Bacterial Association of Smear Layer

The pathological consequences of the smear layer and whether it should be

present or absent under restorations are rather complicated questions. To a great

extent, they seem to be related to the presence of bacteria under restoration.

Brannstrom and Nyborg (1971) first reported the growth of bacteria under

silicate and composite resin restorations. One important question was: Is it

possible that bacteria entrapped in the smear layer survive and multiply under

these restorations?

Similar concern exists regarding the Endodontic smear layer. The bacteria

present in infected root canals are predominantly gram-negative anaerobes. Davis

et al (1972) have shown that the morphology of root canals is very complex and

that mechanically prepared canals contain areas not accessible by currently used

endodontic instruments. Bacteria can be found in all areas of the root canals

system and in the dentinal tubules.

DENTINAL TUBULES

In Restorative Dentistry:

Dentinal tubules originating from a cavity do not only lead to the pulp.

Tubules on the side walls of the cavity may also lead outward toward the enamel

or to the root cementum. These tubules are filled with fluid and viable bacteria

may sometimes enter these tubules from a liquid-filled gap under a restoration. A

grinding debris plug in the tubule apertures does not guarantee a permanent

barrier against bacterial invasion. This plug can be removed by microorganisms.

These are the contributing factors towards secondary caries. This implies that

even the side wall of the cavity ought to be correctly pretreated and lined. The

tubules which lead to the pulp are the ones which are the transportation channels

for irritants such as zinc oxide eugenol, bacterial toxins etc. The more tubules per

square millimeter leading to the pulp, the more permeable the dentin, and the

greater the danger to the pulp.

Garberoglio and Brannstrom (1976) examined under SEM, occlusal dentin

with cross fractured dentinal tubules at various distances from the pulp (refer table

36

Bacterial Association of Smear Layer

2). However, the number of dentinal tubules reaching the pulp varies considerably

on the floor, axial and pulpal walls of buccal/lingual/approximal cavities

depending on the shape and depth of these cavities. For instance, on the floor

(axial/pulpal) of a proximal cavity there may be only 2 tubules/mm2, whereas in

the cervical wall there may be 20/mm2. The number of cross-cut dentinal tubules

increases towards the inner half of the cervical wall. The floor of the cavity, on

the other hand, may sometimes contain a majority of tubules which are obliquely

cut or cut in a parallel direction. Since a local anaesthetic is usually administered

prior to cavity preparation, it is difficult to determine when cross-cut tubules are

open to the pulp. Therefore, all surface should be pretreated in the same manner.

Fig. 15

Axial (pulpal) wall in an approximal cavity, acid etched for removal of smear layer. tubules are cut obliquely in a parallel manner. The tubule apertures in this area number 2.000/mm2

37

Bacterial Association of Smear Layer

Fig. 16

Cervical wall, about half the way to the outer margin in the same cavity as seen in fig. 15. cross cut tubules numbering about 20.000/ mm2

Fig. 17

Cervical wall of buccal cavity. The dentin nearest the enamel (above) has tubules running parallel to surface. Below, 1/5 mm from enamel surface, the tubules are cross cut

38

Bacterial Association of Smear Layer

In Endodontics:

In the root, dentinal tubules extend from the pulp-predentin junction to the

intermediate dentin just inside the cementum-dentin junction. Dentinal tubules in

the root run a relatively straight course between the pulp and the periphery in

contrast to the typical S-shaped contours of the tubules in the crown. They range

in size from approximately 1-3 m in diameter. The density of dentinal tubules

per square millimeter varies from 4900 to 90,000. This density increases in an

apico-coronal direction and similarly in an external to internal direction from the

root surface. At the cementoenamel junction, the number of dentinal tubules has

been estimated to be approximately 15,000/ mm2.

BACTERIA IN THE SMEAR LAYER UNDER RESTORATIONS:

Brannstrom and Nyborg in 1973 prepared facial cavities in 20 contralateral

pairs of human premolars. One cavity, randomly selected after preparation, was

cleaned with water spray, while the other was cleaned with an antiseptic

detergent. Both the cavities were then filled with composite and allowed to set. In

both the teeth, the outer part of the filling was removed and replaced with zinc

oxide eugenol or Cavit cement. In this way, they prevented the growth of bacteria

into the contraction gap between the resin and the cavity walls. The teeth were

extracted after 3-6 weeks. They were coded and histologic evaluation was made.

