Biological and physical properties of calcium hydroxide-based pulp-capping materials and their modifications

Abstract

Purpose: To evaluate the biological and physical properties of calcium hydroxide-containing pulp-capping materials and their modifications with different solutions and antioxidant Resveratrol (RES) addition.

Methods: Calcium hydroxide+distilled-water:C, calcium hydroxide+saline:S, calcium hydroxide+synthetic tissue fluid:STF, Dycal:D, calcium hydroxide+distilled-water+RES:C+RES, calcium hydroxide+saline+RES:S+RES, calcium hydroxide+synthetic tissue fluid+RES:STF+RES, Dycal+RES:D+RES were tested. Cytotoxicity was determined by WST-1. Antibacterial-activity was evaluated by agar-diffusion. The water-absorption and solubility were examined for ISO-6876 and ISO-3107. The color-change was evaluated by spectrophotometer. Radiopacity was evaluated for ISO-6876 and ISO-9917. The normal distribution and homogeneity were determined and comparisons were made with appropriate analysis and post hoc tests (P < 0.05).

Results: The highest cell-viability was determined in the C+RES and the lowest was in D and D+RES after 24 h (P < 0.0001). RES-addition increased cell-viability and the highest rate was detected in C+RES, S+RES and STF+RES after 48 h (P < 0.0001). A limited inhibition-zone against Streptococcus mutans was detected in D and D+RES. RES-addition did not change the water-absorption in S and STF or the solubility in S group.

Conclusion: RES-addition may be used to increase the biocompatibility of calcium hydroxide without any adverse effect on physical properties. Saline may be the first choice as a mixing solution.

Introduction

The pulp tissue has potential to form reparative dentin formation via pulp-capping treatment including the cover of pulp tissue with biocompatible materials [1,2]. Calcium hydroxide has been accepted as a gold standard, since it is the most cost-effective material with the longest history of clinical success for pulp-capping [3]. The two-paste calcium hydroxide-systems have been developed to overcome the disadvantages of one-paste systems, such as non-setting and high solubility, but they are still soluble and dissolve over time [4,5]. Two-paste systems were reported as more toxic than one-paste systems due to additional components such as disalicylate, accelerator/plasticizer [4]. Therefore, there is a need to improve the biological and physical properties of calcium-hydroxide based systems.

The synthetic tissue fluid is a homogeneous mixture used in tests due to the similarity to biological fluids [6,7]. Antioxidants have been used in many fields in dentistry such as the reduction of reactive oxygen species and cytotoxicity. Ascorbic acid, alpha tocopherol, grape seed extract, curcumin, green tea, propolis and resveratrol (RES) are the main antioxidants used in dentistry [8,9,10]. RES is a natural non-flavonoid polyphenol found in at least 72 plants, as well as foods commonly included in the diet such as grapes, cranberries, peanuts and red wine [11].

The aim of this in vitro study was to investigate the biological and physical properties of calcium hydroxide-containing pulp-capping materials after modifications using saline, synthetic tissue fluid and RES.

Materials and Methods

Preparation of extracts and samples

The tested materials are listed in Table 1. The modifications were made using different solutions other than distilled water such as saline and synthetic tissue fluid while mixing the calcium hydroxide (Emsure calcium hydroxide, Merck, Darmstadt, Germany). RES (Sigma-Aldrich, St. Louis, MO, USA) was added as an antioxidant. The materials were classified into eight groups namely C;calcium hydroxide+distilled water, S;calcium hydroxide+saline, STF;calcium hydroxide+synthetic tissue fluid, D;Dycal (Dentsply, Milford, DE, USA), C+RES;calcium hydroxide+distilled water+RES, S+RES;calcium hydroxide+saline+RES, STF+RES;calcium hydroxide+synthetic tissue fluid+RES and D+RES;Dycal+RES. Synthetic tissue fluid was prepared by providing a homogeneous mixture of 1.7 g monopotassium phosphate, 11.8 g disodium phosphate, 2 g potassium chloride and 80 g sodium chloride in 10 liters of water [7]. The reported most effective dose of RES (0.5 µM) was added [12]. Disc-shaped specimens (8 × 2 mm) were prepared using standard plastic molds (n = 6).

