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EXPERIMENTAL CARIES IN YOUNG HUMAN PERMANENT TEETH IMPLANTED IN
REMOVABLE DENTURES
M. BRKNNSTRBM, J. FRISKOPP, G. ISACSSON and L. WICTORIN
School of Dentistry, Karolinska Institute, Stockholm. Sweden
Summary-Carious lesions were produced in young intact permanent teeth placed in dentures. The pulp chamber in one of the teeth in contralateral pairs was treated with fluoride solution or filled with cement with a high concentration of fluorides. This did not reduce the incidence of caries. There was no indication that an internal fluid phase carrying substances from the pulp or periodontium plays a role in the development of caries in uiuo. Examination of the experimental lesions with microradiography and scanning electron microscope revealed that bacteria were present both in enamel and dentine at an early stage. In the dentine, the bacteria attacked predominantly peritubular dentine and the enameldentine junction. Demineraliza- tion in enamel and dentine was slightly in advance of the bacterial invasion. The peritubular area seemed to be the structure first removed. Radiopacity was present in carious dentine. at the surface of several cavities, the centre of the lesion and at the lateral border, indicating a local remineralization process induced by substances originating from caries or from saliva. No sclerosis was found in dentine between the lesion and the pulp chamber. This is considered to support the view that such changes in vital teeth are due to distribution of substances from the pulp.
INTRODUCTION
Various opinions have been expressed whether the tissue fluid in teeth is concerned in the development of caries and in the remineralization process of arrested caries. Wolf and Neuwirt (1941) showed that remineralization of acid-etched enamel surfaces occurred after several days. They compared reminera- lization between vital and non-vital teeth in situ and studied etched enamel surfaces on teeth implanted in removable dentures. They reported that remineraliza- tion was the same regardless of tooth vitality and concluded that the pulp and periodontium was not involved in remineralization.
In-vitro studies by Wachtel and Brown (1963) sup- port the hypothesis that substrates from the pulp which could promote bacterial growth might contrib- ute to the development of caries. In animal experi- ments, Brown and Lefkowitz (1966) found that teeth in which the coronal pulps were replaced with cement or inorganic bone were more resistant to caries than vital teeth. Bergman and Linden (1966) showed that a solution consisting of dicalcium phosphate and potassium fluoride placed in the pulp chambers of human teeth in vitro reduced the demineralization of enamel exposed to acetate buffers. They assumed that “internal” factors might be of significance in incipient caries. Chimside (1961) studied bacterial penetration in the dentine of teeth implanted in removable den- tures. In exposed dentine, bacteria invaded the den- tinal tubules to the pulp within a period of three weeks. In-Go investigations have also shown that bacteria easily invade mineralized dentine (Lundy and Stanley, 1969; Olgart, BrHnnstrGm and Johnson, 1974; Mjor, 1974). On the other hand, Miller (1890) was of the opinion that demineraljzation preceded bacterial invasion in the development of caries. This
view has been supported in later experiments (Dorf- man, Stephan and Muntz, 1940; Sarnat and Massler. 1965; Fusayama, Okuse and Hosoda, 1966). Nygaard- hstby, March and Hals (1967) described the induc- tion of caries in human teeth in viuo. The influence of substances from the pulp and periodontium are eliminated in teeth implanted in removable dentures.
Our aim was to investigate: (1) the induction of caries in young intact human permanent teeth. im- planted in removable dentures; (2) whether fluorides in the pulp chamber could affect the development of caries; (3) microscopic appearance of carious lesions that may develop.
MATERIALS AND METHODS
The material consisted of 20 teeth; 10 contralateral, intact pairs of human teeth. The teeth were young premolars extracted for orthodontic reasons. After extraction, the roots were separated from the crowns approximately 3 mm apically from the cervix. The pulp was removed and replaced with various dress- ings. In 8 pairs of teeth, one pulp chamber of each pair was treated with a fluoride (Table 1). For all teeth, gold overlays were made covering the entire root surface and in most cases extending on the cervi- cal half of the buccal surface overlying the enamel surface by 0.3 mm. The overlays were cemented to the root surface with phosphate cement but the over- lay covering the buccal enamel was left free from cement allowing for the retention of plaque as de- scribed by Nygaard-Gstby et al. (1967).
