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Storage media to preserve dentin and their effects on surface
properties
Shamim Sultana, BDS,a Toru Nikaido, DDS, PhD,a Md Asafujjoha, BDS,b Junji Tagami,
DDS, PhD,a,c and Khairul Matin, BDS, PhDa,caCariology and Operative Dentistry, Department of Restorative Sciences, Tokyo Medical and Dentalb
University, Tokyo, Japan, Department of Conservative Dentistry, Bangabandhu Sheikh Mujib Medicalc
University, Dhaka, Bangladesh, and Center of Excellence (COE) Program for Frontier Research on
Molecular Destruction and Reconstruction of Tooth and Bone at Tokyo Medical and Dental University,
Tokyo, Japan

Purpose: This study assessed the probable changes in surface properties of dentin preserved in four different
storage media: 1) deionized water (Milli-Q), 2) sodium chloride buffered saline solution (NaCl), 3) Hank’sbalanced salts solution (HBSS), and 4) phosphate buffered saline solution (PBS).
Materials and Methods: Equal sized dentin beams were prepared from bovine incisors and stored in the above
mentioned storage media for different time periods (1, 7, and 30 days) which then subjected to nanoindentation
measurements for hardness and elasticity. Surface roughness (Ra) of the samples was analyzed by a confocal
laser microscope. Data were statistically analyzed by one-way ANOVA and Dunnet T3 tests.
Results: Nanoindentation showed that storing dentin in Milli-Q or NaCl media resulted in large decreases in
hardness and elasticity, 14% and 18%, respectively by 1 day. After 7 days due to demineralization processes inMilli-Q and NaCl media, hardness and elasticity decreased about 27% and 31%, respectively relative to thestarting values which were significantly lower than samples stored in the other two media. In HBSS and PBS,
there were no significant alterations in mechanical properties during the 30-day long storage. Ra values of all
stored samples were more or less the same as control samples except for samples stored in PBS for 30 days, inwhich the Ra was increased significantly.
Conclusion: PBS is preferred for short term preservation of dentin, as opposed to NaCl or Milli-Q, to avoid
alterations in the mechanical properties of dentin surfaces. (Int Chin J Dent 2006; 6: 123-129.)
Key Words: dentin, hardness, modulus of elasticity, storage media, surface roughness.

Introduction
Dentin is a hard mineralized tissue whose function is to provide elastic support to the overlying hard enamel,thus enabling the dispersion of mechanical stresses applied during mastication.1 Dentin consists of an organic
matrix made up primarily of collagen and an inorganic phase made up of apatite crystals.2 The apatite phase
contributes to most of the compressive strength that might directly effect hardness, while the collagen phaseprovides elasticity.3 Changes in these two phases might affect alterations in the physical properties; hardness
and modulus of elasticity of these biological composites. The mechanical properties of dentin are largely
determined by the intertubular dentin matrix, which is a complex composite of type I collagen fibers and acarbonate-rich apatite mineral phase.4 By using a nanoindentation tester, the modulus of elasticity which
represents the elastic properties of dentin could be determined.5,6Maintaining the mechanical properties of tooth substrates is important during in vitro manipulations as well as
being important for clinical tooth preparations. The mechanical property concerns regarding the handling ofteeth for research purposes have prompted investigators to evaluate the effects of storage in various storage
media on hard tooth specimens as well as extracted teeth during transportation. An aqueous solution is essential
for maintaining the hydration of the tooth samples prepared for mechanical testing. The mechanical propertiesof calcified tissues are also dependent on their mineral content.7 For in vitro studies, primarily nanomechanicaltesting, bond strength tests, durability studies, caries research and oral biofilm research, storage of samples canbecome a critical issue since chemical reactions such as etching and dissolution affect the surface layer. The

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hardness of enamel and dentin is associated with mineral loss. Obviously, demineralization decreases hardness
and remineralization usually restores hardness by deposition of mineral particles.8-11
In recent years, nanoindentation has become an excellent tool for the determination of the local mechanicalproperties of structural features of biological hard tissues.12-15

