Effects of storage solutions on mineral contents of dentin

Article Outline

• Abstract
• Introduction
• Materials and methods
  • Preparation of dentin slabs
  • Preparation of groups
  • ICP-AES technique
  • Statistical analysis
• Results
• Discussion
• References
• Copyright

Abstract 

Background/purpose

It is important to understand how storage conditions affect the tooth structure for in vitro studies. There is little information regarding the selection of an appropriate storage solution. This study was conducted to determine the influence of storage solutions on the mineral contents of dentin.

Materials and methods

Ninety dentin specimens were obtained from 30 molar teeth. Specimens were divided into two groups of 45 each (storage for 45 and 90 days). Each of the two groups was further divided into nine storage solution groups (n=5). For the control group, freezing was used to store the teeth. The mean percentage weights of calcium, potassium, sodium, and phosphorus in each dentin slab were measured by inductively coupled plasma-atomic emission spectrometry. Two-way analysis of variance and Tukey’s honest significant difference test were used to analyze the data (P=0.05).

Results

There were significant differences in calcium among groups. The potassium level of slabs stored in artificial saliva and the sodium level of slabs stored in buffered solutions and saline solution increased (P<0.05). Potassium, sodium, and phosphorus levels were highest when stored for 45 days (P<0.05).

Conclusion

The storage solution and storage time affected the compositional structure of dentin. The results suggest that storage processes may influence outcomes of in vitro dental research.

Keywords: dentin, inductively coupled plasma-atomic emission spectrometry, mineral content, storage solution, storage time

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Introduction 

It is important to study systems in an in vitro model prior to in vivo use to identify treatment or material variations that might improve clinical performances. Extracted human teeth are used in many areas of in vitro dental research, including studies of dentin permeability and hydraulic conductance, and bond strengths of dentin-bonding agents.¹ Freshly extracted teeth must be stored in a storage solution after extraction. Storage solutions are used to prevent the growth of microorganisms and dehydration of the teeth.², ³ In an in vitro setting, dentin surface moisture can be influenced by the dentin body moisture, which significantly varies with different tooth conditions.⁴

Several storage solutions have been suggested in the published literature, such as glutaraldehyde, ethanol, methanol, formalin, neutral buffered formalin, distilled water with thymol, phosphate buffered saline (PBS) with thymol, sodium hypochlorite, sodium azide, aqueous chloramine, chloramine T, physiological saline solution, PBS, Hank’s balanced salt solution (HBSS), Homofix (70mL H₂O, 30mL glycerol, and 4g phenol), cetylpyridinium chloride, H₂O₂, artificial saliva, mineral oil, and distilled water.¹, ², ³, ⁴, ⁵, ⁶, ⁷, ⁸, ⁹, ¹⁰, ¹¹, ¹², ¹³, ¹⁴, ¹⁵, ¹⁶ A steam autoclave, chemical heat sterilization, dry heat, γ radiation, ethylene oxide, and freezing are also used as methods of storage.², ⁸, ¹⁴, ¹⁷

The period of storage, which ranges from a few hours to years¹, ⁵, ¹², ¹⁵, ¹⁸ and the frequency at which the solution is changed¹⁸ are other factors which may affect the storage process.

Dentin has a content of 70wt% minerals, 20wt% organic substances, and 10wt% water. Whereas enamel has the following composition: 95wt% mineral content, 4wt% organic substances, and 1wt% water.¹⁹ Owing to these differences in structural composition, the effect of a storage solution on dentin should greatly differ from that of enamel. The aim of this study was to evaluate compositional changes [calcium (Ca), potassium (K), sodium (Na), and phosphorus (P)] in dentin slabs kept in different storage solutions for different storage durations using inductively coupled plasma-atomic emission spectrometry (ICP-AES). This study tested the null hypothesis that the storage solution and storage time do not affect the compositional structure of dentin.

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Materials and methods 

The study protocol was reviewed and approved by the Local Ethics Committee of the Faculty of Medicine, Gaziantep University under protocol number: 05-2009/209.

Preparation of dentin slabs 

Thirty lower non-erupted wisdom teeth were cleaned with gauze and a fine brush after extraction. The teeth were then mounted in quadrangular molds with an autopolymerizing acrylic resin (Meliodent, Bayer Dental, Newbury, UK). The enamel of a tooth was removed with a conventional bur under cooling water to expose the dentin surface. The occlusal one-third of the crown was cut with a slow-speed diamond-saw sectioning machine (Isomet, Buehler, Lake Bluff, IL, USA) under cooling water. To prepare the dentin slabs into 0.60-mm thick pieces, cuts were made perpendicular to the long axis of the tooth. Finally, three dentin slabs were obtained from each tooth (Fig. 1).

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• Figure 1 

  The preparation of the dentin slabs.

