0547
Interaction of small crystal form of Hydroxyapatite with mutans streptococci< Results >S. sanguis ATCC10556

**

**

**

2000

Nonheated

1200

**

% of S. sobrinus to HA

Mean

080060

Mean

40200

12000Nonheated

1200

800

800

8060

Mean

40200

1200

200

80080

Mean

60
40200

1200

Nonheated
Heat-treated temperature (℃)

800

8060

Mean

40200

1200

2000

1200

200

800

800

200200

**
**
**

100

Nonheated

8060

Mean

40200

1200

800

200

1008060

Mean

402001200

Nonheated

Heat-treated temperature (℃)

Heat-treated temperature (℃)

Nonheated

Heat-treated temperature (℃)

**

100

Nonheated
Heat-treated temperature (℃)

200

Mean

4020*****8060****
**

100

*
**

100

Nonheated
Heat-treated temperature (℃)

Nonheated
Heat-treated temperature (℃)*80

Nonheated
Heat-treated temperature (℃)

20**

100

**

100

20040*****

1200200

Mean

Heat-treated temperature (℃)

**

100
80
60402080060***% of S. mutans to HA

1200200**

**

HA0**

**

1SANGI Co., Ltd. Japan, 2National Institute of Public Health. Japan,
3National Institute of Infectious Diseases. Japan

20NonheatedHeat-treated temperature (℃)

Heat-treated temperature (℃)

800

Mean

40**

% of S. anginosus to s-HA

2060

80% of S. salivarius to s-HA

4080100****% of S. anginosus to HA

800

Mean

**

% of S. salivarius to HA

120060

**
**

100**
**

**

**
% of S. mitis to s-HA

080

% of S. sanguis to s-HA

Mean

**

% of S. sanguis to HATadayoshi ARAKAWA , Tsutomu ISHIZAKI , Roslyn E. HAYMAN ,23Nobuhiro HANADA and Hidenobu SENPUKU% of S. sobrinus to s-HA

1

**

100
8060
4020100

S. anginosus ATCC33397

**

**

**

**

S. salivarius ATCC9759
**

**

**
**

S. mitis ATCC6249% of S. mitis to HA**% of S. mutans to s-HA

1

S. sobrinus 6715

**

s-HA

1

S. mutans MT8148

800

200

Nonheated

Heat-treated temperature (℃)

Fig. 1 Adsorption Percent of bacteria to s-HA and HA heated at different temperatures[% bacterial adsorption = (O.D.550 before assay - O.D.550 after assay) / O.D.550 before assay × 100]

［Bacteria］(*：Professional Mechanical Tooth Cleaning)

80
*

60
40200.20.5

800℃

200℃

80060
40
200.05 0.1

0.20.500.05 0.1

The amount of addition of Ca ion (mM)
1200℃

Non-heated**000 0.05 0.1

100% of S. mutans to HA

20% of S. mutans to s-HA

40

800℃

200℃

0.20.5

80

1200℃

800℃

200℃

*60

*

402000

The amount of addition of Mg ion (mM)

Non-heated*

Non-heated

0.05 0.10.2
The amount of addition of Mg ion (mM)
1200℃

800℃

200℃

0.5Non-heatedFig. 2 The influence of Ca2+ on the adsorption of S. mutans to s-HA and
HA in adsorption assay

Fig. 3 The influence of Mg2+ on the adsorption of S. mutans to s-HA and
HA in adsorption assay（ Significant difference compared with the value of 0 mM ，* ： P<0.05 ）（ Significant difference compared with the value of 0 mM ，* ： P<0.05 ）【% bacterial adsorption =(O.D.550 before assay - O.D.550 after assay) ／ O.D.550 before assay ×100 】【% bacterial adsorption =(O.D.550 before assay - O.D.550 after assay) ／ O.D.550 before assay ×100 】

200℃-heated (scfHA)[Function of drug-retainer］①②Drug-retainer

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

① Antibacterial agent is placed in drugretainer.
② Drug-retainer with antibacterial agent
is fitted over tooth surfaces.

< Materials and Methods >[1. Adsorption assay]
Streptococcus mutans were mixed with HA or saliva-coated HA (s-HA) in PBS, with and without addition of divalent metal
ions Ca2+ and Mg2+ respectively, and incubated for 90 min 37℃. After standing of mixtures at room temperature, thedifferences in optical density (O.D.550) of the supernatants before and after addition of HA or s-HA were analyzed to assessbacterial adsorption levels.
[2. Clinical trial]
The clinical trial was performed by 25 subjects, and they were divided into 3 groups : scfHA paste (scfHA content 25%, 5subjects), scfHA paste (38%, 15 subjects) and placebo paste (dicalcium phosphate dihydrate;DCPD, content 38%, 5 subjects)
group. All subjects were healthy and the mean age of them was 33.0±6.7 years. Paste treatment consists of drug-retainerthat fit onto teeth and deliver small amounts of paste. Paste treatment was performed to the subjects 5 minutes after toothbrushing following lunch everyday for 1 week. Oral bacteria numbers were determined quantitatively by culture techniquesin saliva collected once a week before and after the application.

