6 . 1 . A b s t r a c t
T his paper describes t he effect of xylito l on demineralized enamel in plaque free and plaque covered condit.ions in vivo.
Fissure-like plaque retention grooves were created in 66 human enamel blocks and demineralized in vitro. The blocks were mounted in a prosthesis of 1 1 participants, who used a 2. 5% xyli
tol , a 2. 5 % sucrose or a water solution extra-orally in a random-ized cross-over design, for three periods of 16 days. The part-icipants submerged the prosthesis twice a day in the solution during 5 min utes. Mineral loss and lesion depth in enamel were measured before and after the in vivo experiment, using quanti
tative microradiography and polarized light microscopy.
L esion depth at the surface enamel was + 4 5 µ m, at the wall o f the grooves ± 3 0 µ m and a t the bottom of the grooves ± 50 µ m be
fore the exp eriment . Aftar 16 days the lcsion depth at the wall o f the gro oves was + 40 µm and at the bottom of the grooves + 7 5 µm. I n the groovcs n o differcnccs were found bctween the xylito l , the sucrose and the water treatmcnt. At the surf ace enamel a significan t rcduction of enamel dcminerali zation was found a ft a r the xylit ol treatment. T h e lesion depth at the
sur-face enamel increased 17 µm after the sucrose treatment and 7 µm after the xylitol t reatment . The mineral loss after the sucrose and the water treatmcnt were bath approximate ly 3x the mineral loss after the xylito l trcatment.
This chapter is submittcd for publication in Caries Research. "The inf luence of extra oral xylitol and sucrose dippings on in vivo enamel demineraliza
tion", M. T. Smits and J. Arends (1987).
C hapter 6 : Enamel demineralization 1n v ivo
6 . 2 . lntroducti on
Xylitol is a sugar substitute aften used in the f ormulation of non-cariogenic sweet foods. Reduction of DMF va l ues was found in severa l f ield studies where xyl i tol containing sweets or chewing gum were added to t he diet [ S c heinin et al., 1975b ; Sc heinin et al. , 198 5]. However, the mechanism of this caries reduction is not fully understood. It is known t hat
(i) xylitol in c hewing gum and sweets inf luences the quantity [ Grenby et al. , 198 2 ; al. , 1983 ; T uompo et and quality of plaque and saliva
Topitsogl ou et al. , 1983 ; Röl l a et al. , 198 3], that
(ii) xylitol reduces ename l demineralization in vitro [ Arends et al. , 1 984] and that
(iii) xylito l f arms complexes with calcium ions [Mäkinen and Soderling, 198 2].
In experiments with xylitol added to the diet, no distinction can b e made between effects on sa liva, effects on plaque and effects on enamel de- and remineralization.
I n the present experiment xylito l is not supplied as food ad di
t i ve but in the f orm of a soaking solution for enamel b locks in (partial) protheses. In this way the effect of xylitol on enamel d emineralization can be examined without major change of the saliva ef f ect.
T he purpose of this experiment is to examine the influence of xy
litol dippings on in vivo enamel demineralization un der plaque free and plaque covered conditions .
6 . 3 . M ater i al s a nd methods
Preparation of the enamel specimens
6 6 permanent human incisors, cuspids and premolars were col lected from denta l practitioners in the Groningen area (non-fluoridated) a n d stored in tap water at 4°C. Roots and pa latinal part of the teeth were removed with a sectioning machine, l eaving only the
7 3
-Chapter 6: Enamel dem 1 nera l iza t 1on 1 n v i vo
bucca l part of each crown. The buccal surface was cleaned w i l h pum 1ce and water, a 1 r dr 1ed and subsequently covered w 1 th na il varn ish except for a rectangular w 1 n dow of
water-cooled d iamond saw (Buehler, 11-424 3) , ±_ 20 mm2• Usi ng a two parallel grooves were created 1 n the ename l. T he depth of a grouve is apprux i -mately 5 00 µm , the w i dth about 200 µm. The spec imens were de-m 1 nera l ized i n d ivi dually in a calc iude-m phusphate buffer solu l ion , 50% undersatural ed w ith respect to HAP, a l pH 4. 5 at 37°C dur ing 30 hours . The solut ion con l a i ned 3. 2 mM ca l c i um and 2 mM phos-phal e. After dem 1neral izat ion one con l rol sec l ion was laken from each enamel spec imen, by cutt ing perpend i cu l ar to the enamel sur
face and perpend icular to the grooves. On each l uuth sect ion en
amel dem ineral i zat ion was exam ined at the enamel surface, at the wall and at buttoms of lwo grooves (f ig. 6. 1) . Th is procedure has previuus l y been described by Smits and Arends [ 1986] (Chapter 5 ) .
