1061

Hyaluronic Acid in Toothpaste Enhances the Remineralization
Effect of Nano-hydroxyapatite
M.OBUKI, R.TAKIKAWA and R.HAYMANSANGI Co., Ltd.Tokyo, Japan

Objectives
Hyaluronic acid, more correctly known as hyaluronan (HY), is a non-sulfated glycosaminoglycan found in human tissues, chiefly as an extracellular matrix
component and lubricant in the skin, cartilage and synovial fluid.
Although often used in dentifrice as a wetting agent, it is known to bind with hydroxyapatite, with which it is frequently combined for use in biomimetic
scaffolds for bone regeneration. We conducted a study to determine whether it would affect, in a toothpaste, the functioning of the remineralizing ingredientnano-hydroxyapatite (nHAP), both as regards nHAP’s remineralization effect on artificial subsurface lesions, and its restoration of damageed enamel rods.

Materials and Methods
1. Investigation of Subsurface Remineralization Effect
1-1. Preparation of artificial subsurface demineralized lesions
Sample blocks (8mm x 10mm x 3mm) cut from bovine tooth enamel were polished under running water, using increasingly fine waterproof abrasive papers.
The mirror polished enamel samples were then coated with nail varnish to leave just a 5x5mm window exposed, and immersed in demineralizing solution(0.1M sodium lactate, 0.1M lactic acid, 3.0mM CaCl2, 1.8mM KH2PO4, 1.0% CMC, pH4.8) for 4 days at 37°C, to prepare the artificial subsurface lesions,after which half of the window was further masked with nail varnish, for use as a control.
1-2. Immersion in treatment solution
Four types of test dentifrice were used: (a) nHAP dentifrice without HY and three nHAP dentifrices with the addition of different concentrations of HY as
sodium hyaluronate, namely (b) HY-0.01%, (c) HY-0.02% and (d) HY-0.2%. The treatment solutions for each group were prepared using 40ml of distilledwater mixed with 25g of the respective dentifrice. Sample blocks (3 for each group) were immersed in the treatment solution for 12 days at 37°C, after which
they were rinsed and the nail varnish removed using acetone.
1-3.Preparation of parallel thin sections
For observation, sample enamel blocks were then sliced to 300-500μm thickness parallel to the tooth axis using a low-speed cutting machine(ISOMET,BUEHLER)(5 sliced sections per group). Sliced sections were then polished by micro grinding machine (MG-400 CS, EXAKT), under runningwater, using increasingly fine waterproof abrasive paper, to a thickness of approx. 100μm, and the sample thickness confirmed by measurement with a linear
gauge (LG-130H, MITSUTOYO).
1-4. Contact microradiography (CMR)
Contact microradiographs were taken using a soft X-ray generator (CMR-3, SOFTEX), under the conditions 14kV, 2mA, exposure time 10 min, using X-ray
films (Konica Minolta High Precision Plate). Thin sections were photographed in conjunction with an internal standard aluminium step wedge (Al-SW, 11μm
thick, 29 steps), followed by film development.
The resulting radiographic images were subjected to computer analysis software (Image-pro Plus, Nippon Roper), using the Al-SW as standard to assess theamount of mineral contained according to the method of Angmar et al1). The mineral loss value ΔZ（% volume mineral ･μm）of the control and treatmentareas on each image was then calculated according to the method of Damato et al2), and the remineralization rate calculated from the following equation.
Remineralization rate(%)={(control area ΔZ-treatment area ΔZ)/control area ΔZ} ×100
Statistical analysis was performed using Student’s t-test. (p<0.05)

2.Observation of Restoration of Enamel Rods
2-1. Preparation of tooth specimens
Four sample blocks（8mm × 8mm × 3mm） cut from bovine tooth enamel were polished, under running water, as in 1-1 above , then treated with an etching
agent (Clearfil, Kuraray-Noritake Dental) for 30 seconds.
2-2. Immersion in treatment solution
Three of the etched samples were immersed, respectively, in one of the treatment solutions described in 1-2 above (treatment solutions (a), (b) and (d)), for 5days at 37°C, and the fourth etched sample was preserved in water for use as control.
2-3. Scanning（electron microscope (SEM) observation
After immersion, samples were dehydrated in ethanol, subjected to t-butylalcohol replacement, freeze-dried, sputter-coated with Pt-Pd and observed by SEM(S-4500, Hitachi).

Results
The analysis of CMR images showed remineralization of the artificial subsurface lesions in all samples treated with the test dentifrices, but not in controls.
Compared with the percentage remineralization seen in the nHAP dentifrice group without addition of HY, each of the dentifrice groups with addition of HY(0.01%, 0.02%, and 0.2% sodium hyaluronate respectively) showed significantly higher remineralization (at 1.51, 1.52 and 1.47 times higher respectively,t-test, p<0.05). However the degree of remineralization promotion by HY was not concentration-dependent.
On SEM observation, all three etched samples treated with dentifrice showed recrystallization and realignment of the enamel rods. But no difference inrestoration of the rods was observed between the different dentifrices, including between those with different concentrations of HY.
2-b)

2-a)
※

50

control

treatment

control

treatment※

remineralization rate（ %）※40nHAP without HY302-d)

2-c)

20

nHAP with HY-0.01%

control

control

treatment

treatment

10※p<0.05

0nHAP

HY0.01

HY0.02

HY0.2

Fig.1 Remineralization rate of enamel subsurface lesions

Fig.2

CMR images of artificial subsurface lesions after treatment with each dentifrice.

after treatment with dentifrice.

3-1)

Untreated etched enamel surface

nHAP with HY-0.2%

nHAP with HY-0.02%

n=5(left side control , right side treatment )

3-2)

3-3)

nHAP without HY

3-4)

nHAP with HY-0.01%

nHAP with HY-0.2%

Fig.3 SEM observation of the etched enamel surface before and after treatment with each dentifrice.

Conclusion
Though further study is required to elucidate the mechanism, the addition of HY to the nHAP dentifrice, even at low concentration, significantly enhanced
nHAP’s subsurface remineralization effect. However within the limitations of this study, no additional restoration or realignment of the post-etchingenamel rods, beyond that observed for the sample treated with nHAP dentifrice without HY, was evident in the samples treated with dentifrices containing
different concentrations of HY.
References
1) Angmar B, Carlstrom D. and Glas J.E. Studies on the ultrastructure of dental enamel. IV. The mineralization of normal human enamel. J Ultrastructure Research 8, 12-23 (1963)
2) Damato F.A., Strang R. and Stephen K.W. Effect of fluoride concentration on remineralization of carious enamel: an in vitro ph-cycling study. Caries Res. 1990; 24(3) : 174-180

94th General Session & Exhibition of the IADR/ June, 2016

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