Biocompatibility evaluation of Jordanian Portland cement for potential future dental application.

BACKGROUND: The demand for novel Portland cement (PC)-based formulations to be used in dental applications is ever increasing in viewing the foregoing knowledge on the favorable effects of these formulations on cellular proliferation and healing, leading to treatment success. AIM: This study investigated the effect of white and gray mineral trioxide aggregate (W-MTA and G-MTA) and white and gray Jordanian PC (W-PC and G-PC) in their raw state on the viability of Balb/C 3T3 fibroblasts using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
MATERIALS AND METHODS: Materials were prepared in the form of disks, with a diameter of 5 mm and a thickness of 2 mm. In the first experiment, Balb/C 3T3 fibroblasts were cultured with the material disks using culture plate inserts. In the second experiment, material elutes were added to cultured cells. The elutes were prepared by adding 2 ml serum-free media to 10 disks of each material and then incubated at 37°C for different time intervals. Material elutes were analyzed using ion chromatography for traces of calcium. The data were analyzed using analysis of variance followed by Dunnett test (α = 0.05) or Tukey test (α = 0.05).
RESULTS: In response to material disks, G-PC had a proliferative effect on cells at day 1 and day 2 with a significant difference from the control at day 1. G-MTA reduced cell viability with a significant difference from the control level at day 2. Elutes of PC showed biocompatible and even proliferative effects on Balb/C 3T3 fibroblasts. Calcium ions were found to leach continuously over the measurement period for all the materials tested in this work.
CONCLUSION: Jordanian PC in its raw state was found to be biocompatible, and the results of this work give promise of its wider use as a base for further development to improve the physiochemical and mechanical properties of the material.

INTRODUCTION

Portland cement (PC) was first reported in dental literature back in 1878 when a case report on the use of PC as a root canal filling material was published.[1] PC-based materials have found their way to dental applications such as vital pulp therapy, root-end filling, perforation repair, apexification, resorption repair, and root canal obturation to name but a few.[23] The initial interest in the use of PC-based materials was based on its hydraulic nature – setting by hydration with water which is of major clinical significance as many dental materials will deteriorate or become ineffective because of troublesome moisture. Consequently, a modified PC was introduced as an endodontic material and was patented as mineral trioxide aggregate (MTA) by Torabinejad at Loma Linda University in the 1990s. It received acceptance by the US Federal Drug Administration in 1998 and became commercially available as ProRoot MTA (Tulsa Dental Products, Tulsa, OK, USA).[3]

The physicochemical basis for the biological properties of PC-based materials has been attributed to the production of hydroxyapatite when the calcium ions released by the material came into contact with tissue fluid.[4] Camilleri et al.[5] showed that MTA and PC had the same constituent elements, except for the bismuth oxide present in MTA. MTA is a refined type 1 PC with a fineness in the range of 4500–4600 cm2/g and a radiopacifier (bismuth oxide) to allow for distinction from the adjacent anatomical structures.[6] Thus, on hydration, both MTA and PC would produce calcium silicate hydrate gel and calcium hydroxide. This would explain the similar mode of tissue reaction to PC-based materials and calcium hydroxide.[578] Despite the favorable biological properties of PC-based materials, there were several concerns associated with its heavy metal content and difficult manipulation and placement into target sites. This is also aggravated by the fact that MTA and PC have a grainy consistency when freshly mixed and require a long period of time until they fully set which collectively limit their clinical suitability for many dental procedures. As regards to the use of bismuth oxide as a radiopacifier, it has been demonstrated that bismuth ions have the potential to interfere with the hydration reaction of the cement by hindering the replacement of silicon from the calcium silicate hydrate structure.[8] In fact, some reports have linked bismuth oxide with tooth discoloration,[9] cytotoxicity,[101112] and reduced physical properties.[13]

Given the clinical impact of these problems, further advances in our understanding of the physiochemical nature of PC-based materials and the subsequent modifications of PC in its raw state are sorely needed and would serve as a basis for researchers to develop novel PC-based formulations with superior biological and physiochemical properties.

The aim of this work is to ascertain the biocompatibility of Jordanian PC in its raw state and explore its potential for possible future modification and reinforcement.