Preparation of groups 

Ninety dentin specimens were divided into two groups of 45 each (with storage for 45 and 90 days). Each of the two groups was further divided into nine storage solution groups of five each [distilled water with 0.1% thymol (DW-T), PBS with 0.1% thymol (PBS-T), saline (S), DW with 10% formalin (DW-F), PBS with 10% formalin (PBS-F), DW, DW with 2% glutaraldehyde (DW-G), PBS with 2% glutaraldehyde (PBS-G), and artificial saliva (AS)] (Table 1). The composition of the artificial saliva was 1.5mmol/L CaCl₂, 8.2mmol/L NaHCO₃, 4.8mmol/L NaCl, 137mmol/L KCl, and 4mmol/L KH₂PO₄ in 250mL.

Table 1. pH measurements and elemental compositions of the storage solutions.

Dentin slabs were placed in their storage solutions. Specimens were kept in a light-proof box for 45 or 90 days at room temperature. For the control group, fresh intact teeth (n=5) were cleaned with distilled water and stored at –20°C until required; dentin slabs were prepared after 45 days.

ICP-AES technique 

Dentin slabs were removed from their storage solutions and stored in plates at 65°C in a cabinet desiccator until they reached a fixed weight. That is to say, the specimens were dehydrated to a constant weight. Their weights were recorded with an electronic balance (Electronic Balance AX200, Shimadzu, Kyoto, Japan). Then, 5mL of nitric acid (HNO₃) and 2mL of hydrogen peroxide (H₂O₂) were added to the specimens. The specimens were heated at 210°C in a microwave (CEM MarsXpress, Matthews, NC, USA) until dissolved. The solutions were then filtered. After calibration of the ICP-AES instrument (Vista AX, Varian, Mulgrave, Australia), 2mL of solution was taken. In this study, three measurements were performed on each element. Levels of the four elements of Ca, K, Na, and P in each specimen were measured by ICP-AES. Mineral contents were calculated as percentage weights (wt%).

Statistical analysis 

Differences between the groups were analyzed by a two-way analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) test. Differences were compared at a significance level of P<0.05 using the statistical program SPSS 13 for Windows (SPSS, Chicago, IL, USA).

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Results 

The two-way ANOVA indicated that the mineral contents of Ca, K, Na, and P of dentin were significantly affected by storage solutions and storage times (P<0.05), and there were significant interactions between the two factors for K, Na, and P (P<0.05). There was no interaction between storage solutions (P>0.05) and storage times (P=0.12) for Ca (Table 2).

Table 2. The results of two-way ANOVA.

Elements	Source	Type III sum of squares	df	Mean square	F	P
Ca	Storage solution	85.354	8	3.223	226.305	<0.001
	Storage time	45.341	1	1.421	99.810	<0.001
	Storage solution×Storage time	55.394	8	1.010	70.897	<0.001
K	Storage solution	25.781	8	3.223	226.305	<0.001
	Storage time	1.421	1	1.421	99.810	<0.001
	Storage solution×Storage time	8.077	8	1.010	70.897	<0.001
Na	Storage solution	23.372	8	2.922	126.306	<0.001
	Storage time	3.200	1	3.200	138.336	<0.001
	Storage solution×Storage time	3.968	8	0.496	21.441	<0.001
P	Storage solution	9.685	8	1.211	5.537	<0.001
	Storage time	82.369	1	82.369	376.710	<0.001
	Storage solution×Storage time	13.436	8	1.680	7.681	<0.001

Mean percentage weights and standard deviations of the four elements for each group are summarized in Table 3. There were no significant differences between the control group and the other groups for Ca (P>0.05, P=0.33), except for DW-T-45, DW-45, S-45, DW-G-45, and AS-45 (P<0.05). There were no significant differences among groups for K (P>0.05, P=1.00), except for AS-45 and AS-90. K levels of dentin slabs in the AS groups significantly increased (P<0.05). There were significant differences between storage times in the AS groups (P<0.05), and the K level was highest at 45 days. There were no significant differences among groups for Na (P>0.05, P=1.50), except for the PBS-T, PBS-F, PBS-G, and S-45 groups. Na levels significantly increased in buffered solutions (PBS-F, PBS-T, and PBS-G) and S-45 (P<0.05). There were also significant differences between storage times in buffered solutions (PBS-F, PBS-T, and PBS-G) and the saline solution (S) (P<0.05). Na levels had significantly decreased at 90 days, whereas it was high at 45 days. There were significant differences in P among groups (P<0.05). There were significant differences between storage times in all groups (P<0.05). P levels significantly decreased at 90 days, whereas it was high at 45 days (P<0.05).

Table 3. Mean percentage weights of the four elements for each group (n=5; mean±standard deviation).

