Antimicrobial Action of New, Proprietary Lining Cements

D. McCOMBI and D. ERICSON2

'Department of Restorative Dentistry, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario, Canada MSG I G6; and
2Department of Cariology, School of Dentistry, Malmo, Sweden

The antibacterial activity of innovative, commercial lining cements

was investigated. A liner which contains calcium hydroxide and is
polymerized by visible light (Prisma VLC Dycal) and a glass-ionomer
lining cement (GC lining cement) were compared with two more established lining cements (Advanced Formula II Dycal and Life). Antibacterial activity and hemolysis-like agar change at 24, 48, and 72
hours were measured on blood agar plates inoculated with Streptococcus mutans KPSK 2 serotypee c), Lactobacillus casei ssp rhamnosus ATCC 11981, and chewing-stimulated saliva.
Prisma VLC Dycal did not affect bacteria or agar. The glass-ionomer lining cement, with an acidic pH at setting, had the most pronounced effect on all test organisms and on the agar. Even after 48
hours' setting, it inhibited growth of S. mutans. The control lining
cement (AFII Dycal) showed antibacterial activity toward both specific micro-organisms as well as some activity against the salivary
organisms. The material Life showed only partial inhibition of microbial growth. For all lining cements, the hemolytic-like agar change
correlated with antibacterial effects.
The surface pH of the freshly-set cements containing calcium hydroxide was alkaline. It would seem that a simple correlation between
high surface pH and antibacterial activity among these cements does
not exist. Also, further biological characterization of new lining cements is required to direct their appropriate clinical use.

J Dent Res 66(5):1025-1028, May, 1987

Introduction.
Proprietary two-paste, quick-setting, lining cements containing
calcium hydroxide are widely used in restorative dentistry because they are easily manipulated, provide protection, and have
generally beneficial effects on the pulp (Shovelton, 1972).
The importance of bacterial contamination as a factor impairing the healing capacity of the injured pulp has been clearly
demonstrated by work with germ-free animals (Kakehashi et
al., 1965; Patterson, 1976; Patterson and Watts, 1981).
Brinnstrbm (1984) has stated that pulpal damage is caused by
bacteria under the restoration and not by the restorative material. Bacteria may either multiply from the smear layer present on the prepared cavity walls (Brannstr6m and Nyborg,
1973) or enter through microleakage from the oral cavity.
Cements containing calcium hydroxide can provide antibacterial activity, and carious dentin can be sterilized in vivo
by exposure to either a calcium hydroxide and water paste
(Fisher, 1972) or to a commercial calcium hydroxide lining
cement, Dycal (Fisher, 1977). Thus, these cements are preferred for lining deep cavities and for direct or indirect pulpcapping procedures (Plasschaert, 1983).
Mjdr (1977) has reported that bacteria could not be demonstrated adjacent to lining materials such as zinc-oxide/eugenol and calcium hydroxide. He suggested that a systematic
evaluation of the antibacterial properties of linings and bases
should be considered as part of the evaluation of their biological properties.
Clinical experience has shown that use of posterior composites is associated with increased pulpal hypersensitivity.
Received for publication July 22, 1986
Accepted for publication November 19, 1986

New lining materials have been developed concomitantly. A

single component liner which contains calcium hydroxide and
is polymerized by visible light, and which has been claimed
to have superior physical properties (Wang, 1986), is available
commercially (Prisma VLC Dycal). Glass-ionomer lining cements have also recently been developed. These are intended
to combine the biocompatibility of the glass-ionomer restorative materials (McComb, 1982), their excellent sealing ability, and fluoride release (Maldonado et al., 1978) in a
radiopaque, flowable consistency.
The new materials are quite different in composition and
properties from materials bearing the same generic name and
are being used clinically without adequate biological characterization. The purpose of the present study was both to assess
the antibacterial properties of such new lining cements and to
aid in their biological characterization.

Materials and methods.

