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									     Journal of Dental Research
             http://jdr.sagepub.com/




         Nutrition and Caries : IV. Histologic Investigation
                     M. Nederveen-Fenenga and L.M. Dalderup
                            J DENT RES 1956 35: 39
                      DOI: 10.1177/00220345560350011401

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                                      NUTRITION AND CARIES
                                      IV. HISTOLOGIC INVESTIGATION
                     M. NEDERVEEN-FENENGA AND L. M. DALDERUP
            The Netherland Institwte of Nutrition, Amsterdam, The Netherlands
INVESTIGATIONS on the effect of diet on the appearance of dental caries
   in rats and hamsters have been in progress for some time at the Netherlands
Institute for Nutrition (Nederlands Instituut voor Volksvoeding). The results
so far have been described by Jansen, Luyken, Dalderup, Nederveen-Fenenga,
Boonstra, and W~istmann,' Nederveen-Fenenga and Dalderup,2 and Luyken,
Dalderup, and Jansen.3 Some histologic changes in the molars of our experi-
mental animals will now be described.
     In order to examine the effect of the composition of the food on the
frequency of caries in rats, the jaws of rats which had received various diets
for different periods of time were compared. All the experiments were con-
ducted with Mus norvegicus which have been bred for the last 20 years in this
laboratory. These white rats, in groups of at least 20, were fed a particular
diet and were sacrificed after observation periods of 21/2 to 6 months.
     The jaws of all the animals were examined under a binocular microscope
at x15 according to the scoring method of Keyes, with our own modification.
The jaws from a number of animal groups were also cut longitudinally into
2 or 4 sections. The caries was then evaluated under a microscope with a magni-
fication of 50, after staining with fuchsin. In addition, sections were made
from the jaws of 2 animals from each group for histologic examination. The
histologic preparations consisted partly of thin sections which were decalcified
and partly of thicker sections which were not decalcified. Clearly, a better
estimate concerning the enamel is possible with the latter than with the former.
     Initially the jaws were examined after the experimental animals had
received a particular diet for a period of 41/2 months. The cavities, however,
were often found to be so large that while they were suitable for the determi-
nation of the caries percentage, they exhibited too much destruction for accurate
differentiation of size and number. The observation period was therefore
reduced for the histologic examination to 21/2 months or less, depending upon
the diet. The changes which consequently appeared facilitated a better com-
parison.
                          TECHNIC FOR THE HISTOLOGIC EXAMINATION
    The following stains were used in the histologic examination.
    1. The methyl blue-cosin stain of Mann, modified according to Dobell.4
       This stain is very useful for visualizing the finer cell structures. The
    Received for publication March 6, 1954.
                                                                  39
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40                            NEDERVEEN-FENENGA AND DALDERUP                                                                          J. D. Res.
                                                                                                                                 February, 1956