Table 1 The composition, manufacturer and lot number of tested materials

Materials	Composition	Manufacturer	Lot number
Emsure	Ca(OH)2 M = 74.09 g/mol	Merck KGaA, Darmstadt, Germany	K48319847 730
Dycal	base paste: disalicylate ester of 1, 3 butylene glycol, calcium phosphate, calcium tungstate, zinc oxide, iron oxide. catalyst paste: calcium hydroxide, ethyl toluenesulfonamide, zinc state, titanium dioxide, zinc oxide, iron oxide.	Dentsply Caulk Milford, DE, USA	00016730
Resveratrol	resveratrol ≥99% (GC)	Sigma-Aldrich Production GmbH, St. Louis, MO, USA	SLBC6832V

Cell culture and viability

The samples were placed in 48 well culture plates with one sample per well and cultured with L929 mouse fibroblast cells (ATCC, CCL-1: NCTC clone 929 Aerolar Fibroblast Mouse, LOT: 70026472) for 24 and 48 h. The wells without samples were used as a control.

Cell viability was determined by the WST-1 method. After incubations, 50 µL of WST-1 at 1:10 dilution was added to each well and incubated for 3 h in an incubator set at 5% CO₂ and 37°C. Then, supernatant was transferred to 96 well plates to measure the absorbance at 440 nm with a microplate reader (Multiskan Go, Thermo Scientific, Ratastie, Finland). The reference wavelength was used as 650 nm. The change in cell viability was determined by the formula absorbance of the test well/absorbance of control well.

Antibacterial activity

The antibacterial activity was investigated by agar-diffusion test. The pulp-capping material specimens (disc-shaped, 8 × 2 mm) were prepared using standard plastic molds (n = 6). The antibacterial activity of each material was examined by culturing in separate petri dishes for each microorganism. The sterile paper discs were used in the control group. Agar medium (5 mm thickness, 20 mL) was poured into 9 cm diameter sterile petri dishes and allowed to solidify. Overnight active liquid cultures of microorganisms (5.8 × 10⁶ cfu/mL) were spread on the surface of the medium. After 1 h at 37°C, standard wells were created in the medium with the blunt tip of the Pasteur pipette, and the samples were placed in the standard wells. Streptococcus mutans (DSM20523), Lactobacillus acidophilus (DSM 20079) and Enterococcus faecalis (ATCC 29212) strains were used. After 24, 48, and 72 h of incubation, the inhibition zone around the each well was randomly measured from two points with the help of a digital caliper (Mitutoyo Absolute Digimatic Caliper, Mitutoyo Corp, Kawasaki, Japan). Two technical replicates were performed in each group for the measurement. The mean value (mm) was obtained for each sample. The absence of antibacterial activity was estimated as 0 (zero) inhibition zone formation.

Water absorption and solubility

The water absorption and water solubility tests were performed in accordance with ISO 6876 and 3107 standards [13]. Each sample was weighed and immersed in 20 mL of distilled water at 37°C to determine its initial mass (I). At 24 h after immersion, the samples were removed and excess water on their surface was removed using a moistened filter paper and the saturated mass was recorded (M). The samples were dried at 37°C until their weight was stable and the final dry mass (D) was recorded. The values were calculated using the following formulas; water absorption (A = [(M − D) / D] × 100) and solubility (S = [(I-D) / D] × 100).

Color assay

The initial color values (L₀, a₀, b₀) were measured with a spectrophotometer (Spectroshade Micro-MHT Optic Research AG). The color measurements were repeated after 24, 48 h, and 1 week. ΔE₁: Color change after 24 h = ( [L₀-L₁]² + [a₀-a₁]² + [b₀-b₁]² ) ^1/2 , ΔE₂: Color change after 48 h = ( [L₀-L₂]² + [a₀-a₂]² + [b₀-b₂]² ) ^1/2 and ΔE₃: Color change after 1 week = ( [L₀-L₃]² + [a₀-a₃]² + [b₀-b₃]² ) ^1/2 were calculated.