F ig. 6.1. Schemal ical drawing of a loolh secl ion showing lwo U-shaped arl ificial grouves. Lesion deplhs were measured at the enamel surface (near positions 1, 4 and 7), at the wall (near 2 and 5) and at the bottom (near 3 and 6). The widlh of a gruove is ap
proximalely 200 µm (E
=
enamel; D=
dentine).Par t i c ipan l s
[ leven pat i ents of the un ivers ity denta l cli n ic part i c ipated volun l ar i l y. T he mean age was 50 ±_ 1 1 (range 3 3 -62) . F ive had a part i a l prosl hesis and six had fu l l dentures. They were all i n good health, used n o med icat iun and had a regular l ifestyle.
Chapter 6 : E n amel dem ineralizati on in vivo
Three test sol ution s
A 2. 5% sucrose sol ution was made by adding 250 g . sucrose to 10 li ter demi nera l i zed wat er . T h i s stock so l ution was remade every four weeks. A 2. 5�� xyli tol soluti on was made by adding 250 g.
xylitol (RocheR) to 10 l i t er demi n erali zed water. T he thi rd test sol uti on was demi nerali zed water.
Outli ne of the experiment
In a ran domized cross-over design each participan t used each test solution dur in g 16 days. A t day D of a test period two enamel specimens were mounted with acryli c resin in the buccal flan ge of the prosthesis adjacent to the cervical margin of the prosthetic teeth ( f i g. 6. 2. ) . Th e speci men s were placed with the enamel surface at the same level as the surface of the buccal flange.
The participan t s were i nstructed to cl ean twi ce a day w i th a fluori de free toothpaste brushin g the enamel speci mens. From day
teeth and appli an ce ( ProdentR) , wi thout 3 untill day 16 the partici pants submerged the appli ance twi ce a day in a l i ttle plasti c contai ner fi lled with the test soluti on during 5 mi nutes.
A fter 16 days two new enamel specimen s were mounted in the appli -ances and one of the other test sol uti ons was provi ded. Thi s procedure was repeated until each participant had used all three test sol uti on s .
E namel speci men anal ysis
A t the end of the experi ment two sections were cut from each enamel bla ck i n t he same direction as the control, giving a total of 132 treated ( t16) and 66 control ( t0) sections. The 198 sec
t i ons were ground p l an oparallel to a thi ckness of approximately 80 µm on waterproof silicon carbi de abrasive paper ( grid 120 0 ) . Polari zed l i ght mi crospy:
The section s were i mbibed in water and each groove was photo
graphed usi n g polarized li ght mi croscopy ( Olympus, Vanox) , giving a total of 396 photographi c sli des.
-75-Chapter 6 : Enamel demineralization in vivo
a
b
Fig. 6.2. (a) Position of two enamel blocks, with two artificial grooves each in the buccal flange of a lower prosthesis.
(b) An enamel black with two grooves (E
=
enamel ; D=
dentin)._Les ion dept h was mea s ure d on project ion s o f the s l i d es , u sin g a m icrometer scale , to an accuray o f + 5 µm. On eac h section the lesion depth was measured at the enamel surface (3 positions) , at the bottom o f the 3 grooves and ha lfway between bottom and top of the 2 grooves ( fig. 6. 1) .
Th e lesion depth at the enamel surface at tO of each specimen is the average value of the 3 measurements on the control sectio n at positio ns 1 , 4 and 7. The depth at the wal! of the grooves at tO i s the average o f the measurements at p ositions 2 and 5. The depth at the bottom of the grooves at tO is the average of the measurements at posit ions 3 and 6.
Chapter 6: Ename l d eminera lization in vivo
The lesion depth at the ename l surface at t16 of each specimen is the average va l ue of six measurements on two sections. The l esion d epth at the wall of the grooves at t16 is the average of four measurements on two sections. The lesion depth at the bottom of the grooves at t16 is a lso the average of four measure
me nts on two sections [Smits and Arends, 1986 ) . Microradiography :
After po lari z e d light microscopy, microradiograms w e re made of the same 198 sections using the procedure previously pub lishe d by Josse lin d e Jong and T e n Bosch [1985 ) . Densitometric tracings with a slit of 30 x 2 µm were mad e on each microradiogram through the same positions as use d in the po lari z e d light l esion depth measurements, giving a tota l of 1386 graphs of minera l % by d epth from the ename l surface.