          chromatin of the nuclei becomes deep blue, the nucleoli red, the cyto-
          plasm violet, the connective tissue red, the dentin blue, and the odonto-
          blasts and their processes red.
     2.   The P.A.S. (periodic acid-Schiff) reaction of lMlacManus.5 Glycogen
          and certain glycoproteins are stained deep carmine by this method.
          Glycogen can be distinguished from other substances which give a
          positive reaction by treating control sections with saliva for 15 to 30
          minutes. If both sections give the same reaction, glycogen is not present.
     3.   The silver impregnation method of Laguesse.6 This stain is used to
          detect reticular fibers. The sections are treated with ammoniacal silver
          solution. On reduction of the silver the reticular fibers become black.
          The cell nuclei are stained red.
     4.   The van Gieson method.7 This is a nuclear staining with Weigert's
          iron hematoxylin followed by picrofuchsin, as a result of which collagen
          and reticular connective tissue are stained red and the epithelial cells
          yellow, while the nuclei become black.
     5.   Gentian violet. This is a nuclear stain which at the same time shows
          gram positive bacteria which are stained violet.
     6.   Methylene blue stain which can be compared with gentian violet stain.
     7.   Thick sections (c. 500 1A in contrast to 8 /u in the case of stains 1 to 6)
          which are not decalcified were stained by fuchsin. This method has
          not been previously described. The staining is carried out as follows.
          The sections which have been preserved in 10 per cent formol are placed
          in a 1 per cent aqueous fuchsin solution. After 15 seconds they are
          washed with tap water and placed on an object glass. Excess water
          is removed after which the preparations are immediately examined in
          the moist state under the microscope.
     8.   Thick sections were also stained by the method of Burnett and Scherp.8
          This reaction is based upon diazotization with sulfanilamide. In places
          where accessible histidine and tyrosine groups have been formed by
          decalcification, an orange-red color is formed.
     9.   Unstained decalcified sections (8 ,) were examined with a polarization
          microscope.
                                                             RESULTS
      The process which is described here was observed in sections of rat molars
in which no caries could be observed macroscopically. The course of the
lamellae described by Gottlieb,9 Bodecker,'0 and other workers could be seen
in these sections.
      Under low magnification (x150) sections from decalcified jaws which have
been stained by the Dobell-Mann method and in which some of the enamel is
still visible, the lamella can be followed in serial sections. This lamella runs
from the surface of the enamel to the boundary between the enamel and dentin
and is stained a definite red by this method. The lamella is sometimes single,
while sometimes more than one can be observed in one section (Fig. 3). The
lamella is generally situated at the deepest point of the fissure, although it



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Volume 3;                              NUTRITION ANI) CARIES.                                TX                                  41,
limberr I

sotietiiiies 0e1181t1s    101re occlisally. The im1)l5ssi01i. is gained that the lainella
is 10o1ind( ini flie (leiitiii ii the waytN in) which a t1e01(111 is attaebe( to Shlarpey-'s
fiblers ill 1b)10e tissile. The lul0C05 (leveloljs at the place -\where such a lamella
irudes into thle (dentin anl(l extends -Ilpallel to tIlie anielodeltinial junction,
generall> on 1)oth sides of' the lamiella and more or less l)(Tpenldicullair to it,
8n1( under the 1)asal memebr-ane oil the amelodetntinral juiction.
      The pr:Xoeess observed coI esonlds to tile pattern of' fissure caries.                                                    The
caxit! on the einamnel suLirface is clinically small while the largest diameter
of tile cavity- -is situlate(I at the enamel.-delit-in bounlary. Severe ntidermininig
of dlentin is slhown. (Figy. 9).
      The p process was not of constant dimensions, larger and smaller lesions
being lound in different animals which were interpreted as earlier and later
 ;taucls of development. If the section is stained
                                                (1) the AMann-I)obell method




                   K'ig.   1.-   M~ethyl      blue cosin stain of
                                                            Ma.nn.Dohol.. .                             (X275 )

 (Fig. I1), tiue I mella anld thle process are both stained red. A few d3entinal
tubuvlles, 'which4 proc(veed froni] the process in1 the (lireetioll of thle pu1ij) are also
stainledl v+ the -r(1 dye in conltrast to thle other (lenltinlal canlals; which are stained
iduelt andcl ontainl red O(lontob~last p~rocesses; inl the lumlen.
       If the1 pr ocess is examined wvithl a mlagnification of 275& (Fig. 2),> it is
observed that the contents of tlue haniella differ from the structure of the walls.
rpth0 warll~s of thle lamellae continue to) the enamnel culticile. Furthler, tile boundary
oAf the enamel and the dentin is dlisl~lac~ed in the region of: the process at the
expense of tile enzamael. Thlis can 1)0 seen in Fig. 2} as a culrve inl the ainelo-
dentinafl juncltionl to tile le-ft 811(1 right next to tile process.