Groups∗	Ca†		K†
	45	90	45	90
AS	33.77±1.73bcd	31.54±1.93abcd	2.73±0.49g	0.79±0.08f
DW	34.75±2.63cd	32.60±1.65abcd	0.10±0.03e	0.03±0.01e
DW-F	31.69±1.62abcd	32.26±2.67abcd	0.07±0.01e	0.04±0.01e
DW-G	33.98±1.05bcd	32.35±1.38abcd	0.09±0.03e	0.01±0.01e
DW-T	35.47±2.43d	31.58±2.07abcd	0.06±0.01e	0.06±0.02e
S	34.13±2.55bcd	31.37±1.93abcd	0.08±0.03e	0.01±0.01e
PBS-F	32.75±1.92abcd	32.05±2.78abcd	0.09±0.06e	0.03±0.02e
PBS-G	32.17±2.33abcd	30.68±1.39abc	0.07±0.02e	0.06±0.03e
PBS-T	29.71±1.68ab	31.21±1.71abcd	0.07±0.02e	0.07±0.03e
Control	29.18±1.01a		0.07±0.01e
Groups	Na†		P†
	45	90	45	90
AS	0.91±0.09k	0.83±0.09k	14.82±0.30stu	12.97±0.40op
DW	0.76±0.14k	0.75±0.06k	15.04±0.61tu	13.75±0.29prs
DW-F	0.71±0.04k	0.72±0.09k	14.12±0.61rst	14.00±0.53prst
DW-G	0.74±0.04k	0.76±0.08k	15.33±0.35uy	13.11±0.20pr
DW-T	0.74±0.04k	0.72±0.08k	16.23±0.84y	13.27±0.49pr
S	1.67±0.09m	1.02±0.09kl	15.30±0.31uy	13.06±0.31pr
PBS-F	2.29±0.38n	1.41±0.16m	16.13±0.46y	13.83±0.77prs
PBS-G	2.42±0.24n	1.34±0.09lm	15.71±0.37uy	13.26±0.18pr
PBS-T	2.22±0.26n	1.54±0.17m	15.52±0.35uy	13.74±0.43prs
Control	0.82±0.04k		11.92±0.52o

∗The groups are artificial saliva (AS), distilled water (DW), distilled water with 10% formalin (DW-F), distilled water with 2% glutaraldehyde (DW-G), distilled water with 0.1% thymol (DW-T), saline (S), phosphate buffered saline with 10% formalin (PBS-F), phosphate buffered saline with 2% glutaraldehyde (PBS-G), and phosphate buffered saline with 0.1% thymol (PBS-T).

^†Same letters were not significantly different at P<0.05.

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Discussion 

Storage conditions are not standardized, and storage solutions and durations widely vary.¹ Hence, storage conditions were shown to affect the results in many previous studies.¹, ⁵, ⁷, ⁸, ¹², ¹³, ¹⁴, ¹⁶, ¹⁷, ¹⁸

The choice of storage solution is important to preserve microelastic tissue properties. Raum et al¹³ inspected dentin slabs stored in three different media for 21 days through quantitative time-resolved scanning acoustic microscopy. They stated that HBSS and artificial saliva did not alter the elastic properties of dentin. Storage in a saline solution resulted in a progressive decrease in the acoustic impedance in dentin by up to 70%. It was found that the concentration of calcium ions was too low to balance the absence of phosphate when specimens were stored in sodium chloride. Those authors noted that demineralization increased with a decreasing pH and that storage in deionized water may be the reason for demineralization due to the absence of calcium and phosphate ions in solution. Chemical dissolution of the mineral phase causes a gradual softening of the surfaces of the specimens. The depth of the affected layer was found to be approximately 300μm. Minerals are rapidly dissolved from dentin when it is stored in a saline solution. Washout gradually occurs and affects only a thin superficial layer.¹³

Goodis et al⁵ tested the most commonly used solutions and found significant changes in permeability, especially in distilled water with thymol and PBS with thymol. Those changes were found at each interval, with dramatic increases in the samples tested at 4–8 days, and with both increases and decreases in samples at 1–3 weeks. They stated that water with thymol and PBS with thymol had no effect on either the organic or inorganic contents of dentin.⁵

Goodis et al¹ found that prolonged storage (6 months) in PBS with 0.02% thymol resulted in significant decreases in dentin permeabilities and increases in bond strengths. They found that long- and short-term storage in 70% ethanol, 10% formalin, distilled water with 0.02% thymol, and distilled water increased the permeability of dentin, but had no effects on bond strengths. Goodis et al¹ stated that decreases in permeability may have been due to redeposition of either mineral or organic components that had been washed out of the tubules earlier. Salt precipitates may occur in dentin stored in PBS, which could explain the decreased permeability over time. Investigators found that fixative solutions of ethanol and formalin were the most stable over time and exhibited the lowest permeability values. The fact that fixative solutions showed lower average permeabilities and less variability suggests that residual material in the tubules may be organic, such as remnants of odontoblastic processes. Water-based solutions exhibited higher permeability values, which may have resulted from the continual washing of the tubules over time.¹ Water is currently the most popular solution used. This storage solution seems to provide a simple, low-cost means of storing teeth.¹⁸

