRG., ORAL MED., AM ORAL PATH., MARCII, 1951

OF IL-TACT NONCAKIOK? HI-MAX TOOTIS CROWVSSBY A;\ ORAI. ATRAIS OF LACTOB.\ClJ,T,I-S ~~cIl~OI~IiII,~-S

wig, 1 _- --.a comparison of the synthctic~ally pro~luced and naturally wcwring pigment assuciate~l \I-itI1 dental caries. The The tooth on the left contains a lesion formed in ViVO. pigmentation in the tooth on the right was produced by the action of methyl glyoxal On the t%posed rnarnal and underlying dentine of H wax-invested caries-free tooth cro~vn prWioUSlY &calcified \vith lactic acid. Fi,q. 2. Photomwrograph shaving the distribution of the yellow color in the involved enamel and dentine. The lesion on the right was formrd in vitro by exposing intact noncarious human tooth crowns to the action of a pure culture of n strain or oral Ltcctobctci77~s ncidophilw. The section on the left is that of a naturally occurring lesion.

DECALCIFICATION

AND

DISCOLORATION

OF

HUMAN

TOOTH

389

CROWNS

The seven teeth were divided into three groups. Group I consisted of three teeth, each of which was incubated in 10 C.C. sterile yeast extract dextrose broth inoculat.ed with one loopful of a twenty-four-hour broth culture of a strain of oral Lactobacillus acidophilus recovered in this laboratory from the saliva of a patient with active dental caries. Group II consisted of two teeth, each of which was incubated in 10 CL of uninoculated test broth to which was added 1 cc. sterile 0.1 N lactic acid. Group III consisted of two teeth, each of which was incubated in 70 C.C. of uninoculated test medium. The inc~uhation temperature was 37’ C. At the end of each seventy-two-hour period, the tubes were removed, the liquid contents decanted and collected, and the broth replaced by an equal amount of sterile test medium. The bacteria remaining in the tubes in Group I following decantation served as the inoculum for the fresh broth. One cubic centimeter of sterile 0.1 N lactic acid was added to each tube in Group II. The pH of the broth collected from each group was determined with a glass electrode. Gram stains were made of the cultures from the inoculated tubes. This procedure was repeated every third day for a period of thirty days. At the end of thirty days, the crowns were removed from the broth, stripped free of the surrounding wax, and examined with a hand lens for evidence of decalcification and discoloration. One crown from each group was decalcified in 50 C.C. of 5 per cent HCl for approximately sixteen hours to determine whether the discoloration, if present, had involved the organic matrix of the enamel and dentine. A second crown from each group was used for the preparation of ground sections. Cross sections through the lesion produced in vitro were examined microscopically and compared with similar sections made from teet.h containing lesions formed in vivo.

Observations The changes occurring in the exposed portions of the was-investctl intact period are sumhuman tooth crowns in each group durin g the experimental marized in Table I. In the teeth in Group I, a light yellow color and a slight etching of the surface enamel were noted by the sixth day. The color deepened t.o light brown by the ninth day, brown by the twelfth day, ant3 deep brown by the fifteenth day. From the sixth day through t,he eighteenth day the exposed enamel surfaces of these teeth Showra macroscopic evidence of etching. On the cighteent h day a gross break was observed in the sllrface continuity of the tbsposed cnamrl whicah became progressively clceper in t lit: rcimaincler of the (~sl)&mental period. No changes either in tht: color or in the c:ont,inuit~~ of the ~x~~~s;cti etlaruet

surfaces

of the :i:Cl.!i

idOlllIi2’iSillg

(-1 t*O:lp~

I I

21!4

1 I I

‘\Vf'I'i'

vnttwj

h!,

inspection throughout the period of obscrratjion. The pH of the broth collected at thrct+tlay intervals E~WII~t,tlc tubes in Group I ranged from 4.02 to 4.29. It, was cibservcld that acid production in these wit,11 a tubes diminished after the twrnty-first day. This finclillg t~Oi~~ciaca change from a uniform tyl,e of l)actt:rial gt’o\rth in the test brotll to a type characterized by a &unpin g of the bacteria it, the deeper I)orl ions of the medium. Gram stains made un loopfuls OFbroth from the t ub~s ill t llis group at three-day intervals did not, disclose the prescncc of any organisms cjthcr than the test

E! 24 27 30

1: 15

TIME (DAYS) 3 6

XI

-

GROUP I (INOCULATED) CONTINUITP OF COLOROF EXEXPOSED TOOTH POSED TOOTH SURFACE SURFACE -Unchanmd Unchanged Slight etching Light yellow Definite etching Light brown Marked etching Br0Wll Marked etching Deep brown Slight cavitation Deep brown Slight cavitation Deep brown Slight cavitation Deep brown Cavitation Deep brown Cavitation Deep brown

-

--

!-

GROUP II (u: NII - iOCULATRD ACII D- CONTROL) COI#R OF EXCONTINUITP OF PH OF POSED TOOTH EXPOSEDTOOTH BROTH -SURFACE SURFACE Unchanged Unchanged 4.95 4.92 Unchanged Unchanged 4.90 Unchanged Unchanied 4.95 Unchanged Unchanged 4.93 Unchanged Unchanged Unchanged 4.98 Unchanged 4.90 Unchanged Unchanged 4.90 Unchanged Unchanged 4.95 Unchanged Unchanged 4.91 Unchanged Unchanged -

-

PH OF BROTH 6.08 6.10 6.15 6.06 6.02 6.07 6.01 6.04 6.06 6.04

GROUP II :1 NINOCULATED -co NTROL) COLOROF EXCONTINUITP OF POSED TOOTH EXPOSED TOOTH SURFACE -SURFACE Unchaneed Unchanged Unchan&d Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchansed -

ZYZ

THE CHANGES IN THE TEST MEDIUM AND IN THE INTACT HUMAN TOOTH CROWNS IN THE THREE GROUPS DURING THE EXPERIMENTAL PERIOD

PH OF BROTH 4.20 4.12 4.09 4.08 4.10 4.02 4.09 4.29 4.28 4.28

TABLE I.

