Reduction of Oral Mutans streptococci by Small-Crystal Hydroxyapatite

2036

1123T. ARAKAWA , T. ISHIZAKI , R. E. HAYMAN , N. HANADA , and H. SENPUKU231SANGI Co., Ltd. Japan, National Institute of Public Health. Japan, National Institute of Infectious Diseases. Japan.

1. Aim of Study
Small crystal hydroxyapatite (scfHA) has been shown to adsorb oral streptococci in vitro andremove mutans streptococci (MS) from the oral cavity in a Dental Drug Delivery System (3DS).
Previous study indicated that oral bacteria adhere principally to rough areas such as minute cracks
in tooth enamel. Other evidence suggests that hydroxyapatite may be effective in restoringsmoothness to minutely surface-damaged enamel*. To assess the effect of scfHA on oral MS bothclinically and experimentally, we conducted (1) a 3DS trial in human subjects and (2) a testcomparing the adherence of Streptococcus mutans to roughened enamel surfaces before and after
treatment with scfHA. (*: 82nd General Session & Exhibition of the IADR posters No. 1919 and 1920)
Dental Drug Delivery System (3DS)[Model of bacteria removalby the combined use of PMTC* and 3DS］

1

[Function of drug-retainer］①①

ToothScalar
Bacteria(MS and total streptococci) were determined quantitatively by culture techniques from saliva samples collected once a week for 4 weeks before and 5weeks after the week of paste application, and the ratio of MS to total streptococci (MS ratio) was calculated. For each subject, the four MS ratio
values obtained prior to application of the test paste were used to determine that subject's average MS ratio and range of MS ratio values prior to thetest. If a subject's final MS ratio, measured 5 weeks after application of the paste, fell below his or her initial range of MS ratios, the MS ratio wasconsidered to have been lowered in that subject. (See Fig. 1)
Enamel from sound extracted human teeth was cut into 4×5×3 mm blocks, and the surface polished with abrasive paper (Buehler Co., Ltd.). One setsof blocks was polished with fine abrasive paper (grit 1200) to produce smoothed enamel, and another set with coarse abrasive paper (grit 600) toproduce roughened enamel. A third set of blocks was prepared by immersing roughened enamel blocks in 10% scfHA slurry at 37℃ for 7 days
(scfHA-treated enamel). Three sets of 3 blocks each were then incubated in whole saliva, and subsequently, in a 1.0ml 3H-labeled S. mutans suspension(OD=0.3, 550nm), at 37℃ for 24h. Adherence of S. mutans to the blocks was measured using a scintillation counter and statistical analysis performed
using ANOVA. The surface appearance of the blocks and bacterial adherence were also observed by SEM (S-5200, Hitachi), and surface roughness (Ra)values for each set measured by scanning probe microscope (SPM) (SPI4000, Seiko Instruments, Inc.) (scanning area: 50×50 μm, dynamic force mode).
3. Clinical Trial and Observation of Enamel Surface and Bacterial Adherence

Drug-retainer
Antibacterial agent

Drug-retainer②③(Group A: scfHA content 25%)
1010

101

1

1ⅠⅡⅢ

0.1

0.01

34

Period of scfHA paste treatment

657ⅠⅡ
Ⅲ

0.1

0.01

83

4WeeksPeriod of scfHA paste treatment

5678MS ratio (%)① Antibacterial agent is placed in drugretainer.
② Drug-retainer with antibacterial agent
is fitted over tooth surfaces.

MS ratio (%)

Only a small amount of antibacterial agent isrequired to penetrate into all corners.

Table 1. Overall effect of the 3 pastes on MS ratio(Group C: DCPD content 38%)

(Group B: scfHA content 38%)

MS ratio (%)① Even after biofilm is physically removed byPMTC, bacteria remain on tooth surface.
② Drug-retainer containing antibacterial agent
is used to eliminate remaining bacteria.
③ Bacteria are completely removed.
(*：Professional Mechanical Tooth Cleaning)

PastesⅠⅡⅢ

Percentages of subjects in which MS ratio
was reduced after treatment

Group A : scfHA 25%

0/5 (0%)Group B : scfHA 38%

8/15 (53.3%)

0.1

*0.01
3Weeks4Period of placebo paste treatment

567

Group C : DCPD 38%

8Weeks

1/10 (10.0%)(*: p<0.05, chi-square test)

2. Materials and Methods

Fig. 1. Representative effects of 3 pastes on MS ratio in saliva samples in 3DS trials【Experiment 1: 3DS】

MS ratio (%) = colony numbers of MS / total streptococci ×100

A 3DS clinical trial was performed with 30 healthy adult subjects (age: 24～41 years). Group A(5 subjects) used scfHA paste containing 25% HA, Group B (15 subjects) scfHA paste containing
38% HA, and Group C (10 subjects) a placebo paste containing 38% dicalcium phosphate dihydrate(DCPD). Preliminary PMTC, bacterial elimination and control of subjects' life-style were not carriedout, but subjects applied a drug-retainer containing a small amount of the test paste to their upperand lower teeth for 5 minutes once daily following tooth brushing for 1 week. Oral bacterial levelsSmoothed enamel****

1000(A)**

Roughened enamel

ScfHA-treated enamel

(B)

800

12

Control period

34

Application oftest paste

5678 (weeks)

Weekly monitoringof oral bacteria(: The collection of saliva sample (11 a.m.))

CPM[Time schedule of clinical trial]

0600
400(D)(E)

2000
Smoothed enamel

Roughened enamel

ScfHA-treated enamel

Fig. 2. Adherence of S. mutans
to different forms of enamel surface(n=9, **: p<0.01)
Drug-retainers applied to the teeth

Roughened enamel

ScfHA-treated enamel

Ra = 11.6±3.0

Ra = 218.9±46.3

Ra = 103.9±25.9【Experiment 2: S. mutans adherence】

Antibacterial agent②Smoothed enamel

Fig. 3. SEM images of S. mutans adhering
to different forms of enamel surface

Fig. 4. SPM images showing different forms of enamel surface(values of Ra are expressed as± mean SD (n=5))

4. Results
4.1. 3DS clinical trial
・Saliva testing 5 weeks after 3DS treatment showed a significant decrease in
MS ratio in subjects using scfHA (HA 38%) paste in comparison with placebocontrols.
・However no effect on the MS ratio was seen with scfHA (HA 25%) paste.
4.2. Bacterial adherence test
・SEM observation showed that S. mutans adherence was much greater onroughened than on smoothed enamel. However on roughened enamel subsequently treated with scfHA, it was similar to the lower level of adherence
seen on smoothed enamel.
・Quantitative analysis showed that the difference in bacterial adherence between
the three forms of enamel surface was statistically significant.
・No clear correlation however was identified between the level of bacterial
adherence and Ra values for the three types of enamel surface as measured by
SPM.
5. Conclusions
The clinical results in this study, though with limited numbers, support our earlierstfinding (81 General Session & Exhibition of the IADR) that scfHA paste is aneffective agent for removing MS from the oral cavity in a 3DS. In addition, therestoration of surface smoothness to roughened tooth enamel after treatment with
scfHA paste, and the reduction in bacterial adherence seen to accompany this,suggest that scfHA may also inhibit MS adherence to the enamel surface, byphysically or chemically filling minute surface fissures, changing the enamelsurface from rough to smooth.

(Magnification, A, B, C: ×500, D, E, F: ×5,000)

82nd General Session & Exhibition of the IADR, MARCH 10-13, 2004
arakawa@sangi-j.co.jp