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Two New Methods for Evaluation of Subsurface Enamel Lesions
11112222K. FUJITA , H. KAWAMATA , T. ISHIZAKI , R.E. HAYMAN , T. UCHIYAMA , M. KIMURA , H. KIBA , and T. IKEMI
12SANGl Co., Ltd, Saitama, Japan, Nihon University School of Dentistry at Matsudo, Chiba, Japan

Introduction

When investigating the remineralization of incipient caries, whether its mechanism or status, it is important clearly
detect the border between sound and demineralized enamel at the base of the subsurface lesion. This border
cannot be clarified by Contact Micro Radiogram (CMR), the test method commonly used in vitro experiments
because in CMR, sample thickness is a factor of data measurement. CMR is also time-consuming, and requires
advanced technical, skill. We sought a simpler, more effective way to assess remineralization and found that theborder between sound and demineralized enamel at the base of the subsurface lesion could be more clearlyobserved when using either (A) a dye-permeation technique or (B) scanning electron microscopy (SEM) ofsamples pretreated with argon-etching.

The subsurface demineralized lesion(control)Fig-2 stereomicroscopy observation of thesubsurface demineralized lesion (control)
and remineralized phase

Objective
To compare the ability of CMR and two previously unreported observation methods to clarify the border between soundand demineralized enamel at the base of the subsurface lesion, namely (A) a dye-permeation technique (which forcomparative purposes we then visualized by microscopy) and (B) SEM using sample pretreated with argon-etching.

The subsurface demineralizedlesion

remineralizedFig-6 CMR observation of the subsurface
demineralized lesion (control) andremineralized lesion

ResultMaterials and Methods(1) Preparation of artificial subsurface lesion
Samples were prepared by common methods of immersing extracted human molars in a lactic acid buffer solution(3.0mM-Ca, 1.8mM-P, pH 4.5) for 7days at 37℃, to create a subsurface demineralized lesion.
(2) Preparation of remineralized lesion
Half of demineralized lesion was coated with nail enamel as control, and the sample then immersed in a remineralizing
solution containing hydroxyapatite for 24 hours at 37℃.
(3) Observation of the border between sound and demineralized enamel at the base of the subsurface lesion.
(A) By dye-permeation technique(a) Sample preparation
An extracted human molar with subsurface demineralized lesion and remineralized lesion was embedded in resin(RigolacTM) after dehydrated in ethanol and degreased in acetone. The embedded molar was cross-sectioned
perpendicular to the tooth axis using a diamond disk to obtain parallel sections, and the section was fine polished to athickness of with 90μm Imperial TM Lapping Film (3M). The mirror-faced parallel sections were then dehydrated inethanol, immersed in acid-red dye for 1minute, and rinsed with water.
(b) Observation of dye-stained samples
The border between sound and demineralized enamel at the base of the subsurface lesion for both the demineralized(control) and remineralized portions of the dye-stained samples was clearly visible by stereomicroscope (OLYMPUS,BX60). The image was incorporated by digital video camera(OLYMPUS,3CCD COLOR VIDEO CAMERA KYF55MD).
(B) By SEM after argon-etching pretreatment(a) Preparation of sample extracted human molar was cut into parallel sections as described in (A)(a). The sections werefine polished to a thickness of 300μm with Imperial TM Lapping Film (3M) then dehydration in ethanol, subject to tbutylalchol replacement and freeze-dry. The surface of fine polished parallel sections was etched flat using anirradiation argon-etching device (HITACHI, E-3200 Flat Milling Device)(Fig-1), under conditions of 3KV, 1mA for1hour.
(b) Observation of samples pretreated with argon-etching
Argon-etched parallel sections were coated with Pt-Pd, and the, and the border between sound enamel and thesubsurface demineralized or remineralized phase examined by SEM (HITACHI, S4500).
(C) By CMR(a) Sample preparation
As in (A)(a),an extracted human molar was cut into parallel sections, and fine polished to a thickness to 100μm with
ImperialTM Lapping Film (3M ).
(b) Observation of samples
Super soft X-ray images were taken on SO-181 film (KODAK) with super soft X-ray supply (Softex Co. Type CMR-3)
under the condition of 12V, 2.5mA for 10minutes and images on the films observed.

remineralized phaseFig-1 HITACHI E-3200 FLAT MILLING DEVICE

When the base of the subsurface lesion was examined by stereomicroscopy for samples prepared by the dyepermeation technique (Fig-2), on the control side of the sample the border between sound and demineralized
enamel was clearly observed. On the remineralized side of the sample, a layer of palely stained tissue wasobserved along the border between sound and demineralized enamel, suggesting that remineralization that had
taken place there.
Samples pretreated with argon-etching, when observed by SEM (Fig-3), were free of the 'smear layer '(Fig-4) ofabrasion debris usually detected on enamel samples observed by SEM. The border between sound anddemineralized enamel at the base of the subsurface lesion was clearly detectable, and the status ofremineralization, could be clearly observed.

The subsurface demineralized lesion(control)

remineralized phase

The sound enamelsound enamel

Fig-3 SEM observation of the subsurface lesion (control) andremineralized phase

When the subsurface demineralized layer was observed at high magnification (Fig-5), its enamel crystals wererough in appearance compared with those of' sound enamel1, and the enamel rods (prisma adamantinum) wereclearly visible. Gaps in the crystals of the enamel rods made the rods appear porous, and gaps were also observed
between the sheaths of enamel rods.
When observed by CMR (Fig-6), the border between sound and demineralized enamel at the base of thesubsurface lesion was not clear. We believe this is because images obtained by CMR reflect all information up to athickness of 100〜120μm.

Conclusion
Detailed examination of incipient caries lesions by the new methods proposed in this study, namely (A) dyepermeation and (B) SEM of samples pretreated with argon-etching, showed that both methods clearly detect theborder between sound and demineralized enamel at the base of the subsurface lesion.
Moreover, the base of the subsurface lesion was lower en the control side of the samples than on theremineralized side, indicating that the remineralization solution used in our study permeated satisfactorily to thebottom of the subsurface lesion. On the demineralized (control) side of the samples, gaps between the sheaths ofenamel rods and among the crystals of the enamel rods themselves could be clearly observed.
We concluded that the two new methods proposed in this study may be useful tools for studying the mechanism ofdemineralization.

Fig-4 SEM observation ofthe smear layer

Fig-5 SEM observation of the subsurface
demineralized layer