The histological evaluation revealed that in 17 of the water-cleaned cavities,

with the smear layer remaining numerous bacteria were present; in the

antiseptically cleaned cavities, bacteria were absent. These results were highly

significant and showed that a few bacteria entrapped in the smear layer may

survive and multiply. There was also pulpal inflammation under these cavities.

The fact that bacteria may multiply on cavity walls if there was no

appreciable communication to the oral cavity seems to indicate that certain

microorganisms get sufficient nourishment from the smear layer and dentinal

fluid. This view was also supported by the results from the experiments with

inlays cemented with phosphate cements without any protective lining of the

cavity walls (Brannstrom and Nyborg I960, 1974, 1977). They used inlays made

39

Bacterial Association of Smear Layer

of lead because it was soft and more easily adapted to the margins of the cavity. In

this way, they got a good seal and minimal communication to the oral cavity. In

the 59 inlays they had cemented in antiseptically cleaned cavities, almost all the

teeth demonstrated no bacteria on cavity walls and no inflammation in the

corresponding pulp, not even when there was a pulpal exposure. On the other

hand, when the cavities had been cleaned only with water before cementation, a

high frequency of inflammation was found in 22 of 25 teeth and in 10 teeth the

inflammation was moderate to severe. There were also indications that in these

teeth, bacteria were present.

Fig. 18

Smear layer with a few microorganisms

These considerations favor the opinion that most of the smear layer should

be removed and any smear layer remaining for instance at the tubule apertures,

should be antiseptically treated before the application of a lining or luting cement.

The presence of a smear layer may also affect the retention of a lining or luting

cement. Their retention was obtained mainly by mechanical interlocking in the

micro undercuts present in the dentin (Oilo, 1978). It is possible that the presence

of a superficial smear layer will weaken mechanical retention between the lining

and the surface of the cut dentin.

40

Bacterial Association of Smear Layer

It has been suggested that bacteria are not present in freshly prepared smear

layers (Mjor, 1974). This suggestion was based on stained sections of freshly cut

intact teeth It seems clear that histologic techniques cannot reveal a few bacteria

entrapped within a smear layer They must multiply for sometime, form a thicker

layer, replace the smear layer, and become attached to the cut dentin if we are to

find them with certainity on the stained sections, because, during the

demineralization of freshly cut and unprotected dentin in preparation for cutting

sections, not only enamel, but also the smear layer disappears At the same time,

microbes entrapped within the smear layer disappear as well.

It is true that a smear layer without bacteria can be produced when intact

teeth are cut experimentally. On the other hand, in normal clinical procedures,

especially when operating on carious teeth, usually with low-speed or hand

instruments in the final preparation, we must consider the great risk of bacteria

surviving in the smear layer. Bacteria may even be left in the narrow gap between

the enamel and dentin at the lateral walls as well in single tubules in mineralized

dentin underneath. There was no evidence that common permanent restorative

materials are sufficiently antibacterial to kill bacteria entrapped within the smear

layer, especially when a fluid filled contraction gap 5-20 m wide, separates the

restoration from the smear layer.

It has been found that bacteria may also enter from the tooth surface into the

fluid-filled contraction gap around composite restorations. Bergenholtz & others

(1982) found that microbial invasion occurred frequently around amalgam

restorations. Thus all cavity walls should not only be cleaned and antiseptically

treated but also protected with a thin lining. This lining, applied to all cavity

walls, should not be placed over a superficial smear layer, as a thin lining may be

insufficiently antibacterial (Brannstrom, 1982, 1984; Brannstrom, Nordenvall

&Glantz, 1983). Moreover, for adequate retention of the lining to the cut enamel

and dentin, a superficial smear layer must not be present. However, Jodaikin et al

(1986) showed that more pulpal inflammation was seen beneath amalgam

restorations wherein smear layer had been removed prior to placement of the

restoration.

