3713

Benign Shift in the Oral Flora Induced by Nano-hydroxyapatite
T. Fujimaru, T. Ishizaki, R. Hayman, SANGI Co.,Ltd.
H. Suzuki, Y. Kono, Nihon University School of Dentistry at Matsudo
H. Senpuku, National Institute of Infectious Diseases, Tokyo, Japan.
Objectives

High oral mutans streptococci (MS) concentration can be associated with caries risk. We previously showed that nano-hydroxyapatite (nHAP) adsorbs oral pathogenic microbes(IADR 2007, 2010 ), and reduced the ratio of MS in the oral flora in a clinical trial (IADR 2013). One unreported finding was a concomitant increase in lactobacillus (LB)（Fig.4）,
which is believed to lessen caries risk. This time we examined nHAP’s ability to induce a benign shift in the oral flora and the term of efficacy of its effect.

Materials and Methods

1. Method of using test pastes
19 subjects applied the test paste (nHAP paste (n = 10) or placebo paste without nHAP (n = 9) to their upper and lower teeth after professional mechanical tooth cleaning (PMTC)
and then after night-time toothbrushing for 6 days, using a retainer tray which they kept on their teeth for 5 minutes（Fig.1）.
Test paste

Drug-retainer

Subjects applied a drug-retainer containing 5ml of the test paste
to their teeth for 5 min daily for 6 days.

Fig 1. Test paste application

2. Method of assessing the MS ratio and LB
Subjects were asked to provide a sample of saliva, at designated times (11:00am before lunch), and each sample was sent to a testing laboratory (BML, Inc.). The number(CFU/ml) of total streptococci, mutans streptococci and LB were assessed by culture from these saliva samples, and the MS ratio for each subject was calculated based on thesebacterial counts (Fig 2).

MS, total streptococci, and LB assessedby culture techniques from saliva samples.

Test laboratory ( BML, Inc. )Saliva sample taken

Fig 2. Examination of MS ratio and LB

3. Clinical trial procedure
Saliva sampling to determine the MS ratio and LB count by culture techniques was done on all subjects immediately before, then the day after PMTC, then after final applicationof the test paste in a retainer tray, i. e. at 7 days, followed by further saliva sampling after 1 and 2 months. The nHAP group alone underwent an additional saliva sampling at 3months, followed by another 6 days home treatment, then successive saliva sampling after another 1, 2 and 3 months, after which a third similar round of home treatment andsaliva sampling was repeated (Fig.3).
Both paste groups(nHAP paste and placebo)Start of trial

Pre trial• Informed consent• Impression taking

Homecare:6days

Examination

1month• Saliva sampling• Saliva sampling• Test paste applied
once daily for 6days⇩• Saliva sampling• Saliva sampling⇩• PMTC⇩• Saliva sampling⇩• Test paste applied⇩• Saliva sampling

Total paste application 7 times

1mnHAP paste group only

Examination

1monthhtno

Start of trial

Examination• Saliva sampling

Homecare:6days

Examination

1month1month• Saliva sampling• Test paste applied
once daily for 6days⇩• Saliva sampling• Test paste applied⇩• Saliva sampling

Examination

1monthExamiination• Saliva sampling• Saliva sampling

Total paste application 7 times

Two - rounds

Fig 3. Time table

Results

1. Re-analysis of previous clinical trial results at Nihon University School of Dentistry at Matsudo
Linear regression re-analysis showed a correlation, over 2 months, between nHAP treatment (n=30) and not only the MS ratio decrease (Y=-6E-07x+0.0026, R2 = 0.4558) but also
an LB increase (Y=3E-04x+3.6912, R2 = 0.5089). In contrast, for the placebo group (n=30), both the MS ratio (R2 = 0.0836) and LB level (R2 = 0.0014) were unchanged（Fig.4）.
These results were further confirmed by the present study.
nHAP group(n=30)

0.0040①②③④⑤Placebo group(n=30)

6.00

0.0040①②③MS比MS ratio④線形(MS比)Linearityof MS ratio

LogLB
Log LB

0.003420

0.0035

5.50

Linearityof Log LB
線形(LogLB)
0.003224

線形(MS比)of MS ratio
Linearity

0.002278

3.90

3.93

0.00254.15
4.00

0.001497

Test paste applied
0200

400

600

0.0010

3.00
800

1000

Times (h)