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42                   |N)ERV| EN 1 EN1NGA
                     X Egg                                          ANI) 1D) a\[)ERIT1                   1. D.
                                                                                                            1).




            Fig. 2.       Methyl blue eosin stain of Mann-Dobell.                              (X275.)




              Fi,-g ".-Methvyl blue-eosin stain                   of Mann-I)obell.              (X5X.)




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Vol une 35                                N U TBI TRf)N A ND1) CARIES.                         TV                                       43
    tiber

      Trlih. (lcestirnil unow Carises wxletlher tiw-el                          is Ca    swewlling(,       or    thickening       of   the
tl(en1till (o1 xxhetlie   l)-h ('ess is situated oiitsite the (deintin aid causes only
                         the
alt aIp)paretilt    thickenilg of, the littler. III orderl to itlvestigate this point,
iticistiiiede setiiios; wexl esxiiiiintLl W/ithl te J)olOIiziiig mieroscwope. The deiitinal
tIiiuiles could h1e allowed through thle cutlie p1 oess up) to the dent inoenanel
*junlleti11.
      JIt 1n11SIt be assumed, therefore, tIhat the whole process actually takes place
if] thle dent-inlland that aI. swelling is caused 1y iyrophl)ilic colloitis (Vig. '2).
TIwo llalalc ean he seen iii the section in Fig. 3, aid 't will lie observed
  XXlN
thllt thle,process is eolinected to bothl of the laliellac in the region of the
deiitmi. TherT is r-io comlliioni coiiiiectiori to be seer litetweeni tle 2 jprocesses
am moic thall thlere is between the 2 lamellac. A verx tlefinite swelling of
flie affeted (ldentin hivc-c can he seen herie. Fi. 4 shows a more adaii          aced
stage. The process lhals extelded inlto the dientin pairallel to the dentinoenamel
jliietiOn anti has uinderminiedl the deniin lat the edges. whjiieh still appear nTiormal.
      The whole process is sharplyv deiiiaeated aid, v,,s it extends further, tlle
p)inlt olf iadvanlice into the dentin can he followed. The odontoblast processes




                                     Fig-. 4. -Ml~etiyhnsllfe bimle: dstJin.         .
                                                                                           (   10. )
 (ired) can be :fol low+ed iii th}e clinlt iii ii tuballe~s Kb uc ) upy to thle boundary- o~f
                                                             l

thle process ix hu h-] mr1Cl indicate its extenlsion B. icr1ena~ Canr be clearly demnlol
str~ated: in tin ioeshr()EX means }inc
                                  of>ll let!}n, ce bluie andf (ventian vriolet. Thwe
sharply tleidi
          (ltahlon of- thle process hlouever v a;es thle qulestionl whether!T theC
hxaeter~ia la 1 important parnt heiC.' The ~pI'ocesiS :ecoinles furlthler extendled
             1an
toevwardel thlle enaml~zel and reachles thle enanilseL-denltinl b~ountldary (Ftig. 5). No..
                                     I
 iiomriil dlent; ur cii be ohiserx ed inl the dliietionz of thze enamel an~d thle process
re.sts as through'} it were al flalt dis;k onI top of thes s.ounde denetin. This can also
lie seent inl the sectionls xvhiehl arel not delewelc fledS (Fig. (3) wheree thle enamel
is entlirl inltact and( thze dlisk is ehlearli iisihile. The dlisk becomes d1ark red
           c
On1 stallining WxitIl fuehslliu, while s~tainling' by the(- melt hod( of Burlnett anld Seherp
gives al more olang>e-nd color. Muchtl seciildatry denetin is; l)1eSetlt on the side
of thec Jut.lp.
        In a furlthler stag~e (Fsigt. 7), a slight s~eparaltionl of thle process friom~ thle
 Ilentiii can.1 bet oblservedl at thle extremlliti;es as al resultl o)f shrinkage dulring
Prel~~lniation oft thle section. This is legardled as an artifact



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44                              AIM )LRV           N F N N Gt ANI 1) 1A)1D)ER12                                                     J. D. Res.
                                                                                                                                February,   1956



                                                               Fit. 5.