MATERIALS AND METHODS

Four PC-based materials were tested in the present study, namely:

ProRoot® MTA tooth-colored (white-MTA [W-MTA]) (Dentsply-Tulsa Dental, Johnson city, TN, USA)

ProRoot® MTA regular (gray MTA [G-MTA]) (Dentsply-Tulsa Dental, Johnson city, TN, USA)

White PC (W-PC) (Arab Company for White Cement Industry, Amman, Jordan)

Gray PC (G-PC) (Jordan Lafarge Cement Factories Company, Amman, Jordan).

Table 1 represents the constituents of both variants of Jordanian PC (gray and white).

Table 1

The constituents of Jordanian Portland cement

	W-PC (%)	G-PC (%)
CaO	66.1	63.4
SiO2	22.92	20.01
Al2O3	4.2	4.9
MgO	<0.42	2.7
SO3	2.35	2.98
Cl−	<0.11	0.02
FeO	<0.30	3.22
K2O	<0.11	0.82

Data derived from safety data sheets of W-PC (MSDS#1651) and G-PC (MSDS#11876). W-PC: White Portland cement, G-PC: Gray Portland cement

Preparation of material specimens

Material disks were aseptically prepared inside the laminar flow hood (ESCO, Changi, Singapore). ProRoot® MTA materials were mixed according to manufacturers' instructions while PCs were mixed to a consistency similar to MTA and placed into autoclaved plexiglass (polymethyl methacrylate transparent plastic) rings used as molds, with an inner diameter of 5 mm, and a thickness of 2 mm to produce material specimens in the form of disks. Excess material was removed using a sterile blade, and materials were left to set for 24 h. Material disks were detached from Plexiglas rings using a custom-made plugger.

Cell culture

Balb/C 3T3 fibroblasts Cells (Clone A31, European collection of cell culture, Salisburg, Wilts, UK) were routinely maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum, 5% new born calf serum, and antibiotic/antimycotic mixture (GIBCO Invitrogen Life Technologies, Grans Island, NY) at 37°C in an atmosphere of 5% CO2. They were routinely passaged by trypsinization.

Cellular response to the material disks

The measurement of cell proliferation in the presence of the test materials was performed using 24-well culture plates, each well having a culture insert with a porous bottom (3 μm pore size) (Nunc, Roskilde, Denmark). Balb/C-3T3 fibroblasts (500 μl) were seeded at a concentration of 5 × 105 cells/ml onto the 24-well culture plate, and then, a culture plate insert with one disk of test material was placed in each well for an incubation period of 24 h at 37°C. Cell culture inserts containing Teflon disks served as controls.

Directly after the end of the incubation period, the culture plate insert and the disk contained within were removed using sterile forceps and the insert containing the disk placed into another 24-well culture plate that had been previously seeded with Balb/C-3T3 fibroblasts (5 × 105 cells/ml) for an additional 24 h of incubation at 37°C and 5% CO2.

The effect of material disks on cell viability was evaluated by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Eight replicates were made for each group of materials.

Cellular response to the material elute

For each material, four replicates were used, each replicate was incubated in DMEM for a different time interval, namely 1 day; 2 weeks; 4 weeks; and 6 weeks. For each replicate, ten material disks were placed in a 4.5 ml tube (Nunc, Roskilde, Denmark) containing 2 ml D-MEM, and then incubated at 37°C. After the incubation period, the disks were removed using sterile forceps. For the control, four tubes of 2 ml D-MEM each were incubated for the same time intervals at 37°C. At the end of the incubation period, the contents of each vial of the elutes and the control were refrigerated until use.