41

Bacterial Association of Smear Layer

Fig. 19 & Fig. 20

LINER ON THE SURFACE WITH AND WITHOUT SMEAR LAYER

Smear layer left – Bacteria enclosed in smear: Antibacterial effects from liner or filling may not reach bacteria enclosed deep in smear in which they may multiply.

Smear layer removed – A few bacteria contaminating the surface. Antibacterial effects from liner may eliminate bacteria still present or contaminating after cleaning.

42

Bacterial Association of Smear Layer

Bases of zinc oxide and eugenol and calcium hydroxide may have good antiseptic

effects but, unfortunately, under permanent restorations these bases cannot be

placed on all cavity walls. Also, bases of calcium hydroxide, such as Dycal, may

disappear when leakage occurs, leaving a fluid space for bacteria to enter. Bases

of fast setting calcium hydroxide may attach poorly to the cut surface and there

was the risk that a fluid filled gap may develop on both sides of the lining. Pure

calcium hydroxide is an excellent antibacterial temporary dressing and should be

applied under temporary fillings. This has been confirmed in many studies of pulp

capping. It is also possible-but not proved-that calcium hydroxide may reinforce

the remaining smear plugs in the outer apertures of the dentinal tubules.

To summarize, the cut dentin surface with its smear layer should be regarded

as an infected wound. Non-irritating antibacterial detergents should remove the

main part of the smear layer thereby reducing or eliminating the infectious

material at the cut surface. The reduced dentin permeability created by the smear

layer should then be retained by the residual smear plugs in the dentinal tubule

apertures.

SMEAR LAYER ON DENTIN EXPOSED TO THE ORAL CAVITY:

Another question concerns what may happen to the smear layer on surfaces

exposed to the oral cavity and left unrestored, for example in root planing after

superficial grinding, or under poorly fitting temporary crowns. It was found that

when a smear layer is produced experimentally on the human dentin and left

exposed it disappears after a couple of days and was replaced by bacteria and after

a week almost all the tubules are opened and some even widened (Brannstrom,

1982). There may be 10,000-20,000 tubules per square millimeter exposed on a

superficial hypersensitive exposure. The consequence is the invasion of bacteria.

Dentinal smear layers are very acid labile, and it is possible that acids present in

the diet may have caused dissolution of the smear layer and invasion of bacteria.

Cariogenic plaque may also cause dissolution of the smear layer. In single tubules,

bacteria can be found to have penetrated rather deeply (Olgart et al, 1974).

Vojinovic et al (1973) reported that dentinal smear layer beneath restorations

significantly prevented bacterial penetration into dentinal tubules by occluding the

43

Bacterial Association of Smear Layer

superficial portion of the tubule. Bacteria may also plug the tubular apertures.

However after 2 weeks, occasionally a mineralized pellicle was seen blocking the

aperture of the tubules (Brannstrom, 1982).

FATE OF SMEAR LAYER UNDER RESTORATIONS:

We cannot expect a mineralized pellicle to develop under a restoration where

saliva does not circulate. However, we know that the outward flow of fluid in

dentinal tubules and around the fillings may be reduced with time. The pulpal

ends of the tubules may be partly blocked by irregular dentin. As reported by

Pashley (1984), accumulation of solids in tubules and at their outer apertures may

contribute to a reduced flow of fluid. Under favorable conditions, a mineralized

pellicle may develop at the outer aperture of the contraction gap. The same has

been observed in the aperture of tubules of cut dentin left unprotected.

Little research is available to indicate what happens to the smear layer left

under a restoration. The smear layer may be detached and follow the outward

flow of fluid in the contraction gap. In a vital tooth, this flow is directed outward

due to the pressure gradient - a higher pressure of fluid in the pulp. The size of the

gap around the restoration may vary from 5-20 m. Johnson & Brannstrom

(1971) noticed that parts of the smear layer had been removed from the floor of a

cavity containing a poorly fitting temporary restoration of gutta percha for 3 days.

Certain bacteria may remove atleast parts of the smear layer. Histologic sections

reveal that the bacterial layer is closely oriented to the surface of cut dentin; the

bacteria have, in other words, occupied the smear layer.