The fo l lowing parameters w ere d e rive d from each tracing graph:
(1) min era l loss and ( 2) l esion depth (µm) . The measurements on a l l 21 positions on the thre e sections of each ename l specimen w e re averaged in the same way as in dicated above for po lariz e d l ight lesion d epth measurements. This results in averaged para
meters for each enam e l specimen for the surface ename l at t0 and t16, for the wal l of the groove at t0 and t16 and for the bottom of the groove at t0 and t16.
No different findings w e re obtain e d between the partia l and the ful l d e nture participants (t-test, p> > D. 05). Therefore the re
sults of this experiment wil l be presented ave raged over a l l participants.
6.4 . R e sults
The results of this experiment wil l be presented in 2 parts.
( 1 ) The min era l loss from surface ename l, wal l and bottom of the grooves before an d after the in vivo demin era lization. These re sults w e re ca lculate d from the d e nsitometrica l tracings on the
-
77-Chapl or 6 : Ename l dominora liza l ion in vivo
mic roradiograms (öZ ) .
(2) T ho losion dopl h b o f oro and af tar tho in vivo po riod of 16 days. L osion dopths woro moasurod a l tho onamo l surf aco, at L ho wall and at tho bottom of tho artif icia l groovo. Thoso va l uos wo ro dorivod f rom po l ariz od l ight mic roscopy and f rom microradio
graphy.
Fig. 6.3. Polarizod light microscopie picture showing lho basolino domin
oralization at tho sur face onamol, tho wall and tho bottom of tho artificial groovo. Tho width of the groove is approximately 200 µm.
Mineral loss
Tabla Vl. l shows the mineral loss (öZ ) in vol�ó. µm f rom the sur
f ace enamel, f rom the wall and f rom the bottom of the grooves, averaged over 22 enamel blocks in 11 participants. 6 Z - t0 is the
Chapler 6: E namel demineralization in vivo
average mineral l oss from the enamel specimens after 30 hours de-mincralizal i on in vitro. A Z-t16 is the avcragc mincral l oss af-ter the usc of waaf-ter , sucrose or xylito l during 16 days in vivo.
During the in vivo pcriod thcre is a significant increase in min
cral loss at the surface enamel , at the wal ! of the gro oves and at the bottom of the gro oves , for cach trcatmcnt (p<O . D l , paircd t-test) . During the in vivo period the mineral l oss (ave ragcd over al l trcatments) from the surfac c enamel is .±_ 5 5 0 vol?�. µm , fro m the wal ! of the gro ovcs .±_ 600m vo l %. µm and from the b ottom of the gro ovcs .±_ 1050 vo l %. µm. During the in vivo pcriod , aver-aged over al l treatmcnts , signifi cantly more mineral is l ost from the botto m of the gro oves lhen from the wal! of the gro oves and from lhe surface enamel (p<0. 0 5 , analysis of variance) .
Table V I . 1 . Average mineral loss (AZ in vol%. µm) from the surfacc enam-cl , from the wal ! and from the bottom of the groovcs, be-fore ( to) litol. and after tl6) the use of water, sucrose and
xy-lest- M-t0 M-tl6 diffcrence
position solulion mcan SE mean SE mean SE (N) surf ace water 1558 182 2487 171 929 218 (16)
sucrose 2009 139 2609 171 600 123 (19) xylitol 2191 203 2371 175 180 182 ( 21)
wall water 1706 112 2164 176 458 202 (14) sucrose 1763 132 2445 167 682 209 (15) xylitol 1526 59 2145 173 619 177 (17) bottom water 2840 138 3685 344 845 357 (16) sucrose 2661 163 3962 264 1301 303 (17) xylitol 2556 106 3596 302 1040 298 (20) SE denoles lhe standard error of the mcan; N dcnotes the number o f enamel blocks.
-79-Chapter 6: Enamel demineralization in vivo
Dif ferences between the treatments
The mineral loss at the surface enamel is for xylitol signi
ficantly less then the loss after water or sucrose treatment ( p<0 . 0 5, Analysis of covariance, 6 Z -t0 as covariate).
