1121

Adsorption of Oral Pathogenic Microbes by Small Crystal Hydroxyapatite
1112T. FUJIMARU , T. ISHIZAKI , R. HAYMAN , and H. SENPUKU
12SANGI Co., Ltd., Tokyo, Japan, National Institute of Infectious Diseases, Tokyo, Japan

Objectives

Data and Observation
Table 1. Adsorption rate of periodontal and opportunistic microbes to HA

Small crystal hydroxyapatite (scfHA), developed for use in a dental drug delivery system (3DS), has beenshown to adsorb cariogenic bacteria in the oral cavity. By radioisotopic liquid scintillation counting, weevaluated its capacity to adsorb microbes associated with caries, periodontitis, and oral opportunistic infection,
both individually and in two-species solutions, and examined the effect on this adsorption of both saliva andthe bacteria-adsorbing salivary agglutinin peptide SRCRP2 (scavenger receptor cystein-rich domain peptide 2).

HA

Rate of adsorption (%)

100

Materials and Methods
Preparation of saliva or SRCRP 2-coated HA and non-coated HA
The HA preparations used in this study were synthesized by SANGI Co., Ltd. 20mg of scfHA was equilibrated
with phosphate buffer saline (PBS) and soaked in 1.5ml of whole saliva or 10 or 100 μg/ml SRCRP2 (aminoacid sequence: -QGRVEVLYRGSWGTVC-) solutions, then incubated for 60 minutes at room temperatures
and washed twice with PBS remove the unbound components.

s-HA

SRCRP2:10

SRCRP2:100

8068.8 69.1

60

82.1

80.0

75.5

75.5 72.5

StrainLog CFU/ml
ATCC33277
9.5
P. gingivalis
W83
9.6
ATCC25922
8.6
A. actinomycetemcomitans
Y48.4
SC5314
7.4
C. albicans

83.6 84.0

79.4

84.7
71.7 71.773.4

72.2

62.3

70.9

67.0

59.3

Rate of Adsorption (%)
HAs-HA
86.1±6.872.5±9.4
81.9±10.1
24.7±4.4
65.4±4.2
54.7±2.439.1±4.524.3±4.554.9±7.5
51.7±7.3

67.5

40
200

7.2

C. albicans SC5314

A. actinomycetemcomitans Y4

P. gingivalis W83

35.3
22.8
4.2

S. sobrinus
6715

S. mutans
MT8148S. sanguinis
ATCC10556

S. mitis
ATCC6269

S. salivarius
ATCC9759

S. anginosus
ATCC33397

Figure 2. Adsorption of oral streptococci to HA, s-HA or SRCRP2-HAA

S. mutans MT8148

Oral microbe strains and culture conditions
Microbes used were as shown in Figs. 2, 4 and Table 1. All bacteria were cultured in Brain Heart Infusion(BHI) broth at 37℃ under anaerobic conditions. C. albicans SC5314 was grown in Yeast Peptone Dextrose(YPD) broth at 37℃ under aerobic conditions.

BAB

CFigure 5. SEM photographs showing adsorption of oral pathogenic microbes to HA
Assays of oral microbes' adsorption to HA
Oral streptococci were cultured at 37℃ for 24h in BHI broth containing [methyl-3H] thymidine at final
concentration of 2μCi/ ml. After removing broth, the [3H]-labeled bacteria were washed 2 times with PBS.
Bacterial suspensions were adjusted to OD 1.0 at 550nm. Non-treated scfHA (HA), saliva-treated scfHA (sHA) or SRCRP2-treated scfHA (SRCRP2-HA) respectively were suspended in 1.5 ml [3H]-labeled bacterial
solutions which were then incubated with shaking for 90 minutes at 37℃. The amount of adsorbed bacteria
was determined with a liquid scintillation counter and the rate of bacterial adsorption was calculated. (Fig. 1)
The same assay was repeated for HA and s-HA using suspensions of P. gingivalis, A. actinomycetemcomitans
and C. albicans respectively. Similar adsorption assays were also performed using HA and mixed microbialsolutions, one series containing S. mutans MT8148, S. sanguinis ATCC10556 or S. mitis ATCC6249
respectively with [3H]-labeled C. albicans SC5314, and another series containing C. albicans SC5314 with
[3H]-labeled forms of the respective streptococci.