Kitasako et al¹⁸ noted that changing the storage solution might induce the loss of calcium from dentin. Conversely, an equilibrium of calcium ion transfer between the dentin and unchanged storage solutions would be established in the solution. They found that the mean shear bond strengths did not show significant differences between water and PBS storage solutions and were significantly higher in the unchanged storage solution. The solutions were unchanged in our study.

Francescut et al¹¹ evaluated the effect of storage solutions on the infrared laser fluorescence response. Fluorescence decreased for samples stored in formalin (–60%), chloramine (–72%), and thymol (–54%) after 2 years. Frozen teeth showed a non-significant increase in fluorescence of 5%.

Titley et al⁸ found that fresh teeth are required for the highest possible resin–dentin bond strength. They also noted that postmortem changes can occur in dentin, and that freezing the teeth immediately after extraction prevents such changes. In their study, storage in neutral buffered formalin, sodium hypochlorite, chloramine, distilled water, and homofix produced statistically similar results, although the shear bond strengths were lower than those obtained with frozen teeth. They stated that should insufficient numbers of teeth be available at one time, freezing teeth in distilled water as soon as possible after harvesting would be the next preferred method of storage.⁸

In this study, the mineral contents of Ca, K, Na, and P of dentin were significantly affected by storage solutions and storage times. There were no significant differences between the control group and other groups for Ca (P=0.33), except for DW-T-45, DW-45, S-45, DW-G-45, and AS-45. There were no significant differences among the groups for K (P=1.00), except for the AS groups. K levels of dentin slabs stored in AS, which contained a high concentration of K, significantly increased. Similarly, it was found that slabs stored in S-45 and buffered solutions (PBS-F, PBS-T, and PBS-G) had the highest Na content. Solutions containing sodium significantly influenced the amount of sodium in the dentin slabs. This may have been due to its features. Sodium is highly reactive because it has a single valence electron. With a smaller molecular size, the diffusion rate increases.

Ca values were affected by storage times; there were significant differences between storage times in the PBS-T group. There were significant differences between storage times in the AS groups for K. It was found that the K value was lower at 90 days than at 45 days. There were significant differences between storage times in buffered solutions (PBS-F, PBS-T, and PBS-G) and the saline solution (S) for Na. It was observed that values were lower at 90 days, whereas Na values for slabs stored in saline and solutions containing PBS were significantly higher at 45 days. There were significant differences between storage times in all groups for P. P levels significantly decreased at 90 days, whereas it was high at 45 days. However, P values of specimens were found to be unexpectedly low in spite of the high P levels of the PBS and AS groups.

These results require rejection of the null hypothesis, namely that the storage solution and storage time do not affect the compositional structure of dentin. Therefore, further studies are needed to clarify postmortem changes in dentin and the effect of the storage process (storage solution or method, storage time, pH, and temperature) on the compositional structure of dentin. To obtain more realistic results with in vitro tests, short storage times should be used, thus minimizing the negative influence on the dentin structure.¹⁵ One limitation of the current study was the difference in elemental contents from tooth to tooth and from one region of a tooth to another. The groups were not completely standardized because of this limitation.

Teeth obtained from humans and bovines are contaminated with bacteria. Thus, the potential for transmission of communicable diseases via blood-borne pathogens is a concern.²⁰ Storage solutions are also used to prevent dehydration of teeth.⁴ Generally, researchers make a decision based upon their objectives, ease of use, and personal experience. However, the nature of the storage method can affect properties of adsorption, diffusion, and dissolution, and therefore possibly alter the physical properties of dentin. It is important to preserve tissue properties for standardization, availability, and reproducibility of results. An ideal storage solution or method does not affect either the organic or inorganic contents of dentin.

Within the limitations of this study, the following conclusions were drawn. (1) Storage procedures can affect the mineral contents of Ca, K, Na, and P of dentin. (2) There were no significant differences between the control group and the other groups for Ca (P=0.33), except for DW-T-45, DW-45, S-45, DW-G-45, and AS-45. (3) K levels of slabs stored in AS and Na levels of slabs stored in buffered solutions (PBS-F, PBS-T, and PBS-G) and S-45 significantly increased. K, Na, and P levels were highest at 45 days. (4) Freezing in wet gauze is recommended to preserve teeth because different storage solution–time combinations lead to changes in different elements in dentin.

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