The pairs of teeth were implanted in full upper den- tures in 4 patients who had slight to moderate caries activity in their remaining lower natural teeth (Table
571
Tab
le
1
Patie
nt
Tes
t T
ooth
pe
riod
pa
ir
(yr)
N
o.
Pulp
ca
vity
dr
essi
ng
Too
th
posi
tion
in d
entu
re
Vis
ual
exam
inat
ion*
E
nam
el
cari
es
Den
tme
cari
es
I 1
1 2
2.5
3 4
II
2.5
5 6
III
3.5
7 8
IV
3.5
9 10
Ros
in
+
gutta
perc
ha
Ros
in
+
gutta
perc
ha
Ros
in
t gu
ttape
rcha
R
osin
+
gu
ttape
rcha
N
aFt
- R
osin
+
gu
ttape
rcha
R
osei
n +
gu
ttape
rcha
Z
nO
+
lO”/
,SnF
, Z
nO
ZnO
+
lO
%Sn
F,
ZnO
Z
nO
+
10%
SnF
,j Z
nOf
Cav
itg
+
10%
SnF2
C
avit
Cav
it +
lO
%C
aPO
,F
Cav
it C
avit
+
lO%
CaP
O,F
C
avit
Cav
it +
lO
%C
aPO
,F
Cav
it
+4
4+
+5
5+
+4 +5
5+
4+
5-t
4+
+4
+5
+5
+4
51
4+
5+
4-k
+5 +4
But
. 1;
Dis
t. 2;
M
es.
3 In
tact
B
ut.
1; M
es.
1 B
ut.
1 M
es.
2 In
tact
In
tact
M
es.
2 In
tact
M
es.
2 B
ut.
1
But
. 1
But
. 3;
Dis
t. 3
Inta
ct
Lin
g.
1 L
ing.
1
But
. 1;
Mes
. 1;
Lin
g.
1 M
es.
1; D
ist.
1.
Mes
. B
ut.
But
.
But
. M
es.
Lin
g.
Mes
. M
es.
Dis
t. B
ut
* E
nam
el
cari
es
grad
e 1:
glo
ssy
whi
te
spot
; 2:
du
ll or
rou
gh
whi
te
spot
; 3:
cav
ity
in e
nam
el.
t4%
N
aF
sol.
encl
ose
d
in t
he p
ulp
cavi
ty
for
14 d
ays
befo
re
plac
emen
t in
pro
sthe
sis;
to
oth
in s
ynt.
saliv
a.
$ Sm
all
enam
el
cavi
ty w
as d
rille
d w
ith
a ro
und
diam
ond
to d
entin
e bo
rder
un
der
gold
pla
te.
4 C
avit:
te
mpo
rary
ce
men
t. (E
SPE
, G
mbH
., Se
efel
d,
Obe
rbay
, G
erm
any)
.
Young human permanent teeth implanted in removable dentures 573
1). The pairs of teeth were fixed in premolar positions with self-polymerizing resin. During the entire pro- cedure, the teeth were stored in saline. Before inserting the dentures, attempts were made to eliminate bac- teria on the denture by applying a 30 per cent solu- tion of hydrogen peroxide, followed by tincture of iodine, each applied for 5 s.
The patients were instructed to wear the dentures continuously and only to remove them for cleansing purposes. The pairs of teeth were removed from the dentures after 1, 2.5 and 3.5 yr and placed in a 10 per cent neutral buffered formalin (Lillie, 1954) and stored for two weeks. After removal of the gold over- lays and plates, the teeth were examined in a dissect- ing microscope to record the caries. Eight teeth with caries were studied with scanning electron microscopy (SEM). These teeth were fractured through the centre of the carious lesion. A groove was cut on the oppo- site surface to determine the fracture line. The teeth were placed in liquid nitrogen for two minutes and after removal the fracture was accomplished by lever- age within the groove.
One fractured half was freeze-dried for 16 h and then covered with a 200 A thick layer of gold under vacuum and studied in a Cambridge scanning elec- tron microscope. Both the half used for SEM and the other half were used for preparation of a ground section close to the fractured surface. Similar ground sections were prepared for all other teeth with caries after dehydrating in absolute alcohol and embedding in methylmethacrylate. Plane-parallel sections ap- proximately .lOO pm thick were prepared through the centre of the lesions. These were microradiographed to study the inorganic component by placing in close contact with fine-grained photographic emulsions (Kodak High Resolution Plates) and exposing to Nl- filtered CuK u radiation (1 = 1.54A).