Before sectioning of a tooth in the laboratory, a composite or amalgam

restoration is removed. Under the scanning electron microscope, we can see the

bacterial layer attached also to the inner surface of the restoration. Sometimes,

the whole bacterial layer is detached from the cavity and no bacteria are seen in

the dentinal tubules because of the presence of smear plugs in the tubular

apertures. This is one reason why we may not always find a correlation between

pulpal inflammation and the presence of bacteria on the cavity walls.

Inflammation may be present in the pulp, but a bacterial layer may not appear on

44

Bacterial Association of Smear Layer

the actual sections because it has been detached. Another reason for this failure is

that usually the sections examined in the microscope cover only a small part of the

total area of cavity walls. Bacteria might multiply on a lateral wall and the

concentration of toxins may increase in the fluid filled gap, but in the area

sectioned, or microbiologically sampled, the bacteria may not be attached to the

dentin.

Conversely, bacteria may be present on the sections but no inflammation

seen in the corresponding pulp because of the presence of atubular, irregular

dentin blocking the pulpal ends of the tubules. This "reparative" dentin may

develop after two weeks in monkeys and dogs, animals often used in experimental

studies. In humans, usually, 2-3 months are needed for developing this barrier.

This fact makes it difficult to interpret results from such animal experiments and

correlate them with the clinical situation in humans (Brannstrom 1982).

PRESENCE OF BACTERIA IN ENDODONTIC SMEAR LAYER:

It is generally accepted that one of the main causes of periapical disease is

bacterial infection of the root canal system (Yawata, 1973; Bystrom, 1987). Thus

eradication of bacteria form the root canal system is important for a successful

outcome. The bacteria that are most frequently found inside the root canals of

infected teeth are:

Obligate anaerobes:

Fusobacterium nucleatum

Porphyromonas gingivalis

Prevotella melaninogenica

Actinomyces odontolyticus

Facultative anaerobes:

Enterococcus faecalis

Escherechia coli

Pseudomonas aeruginosa

Streptococcus mitis

Streptococcus mutans

45

Bacterial Association of Smear Layer

Streptococcus sanguis

Streptococcus salivarius

Microaerophiles:

Actinobacillus actinomycetemcomitans

These bacteria may also be incorporated in smear layers during preparation

or enlarging of root canals with files and burs because it consists mainly of debris

of dentin and pulp tissue (McComb et al, 1975; Norman et al, 1980; Mader et

al, 1984; Garberoglio & Becce, 1994; Sen et al, 1995). Baker et al (1975) and

Yamada et al (1983) observed that bacteria could remain in the smear layer and

in the dentinal tubules despite instrumentation of the root canal and thus they may

survive and multiply (Brannstrom and Nyborg, 1973).

Fig. 21

ROOT CANAL SURFACE WITH AND WITHOUT SMEAR LAYER

Persistence of this smear layer with viable bacteria surviving in it may result

in bacterial penetration into the dentinal tubules of the root canal which in turn

depends upon several factors that act by increasing or decreasing the depth of

penetration. Michelich et al (1980) and Diamond & Carell (1984) stated that

bacteria could not penetrate into dentin in the presence of smear layer. It has been

shown that removal of smear layer facilitated passive penetration of bacteria

(Olgart et al, 1974; Michelich et al, 1980; Haapasalo & Orstavik, 1987; Safavi

46

Bacterial Association of Smear Layer

et al, 1989; Orstavik & Haapasalo, 1990). This layer though is not a strict

barrier to bacteria.

1. Type of microorganism:

The existence of bacteria in the dentinal tubules can cause a possible

pathology to the pulp and/or periapex. It is therefore of extreme importance to

eliminate these microorganisms during the accomplishment of the endodontic

treatment. It has been demonstrated in studies that bacteria are able to colonize

and survive inside the dentinal tubules of root canals and sometimes does not

obtain it elimination with instrumentation. Studies have been done with different

microorganisms under different circumstances to determine the degree of depth of

bacterial invasion inside the tubules. The extent of bacterial invasion is dependant

on the type of bacterial species and on time (Akpata & Blechman, 1982;

Meryon & Brook, 1990; Orstavik & Haapasalo, 1990).