Streptococcus mutans KPSK 2 (serotype c) and Lactobacillus casei ssp rhamnosus ATCC 11981 were maintained on
blood agar plates, and a loopful of the cultures was inoculated
in 10 mL of Todd-Hewitt broth (Difco Laboratories, Detroit,
MI) and incubated at 370C in 10% C02, 10% H2, and 80%
N2 for six hours. Three drops of this culture were then used
to inoculate duplicate Petri dishes containing 20 mL of Tryptic
Soy Agar (Difco) supplemented with 5% sheep blood, of approximately 3 mm depth. Similarly, three drops of freshly
collected, chewing-stimulated saliva were spread on identical
blood agar plates.
Ten wells, each with a diameter of 3 mm, were punched in
the agar of all inoculated and two sterile plates. The wells
were filled in duplicate with four commercial lining cements
(Table 1), the remaining two wells with phosphate-buffered
saline (0.1 mol/L phosphate, 0.14 mol/L NaCl. pH 7.4, PBS).
All two-component cements were mixed according to the manufacturer's instructions. The standard powder/liquid ratio 1.2
gig was used for the glass-ionomer lining cement. The visiblelight-cured (VLC) material was polymerized with a HeliomatSF light unit (Vivadent, Schaan, Liechtenstein), having a 4.5mm-diameter retruded tip, for 40 sec both above and below
the appropriate wells. One PBS-filled well on each plate was
similarly treated with the light source. To investigate any change
TABLE 1
LINING CEMENTS TESTED
Material
Batch
Manufacturer
Prisma VLC Dycal
121685
L.D. Caulk Co/Dentsply
072484
L.D. Caulk Co/Dentsply
Advanced Formula II

Dycal
Life

Catalyst 4 121 1
Base 4 1131

Kerr Co./Sybron

GC Glass-ionomer
Lining Cement
(Standard P/L ratio)

Liquid 250151

G-C Dental Ind. Corp.

Powder 230151

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1025

1026

McCOMB & ERICSON

Prisma VLC Dycal

Advanced Formula II
Dycal
Life
GC Glass-ionomer
Lining Cement

J Dent Res May 1987

TABLE 2
MEAN SURFACE pH OF LINING CEMENTS
Surface pH of
Freshly Set Cement + S.D.
11.9 0.4
11.1 0.6

Surface pH of Test Cement

Samples after Four Days in Agar + S.D.

7.5 0.4
8.2 + 0.5

11.4 0.4
2.6 0.4

in antibacterial properties with time, we seeded the uninoculated plates containing the set cements with S. mutans KPSK
2 after 48 hours of pre-incubation.
The blood agar plates were incubated as broth cultures and
examined after 24, 48, and 72 hours. At each time period, the
effects of the different materials on the growth of the organisms
were noted as circular zones, the diameter of which could be
measured. Colony-free zones were labeled "total inhibition",
and zones of impaired growth, "partial inhibition". The degree of hemolysis-like agar change was also noted and measured, with change of agar translucency used as the only
criterion. The whole experiment was repeated once with the
same organisms and carried out in the same manner. Mean
values from four plates, i.e., eight cement samples, were calculated for each organism and condition. The procedure was
modified from Welker et al. (1984).
The surface pH of freshly set cement specimens, out of agar,
and specimens removed from the blood agar plates at four days
were measured with a pH meter [Accumet pH meter model
620 - Fisher Scientific Ltd. (Ottawa, Canada)] and a combination flat surface-polymer body electrode, Cat. No. 13-63983 (Fisher). The electrode was calibrated with standard buffer
solutions of pH values 4, 7, and 11. One drop of distilled
water was placed on the surface of the cement to obtain a pH
reading.

Results.
The mean diameters of zones of complete and partial inhibition of bacterial growth at 48 hrs are shown in Fig. 1. The
mean widths of hemolytic-like zones are also included. The
antibacterial zones and agar changes varied with the different
micro-organisms and with the different lining cements. A typical S. mutans plate at 48 hrs is shown in Fig. 2. Similar results
were shown after 24 and 72 hrs. The Prisma VLC Dycal did
not affect any parameter. The glass-ionomer cement had the
most pronounced effect on both bacterial growth and agar. This
cement was the only material to show zones of total inhibition
of bacterial growth to all test organisms, even when inoculated
with S. mutans after setting for 48 hrs. Agar changes were
pronounced and exhibited a wide halo effect of approximately
22 mm (Fig. 3).
The (AFTI) Dycal showed antibacterial activity toward all
organisms. The antibacterial activity toward S. mutans was
eliminated when plates were inoculated after the set cement
was incubated for 48 hrs in agar. The material Life showed
only partial inhibition of salivary micro-organisms. Both Life
and (AFII) Dycal exhibited a similar degree of effect on the
blood agar. The antibacterial effect of a material was generally
correlated with its hemolytic-like activity (Fig. 1).
The less-aciduric S. mutans was most sensitive to the acidic
G.C. lining cement, as compared with L. casei (p < 0.0005,
Student's t test). The opposite was true for (AFIL) Dycal (p <

8.1 0.4
7.4 + 0.6

PRISMA
VLC DYCAL

21
3

51
ADVANCED
FORMULA II DYCAL

LIFE

l.S.mutans

2
3
4
5.