Radiopacity measurement

The specimens were placed on a phosphor plate (Digora-Soredex, Orion Corporation, Helsinki, Finland) adjacent to an aluminum (99.5% pure) stepwedge (1-10 mm). Radiographic images were obtained with the X-Ray unit (Trophy Radiologie, Vincennes, France) at 250 kV. Digital images were transferred to a computer and analyzed with software (Adobe Photoshop 8.0 CE, Adobe Systems Inc. San Jose, CA, USA). The mean grey values of the materials and each step of stepwedge were measured with histogram analysis (Fig. 1). Then, the mean value for each material in mm Al was calculated.

Fig. 1 The measurement of the mean grey values of the materials with histogram analysis (n = 6)

pH measurement

The specimens (after the completed setting reactions) were immersed in 20 mL of distilled water (pH: 7.4) in separate closed tubes and incubated at 37°C. At 24, 48, 72, 168 (7 days), 336 (14 days), and 504 (21 days) h after immersion, the pH values of all solutions were measured with the pH meter (inoLab pH 720, WTW, Weilhem, Germany).

Statistical analysis

Statistical analysis was performed with GraphPad Prism 5 for Windows Version 5.05 (La-Jolla). The significance level was considered as P < 0.05. In the statistical analysis of cell viability, water absorption and water solubility data, there was no normal distribution according to the Shapiro-Wilk test (P < 0.05). The homogeneity of variances (homoscedasticity) is tested via Levene test (P > 0.05). Therefore the non-parametric tests, Kruskal-Wallis test and post hoc Dunn’s test were used for comparisons between the groups. Mann-Whitney-U test was used for pairwise comparisons. In the statistical analysis of the data on color assay and radiopacity, there was normal distrubution (P > 0.05). The homogeneity is tested via Bartlett test (P > 0.05). Therefore the parametric tests one way ANOVA and post hoc Tukey test were used for comparisons.

Results

Cell viability

Considering the 24-h cell viability (Fig. 2), the highest rate was found in the C+RES (P < 0.0001). The lowest cell viability rate was found in the D and D+RES (P < 0.0001). There was a significant difference between the groups as; control vs D, control vs D+RES, C+RES vs STF+RES, C+RES vs D, C+RES vs D+RES, S+RES vs D and S+RES vs D+RES (P < 0.0001). RES increased the cell viability in C and S groups. The difference between C vs C+RES and S vs S+RES was significant (P < 0.0001).

Considering the 48-h cell viability (Fig. 2), the highest rate was found in C+RES, S+RES and STF+RES (P < 0.0001). The lowest cell viability was found in the D and D+RES (P < 0.0001). There was a significant difference between the groups as; control vs D, control vs D+RES, C vs S+RES, C vs STF+RES, C+RES vs D, C+RES vs D+RES, S+RES vs D, S+RES vs D+RES, STF+RES vs D and STF+RES vs D+RES (P < 0.0001). RES addition provided an increase in cell viability for C, S and STF groups. The difference between C vs C+RES, S vs S+RES and STF vs STF+RES was significant (P < 0.0001).

Fig. 2 Cell viability rate (%) after 24 and 48 h by WST-1 assay (n = 6)

*vs all groups (P < 0.0001); **vs all groups (P < 0.0001); ^#S vs S+RES (P = 0.0003); ^aC vs C+RES (P = 0.0003); ^bS vs S+RES (P = 0.0003); ^cSTF vs STF+RES (P = 0.0003); **vs all groups (P < 0.0001)

Antibacterial activity

A limited inhibition zone (16.6 ± 0.57 mm) against Streptococcus mutans was detected only in the D and D+RES (Fig. 3). No inhibition zones were observed in other groups. Additionally, a limited diffusion zone formation was detected in S and STF against Lactobacillus acidophilus, but no inhibition zone formation was observed in these groups.