DECALCIFICATION

AND

DISCOLORATIOn’

OF

HUMAX

TOOTH

CROWXS

391

organism. Morphologic changes in the test organism similar to those seen in old cultures of lactobacilli were noted during the last ten days of the study. The PI-I of the seventy-two-hour broth from the tubes in Group II ranged from 4.90 to 4.95 and that from t,he tubes in Group III ranged from 6.01 to 6.15 (Table I). The tooth crowns from each group exposed to the action of 5 per cent HC1 for approximately sixteen hours underwent rapid decalcification. The brown discoloration present in the crown from Group I was retained in the organic matrix of the enamel and dentine following the dissolution of the inorganic salts. In cross section, the discoloration involved a triangular area with the apex toward the pulp and the base at the periphery. In color, location, and solubility, the pigment resembled t,hat seen in naturally occurring carious lesions. No discoloration was observed in the organic matricc>s of the decalcified tooth crowns from Groups II and III. Ground cross sections from a tooth in Group I and from a tooth containing a naturally occurring lesion of the smooth surface variety are shown in Fig. 2. The sites of the lesions are the labial surface of a lower lateral incisor and the distal surface of a lower first premolar, respectively. Each section shows decalcification of the enamel and dentine and yellow discoloration in the involved and underlying areas. The discoloration follows the path of the dentinal tubules. The penetration of the yellow color in the tooth containing the lesion produced in vitro is more shallow and more uniform than that in the tooth containing the lesion produced in vivo, a difference attributable in part to differences in the time of exposure and the conditions under which the two pigments were produced. Ground sections made from the teeth in Groups II and III did not, show any microscopic evidence of decalcificat,ion and discoloration.

Discussion The mechanism of the format,ion of the yellow color in the carious lesions of human teeth has long been t,he subject of speculation. In the past,, this discoloration has been attributed primarily to the action of “ proteolytic” bacteria. It was postulated that organisms capable of splitt,ing proteins invade tooth structure along the organic pathways and yield metabolic products which react with the dental proteins or their derivat,ivcs to form the characteristic pigment.6 The findings obtained in this study indicate that, organisms other than . ’ proteolytic” bacattria ma>- be c’onccrned Cth pigment formation ill the carious lesion. The lesions pmduccd in vitro in intact human tooth t~owns exposed to :I10 artion ilt' a plircl r*tlltllr.c :-rf it strain of orH1 i,trc,ioljric,;ll’rts r7c’iiftjp:til~l.y ill a. synthetic i~rrtlium c+onlainin y plliroScl ,Wsr~~Irl~lt?~lthosc~ Seen in viva both grossly These observations are in accord with those recently reand microscopically. ported b,- Wcisberger$ who produced a pigmented rarious change in the exposed enamel surface and underlying dentine of intact human tooth crowns in a glucose-containing medium consisting primarily of known chemical substances and inoculated with mixed oral flora. In both instances, the pigment associated with dental caries was produced in a highly acid medium by organisms capable of fermenting carbohydrates. It would appear, therefore, that the pigment

32

SAMUEL

DREIZEK

-4ND

TOM

D.

SPIES

produced in the carious lesions of human teeth may be the result of a chemical reaction between certain degradation product,s of glucose and the amino acids or intact proteins present, in toot,h structure.

Summary and Conclusions 1. Exposed enamel surfaces and the underlying dentine of wax-invested intact caries-free human toot,h crowns have been decalcified and discolored when incubated in yeast extract dextrose broth and inoculated with a strain of oral The broth was replaced every third day for a period Lactobacillus acidophilus. of thirty days. 2. The findings indicate that degradation products elaborated during the fermentation of glucose by the Lactobacillus acidophilus and the amino acids or intact proteins present in tooth structure may be involved in the formation of the yellow color associated with human dental caries.

References 1. Deakins, M.: The Isolation of a Melanin-Like Substance From Carious Organic Matrix of Human Dentin, J. D. Res. 20: 39, 1941. 2. Leicester, H. M.: Biochemistry of the Teeth, St. Louis, 1949, The C. V. Mosby Company, p. 270. The In Vitro Production of 3. Dreizen, S., Greene, H. I., Carson, B. C., and Spies, T. D.: in the Organic Matrix of Non Carious a “Yellow Brown Melanin-Like Pigment” Human Tooth Crowns by Methyl Glyoxal (Pyruvic Aldehyde) and Acetol (Acetyl Carbinol), J. D. Res. 28: 26, 1949. A Comparison of the Chemical Nature of a 4. Dreizen, S., Greene, H. I., and Spies, T. D.: Synthetically Produced Pigment and the Naturally Occurring Pigment Associated With Dental Caries, Oral Surg., Oral Med., and Oral Path. 2: 922, 1949. A Note on the Production of a Yellow Brown Pigment in 5. Dreizen, S., and Spies, T. D.: the Organic Matrices of Non Carious Human Teeth by Oral Lactobacilli, Oral Surg., Oral Med., and Oral Path. 3: 686, 1950. 6. Gottlieb, B.: Dental Caries, Philadelphia, 1947, Lea & Febiger, p. 68. A Role of Glucose in the Production of Artificial Caries, J. D. Res. 29: 7. Weisberger, D.: 14, 1950.