Figure 3. SEM photographs showing adsorption of S. mutans MT8148 to HA

A

50
40

56.3 57.1

C
35.3

20

20.5 21.6

24.4

2.7

0H-labeled microbes

58.0

30

10

Specified quantity

62.3

ATCC25175

20.7
12.5

9.4

9.9

1.2

GS5

UA159

70
60
50

64.2

65.9

58.8

61.1

40
30
20

B

70
77.2
60
50

80

62.1

20

S. mutans MT8148

coexistence

65.5

300single

68.5

40090

coexistence

80

10

C. albicans SC5314

single90

10C. albicans SC5314

S. sanguinis ATCC10556

D70
74.7

60
50
40

67.7

63.0
55.8

30
20
100MT8148

3coexistence

Adsorption rate by HA (%)

60

68.8 69.1

Adsorption rate by HA (%)

Rate of adsorption (%)

Suspensions of microbes(OD550 = 1.0)

HAs-HA
SRCRP2:10
SRCRP2:100

single80
Adsorption rate by HA (%)

80
7090

C. albicans SC5314

S. mitis ATCC6269

Figure 4. Adsorption of S. mutans strains to HA, s-HA or SRCRP2-HA

Figure 6. Adsorption ability of HA under C. albicans and streptococci coexistence

1. Adsorption of oral streptococci to HA, s-HA or SRCRP2-HA
The adsorption of HA, s-HA or SRCRP2-HA to various streptococci strains was compared. HA showedsignificant adsorption to all the tested oral streptococci. However, adsorption was inhibited by salivary
treatment of HA. On the other hand, adsorption of HA to S. mutans MT8148 was slightly increased bytreatment of HA with SRCRP2 (Fig. 2). Adsorption of HA particles on S. mutans was observed by SEM(Fig. 3A). HA clusters including bacteria were also observed (Fig. 3B). Adsorption activities of HA toother S. mutans strains were also inhibited by treatment of HA with whole saliva but not increased bytreatment with SRCRP2 (Fig. 4).

3. Adsorption ability of HA under C. albicans and oral streptococci coexistence
Adsorption of HA under conditions of two-species co-existence was investigated. C. albicans showedsimilar HA adsorbance in co-existence with S. mutans, S. sanguinis or S. mitis as in single culture (Fig. 6).
Furthermore, each streptococci showed a similar level of HA adsorbance in co-existence with C. albicans
as in single culture. In co-existence culture of S. mutans and C. albicans, SEM observation showedadsorbance of HA around the C. albicans cell surface which was not observed in S. mutans (Fig. 6D).

2. Adsorption of periodontal and opportunistic microbes to HA and s-HA
Adsorption to HA or s-HA was compared in periodontal bacteria (P. gingivalis andA. actinomycetemcomitans) strains and C. albicans strain. HA showed significant adsorption to all thetested microbes. Adsorption of HA to P. gingivalis W83 was remarkably inhibited by treatment of HAwith whole saliva, but the inhibition levels in other bacteria were slight (Table 1). In contrast, adsorption
to C. albicans was not inhibited by salivary treatment of HA (Table 1). Adsorption of HA particles tobacteria and C. albicans was observed by SEM (Fig. 5).

ScfHA is a powerful agent for adsorbing oral pathogenic strains in conditions without whole saliva.
Moreover, scfHA shows the same levels of adsorption to individual microbe species in co-existence
cultures of C. albicans and S. mutans, S. sanguinis or S. mitis, as in single cultures.
Therefore scfHA may be a useful material for removing oral microbes, especially if a drug-retainer
system that avoids the influence of saliva is used on the tooth surface.

Mixture withscintillation cocktail
Radioactivity measurement
andcalculation of adsorption rate

HA etc.
Specified quantity
Specified quantity

Shaking
90min at 37℃

PBS washingand sample preparation

Figure 1. Assay of microbe adsorption ability of HA

SEM observation of HA adsorption in mixed microbial suspensions
The adsorption of oral pathogenic microbes to HA in mixed suspensions was observed using SEM
photographs. The specimens were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde for 30 minutes,
then washed 3 times with PBS, re-fixed with 1% osmium tetraoxide for 20 minutes, and re-washed 3 times
with PBS. The samples were dehydrated using increasing concentrations of ethanol (50% 〜 100%) andcritical-point-dried with liquid CO2.

Results

Conclusion

85th General Session & Exhibition of the IADR/ March, 2007
E-mail : fujimaru@sangi-j.co.jp