The microradiograms were evaluated densitometri- tally with a Leitz Photometer (Lindstriim and Phi- lipson, 1969) which had a light field diameter of 15 pm. For some specimens with large dentinal lesions, the densitometric values were coordinated with the morphologic appearances of the same areas in the SEM. The measuring points were situated in the areas with high relative radiodensity. Radiolucent areas were also determined. Two values were obtained from each measured point. The mean value of each area was compared with the mean value obtained from intact areas.
The area corresponding to the microradiograph was not exactly identical to the fractured surface, even if the ground section was obtained as near as possible to this surface. Therefore, correlations between mic- roradiographs and SEM were limited to larger lesions were the extents of the lesion were judged to be simi- lar.
RESULTS
Visual examination
Carious lesions were detected in 15 of the 20 teeth implanted in the dentures. Three carious lesions were under the pinned buccal plates and 6 under the over- lays. Eighteen lesions appeared adjacent to the acry- late. Fewer lesions appeared in 4 teeth where the pulp cavity was fluoride-treated or filled with a fluoride
dressing, in comparison to the untreated controls. In 4 paired teeth, there were more lesions (Table 1). In one denture, deep carious lesions were found in both teeth placed on the same side, whereas the contra- lateral pair of teeth, obtained from another individual. revealed insignificant enamel lesions.
Microradiography and scanning electron microscop)
Incipient carious lesions with glossy and white sur- faces (grade 1. Table 1) showed a marked radiopaque surface and a strong radiolucent subsurface area. The SEM of these areas showed relatively shallow pen- etration and, m the subsurface area. granular struc- ture with tags or cylindrical mvagmations from the surface.
.The dull and rough enamel lesion (grade 2) also exhibited a distinct radiopaque surface, with a radio- lucent area beneath which. in two cases, extended to the enamel-dentine junction and spread there to pro- duce a conic radiolucency in the dentine (Figs. 1 and 2). This type of lesion showed a surface that was rough or with pits 2@40pm in diameter (Figs. 3-5). Bacteria lying in small lacunae between the enamel prisms were observed on the surface of fracture through the lesion (Fig. 6). In places, intact enamel prism structure was present immediately under the surface (Figs. 5 and 6) but nearer the dentine the en- amel was in the process of dissolution.
In 2 lesions. the underlying dentine was demmera- lized despite the absence of a cavity in the enamel (Figs. 1 and 2). A few bacteria were related to dentinal tubules. Prior to the gold evaporation of the prep- aration, it was noticed that the dentme m this area was brown. Some dentinal tubules showed a lack of peritubular dentine. but the intertubular dentine remained without evident mineral loss.
Teeth in which the cavity had reached the dentine. cocci and some rods filled the gap between the enamel and dentme at a considerable distance from the cavity (Fig. 7). In these areas, bacteria were lying in small lacunae on the dentme side, as well as on the enamel side of the junction (Figs. 7 and 8).
Where demineralization of the dentine was marked, there was a total absence of peritubular dentine. and bacteria were invading intertubular dentine (Figs. 9 to 11). As a rule m these areas. the intertubular den- tine was relatively dense and contained rounded bodies. The fibrillar structure was not seen. In some tubules closer to the pulp and in areas which in the microradiogram was less demineralized, the peritubu- lar dentine was completely removed. The dentinal tubules m certain parts of the carious dentine were filled with a smooth or granulatar substance indlcat- ing mineralization (Figs. 10 and 12).
Local radiopacity was present m some areas and sometimes followed the direction of dentinal tubules in the radiolucent area (Fig. 2). In 4 lesions a lateral radiopacity bordered the lesion towards the untiec- ted dentine and seemed to correspond to type I scler- osis described by Levine (1974). Densitometry showed that this border zone had a higher density than the surrounding intact dentine. In 7 lesions. there was a radiopaque layer at the surface of the cavity (Fig. 2). In 5 lesions, there were thin bands along the in- cremental lines m the radiolucent areas. Levine type
574 M. Brtinnstriim et al.
II sclerosis, which is a radiodensity between the pulp and the carious lesion, was not observed.