                                                               Fig.    6.


     Fig. ,i.-PAS reaction Of MacAImbnus.                     (X,50.)
     Fig. fi.-Fuchsin stain. (X;0).)




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\utluine :;i                               SNUTRITIONt A.NI-) CGARIES. IV                                                          45
\N umber




                               Fig. 7.-PAS reaction of MacTleans.                                 (X 0. )




                       Mig. 8.-Silver impregnation method of Laguesse.                                      (X50.)




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46                          N- I' )ERlYVI, .1N-Ii'ENE'NGA ANI) D.A-LDEI-RUP                                                            .1,   1J. R-S.
                                                                                                                               Fe
                                                                                                                                (li)         3, 1956

      If the prlcess is stained at this stage by tie mettiohd (01f' MaeManus, th]e
glycogen reaction within carmine is foind to be positive. The control treated
with saliva, however, gives a reaction of equal intensity so that the I)p0res11c
of g1ycogen cannot 1)e assumed at this stage and( the intense loeal staining
along the lower a11( upper edges of the process must be regar(le(l as (lecalci-
fication. Whlen bone is decalcified, thle method of Mac.A.tanus gl-ives an equally
intense staining of the osteocytes at the place wh-lere extensive bleakdown takes
plzlec and ela-cium has dlisaJppearedl locally- as a result of depolynerization of




                  Fig. 9.       Methyl blue eosin staLin of 1'rann-Dobell.                             (Xl5.)

nluneopolysace-liarides (giyeoproteins) As tile process becomes older., a less
intense color is oIwodilced.
      When. sections whliclh are not decalc{dified atre stained lby the met-hod of
Bullretf and Scherp, the (liis referred to 1)ecomes clear>11, visible inI th1e Prep-
aration. This l)ints to a decalcification in whichl lieessilile ty.rsine and
Iiistidine groups are formed tliirough rupture o f t lie linkage lictweeni organic
and inorganic iitaterial or through spatial rearrangement of the proteini.
Proteolysis also is a possibility here.



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Volume 35                               NUTRITION AND CARIES. IV                                                                47
Number I

     If decalcified sections are made and stained by the method of Laguesse
(Fig. 8), definite fibers, which are stained black by silver impregnation, are
seen running crisscross in the middle of the process and in the middle of the
lamella. This indicates the presence of reticular fibers. This silver impreg-
nation is seen at the same time at the enamel-dentin boundary.
     If the sections are stained by the method of Gieson, then the disk is
colored yellow with red, with black streaks in the center, so that the presence
of collagen and reticular fibers can be assumed.
     Staining of thick sections with fuchsin is not specific. The various stain-
ings give, as yet, no clear picture of the course of the reactions at the site
of the process, so that attempts will have to be made by further investigation
to obtain some insight into the different structures. It has been demonstrated,
however, that a decalcification with depolymerization of mueopolysaccharides
and production of accessible tyrosine and histidine groups in the disk-shaped
process takes place. Many reactions take place, especially on the edges of
the process.
                                                         DISCUSSION
     In our view, the process described represents a very early stage of caries.
A practically identical process in human teeth has been described by Barker,1'
Rosebury and Karshan,12 and others. The reactions described by Burnett
and Scherp were carried out with human teeth.
     The relation of the lamellae to the process is not entirely clear. The
question whether the lamellae are present in the enamel before there is a
reaction in the dentin will have to be investigated. No disk in the dentin
should then be visible in the first stage of a process beginning in the lamellae.
     A further point to be investigated is whether the lamellae are more
readily stained as a result of the process which is beginning or can only be
stained when the process has reached the dentinoenamel junction. If the
lamellae are, in fact, the earliest stage of caries, all foods with a known
cariogenic effect should cause the lamellae to appear.
     At the present time, the development of the process in the lamellae is
being investigated with very young rats, whose mothers had received a cario-
genie diet.
     The walls of the lamellae are different from the contents, the wall being
doubly refracting but the contents not being so.
     The question remains whether the swelling begins as a reaction to the
hydrophilic properties of the connective tissue itself or whether this, which
is much more probable, takes place as a result of the depolymerization of
mucopolysaceharides in combination with the reaction described by Burnett
and Scherp. It is also possible that the two latter processes are closely related
to one another. It is, in any case, very likely that the accessible tyrosine and
histidine groups are formed in the depolymerization of mucoid in which the
polysaccharide part splits off, leaving intact the protein part which is possibly
hydrolyzed later.