The effect of the material elutes on cell viability was evaluated by means of the MTT assay. Four sets of experiments were performed, which represent the effects of 1-day, 2-week, 4-week, and 6-week elutes on cell viability. In each experiment, a 96-well culture plate was used. Each well received 100 μl of cell suspension (5 × 105 cells/ml) and 10 μl of the material elute or the medium control. Ten replicates were made for each group. Cells were incubated with the elutes for 24 h at 37°C and 5% CO2.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Following exposure of the cells to the materials and their elutes, the effect on cells was assessed using the MTT assay (Sigma Chemical Co, St. Louis, MO). Briefly, MTT (5 mg/mL in Hanks balanced salts solution) was added in amounts equal to 10% of the original volume of the cell suspension (i.e., 10 μl for the 96-well plate and 50 μl for the 24-well plate). The cells were incubated for 3 h at 37°C. The resulting formazan crystals were then dissolved by adding 100 μl of dimethyl sulfoxide (ScharlauChemie SA, Sentmenat, Spain) for the 96-well plates and 500 μl for the 24-well plates. The absorbance of the solubilized reaction products was then determined using an ELISA plate reader (Ultra Microplate Reader Elx 808 IU, Bio-Tek instruments Inc., Winooski, VT, USA) at a wavelength of 570 nm.

Leached calcium ion analysis

For each replicate, ten material disks were placed in a 4.5 ml tube (Nunc, Roskilde, Denmark) containing 2 ml D-MEM (serum free) and then incubated at 37°C for 24 h (day 1). After the incubation period, the disks were removed using sterile forceps. Following initial elution of the materials for 24 h, the disks were removed and then re-eluted in fresh serum-free media for another 24 h (day 2). After the second elution of the materials, the disks were removed again and re-eluted in fresh serum-free media for an additional 24 h (day 3). Finally, the resulting day-1, day-2, and day-3 elutes were subjected to analysis for calcium ions using ion chromatography (Dionex ICS-1000, USA).

Since the elution volume for each elution period was 2 ml, two-fifths that required for ion chromatography, only two samples from each elution period were analyzed for this work by adding 1 ml of each elute into 4 ml of diluting ultradistilled water (Advantec, Tokyo, Japan) to a final volume of 5 ml and a 1:5 dilution factor.

Statistical analysis

Absorbance values obtained for each well represent the amount of MTT reduction, which is proportional to the number of viable cells. To assess the percentage of viable cells present in each well, the absorbance values were related to those of the control. This was achieved by setting the mean absorbance of the control at 100%.

Formula 1: Percentage of viable cells calculation

Minitab software (version 15.1, Minitab Inc., State College, PA, USA) was used for the statistical analysis. The effects of the materials and their elutes on cells were compared to that of the control using the percent viability values as indicators of cell numbers, and the data were analyzed using analysis of variance (ANOVA) followed by Dunnett's test (α = 0.05) or Tukey's test (α = 0.05).

RESULTS

Experiment 1: Cellular response to material disks

On the 1st day, cells exposed to PC, whether W-PC or G-PC, showed an increase in viability while cells exposed to MTA, whether W-MTA or G-MTA, showed a reduction in viability relative to the control [Figure 1]. The increase in cell proliferation was obvious with the G-PC group (i.e., 19.5%), while the other effects were relatively minimal in other groups. Statistical analysis (ANOVA) showed highly significant effects between the groups (P < 0.001) on cell viability. Follow-up comparison by Dunnett's test (α = 0.05) showed that only G-PC was significantly different from the control (P < 0.05), while with the other groups, the difference was not statistically significant (P > 0.05).

Figure 1

Percentage of cell viability (mean ± standard deviation) relative to the control (100% viability) over 2 days for the cellular response to material disks. Bars carrying the asterisk sign (*) are significantly different atP< 0.05

On the second day, the materials had little if any effect on cell viability relative to the control, except for the G-MTA, showed a reduction in cell viability of about 8% [Figure 1]. Statistical analysis (ANOVA) showed significant differences between the groups (P = 0.013) on cell viability. Follow-up comparison by Dunnett's test (α = 0.05) showed that G-MTA was significantly different from the control (P < 0.05), while the differences of the other materials from the control were not statistically significant (P > 0.05).

Overall, there was a reduction in cell viability on day 2 relative to day 1 for all the materials tested. Statistical analysis (ANOVA) followed by Tukey test showed that a significant difference was found only in the G-PC group (P < 0.05) while for the others, the reduction in cell viability on the second day relative to the 1st day was not statistically significant (P > 0.05). Moreover, statistical analysis (ANOVA) followed by Tukey test revealed significant differences between W-MTA and G-PC, G-MTA and G-PC, and G-MTA and W-PC over the 2 days of testing (P < 0.05). However, the differences between W-MTA and W-PC, W-MTA and G-MTA, and W-PC and G-PC were not statistically significant (P > 0.05).

