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Microhardness Testing to Evaluate Remineralization of Tooth Enamel
1112T.FUJIMARU , T.ISHIZAKI , R.E.HAYMAN , and K.NEMOTO
12SANGI Co., Ltd. Saitama, Japan, Nihon University School of Dentistry at Matsudo, Chiba, JapanINTRODUCTION

Lesion morphology (qualitative assessment) :The pattern of indentations observed in microhardness testing indicated that the enamel was hardest in control areas andin the immediate surface and deeper sound enamel surrounding the artificial subsurface lesion, falling to its lowest level ofhardness within the body of the artificial lesion. This pattern of mineral distribution, or lesion morphology, resembled thatobserved for the same samples by CMR.

CMR is a time-consuming indirect method of observing remineralization. It is inefficient, requiring advanced techniquesof sample preparation, and also subject to error, as it evaluates changes in mineral content on the basis of aradiographic image. We considered that the physical characteristic of tooth hardness may be a potentially valuable
indicator of subsurface changes in the mineral density of tooth enamel, both at the clinical and laboratory level.

Area of ΔH

300250

ΔH200150
100Hardness of sound enamel

50Hardness change0MATERIALS AND METHODSRemineralization(%) =050

100150200250

ΔHc - ΔHe×100ΔHc

ΔHc: Area of control layer
ΔHe: Area of remineralized layer300

Depth from Surface (μm)

Sample Preparation :Observation & Calculation of Remineralization :100400350
300
250200150
100

Remineralized layer

50Contorol layer0(1) Artificial subsurface lesions were formed on extracted human molars using a demineralizing solution of 0.1M-lactic acidbuffer, 3.0mM-Ca, and 1.8mM-P, at pH4.5. Half the lesion area was then masked with resin as a control. (2) One sample each
was subjected to remineralization by immersion for 72 hours below 37℃ respectively in (a) artificial saliva (20mM-HEPES,3.0mM-CaCl2, 1.8mM-KH2PO4, 150mM-NaCl) or (b) artificial saliva containing 250ppm fluoride (NaF). (3) The lesion area ineach case was then reinforced with photopolymerized resin, and a horizontal section cut and polished for hardness testing.
(4) A thin section from the same lesion area of each tooth was also prepared for examination by CMR. (5) A further section
from each sample was prepared for SEM observation, using argon ion etching as a surface pretreatment to ensure that theshape of each lesion could be distinctly observed.

Change in mineral density
due to remineralization - CMR -Change in hardness due to remineralization- Knoop Mineral volume percentages (vol%)The purpose of the study was to test the value of tooth hardness as a measure of enamel remineralization by comparing dataon changes in tooth enamel obtained from both microhardness testing and CMR. SEM observation was also used to evaluate
the microhardness testing method.

350

Knoop Hardness (Kg/mm2)

PURPOSE

Knoop Hardness (Kg/mm2)

400050100150200250300806040
Remineralized layer

20Contorol layer

0050

100150200250

Depth from Surface (μm)

Depth from Surface (μm)

RESULTS
Microhardness test indentations :Remineralization (quantitative assessment) :SEM observation before and after microhardness testing showed that the test indentation marks differed insize and shape between sound areas of surface and deep enamel and the demineralized area in the body ofthe subsurface lesion. Indentation marks were smallest and tended to be linear in areas of sound enamel but
increased progressively in both width and breadth, showing an increasingly diamond-like shape, towards thebody of the demineralized area.

Microhardness testing showed that the hardness of sound enamel was 350Kg/mm2. Remineralization for the two samples,
according to the remineralizing solution used, was 6.21% (for artificial saliva) and 11.24% (artificial saliva plus fluoride)measured by microhardness testing and 10.79% (artificial saliva) and 14.74% (artificial saliva plus fluoride) measured by CMR.

Rate of remineralization according to treatment used

Microhardness Testing: Hardness was tested at 20μm intervals on the section surface in the direction of the depth of theenamel, using a Microhardness tester, and Knoop hardness calculated by digital image processing. Remineralization wascalculated by comparing the hardness of the remineralized and control layers with that of adjacent sound enamel. First thedifference in enamel hardness at 20μm intervals on the section surface was plotted and the area between this line and aline showing the hardness of sound enamel was calculated and described as ΔH. Remineralization was expressed as thedifference in ΔH between the remineralized and control areas, shown in percentage terms.
CMR: A thin section from the same lesion area of each tooth was examined by CMR and remineralization calculated by thestandard method of computerized image analysis.
SEM: Sections prepared for SEM were examined using a SEM (HITACHI S-4500), then submitted to microhardness testing
and reexamined to assess the appearance of the artificial subsurface lesions before and after the hardness test.

14.74

Remineralization(%)

151211.24

10.79

9CMRHardness testing

6.21

6050

100

150

200250

Fluoride Concentration(ppm)

Subsurface lesions
Length of Indentation

CONCLUSION
20μm
Sound enamel layer

SHIMADZU Microhardness Tester

Image of hardness testing

BeforeAfter
SEM Image of hardness testing

Results from both microhardness testing and CMR showed a similar tendency as regards lesion morphology andquantitative remineralization assessment, and both showed higher remineralization when fluoride was added tothe remineralizing solution. We concluded that hardness testing could be a valuable method for evaluating enamel
remineralization and an effective substitute method for CMR. We intend to further study the value of both methods
in greater detail.