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48                           NEDERVEEN-FENENGA AND DALDERUP                                                                         J. D. Res.
                                                                                                                               February, 1956

    We were able to examine some human teeth in which we demonstrated
the same process in its entirety. The process was also observed in hamsters.
                                                          SUMMARY
     A process similar to that which had been demonstrated in human teeth is
described, with the aid of histologic reactions in the molars of Mus norvegicus.
The process proceeds in the dentin from the lower edge of a lamella and parallel
to the enamel-dentin boundary. It was shown by means of different histo-
logic stains that the process is accompanied by decalcification and depoly-
merization of mueopolysaccharides. Accessible tyrosine and histidine groups
were demonstrated. Connective tissue and reticular fibers can be observed in
the center of the lamella and in the center of the process.
      We wish to express our thanks to the T.N.O. organization for the provision of a
grant which made this investigation possible.
      The histologic preparations were made by Mrs. L. Bult-Vreugde, Histological
Laboratory, Amsterdam.
                                          REFERENCES
 1. Jansen, B. C. P., Luyken, R., Dalderup, L. M., Nederveen-Fenenga, M., Boonstra, J. P.,
         and W6stmann, B.: Voeding en Caries I (Nutrition and Caries I), Tijdschr.
         tandheelk. (The Netherlands) 58: 2, 1951.
 2. Nederveen-Fenenga, M., and Dalderup, L. M.: Voeding en Caries II (Nutrition and
         Caries II), Tijdschr. tandheelk. (The Netherlands) 58: 321, 1951.
 3. Luyken, R., Dalderup, L. M., and Jansen, B. C. P.: Nutrition and Caries, Part III. The
         Reproducibility of the Caries Scoring Method for Rats and the Influence of Gluten
         on Experimental Rat Caries, Int. Rev. Vitamin Res. 25: 42, 1953.
 4. Dobell, C.: The Amoeba Living in Man, London, 1919.
 5. MacManus J. F. A.: Histological Demonstration of Mucin After Periodic Acid, Nature
          158: 202, 1946.
 6. Heringa, G. C.: Reticuline und Kollageen, Ztschr. mikr.-anat. Forsch. 34: 4 and 459,
         1933; or BeIg. tijdschr. geneesk. 10: 7, 1948.
 7. Romeis, B.: Taschenbuch der mikroskopischen Technik, 167 and 195, 1948.
 8. Burnett, G. W., and Scherp, H. W.: The Accessibility of the Organic Dentinal Matrix,
         J. D. Res. 31: 776, 1952; 32: 46, 1953.
 9. Gottlieb, B.: Dental Caries, Philadelphia, 1939, Lea and Febiger.
10. Bodecker, C. W.: Enamel Lamellae and Their Origin, J. D. Res. 32: 239, 1953.
11. Barker, F. J.: Normal and Abnormal Tooth Structures, J. A. D. A. 18: 17, 1931.
12. Rosebury, T., and Karshan, M.: Studies in the Rat Susceptibility to Dental Caries,
          J. D. Res. 13: 379, 1933; J. A. D. A. 21: 1599, 1944.




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