T he mineral losses from the surface enamel during the sucrose treatment and the water treatment do not differ ( p> D. 0 5 , analysis of variance) .
There are no significant differences between the treatments in mineral l oss from the wall and bottom of the grooves ( p> D . 0 5, analysis of variance) .'
Table VI . 2 . Lesion depth measurements and lesion depth di fferences (in
position surface
wall
bottom
µm) at the enamel surfaces, at the wall and at the bottom of the grooves, before (t0) and after 16 days in vivo (t16), treated with water, sucrose and xylitol solutions. Polarized light and microradiographical data.
LESION DEPTH IN LESION DEPTH IN POLARIZED LIGHT MICRORADIOGRAPHY test to tl6 difference to tl6 di fference solution µm SE µm SE µm SE ( N) µm SE µm SE µm SE ( N ) water 37 5 59 3 23 6 (18) 40 4 60 2 20 4 (16) sucrose 49 2 65 2 1 7 3 ( 21) 49 2 62 3 12 3 (19) xylitol 50 4 58 3 7 4 ( 21) 50 3 57 2 7 3 ( 21) water 27 2 41 2 13 3 ( 17) 29 3 39 3 11 3 (14) sucrose 27 1 43 2 17 2 ( 20) 30 2 44 3 14 4 (15) xylitol 32 2 40 3 6 3 ( 20) 28 1 41 4 13 5 (17) water 46 3 78 4 31 6 (17) 45 2 71 6 27 7 (16) sucrose 43 2 74 5 31 5 ( 21) 42 2 75 5 33 6 (17) xylitol 50 2 72 7 19 6 ( 21) 47 2 76 7 25 6 ( 20) SE denotes the standard error of the mean; N denotes the number of enamel blocks
Chapter 6: Enamel demineralization in vivo
Lesion depth
Table VI. 2 shows the average lesion depths at t he enamel surface, at the wall and at the bottom of the groove before ( t0) and after ( t16) the use of each lest solulion ( 22 enamel specimens worn by 11 parli cipant. s) . L es ion depth differences aft er 16 days are calculaled as the mee n difference bet ween t he lesion depth at t0 and t.16 for each enamel black.
After 16 days demineralization in vivo there is a significant in
crease in lesion depth at all measuring positions ( p<0. 01, paired t -t. est). This was found bath with polarized light techniques as well as by microradi�graphy.
Differences between treatment s
Th e lesion depth at the su rface enamel aft er xylitol t r eatment is significant ly less then t he lesion depth aft er water and or su crose treatment ( p<O . 05, analysis of variance) . However , for walls and bottoms of the grooves no significant differences were found for the t hree trealment s ( p> 0. 05, analysis of variance) . 6 . 5 . Dis cu s sion
In the present experiment enamel blocks wit h arlificial grooves were demineralized in vitro and subsequ ently mou nled in t he prostheses of 11 participants. In lhis experiment.al model enamel de- and remineralizat.ion can be examined in t. wo different condi
t.ions in each enamel specimen:
( 1) in a fissure like, plaque covered sit. uat.ion in t he grooves and
( 2) on the fr ee enamel su r faces next to these grooves, where less plaque accu mulation is fou nd during the t est .
l t was found that. ( i) demineralizat ion of enamel under plaque in a groove takes place withou t any measurable influence by ext ra
oral treat ment s wit h xylit ol, sucrose or wat er. ( ii) Demineral
ization of t he enamel surface is significantly reduced by the xy-- 8 1xy--
1-Chap ter 6 : Enamel demineral iat ion i n vivo
l i t ol treatment.
Several fac tors should be consi dered to exp lain why xyli tol does not influence t he dem ineral izat ion in t he grooves.
The d i ffusion ra te for sucrose i n sali va and plaque flui d is ap -- 5 - 1
prox ima tely 10 cm2 sec [Tatevoss ian, 197 9]. I t can b e cal-culated from X "' S QR (D
*
t),. (X = d istance ; D = d iffusion rat e ; t = t ime) and a d i ffus ion rate D = 1 0- S cm2 sec-l t hat in 5 m i nutes xyli tol d i ffuses up to a dep t h of ap proxi mately 600 µm .i n t he plaque-fi lled grooves. Because t he average dep t h of a groeve .is approx i mately 500 µm, i t is reasonable to assume t hat xyli tol d i ffuses at least halfway down t he groeve. The fact t hat no x y l i tol effect was measured a t t his d i s t ance i n t he wall of t he grooves ind i cates t hat lack of xyli tol penetrat ion i n t he grooves cannot expla i n t he absence of a xyli tol effect in t he grooves.
