2036 Reduction of Oral Mutans streptococci by Small ...
1
1. Aim of Study Small crystal hydroxyapatite (scfHA) has been shown to adsorb oral streptococci in vitro and remove 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 restoring smoothness to minutely surface-damaged enamel * . To assess the effect of scfHA on oral MS both clinically and experimentally, we conducted (1) a 3DS trial in human subjects and (2) a test comparing 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) 【Experiment 1: 3DS】 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 carried out, but subjects applied a drug-retainer containing a small amount of the test paste to their upper and lower teeth for 5 minutes once daily following tooth brushing for 1 week. Oral bacterial levels Table 1. Overall effect of the 3 pastes on MS ratio Pastes 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%) Group C : DCPD 38% 1/10 (10.0%) * (*: p<0.05, chi-square test) 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 placebo controls. ・However no effect on the MS ratio was seen with scfHA (HA 25%) paste. (B) (E) Fig. 2. Adherence of S. mutans to different forms of enamel surface Fig. 3. SEM images of S. mutans adhering to different forms of enamel surface (Magnification, A, B, C: ×500, D, E, F: ×5,000) Smoothed enamel Roughened enamel ScfHA-treated enamel 2036 Reduction of Oral Mutans streptococci by Small-Crystal Hydroxyapatite T. ARAKAWA 1 , T. ISHIZAKI 1 , R. E. HAYMAN 1 , N. HANADA 2 , and H. SENPUKU 3 1 SANGI Co., Ltd. Japan, 2 National Institute of Public Health. Japan, 3 National Institute of Infectious Diseases. Japan. Dental Drug Delivery System (3DS) ① Drug-retainer Antibacterial agent Drug-retainer ① ② ③ ② Tooth Scalar Bacteria Antibacterial agent ① Even after biofilm is physically removed by PMTC, 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) Only a small amount of antibacterial agent is required to penetrate into all corners. ① Antibacterial agent is placed in drug- retainer. ② Drug-retainer with antibacterial agent is fitted over tooth surfaces. [Model of bacteria removal by the combined use of PMTC * and 3DS］ [Function of drug-retainer］ Fig. 1. Representative effects of 3 pastes on MS ratio in saliva samples in 3DS trials (Group B: scfHA content 38%) (Group C: DCPD content 38%) (Group A: scfHA content 25%) ** ** ** (n=9, **: p<0.01) (A) (D) 4.2. Bacterial adherence test ・SEM observation showed that S. mutans adherence was much greater on roughened than on smoothed enamel. However on roughened enamel subse- quently 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. The clinical results in this study, though with limited numbers, support our earlier finding (81 st General Session & Exhibition of the IADR) that scfHA paste is an effective agent for removing MS from the oral cavity in a 3DS. In addition, the restoration 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, by physically or chemically filling minute surface fissures, changing the enamel surface from rough to smooth. 82nd General Session & Exhibition of the IADR, MARCH 10-13, 2004 [email protected] MS ratio (%) = colony numbers of MS / total streptococci ×100 Smoothed enamel Roughened enamel ScfHA-treated enamel Ra = 11.6±3.0 Ra = 218.9±46.3 Ra = 103.9±25.9 Fig. 4. SPM images showing different forms of enamel surface 2. Materials and Methods 0.01 0.1 1 10 3 4 5 6 7 8 Ⅰ Ⅱ Ⅲ MS ratio (%) Weeks Period of scfHA paste treatment 0.01 0.1 1 10 3 4 5 6 7 8 Ⅰ Ⅱ Ⅲ Weeks MS ratio (%) Period of placebo paste treatment 3. Clinical Trial and Observation of Enamel Surface and Bacterial Adherence 4. Results 5. Conclusions 0 1 2 3 4 5 Application of test paste Control period Weekly monitoring of oral bacteria 8 ( weeks) [Time schedule of clinical trial] Drug-retainers applied to the teeth ( : The collection of saliva sample (11 a.m.)) (values of Ra are expressed as± mean SD (n=5)) 0.01 0.1 1 10 MS ratio (%) Ⅰ Ⅱ Ⅲ 3 4 5 6 7 8 Weeks Period of scfHA paste treatment 0 200 400 600 800 1000 Smoothed enamel Roughened enamel ScfHA-treated enamel CPM 6 7 (MS and total streptococci) were determined quantitatively by culture techniques from saliva samples collected once a week for 4 weeks before and 5 weeks 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 the test. 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 was considered to have been lowered in that subject. (See Fig. 1) 【Experiment 2: S. mutans adherence】 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 sets of blocks was polished with fine abrasive paper (grit 1200) to produce smoothed enamel, and another set with coarse abrasive paper (grit 600) to produce 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 3 H-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).