This depth-sensing technique allows theperformance of nanomechanical testing, including those for nanohardness and elasticity, of dentin to minimize
artifacts caused by processing during sample preparation. There are, however, varying reports on the effects ofstorage media on the mechanical properties of dental hard tissues.16 In the present laboratory oriented study,
four different storage media: 1) deionized water (Milli-Q), 2) sodium chloride buffered saline solution (NaCl), 3)
Hank’s balanced salts solution (HBSS) and 4) phosphate buffered saline solution (PBS) were assessed using
bovine dentin.

Previously, HBSS (which is used as a control medium in the present study) has been

recommended for short-term preservation in order to maintain the physical properties of human dental tissues.16
Therefore, this study was designed to investigate the changes in nanomechanical properties of the dentin
preserved in the above mentioned storage media using a nanohardness tester and followed with testing forsurface roughness.

Materials and Methods
Specimen Preparation
Approximately 1 mm thick and 2 mm wide beam shaped specimens were prepared from the mid-labial portionof twenty sound bovine incisors with a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA). Fourequal sized beams were obtained from each tooth as shown in Fig. 1. The specimens were then flattened using a
low-speed polisher (Buehler) with silicon carbide paper under running water using grit sizes 1,000, 1,200, 1,500
and polished with water based diamond paste to 0.25 µm and ultrasonically cleaned for 15 s.

Fig. 1.
Locations of indentation imprints are displayed in the
diagrams; E, D and DEJ indicate enamel, dentin anddentino-enamel junction respectively. Four equal sized
beams were obtained from mid-labial part of each tooth(two beams from each half). Indentations were appliedhorizontally and approximately 100 µm inside from DEJ
shown in the figure. SEM view of a typical indentation
imprint on a control dentin surface is shown on the right.
Indentation was programmed and set to place on adentin cross section where dentinal tubules wereperpendicularly cut with the indenter tip aiming at
intertubular spaces. Sharp and prominent imprint of theindenter tip, walls and edges are visible.

Preservation in storage media
Before storage all specimens were subjected to nanoindentation tests and the baseline measurement was usedas ‘0’ day or control data. The specimens were then stored separately in 4 mL of the following storage media: 1)
NaCl, 2) HBSS, 3) Milli-Q and 4) PBS. Chemical components of the storage media are shown in Table 1. The
pH of all storage media was measured with a pH-meter (pH/ION Meter F-53, Horiba Ltd., Kyoto, Japan) and thesolutions were filtered with Millex-GS 0.22 µm filter units (Millipore Corp., Bedford, MA, USA) before use.
One beam from each tooth was stored in each medium at 4ºC for 1, 7, and 30 days time and were then subjected
to nanoindentation testing to measure hardness and modulus of elasticity.

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Table 1.

Int Chin J Dent 2006; 6: 123-129.

Chemical components of storage media.

Storage media

pH

Composition (mg/L)

Manufacturer

Normal saline

5.9

900 NaCl

Otsuka PharmaceuticalCo., Ltd., Tokyo, Japan

Hank’s balanced
salt solution (HBSS)

8.0

400 KCl, 60 KH 2PO4, 8,000 NaCl, 1,000 glucose,
90 Na2HPO4 7H2O, 350 NaHCO3, 1,400 CaCl2,
100 MgSO4 7H2O, 100 MgCl2 6H2O

Sigma Aldrich Co.,
St. Louis, MO, USA

Milli-Q

6.5

Deionized by water purifier (Millipore)

Japan Millipore Corp.,
Tokyo, Japan

Phosphate bufferedsaline solution (PBS)