Surface plaque is of course more easi l y access ible for t he test solut ions t han t he plaque i n t he grooves . Consequen tly d i ffer
ences in xyli tol leaching out of t he plaque as well as d iffer
ences i n t he concen tra t ions of m i neral ions and m i crob iologi cal substrates are presumably responsible for t he d ifference in t he xyli tol effect.
In ep i demiologi cal studies a consi derable reduct ion i n buccal -free surface- cari es was found after xyli tol add i t ion to t he d i et [Sche i n i n and Banoczy, 1985 ; Re kola, 1986]. In v i ew of t he results of our experiment, this effect cannot be due only to t he accessib i l i t y of t hese surfaces to sal i va.
A d i rect effect of xyli tol on t he process of enamel d e- and re
m i neral izat ion [Mä k i nen and Soderl i ng, 1982 ; Arends e t al. , 1984]
might also explai n t he xyli tol effec t on m ineral loss and lesion dep t h · i n t he present experimen tal set -up. Dem ineralizat ion of t he enamel spec imens can lead to two m ineral d istribut i ons. I n vi tro demi neral izat ion of t he i ntact surface enamel results i n lesions w i t h a surface layer clearly v i s i ble on m i croradiograph-i cal trac croradiograph-i ngs. Lesion format .ion i n t he grooves result s in a m i n
-Chapter 6 : E name l demineraliz ation in vivo
eral dislribution without a cl early visib l e surface l ay er [S mits and Arend s, 1986 ] . The finding that xy litol has no effect on le
sion depth and mineral loss in the grooves may also be d ue to diff erent e f f ects of xy litol on these two mineral d istributions.
The present e xperiment provid es no information on th is point .
O ther studies, using the Intraoral Cariogenicity T est (I CT) have also shown d emineraliz alion in enamel slabs afler 5 or 7 days, using microhardness techniques [ K oulourid es et al. , 1976; Pearce and Gallagher, 1979 ] . I n the I CT p laque format ion is heavil y induced on the ename l surf ace und er gauz e. In the present ex
periment plaque accumulated rapidly in the grooves. Svanberg and Loesche [1977] showed that
(i) coloniz ation of articial fissure inserts in the human mouth occurs within 1 day and that
(ii) the total number of micro- organisms was reasonably constant during 2 or 3 week experimental period s and that
( iii) the microorganisms remained viable for a long period [ L b e et al. , 1 973; Theilad e el al. , 1974; Minah and Chu, 1984 ] .
In the present experiment we consid er the situation in the grooves as a p l aque covered situation and the situation at the sur f ace ename l as a rel atively plaque free situation . I n this experiment the small amount of plaque on the ename l sur faces was not determined , because it has been shown in the l iterature that a red uction of the plaque covered area is only achieved if larger amounts of xy litol are administered [Rekol a, 1982] or if xy l itol is given in combination with a sucrose free diet [ Plü ss, 1978].
No standard iz ed d iet was prescribed and no d ietary checklists were used in this stud y . All participante had a normal l i festy l e and dietary habits, including the use o f sucrose containing food s and drinks. D i f f erences in
pants and treatment groups ind ications were found that
sucrose consumption between partici
influence individual results, but no individ ual differences in ( sucrose) consumption have had a major e ffect on the present resul ts.
-83-Chapt er 6: E namel demincralizat ion in vivo
The fact that sucrose and water have a similar influence on en
amel demineralization is an int eresting aspect . T his was found in t he grooves as well as in t he surface enamel an d shows t hat, under t he condit ion s of t his experiment , t he sucrose from t he 2. 5�� solution does n ot produce a more n oticible cariogenic at t ack, t han t hat result in g from sucrose an d ot her subst rat es in t he dail y food an d drinks.
lt can be con cluded t hat
- enamel dcmineralizes at both free surfaces and in grooves dur
ing 16 day s in vivo, if a solution cont ainin g 2. 5�� sucrose or 2. 5% xylit ol or wat er is applied t wice a day .
- application of x y lit ol t wice a day in a solution significant ly
- application of x y lit ol t wice a day in a solution significant ly