Experiment 2: Cellular response to material elutes

When cells were exposed to the 1-day elute of the materials, W-PC and G-PC caused an increase in the cell viability while W-MTA and G-MTA caused a reduction in the cell viability relative to the control. Cell viability mean values for all the material elutes tested for the 2-week group was reduced by a range of 2.7%–8%. Whereas, cell viability mean values for all the material elutes tested for the 4- and 6-week groups were very close to that of the control [Figure 2]. Statistical analysis (ANOVA) showed no significant differences between the groups (P > 0.05). The effects of the material, time, and their interaction were also nonsignificant (P > 0.05).

Figure 2

Percentage of cell viability (mean ± standard deviation) relative to the control (100% viability) over four time periods for the cellular response to material elutes

Leached calcium ion (Ca+2) analysis

The results of the elute analysis of calcium using ion chromatography are presented in Table 2. Each concentration represents the mean of two readings. For the day-1 elutes, the highest concentration of calcium was found with the elute of W-PC followed by G-PC, G-MTA, and W-MTA. When these materials were re-eluted in day 2, the calcium levels decreased, and the highest levels were found in the elute of G-PC followed by G-MTA, W-PC, and W-MTA. Calcium levels dropped further in the day-3 elutes of all materials and the highest levels were found in the elute of G-PC followed by W-PC, G-MTA, and W-MTA.

Table 2

Leaching amounts of calcium ion (mM)

Materials	Day 1		Day 2		Day 3
	Mean	SD	Mean	SD	Mean	SD
W-MTA	0.67	0.02	0.58	0.01	0.14	0.03
G-MTA	1.03	0.01	1.21	0.01	0.27	0.03
W-PC	4.52	0.02	0.76	0.03	0.74	0.04
G-PC	2.07	0.02	1.46	0.05	1.24	0.02

SD: Standard deviation, W-MTA: White mineral trioxide aggregate, G-MTA: Gray mineral trioxide aggregate, W-PC: White Portland cement, G-PC: Gray Portland cement

Overall, the levels of calcium in elutes of PC showed higher calcium ion elution in day 1 compared to that of MTA. Moreover, the elutes of PC showed a more consistent calcium elution pattern over day 2 and day 3, in contrast to that of the elutes of MTA which showed an obvious reduction in calcium ion elution after the second re-elution.

DISCUSSION

Novel PC-based formulations have shown some superior performance in dental applications, nevertheless, they fall short in certain aspects limiting their use to just a few clinically relevant situations. Therefore, the development of new materials and formulations that can improve these aspects is crucial and has numerous positive implications in dentistry.

PC is generally composed of three principal components: tricalcium aluminate, tricalcium silicate, and dicalcium silicate. It is widely preferred in engineering and construction applications because of its low cost and availability. Commonly, type I PC is the core material of MTA-like cements.[13] The composition of Jordanian PC is presented in Table 1. More than understanding the composition of PC, for safe clinical use, it is crucial to study its biological effects at a cellular level.

Herein, two forms of MTA and PC were investigated for their biocompatibility through their effect on cellular viability. Furthermore, the leached calcium ions from these materials were analyzed to help better understand the biological effects of the materials.

In this work, PC was found to be biocompatible and exerted a proliferative effect on Balb/C 3T3 fibroblasts. It is difficult to compare the present results with those of previous studies owing to different experimental conditions, such as cell type or culture conditions. However, MTA and PC were suggested to be equally biocompatible and share similar biological properties.[1415] Moreover, Holland et al.[7] have shown similar mechanisms of action of PC, MTA, and calcium hydroxide.