Type of bacteria: Williams & Goldman (1985) showed that smear layer delayed

the penetration of Proteus vulgaris, but was not a complete barrier to these

bacteria. Meryon et al (1986) also found that Pseudomonas aeruginosa

penetrated even thicker dentin slices, by removing the smear layer itself and by

opening the orifices of dentinal tubules after possible collagenase production.

Meryon & Brook (1990) observed that Actinomyces viscosus, Corynebacterium

species and Streptococcus sanguis digested the smear layer and facilitated their

penetration. After degradation of the smear layer by proteolytic enzymes released

by certain bacteria (Uitto, 1988) a gap will develop between the filling material

and the canal wall, permitting the leakage of other bacterial species and their

byproducts along the canal walls into dentinal tubules and the periradicular

tissues.

Depth of penetration: Armitage et al (1983), Ando & Hoshino (1990) have

reported the presence of bacteria in the dentinal tubules of infected teeth at

approximately half the distance between the root canal walls and the

cementodentinal junction. Sen et al (1995) reported bacterial penetration into the

47

Bacterial Association of Smear Layer

tubules upto 150 m in the apical 2/3rd of the roots of teeth with necrotic pulps.

Horiba et al (1990) found endotoxin within dentinal walls of infected root canals.

Siqueira et al (1996) removed the smear layer and inoculated with 5 species of

bacteria and showed that all the test bacteria were able to penetrate into dentinal

tubules to varying depths. Perez et al (1993) found a mean penetration depth of

479 m for Streptococcus sanguis after 28 days of incubation, with maximum

penetration of 737 m. Peters et al (2001) reported the presence of bacteria in

more than half their samples (teeth with periapical lesions) close to the cementum.

Drake et al (1994) showed that removal of smear layer opened the tubules,

allowing bacteria to colonize in the tubules to a much higher degree (10-fold)

compared with roots with an intact smear layer. This indicated that smear layer

may inhibit bacterial colonization of root canals. Love et al (1996) showed that

Streptococcus gordoniii penetrated all non-smeared samples while 9 out of 10

smeared samples showed no bacterial penetration. This suggests that dentinal

smear layer is an effective barrier to dentinal tubule invasion by S. gordonii. Thus

factors such as the number and type of bacteria in addition to the length of

exposure and the presence or absence of smear layer, could influence the depth

of penetration of bacteria into the dentinal tubules.

Adherence of bacteria: Love (1996) investigated the adherence of S.gordonii to

smeared and non-smeared dentin and assessed the influence of patent dentinal

tubules on bacterial retention. He found that smear layers do not impede bacterial

adherence to dentin matrix and that surfaces with patent dentinal tubules retain

more bacteria than a smeared surface. Bilge Hakan Sen et al (2001)

demonstrated that the number of adherent Candida albicans cells to dentin

decreased after removal of the smear layer. This may be because C. albicans

retains poorly to clean dentinal surfaces and always requires a pellicle of proteins.

Here the smear layer acted as the pellicle of proteins. Yang & Bae et al evaluated

the effect of presence or absence of smear layer on the adhesion of Prevotella

nigrescens (a black-pigmented gram negative anaerobic bacteria considered to be

the most virulent bacterial strains of the root canal system) to the dentinal walls

and concluded that removal of smear layer inhibited bacterial adherence. They

48

Bacterial Association of Smear Layer

applied the same methodology as this study except that they used a different

bacterial strain, Staphylococcus aureus and showed the opposite result. Teeth with

smear layer contained significantly fewer bacteria than those in which the smear

layer has been removed. They cncluded that different results have been obtained

due to different growth rates, physiological characteristics and motility status of

the test organisms.

2. Vitality of the teeth:

Although some authors defended that the bacterial invasion is greater in vital

teeth than in non-vital ones by the contribution of nutrients, it has been

demonstrated that the depth, extension of invasion and bacterial proliferation were

greater in non-vital teeth, since odontoblastic processes act like defensive barrier

in vital teeth.

3. Age of the teeth:

In mature teeth, the depth of invasion is smaller since the number and

diameter of tubules diminish with age.

4. Dentinal permeability:

According to Pashley & colleagues, a greater bacterial invasion exists with

a higher permeability. For example, in a non-vital tooth, where the diameter of the

tubules remains constant and does not diminish, maintaining the permeability, the

invasion is much greater.

49