Fig. 3 Inhibition zones of tested materials at 24, 48, and 72 h incubation by agar-diffusion test (n = 6)

Water absorption and solubility

Considering the water absorption (Fig. 4), the highest value was determined in the C+RES and the lowest value was obtained in D and D+RES (P < 0.0001). There was a significant difference between the groups as; C+RES vs D, C+RES vs D+RES, S vs D+RES, S+RES vs D, S+RES vs D+RES, STF vs D, STF vs D+RES, STF+RES vs D and STF+RES vs D+RES (P < 0.0001). There was a significant difference between the C and C+RES. RES addition caused an increase in the water absorption for C group (P = 0.0056). It did not change the water absorption values for S and STF groups.

Considering the water solubility (Fig. 5), the lowest water absorption was found in the D and D+RES (P < 0.0001). There was a significant difference between the groups as; C vs D, C vs D+RES, C+RES vs D, C+RES vs D+RES, S+RES vs D, S+RES vs D+RES, STF+RES vs D and STF+RES vs D+RES (P < 0.0001). There was a difference between C and C+RES (P = 0.0495). The difference between STF and STF+ RES was also significant (P = 0.0057). RES addition caused an increase in water solubility in these groups. RES addition did not change the water solubility values for S group.

Fig. 4 Water absorption rate (%) of tested materials (n = 6)

*vs all groups (P < 0.0001); **C vs C+RES (P = 0.0056)

Fig. 5 Water solubility rate (%) of tested materials (n = 6)

*vs all groups (P < 0.0001); **C vs C+RES(P = 0.0495); ^#STF vs STF+RES (P = 0.0057)

Color assay

The color change was observed only in the D and D+RES (P < 0.05) (Fig. 6). It increased after 24 h and remained the same after 48 h and 1 week. The obtained color change was clinically higher than the color mismatch value (ΔE ≥ 3.7) [14].

Fig. 6 ΔE values of tested materials after 24, 48 h, and 1 week (n = 6)

Different superscripts indicate statistical significant differences (P < 0.05). ΔE₁= Color change after 24 h; ΔE₂= Color change after 48 h; ΔE₃= Color change after 1 week

Radiopacity

RES addition did not change the radiopacity value for all materials (Fig. 7). The highest radiopacity value was observed in D and D+RES groups (P < 0.05).

Fig. 7 Mean radiopacity values of tested materials (mm Al) (n = 6)

*Significant difference compared to other groups (P < 0.05)

pH values

RES addition caused a slight decrease in the tendency for pH values in all groups except the D. The pH value of the calcium hydroxide-containing groups was higher than the Dycal groups (Fig. 8).

Fig. 8 pH changes of tested materials after immersion in distilled water in predetermined time intervals (n = 6)

Discussion

The effects of different modifications on the biological and physical properties of calcium hydroxide-based pulp capping materials were investigated in this study. The liquids were modified using saline and synthetic tissue fluid. Saline is distilled water solution containing certain amounts of sodium chloride. Synthetic tissue fluid has been used as a medium to evaluate the solubility and microhardness of materials [7,15,16,17]. In this study, it was used as a mixing solution to investigate the effect on the solubility.

The ideal pulp-capping material is expected to be biocompatible to maintain the pulp vitality [18]. According to the WST-1 analysis results after 24 h, the highest cell viability was detected in the C+RES group and the addition of RES increased the cell viability rate in the C and S groups. The lowest viability was observed in the D and D+RES groups. This finding may be related to the effects of accelerator and plasticizer components that exist in Dycal [5]. While there was no significant difference between the liquids at the end of 48 h, the addition of RES increased cell viability in all of the C, S and STF groups. RES addition increased the cell viability consistent with the previous findings about pulp-capping materials [19]. The higher cell viability compared to the control group in the C+RES group at 24 h is also noticeable. This indicates that the material intensely induces proliferation in cells in the first 24 h. On the other hand, the cell viability rate of C+RES was similar to the control group and other RES groups at 48 h. This shows that although the cell proliferation-inducing effect of the C+RES group seems to be higher in the early cell culture time period, it may be limited at some point and reaches a similar level to other groups in the later stage.