7.2-7.4

8,000 NaCl, 1,280 Na2HPO4, 350 NaH2PO4

Wako Pure ChemicalInd., Ltd., Osaka, Japan

Nanoindentation
The nanoindentation tester (ENT-1100, Elionix Co., Tokyo, Japan) used in this study was a depth sensing
computer controlled instrument which has Berkovich indentor, which consists of a three-sided pyramid diamondprobe. Just before testing, the specimen was fixed on a flat metallic dais using utility wax to stabilize thespecimen and to orient the surface parallel to the stage of the nanoindentation tester with the polished surface
facing upwards for indentation. The specimen was kept under dry conditions during measurements since thisdevice can only maintain the temperature in the chamber at 27°C but not the humidity. During indentation, theapplied indenter force load and the depth of penetration into the sample are continuously monitored. An
indentation load of 0.049 N (5 gf) and 10 indents at 30 µm intervals per specimen were applied 100 µm from thedentino-enamel junction (DEJ) towards the pulp at a speed of 4.9×10-6 N/ms (0.5 mgf/ms). Nano-hardness(GPa) and Young’s modulus of elasticity (GPa) were calculated and recorded by an attached computer.
Surface roughness
The specimens stored for 30 days were used for the measurement of surface roughness (Ra, µm) by a confocal
laser scanning microscope and analyzed by a computerized roughness tester (Keyence Corp., VK-8500, Osaka,
Japan). The specimen was fixed on a stage and laser light was applied to visualize the surface. The applied
pitch was 0.05 µm and the scan time was 4 minutes. Data were recorded from four corners and the central partof the selected area for each specimen.
Statistical Analysis
All data were statistically analyzed by one-way ANOVA and Dunnet T3 testing using the Statistical Package
for Medical Science (SPSS for Windows release11.0, SPSS Inc., Chicago, IL, USA). The data were analyzed byrepeated measurement analysis of variance at the 95% confidence level and the two variables assessed were“storage media” and “time period”.

Results
Hardness and modulus of elasticity
The results of this study showed that teeth stored in NaCl or Milli-Q resulted in a large decrease in hardness
and modulus of elasticity (Figs. 2a and 2b). At 1 day, a decrease in the mechanical properties values of up to
14% and 18% was observed in case of NaCl and Milli-Q respectively. After 7 days, due to the demineralization
process during storage in NaCl and Milli-Q solutions, the mechanical properties dropped to 27% and 31%
respectively of their starting values, which were significantly lower than those with HBSS and PBS. In HBSS

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and PBS, there were no significant alterations in the mechanical properties for a time interval of 7 days.

ab

Figs. 2a (left) and 2b (right).
a) Reduced nanohardness of bovine dentin versus time stored in NaCl, Milli-Q, HBSS and PBS. Reduction was not significant
when stored in HBSS or in PBS (SDs are indicated by error bars, n=10). b) Reduced Young’s modulus of bovine dentin versus
time stored in NaCl, Milli-Q, HBSS and PBS. Reduction was not significant when stored in HBSS and PBS (SDs are indicated byerror bars, n=10).

After 30 days, NaCl and Milli-Q showed 47% and 49% loss in hardness and Young’s modulus elasticity. In
the case of Milli-Q, hardness and elasticity were 0.29 GPa and 14 GPa, respectively. Also for NaCl, these values
were 0.31 GPa and 15 GPa. For PBS, it was found that there were no significant changes in hardness andmodulus elasticity after 30 days. Also, in HBSS there were no significant changes in hardness and modulus
elasticity after 30 days. These results indicated that the effects on sample hardness for each storage media weredifferent. The hardness of dentin stored in PBS and HBSS was not decreased following storage up to 30 days(p>0.05). However, with Milli-Q and NaCl hardness was decreased significantly during that same period.
Surface roughness
Surface roughness of the samples after 30 days was mapped by a confocal laser scanning microscope, and thesummarized Ra data (mean and SD) are depicted graphically (Fig. 3). No significant differences could be
detected between the control dentin surface and dentin surfaces stored in HBSS, Milli-Q or NaCl. Some
scattered high and low picks were visible probably because of salt sediments and open dentinal tubules,
respectively. Samples stored in PBS significantly increased in surface roughness compared with all other stored
and control samples with more high picks and crystalline precipitations detected.