3.80

3.59

3.39

0.0005y = -2E-05x + 3.7189
R² = 0.0014

0.00153.500.0010

0.002146

4.00

y = 0.0003x + 3.6912
R² = 0.5089

4.500.002117
0.00204.03

5.000.002561

4.25

y = -6E-07x + 0.0026
R² = 0.4558

0.002000

0.0015

4.50

1200

1400

5.50

Log LB

0.0025

MS ratio

5.000.002719

0.0020y = 3E-07x + 0.0025
R² = 0.0836

0.0030

Log LBMS ratio

線形(LogLB)
Linearityof Log LB

0.002938

0.00306.00

LogLB
Log LBMS比
MS ratio

0.0035⑤3.50

Test paste applied
0200

400

3.00

600

800

1000

1200

1400

1600

② After PMTC
③ After home care

3.380.0005

1600

3.62

① Initial④ 1 month after home care
⑤ 2 months after home careTimes (h)Fig 4. Re-analysis of results of previous clinical trial (Nihon University School of Dentistry at Matsudo, n=60)
2. Influence of repeated nHAP treatment on the MS ratio and LB level
Linear regression analysis showed a similar correlation, over 2 months, between nHAP treatment and the MS ratio decrease (Y=-2E-07x+0.0026) and LB increase(Y=3E-04x+4.5878)(Fig. 5-A). In contrast, the placebo group MS ratio increased (Y=3E-06x+0.0013) and LB was unchanged (Fig. 5-B). The nHAP group showed a tendency
toward recovery of the MS ratio and decline in LB after 2 months. But after a second round of home treatment, even without prior PMTC, further reduction in the MS ratio and anincrease in LB were observed. The same tendency was confirmed by the third round of treatment. Ultimately, over three rounds of treatment, an MS ratio decrease(Y=-1E-07x+0.0023) and a concomitant LB increase (Y=9E-05x+4.7059) were confirmed (Fig. 5-C).

Conclusion

Our results indicate that treatment with nHAP both reduces the MS ratio and increases LB, causing a shift in the oral flora that may lessen caries risk. Signs of regression after 2months suggest that the treatment interval should be 2-3 months.

MS ratio⑤5.69

Log LB
Linearity of MS ratio

MS ratio

⑥4.64

②4.04

0.0025

5.50
⑥5.45

④5.26

0.0045⑤0.004000
0.00354.50
MS比MS ratioy = 3E-06x + 0.0013
R² = 0.9845

0.00254.00

1st round① Initial

5.00

Log LB
LogLB
4.00

線形(MS比)Linearityof MS ratio④0.002198
0.00153.50

3.50①0.001333③0.001267②0.000933

Test paste applied

0.0005
0400

600

800

Times (h)

1000

1200

1400

01600

200

400

3.00
600

800

1000

Times (h)

1200

1400

1600

0.0065⑦5.47

MS ratio

0.0045y = 9E-05x + 4.7059
R² = 0.2235

⑧5.35

⑪5.14

①4.94

③0.003591
0.0035

③4.49

④4.55

⑭5.04

⑩4.72

⑥4.64

Linearity of Log LB⑫0.002540

4.00⑮0.002017⑧0.001823⑤0.001910

3.50⑭0.001387⑯0.001346⑨0.001222

Test paste applied

0.0005
0⑪0.000895

Test paste applied
1000

2000

1st round

3000

Times (h)2nd round4000

y =-1E-07x+0.0023
R² = 0.2077

Test paste applied
5000

6000

3rd round⑬ After first paste use⑭ After home care
⑮ 1 month after home care

⑰0.002019⑬0.001610⑩0.001463⑦0.001654

0.00154.50⑫ 3 months after home careLinearity of MS ratio①0.002379④0.001857⑪ 2 months after home care
5.00

⑮5.01

Log LB⑥0.002480②4.04②0.002473⑯5.17

MS ratio⑨4.33

0.0025⑩ 1 month after home care

5.50

⑰5.34
⑫5.19

⑥ 2 months after home care

⑨After home care

⑬5.620.0055⑤ 1 month after home care

2nd round⑧ After first paste use

6.00
⑤5.69

③ After first paste use⑦ 3 months after home care

Log LBC) nHAP group(3rounds, n=10)

200

Test paste applied

0.00053.00② After PMTC

④After home care

線形(LogLB)
Linearityof Log LB⑤0.001910④0.0018570.00154.50⑥0.002480

y = -2E-07x + 0.0026
R² = 0.0634②0.002473①0.002379

0.0055

5.00

④4.55

⑤5.54

y = 5E-05x + 5.4044
R² = 0.047②5.41

y = 0.0003x + 4.5878
R² = 0.1376③4.49⑥0.006000③5.26③0.003591
0.0035

6.00
①5.65

5.50

Linearity of Log LB①4.94

0.0065

MS ratio

0.00550.0045B) Placebo group(1st round, n=9)

6.00

Log LB

0.0065

Log LBA) nHAP group(1st round, n=10)⑯ 2 months after home care
⑰ 3 months after home care

3.00
7000

3rd roundFig 5. MS ratio and LB over time in nHAP group and placebo group
93rd General Session & Exhibition of the IADR/ March, 2015

E-mail : fujimaru@sangi-j.co.jp