1200℃-heated (lcfHA)(ｘ 60,000)[Qualitative and quantitative analyses of oral bacteria]
Whole saliva was collected by biting paraffin gum for 5 minutes, and the followingnumbers of oral bacteria in the saliva were measured.
・Total streptococci
・Mutans streptococci
・MS ratio (%)

(A)

(B)

(C)

4

4

10subjects

subjects

3ABCDE2
1

1

Period of scfHA paste treatment

To obtain and enumerate total bacterial count, Mitis-salivarius agar (MTS) plates
was used. For detection and counting of mutans streptococci colony, modified MTSB
agar (MMTSB) plate was used MMTSB contains 20% sucrose, 2μg/ml gramicidine,
10μg/ml nalidixic acid, 10μg/ml colistin sulfate and 2 mg/ml yeast extract. Afteranaerobic incubation for 48 hours at 37℃, the colony-forming units (CFUs) werecounted. S. mutans and S. sobrinus were identified by their characteristic appearance.

FGHIJ

2
12

345KLMNO

64
20

000

(MS ratio = colony number of mutans streptococci / total streptococci×100)

3subjects

8MS ratio (%)

800℃-heated (mcfHA)

Antibacterial agent③① Even after biofilm is physically removed byP.M.T.C., bacteria remain on tooth surface.
② Drug-retainer containing antibacterial agent
is used to eliminate remaining bacteria.
③ Bacteria are completely removed.

*1200℃

Non-heated (scfHA)②Drug-retainer
Antibacterial agent

60MS ratio (%)

ToothScalarBacteria*0MS ratio (%)①*The amount of addition of Ca ion (mM)

Dental Drug Delivery System (3DS)[Model of bacteria removal by the combined useof P.M.T.C.* and 3DS］*

80

［Different forms of HA prepared by heating at different temperatures］

100

100

100% of S. mutans to HA

Streptococcus mutans MT8148〃sobrinus 6715〃
sanguis ATCC 10556〃mitis ATCC 6249〃
salivarius ATCC 9759〃
anginosus ATCC 33397% of S. mutans to s-HA

< Aim of study >Hydroxyapatite (HA) is a component of biological hard tissue, including tooth enamel. Small crystal forms of
HA (scfHA) shows stronger adsorption to all streptococci than moderate (mcfHA) and large crystal forms(lcfHA). We examined the potential effectiveness of scfHA as an agent for removing mutans streptococci fromthe oral cavity in a Dental Drug Delivery System (3DS) employing a drug-retainer formed to fit on
individual dentition. First, bacterial adsorption to mcfHA and lcfHA formed by heating at 800 and 1200℃
respectively, and to two types of scfHA formed at 0 and 200℃ respectively was investigated in human saliva.
Second, clinical trials of using scfHA and placebo pastes were performed in 25 human subjects.

01

Period of placebo paste treatment

Weeks

23Weeks4501

Period of scfHA paste treatment

235Pastes

Percentages of subjects in which MS
ratio reduced after the treatment

A : scfHA 38%

8/15 (53.3%)

B : DCPD 38%

0/5 (0%)C : scfHA 25%

0/5 (0%)Weeks

Fig. 4 The changes of MS ratio by the application of 3 pastes in clinical trials
A：scfHA content 38% (Typical data in decreasing of MS ratio)
B：DCPD content 38% (Placebo)
C：scfHA content 25%

4

Table 1 Effects of 3 pastes to mutans streptococci[Time schedule of clinical trial]< Results and Discussion >1

34Control period

5Application oftest paste

14 (weeks)

6
The check of changeof the number of oral bacteria[Application of scfHA paste]

Drug-retainer with scfHA paste

Fit a drug-retainer

・Two types of scfHA formed at 0 and 200℃ respectively showed the strong adsorption to all streptococci.
・Bacterial adsorption to s-scfHA was reduced by addition of Ca2+ in a dose-dependent manner, while Mg2+ showed no effectto the adsorption.
・The positive effect of 3DS with scfHA paste was supposed in removing of mutans streptococci from oral cavity.
・The removing effect to mutans streptococci of scfHA paste may be up-regulated in subject pre-treated by P.M.T.C..

< Conclusion >・ScfHA paste is an effective agent for removing mutans streptococci from the oral cavity in 3DS using drugretainer that keeps the agent saliva-free, because saliva may disturb the bacterial adsorption effect of scfHA.