Fig. 3.
Graph represents mean Ravalues obtained after
preservation in four storage
media for 30 days
compared with the control(SDs are indicated by error
bars, n=15. Broken bars
indicate significant
differences, p<0.05,
F=2.647).

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Discussion
Storage solutions sometimes are used for preservation of tooth substrates in different biological studies.
Several available storage media either manufactured or prepared in situ with different compositions may affectthe expected results of the experiments. Hence, selection of the storage medium for preservation of the samples
is a very important factor. To date several methods have been employed to investigate demineralization andremineralization of teeth such as hardness tests,8 microradiography,17 contact micrography,18 electron probe
microanalysis (EPMA),19 QLF,20 Diagnodent, and many more in vitro or in vivo assays.

The use ofnanoindentation is not limited to characterization of the mechanical properties of tooth tissues but is also used
for dental materials.

The tooth-colored materials are subjected to evaluation using different International

Standards Organization (ISO) Testing.21
On the other hand, for restorations dentin is acid conditioned and primed and an adhesive agent applied.
Nakabayashi et al.22 reported that resin-impregnation creates a transitional "hybrid" layer, that is neither resin nor
tooth, but a hybrid of the two. The thin layer of resin-reinforced dentin locks the two dissimilar substances
together at the molecular level, sealing the surface against leakage and imparting a high degree of acid resistance.
The ideal thickness of a well constructed hybrid layer has proven to be less than 10-µm, which involves a
submicroscopic undisturbed dentin surface layer. Inappropriate storage could cause damage to this very delicate
submicroscopic tooth structure which can influence the data obtained during mechanical testing.

Hence,

selecting a perfectly matched storage medium with appropriate preservation properties was assessed. In thisstudy bovine teeth were used as an acceptable substitute for human teeth because they are large enough toprovide consistent dentin surfaces of sufficient size even in the deep layers and equal sized beams could be
prepared in different preservation solutions.

Most importantly, controlled base line data in terms of themechanical properties of tooth substrates could also be obtained which is important for a comparative study.
During indentation, the applied indenter force load and the depth of penetration into the sample arecontinuously monitored. If the specimen was affected by the solution, loads on the stored specimens weredecreased to maintain approximately constant indentation depths. In our study the hardness and modulus ofelasticity of dentin were altered within 1 day of storage when preserved in physiological saline (NaCl) andMilli-Q.

After 7 days the hardness and modulus of elasticity of dentin were reduced to 27% and 31%

respectively of their starting values and further decreased to 47% and 49% after 30 days. However, with PBS or
HBSS there were no significant changes of hardness and Young’s modulus of elasticity after 30 days.
Apparently, there was an exchange of minerals or electrolytes during 30 days storage in all media with PBS andHBSS exhibiting minimum variability.
The pH of the NaCl solution was 5.9 and sufficiently acidic for some demineralization to occur. On the other
hand, it lacks calcium and phosphate ions for remineralization. It has been shown that the hardness and Young’s
modulus of elasticity of dentin decreased when the specimens were stored in physiological saline. This ispresumably due to the loss of surface calcium resulting in further exposure of the dentin collagen, which could
have an important effect on the hydrolysis of unprotected collagen fibrils. The pH of fresh Milli-Q usually is 6.5,
which is not acidic enough for surface demineralization. Habelitz et al.16 reported that deionized water lacks
calcium and phosphate ions and therefore the chemical potential for dissolution of the mineral phase of dentin
and enamel is high and is assumed to be the major reason for demineralization and softening of the tissues. Also
the hardness was reduced due to the increased permeability of dentin.