3.Saliva

12-

4.S.mutans
(Post inoculation*)

2.L.casei

3--,,z,~,z,

5.Agar change

5T
G.C.LINING CEMENT

/zz~zfxz2Jz

BUFFER CONTROL
Total inhibition

Partial inhibition

__ Agar change

2-3
4- 4
0

10
15
20
DIAMETER (mm)

25

Fig. 1 - Diameters of zones of antibacterial activity and agar change

at 48 hours. *Incubated plates inoculated with S. mutans 48 hours after
insertion of test cements. Bar indicates S.D.

0.0025). The PBS controls caused no inhibition of bacterial

growth.
Table 2 shows the surface pH of the freshly set cement (three
minutes) and the surface pH of the test cement samples after
four days in agar.

Discussion.
A significant difference in antibacterial effect has been shown
in this study between a conventional two-paste calcium hydroxide cement (AFII Dycal) and a new visible-light-cured
liner containing calcium hydroxide (Prisma VLC Dycal). The
former shows definite early antibacterial activity, particularly
against L. casei, the bacteria most likely to be present on cavity
floors (Fisher, 1972), and also to S. mutans and to the salivary
micro-organisms tested. The Prisma VLC Dycal showed no
antibacterial effect. Bacteria were even able to grow in contact
with excess material on the agar surface (Fig. 2). The material
appeared to be inert, and this opinion was reinforced by the
total lack of agar change.
It is interesting that, in a pulp-capping study utilizing Prisma
VLC Dycal (Stanley and Pameijer, 1985), the material induced
dentinal bridging over the exposures without creating the
chemical cautery or intermediate necrotic layer normally associated with most calcium hydroxide products. Such pulpcapping studies in monkeys, however, give the pulpal response
uncomplicated by the bacterial contamination that is both pres-

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ANTIMICROBIAL ACTION 01 NEW LINING CEMENTS

Vol. 66Noe.5

Fig. 2
bacterial

Typical S.
inhibition

plate at 48 hours showing zones of

cements clockwise froml A (one o'clock) in

mutans agar

Lining

duplicate: Prisma VLC Dycal, Advanced Formula 11 Dycal. Life, and GC

Glass-ionomer Lining Cement.

10)27

contain calcium hydroxide and record a similar high pH when

immersed in distilled water for up to one week (McComb,
1983). The release of calcium hydroxide in a particular environment will depend on the composition of the material and
its solubility (Schrdder, 1985). The third liner in this study
that contains calcium hydroxide (Prisma VLC Dycal) also recorded a similar high surface pH upon initial setting. The antimicrobial activities of these three cements, however, differed
markedly.
Thus, simple analysis of cement surface pH is not a good
indicator of the antimicrobial activity of the material. It has
generally been understood that the high pH of such commercial
products is indicative of free calcium hydroxide and reflects
their antibacterial activity (Craig et al., 1985). It would seem,
therefore, that a simple relationship between high surface pH
and antibacterial activity may not exist for cements containing
calcium hydroxide. Rather, the total capacity and rate of release of hydroxyl ion will affect this property, as will other
possibly unrelated antimicrobial components.
The glass-ionomer lining cement was markedly antibacterial
to all test organisms, with further areas of influence in the
form of partial inhibition. Antimicrobial activity was present,
although slightly lower, even after pre-incubation for 48 hours
prior to inoculation. Agar changes were more marked with this
cement, further indicating high biological activity. The degree
of antimicrobial activity may be related both to the low pH of
the cement before setting and to the high fluoride content. The
higher sensitivity of S. futntns as compared with L. c(a5sci to
this cement may reflect this (Hamilton et al., 1985). Antimicrobial effects and, more particularly, hemolytic effects of restorative materials have been inversely correlated with
biocompatibility. Welker et ol. (1984) have indeed suggested
such methods for evaluation of relative biocompatibility of
dental materials. Significant evidence exists to suggest that the
glass-ionomer restorative materials have good biocompatibility
(McComb, 1982). The new lining cements, however, have a
different composition and a lower powder-to-liquid ratio, to
allow for a more fluid consistency. More research is needed
to investigate the effects of these changes. Pulpal studies are
necessary to assert that the degree of biological activity shown
by the glass-ionomer lining material does not have the potential
to cause further damage or impair healing when placed in proximity to the pulp.
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Fig. 3 - S. mutans agar plate at 48 hrs from Fig. 2, showing zones of

hemolysis-like agar change when viewed by transmitted light.
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presence of buffering components in the agar, causing insoluble calcium phosphate to be formed around the pellet of test
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