In response to material disks (experiment 1), G-PC had a proliferative effect on cells at day 1 and day 2 with a significant difference from the control at day 1. Furthermore, W-PC also induced a slight increase in proliferation of cells. However, G-MTA reduced cell viability with a significant difference from the control level at day 2, in accordance with other studies that reported that MTA exerts some level of initial cell toxicity.[1617] This initial cytotoxic effect might occur because of the high surface pH of the cements that cause denaturation of proteins in the adjacent cells and the medium.[17] Similar results were reported by De Deus et al.[14] who concluded that initially, both MTA and PC significantly inhibited cell viability and these cytotoxic effects decreased gradually over time. Saidon et al.[18] reported that as the cement set, the pH decreased, and the cell injuries subsided.

Furthermore, in response to material elutes (experiment 2), elutes of PC showed biocompatible and even proliferative effects on Balb/C 3T3 fibroblasts. The results are in agreement with a previous study which reported that exposure to PC elutes did not affect cell viability.[19] Camilleri[20] reported that cements that had been left to elute for 4 weeks resulted in poorer cell growth as compared to early stages of elution. However, this phenomenon was not reported in this work.

The cytotoxicity of PC mixed with bismuth oxide was found to be statistically higher than that of PC alone in short-term cultures, and this reduced in a time-dependent manner. However, this cytotoxic effect was restored to that of raw PC in long-term cultures.[11] In contrast, in another study, addition of bismuth oxide to PC did not interfere with the biocompatibility of the material.[8] It was presumed that the high concentration of calcium ions released from the material made up for the lack of cell proliferation on bismuth oxide. However, several studies reported that bismuth has low to moderate cytotoxicity against different kinds of cells.[1012]

Calcium ions were found to leach continuously over the measurement period for all the materials tested in this work, in correspondence with the literature.[8212223] PC and MTA have abundant amounts of calcium, and the presence of this ion will play an essential role in the bioactive response of these materials in vital tissues such as pulpal and periapical tissues.[2321] It has been shown that the presence of large quantities of calcium ions in vivo can activate adenosine tri-phosphate (ATP), which plays a significant role in the mineralization process.[24] Calcium ions can also modulate osteopontin and bone morphogenetic protein-2 levels during pulp calcification.[25]

In the present study, both variants of PC leached relatively higher amounts of Ca initially on day 1 compared with day 2 and day 3 for the same material. In other words, the values for calcium ion release from PC were higher during the 1st day and tended to subsequently decrease. This result reflected the conclusion of Duarte et al.[23] that slightly higher values of calcium were recorded during the initial setting period. This is also in accordance with the findings of Camilleri et al.[10] who reported that calcium leached at a fast rate initially and then reduced in level with time. This fluctuation was also observed by Bozeman et al.[22] On the other hand, both variants of MTA leached comparable calcium levels throughout the first 2 days of elution before leaching lesser amounts on day 3. It is worth mentioning, however, that the leached calcium levels from each variant of PC were always higher than those of the corresponding variants of MTA at any day of the three time points tested (i.e., white vs. white and gray vs. gray).

The results revealed a higher initial calcium ion release for PC than for MTA. These higher values can probably be attributed to the higher content of calcium-releasing products in raw Jordanian PC than in ProRoot MTA. These findings are in contrast to those of Camilleri[8] who reported that greater levels of calcium ions were leached from MTA compared to those of raw British W-PC. The proliferative effect of MTA and PC may be due to their release of abundant and continuous amounts of calcium ions, which in turn interact with phosphate groups in the surrounding tissue fluid to form hydroxyapatite crystals on their surfaces.[4]

In view of the results of our experiment, PC overall showed more cellular proliferative effects compared to MTA, especially at early time periods. This however might be attributed to the absence of bismuth oxide in raw PC. The results from our study support the idea that raw Jordanian PC has the potential to be used in clinical situations similar to those in which MTA is being used.

CONCLUSION

Within the limitations of this study, we report that raw Jordanian PC is a biocompatible material to be used for various dental clinical applications, provided it gets modified and/or reinforced on a micro- or nano-scale to meet the requirement of being radiopaque and probably enhance its physiochemical properties while maintaining the favorable biological properties reported herein. Further work is required (and planned to be undertaken in the future) based on the findings of this study.

Financial support and sponsorship

This study was financially supported by Deanship of Research at Jordan University of Science and Technology.

Conflicts of interest

There are no conflicts of interest.