The pulp-capping material is expected to have and maintain the antibacterial properties [20]. The fundamental method for determination of antibacterial activity agar-well test, [21,22] was applied and an inhibition zone against Streptococcus mutans was detected only in Dycal as in a previous study [20]. RES addition did not change the antibacterial activity of tested materials in this study. Further studies evaluating antibacterial activity of these materials by different methods may be useful.

The high solubility is considered as one of the disadvantages of calcium hydroxide [23]. Although the lowest solubility was observed in the STF group, the difference between the groups was not significant among the C, S, and STF groups. The absence of a significant difference in the STF group was thought to be due to the fact that STF has a neutral pH and distilled water was preferred as the medium. RES addition increased the water absorption in the C group and water solubility in the C and STF groups. The lowest water absorption and water solubility were observed in Dycal groups consistent with previous studies [24,25] and it may be attributed to the catalyst initiating the setting reaction [4,5].

The color change was not observed in the groups except Dycal. The color change of Dycal as becoming light over time and presenting higher value than the clinical color incompatibility point [14] may be attributed to its own color. Therefore RES addition seems to have no negative effect on the color of the tested materials.

The radiopacity should be equivalent to not less than 3 mm Al according to ISO-6876:2002, entitled “dental root canal sealing materials”, and at least 1 mm Al according to ISO 9917:2007, entitled “water-based cements”. Calcium hydroxide is a material used as root canal seal and pulp capping, but Dycal is just used for pulp-capping. ISO 9917:2007 was used for Dycal and calcium hydroxide groups, and ISO 6876 was used for only calcium hydroxide groups in this study. The use of both standards together did not change the radiopacity assessment. In reference to both standards, while calcium hydroxide groups had low radiopacity values, Dycal groups had enough radiopacity values. There are no studies in the literature that evaluate the radiopacity of pure calcium hydroxide. However, the radiopacity of Dycal is consistent with the previous studies [24,26]. The modification of the solution and RES addition did not change the radiopacity of values.

The cellular functions can be affected by pH and are also relevant to the antibacterial activity [27]. The alkaline pH plays an important role in hard-tissue formation [28]. Considering the pH measurements in this study, all materials have showed alkaline pH. However, the pH values of all groups presented a tendency to decrease over time. RES addition decreased the pH values in the calcium hydroxide groups. The pH variations of D group were consistent with previous studies [13,24].

Calcium hydroxide was used as pulp-capping material in this study. However, it is also used in Cvek pulpotomy and cervical pulpotomy, intra-canal medicament, apexification, apexogenesis, root resorption in the area of pediatric endodontics [29]. In these applications, the biocompatibility is very important especially in case of the overflow of the material into periapical tissues. RES seems to have a significant advantage in this respect due to the properties such as increasing cell viability without any negative effect on the mechanical properties of the material.

In the limitations of this study it can be inferred that, the liquid modifications did not improve the physical properties of calcium hydroxide and the addition of RES had a positive effect on cell viability. The saline was the only liquid that did not have a negative effect on physical properties among the groups with RES. Thus, it was thought that saline could be the first choice as a mixing solution among the tested solutions.

Conflicts of Interest

None of the authors have any conflict of interest to disclose.

Funding

No funding

ORCID iD

¹⁾DA: [e-mail], https://orcid.org/0000-0003-1713-7508

¹⁾CAO*: [e-mail], https://orcid.org/0000-0003-4144-4233

²⁾GA: [e-mail], https://orcid.org/0000-0001-6466-2263

²⁾DB: [e-mail], https://orcid.org/0000-0002-5094-9949

³⁾MA: [e-mail], https://orcid.org/0000-0002-8871-5638

¹⁾HT: [e-mail], https://orcid.org/0000-0002-2376-5984

Acknowledgments

The authors thank the participants for all their support and contributions.

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