DISCUSSION
Brown and Lefkowltz (1966) suggested that pulpless teeth may have a greater resistance to caries. Accord- ing to Wachtel and Brown (1963). this greater resist- ance could be due to a reduction in the exudation from the pulp of substances which could act as bac- terial substrates.
Two of our pairs of teeth from two different mdivl- duals revealed different resistance to caries when placed in the same denture beyond the influence of the pulp, suggesting that the structure enamel and its chemical composition, e.g. fluoride concentration, may be of greater significance for the development of caries than t,he flow of substances from the pulp or periodontium.
The fluoride dressing placed in the pulp chamber in one tooth of each of the 8 tooth pairs did not reduce caries significantly. The passage of fluorine from the pulp chamber to the enamel surface may have been insufficient but definitive conclusions are impossible. The microradiographic appearance of enamel caries was similar to that of incipient caries in uiw so that fluid flow from the pulp appears not to be an influence.
The lateral radiopacity as well as parallel radio- paque bands parallel with the incremental lines of dentine caries support the view of Levine (1974) that lateral sclerosis might be “due to passive deposition at the lateral borders of the lesion of mineral pre- viously removed by the carious process from the body of the lesion”, a view also held by Helmcke (1955) and Frank, Wolff and Gutman (1964). In the SEM. densely packed round bodies in dentmal tubules where the peritubular dentine had been previously removed (Fig. 12), suggested secondary mineralization. Inorganic components and other substances necessary for mineralization may also come from the saliva and contribute to radiodensity. as suggested by Bergman and Engfeldt (1955). This might account for the radio- pacity at the surfaces of lesions. There were no examples of Levine Type II sclerosis. These observa- tions differ from microradiographic investigations of carious lesions with vital pulps. Bradford (1958, 1960) assumed that the sclerosis is caused by activation of the odontoblasts which then induce mineral preapi- tation. According to Brlnnstrijm and Lind (1965) the original odontoblasts in most cases disappear at an early stage of caries leading to the differentiation of new cells which produce irregular secondary dentine. The substances involved in forming irregular dentine might also be available for the older dentine near the pulp.
The presence of bacteria in small lacunae in the enamel prisms (Fig. 6), as well as deep in the enamel under white spot lesions, together with the presence of pores in the enamel surface, would seem to indicate that bacteria may penetrate the enamel at an early stage. With SEM. dissolution of peritubular dentine was observed under white spot lesions without cavi-
tation. Bacteria could even be seen m the dentine un- derneath these lesions and at sites in which the loss of mineral judging from microradiography was mini- mal. These observations indicate an early penetration of microorganisms through the enamel spreading at the enamel-dentine junction and growth in the tubules towards the pulp.
REFERENCES
Bergman G. and Engfeldt B. 1955. Studies on mmerahzed dental tissues. IV. The distribution of mineral salts m the dentine with special reference to the dentmal tubules. Acta odont. stand. 13, l-7.
Bergman G. and Lmdin L. A. 1966. Effect of an “internal factor” on enamel decalcification Archs oral Bio[ II, 943-945.
Bradford E. W. 1958. The maturation of dentine. Br. dent. J. 105, 212-216.
Bradford E. W. 1960. Dentine. A barrier to caries. Br. dent. J. 109, 387-398.
Brown L. R. and Lellcowitz W. 1966. Influences of dental fluids on experimental caries. J. dent. Res. 45, 1477-1497.
Brlnnstrdm M. and Lmd P. 0. 1965. Pulpal response to early dental caries. J. dent. Res. 44, 1045-1050.
Chirnslde J. M. 1961. Bacterial Invasion of non-vital den- tine J. dent. Res. 40, 134-139.
Dorfman A.. Stephan R. M. and Muntz J. A. 1943. In cilro stumes on sterilization of carious dentin. II. Extent of infectlon in carious lesions J. Am. dent. Ass. 30, 1901-1904.
Frank R. M.. Wolff F. and Gutman B. 1964. Mlcroscopie Clectromque de la carie au mveau de la dentine humaine. Archs oral Biol. 9, 181-192.
Fusayama T., Okuse K. and Hosoda H. 1966. Relationship between hardness, discoloration and microbial invasion m carious dentme. J. dent. Res. 45, 1033-1046.
Helmcke J. G. 1955. Elektronenmikroskoplsche Struktur- enuntersuchungen an gesunden und kranken Zlhnen D. -_ahniirztl. Zty 10, 1461-1478.