Transcript of 2036 Reduction of Oral Mutans streptococci by Small ...
1. Aim of StudySmall 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 cracksin tooth enamel. Other evidence suggests that hydroxyapatite may be effective in restoring smoothness 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 test comparing the adherence of Streptococcus mutans to roughened enamel surfaces before and aftertreatment with scfHA. (*: 82nd General Session & Exhibition of the IADR posters No. 1919 and 1920)
【Experiment 1: 3DS】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 containing38% 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 upper and lower teeth for 5 minutes once daily following tooth brushing for 1 week. Oral bacterial levels
Table 1. Overall effect of the 3 pastes on MS ratio
Pastes 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%)
Group C : DCPD 38% 1/10 (10.0%)
*
(*: p<0.05, chi-square test)
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 placebo controls.・However no effect on the MS ratio was seen with scfHA (HA 25%) paste.
(B)
(E)
Fig. 2. Adherence of S. mutans to different forms of enamel surface
Fig. 3. SEM images of S. mutans adhering to different forms of enamel surface (Magnification, A, B, C: ×500, D, E, F: ×5,000)
Smoothed enamel Roughened enamel ScfHA-treated enamel
2036 Reduction of Oral Mutans streptococci by Small-Crystal Hydroxyapatite T. ARAKAWA1, T. ISHIZAKI1, R. E. HAYMAN1, N. HANADA2, and H. SENPUKU3
1 SANGI Co., Ltd. Japan, 2 National Institute of Public Health. Japan, 3 National Institute of Infectious Diseases. Japan.
Dental Drug Delivery System (3DS)
①
Drug-retainerAntibacterial agent Drug-retainer
①
②
③
②
ToothScalarBacteria Antibacterial agent
① Even after biofilm is physically removed by PMTC, 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)
Only a small amount of antibacterial agent is required to penetrate into all corners.
① Antibacterial agent is placed in drug- retainer. ② Drug-retainer with antibacterial agent is fitted over tooth surfaces.
[Model of bacteria removal by the combined use of PMTC* and 3DS］
[Function of drug-retainer］
Fig. 1. Representative effects of 3 pastes on MS ratio in saliva samples in 3DS trials
(Group B: scfHA content 38%) (Group C: DCPD content 38%) (Group A: scfHA content 25%)
**
**
**
(n=9, **: p<0.01)
(A)
(D)
4.2. Bacterial adherence test・SEM observation showed that S. mutans adherence was much greater on roughened than on smoothed enamel. However on roughened enamel subse- quently 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.
The clinical results in this study, though with limited numbers, support our earlierfinding (81st 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 withscfHA paste, and the reduction in bacterial adherence seen to accompany this, suggest that scfHA may also inhibit MS adherence to the enamel surface, by physically or chemically filling minute surface fissures, changing the enamel surface from rough to smooth.
82nd General Session & Exhibition of the IADR, MARCH 10-13, 2004 [email protected]
MS ratio (%) = colony numbers of MS / total streptococci ×100
Smoothed enamel Roughened enamel ScfHA-treated enamel
Ra = 11.6±3.0 Ra = 218.9±46.3 Ra = 103.9±25.9
Fig. 4. SPM images showing different forms of enamel surface
2. Materials and Methods
0.01
0.1
1
10
3 4 5 6 7 8
Ⅰ
Ⅱ
ⅢMS
ratio
(%)
WeeksPeriod of scfHA paste treatment
0.01
0.1
1
10
3 4 5 6 7 8
Ⅰ
Ⅱ
Ⅲ
Weeks
MS
ratio
(%)
Period of placebo paste treatment
3. Clinical Trial and Observation of Enamel Surface and Bacterial Adherence
4. Results
5. Conclusions
0 1 2 3 4 5Application of test paste
Control period Weekly monitoring of oral bacteria
8 (weeks)
[Time schedule of clinical trial]
Drug-retainers applied to the teeth
( : The collection of saliva sample (11 a.m.))
(values of Ra are expressed as± mean SD (n=5))
0.01
0.1
1
10
MS
ratio
(%)
Ⅰ
Ⅱ
Ⅲ
3 4 5 6 7 8WeeksPeriod of scfHA paste treatment
0
200
400
600
800
1000
Smoothed enamel Roughened enamel ScfHA-treated enamel
CPM
6 7
(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 the test. 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 was considered to have been lowered in that subject. (See Fig. 1)
【Experiment 2: S. mutans adherence】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 performedusing 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).