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Our results showed that there were no significant differences in the hardness and Young’s modulus forintertubular dentin when preserved in HBSS. This is consistent with the findings by Habelitz et al.16 The reason
for this is due to the fact that the pH of HBSS is 8.0 which makes it a basic solution. Furthermore, it is highly
concentrated in Ca2+, Mg2+, Na+, PO43- and Cl-. These molar concentrations are sufficient to maintain ionic andmineral exchange between the liquid phase and dentin. This means that surface demineralization is prevented.
On the other hand, there were no significant changes in the mechanical properties of the dentin when preservedin HBSS. It is essential to point out that using HBSS as a preservative solution is also relatively expensive. In
addition, it is relatively unstable when exposed to light for short time periods. PBS is a buffered solution with a
pH of 7.2- 7.4 containing Na+, PO43-, and Cl- ions. This buffer is commonly used in the laboratory especially forbio-medical studies. As a buffer, PBS maintains the electrolytic imbalance in the solution and there was nochange in the pH after 1 month storage.
Carvalho et al.25 reported that long-term storage of EDTA-demineralized human dentin in PBS solution did
not cause any significant reduction in its mechanical properties. The present study indicated that storage of solid
dentin in PBS solution did not cause any significant changes in its mechanical properties. The Ra value wasfound to be significantly high due to crystallization of PBS salts on dentin surfaces. Stationary storage in the
refrigerator for long periods of time may have allowed the PBS salts to crystallize on the dentin surfaces.
Hayakawa et al.26 have found that there was no precipitation after one day immersion in HBSS in a study onto
phosphorylated polymers. After 3 days, the formation of precipitated globules was observed scattered on thephosphorylated polymer surface. After 7 days immersion in HBSS, the phosphorylated polymer surface wascompletely covered with calcium phosphate globules. In our study, there were very negligible precipitations on
dentin while immersed in HBSS. However, comparatively large precipitation globules, rather than salt crystals,
were present.
From the results of surface roughness measurements it can be concluded here that PBS contains sufficientsalts so that during long term preservation the salts were deposited on to the dentin surface. On the other hand,deposition of the salts onto the surfaces may interfere with some results of the experiments. Therefore, thesurface can become rough compared to samples in the other three preservative solutions. On the other hand, thesamples preserved in NaCl and Milli-Q showed smooth surfaces like the control. This may have resulted fromthe sedimentation of the dissolved mineral particles back onto the demineralized dentin surfaces. Opening of thedentinal tubules of these samples resulted in a widened but partially occluded aperture resulting in low Ra values
after laser detection. Therefore, our study suggests that both hardness and Young’s modulus of elasticity werereduced as a result of several media properties including the pH, buffering capacity and the ion content of thestorage media.
Previous investigators have found that formalin storage was effective for infection control purposes.27 It
cannot, however, be recommended as a storage medium for dentin bonding studies due to the alterations in
dentin bond strength resulting from its use.28 Other possible alterations can be due to variations in specimen
wetness, mineralization, microtubule density and orientation.29 The significant influence of various parameters
including the origin of the dentin, types of teeth, tooth storage temperature, maximum storage time of teeth,
dentin depth, as well as the storage of bonded specimens (medium, temperature, and time) need to be further
evaluated.

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Acknowledgment
This work was supported by the Center of Excellence (COE) Program for Frontier Research on Molecular Destruction andReconstruction of Tooth and Bone at Tokyo Medical and Dental University, Tokyo, Japan, and a Grant-in-Aid for Scientific
Research (B) from the Japan Society for the Promotion of Science (No. 16390544). Also, the authors would like to express
their gratitude to Prof. Howard K. Kuramitsu, State University of New York at Buffalo for his kind contributions in editing
the manuscript.

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Correspondence to:
Dr. Shamim Sultana
Cariology and Operative Dentistry, Department of Restorative Sciences
Graduate School, Tokyo Medical and Dental University
1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan
Fax: +81-3-5803-0195 E-mail: rumasultana2000@yahoo.com
Received November 8, 2006. Accepted November 30, 2006.
Copyright ©2006 by the International Chinese Journal of Dentistry.

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