Levine R. S. 1974. The nncroradlographic features of den- tine caries. Br dent. J. 137, 301-306.
Lillie R. D. 1954. Histopathologic Technique and Practical Histochemlstry. The Blackstone Co., New York.
LindstrGm B. and Philipson B. 1969. Microdensitometer system for microradiography. Histochemie 17, 187-193.
Lundy T. and Stanley H. R. 1969. Correlation of pulpal hlstopathology and clinical symptoms m human teeth subjected to experlmental irritation. Oral. Surg. 27. 187-201.
Miller W. D. 1890. Microorganrsms of the Human Mouth S S. White Dental Manufacturing Co., Philadelphia.
Mjijr I. A. 1974. NordIsk Klmrsk Odontologi 2-V-l-2-V-10 A/S Forlaget For Faghtteratur. Copenhagen.
Nygaard-&tby B., Mijrch T. and Hals E. 1967. A method for caries production on selected tooth surface In UOO. Acta odont. stand. 15, 357-363.
Olgart L., BrPnnstrijm M. and Johnson G. 1974 Invasion of bacteria into dentinal tubules. Acta odont. stand. 32, 61-70.
Sarnat M. and Massler W. 1965 Mlcrostructure of active and arrested dentinal caries. J. dent Res. 44, 1389-1401.
Wachtel L. W. and Brown L. R. 1963 In utro caries. Fac- tors Influencing the shape of the developing lesion. Archs oral Biol. 8, 99-107.
Wolf J. and Neuwlrt F. 1941. Reparative Vorglnge und der Oberfllche natiirlicher und kiinsthch hervorgeru- fener Schmelzdefekte Deutsche Zahn- Mund- a. Ktefer- heilk. 8, 222-382.
Young human permanent teeth implanted in removable dentures 575
Plates l-2 overleaf
M. Brlnnstrkn et al.
Plate 1 Fig. I. Microradiogram showing the mineral drstribution. Enamel caries (grade 2) without cavitatton.
Observe the demmerahzation in the adjacent dentine.
Fig. 2. Microradiogram showing the mineral drstribution. Enamel canes (grade 2) with extension of the lesion along the dentine-enamel junction and radiolucency in the denture. Below there is cavitation with dentine cartes Local radiopacrties along the dentmal tubules and upper lateral border as well
as on cavity surface.
Rg 3. SEM of the enamel caries m Fig. 2. Arrow mdrcates enamel surface in Fig. 4. x 53.
Fig. 4. SEM of pits in the enamel surface of Figs. 2 and 3. x 1100.
Fig. 5. SEM of enamel caries (grade 2). From left to right: A. fracture through the lesion; B. rough enamel surface; C. smooth enamel surface. x 200.
Ftg. 6. Details of the quadrangle outlines m Fig. 5. Relatively Intact enamel prrsms with bacteria. some in lacunae m the prisms. x 7200
Plate 2.
Frg. 7 SEM of dentine facmg a gap at the enamel-dentine junction at some distance from a cavity. Bacteria of coccal type and some rods invading the dentine. x 7200
Frg. 8. SEM of bacteria m the enamel at some distance from the enamel-denture junction. x 11,500.
Fig. 9. SEM of denture caries showing three dentinal tubules without perttubular wall. Some bacteria m lacunae at the tubule wall (arrow). In the lower tubule. bacteria are also mvadmg intertubular
denture. whtch IS fairly intact. x 7000
Fig. 10. SEM of area near the pulp underneath a deep lesion in dentine The densitometric values of the microradrogram in the correspondmg area indicated insignificant mmeral loss. Tubules with partial loss of peritubular denture. some tubules are obliterated with either homogeneous or granular
material. A few bacterta are present. x 7000.
Fig. 11. SEM of a dentrnal tubule with perrtubular denture still present but bacteria are penetrating the tubule wall. Bacteria are also present in the adjacent intertubular denture. The corresponding
microradtogram showed no significant mineral loss. x 13,ooO.
Fig. 12. SEM of dense material composed of round bodies m a dentinal tubule devoid of peritubular wall. The intertubular dentine adjacent to the tubule IS disintegrated. x 26,000.
Young human permanent teeth implanted in removable dentures 577
Plate 1
578 M. Brbnstrtim et al.
Plate 2
