Cellular interaction and antibacterial efficacy of two hydraulic calcium silicate-based cements: Cell-dependent model.

BACKGROUND: This study investigated cytotoxic probability, osteogenic potential, and antibacterial efficacy of two pulp-capping hydraulic calcium-silicate cements.
MATERIALS AND METHODS: For osteogenic potential and cytotoxicity evaluation, mesenchymal stem cells (MSCs) and materials disc-shaped specimens were used. Increase or decrease in a number of proliferating MSCs was calculated after three intervals. Alkaline phosphatase (ALP) levels in osteogenic media were normalized to the total protein content of cells and measured spectrophotometrically. Antibacterial efficiency through growth curves of Streptococcus mutans in direct contact with tested materials.
RESULTS: Biodentine showed the highest number of proliferating MSCs (278000.41 ± 4000.06, after 72 h) and the highest concentration of ALP after 12 days (209.26 ± 7.17 μU/μg protein). It showed the lowest slope (0.003 ± 0.0005) of S. mutans strains growth curves after 18 h.
CONCLUSION: Biodentine proved a highly significant osteogenic ability and gave a significant reduction of S. mutans growth.

INTRODUCTION

The hydraulic calcium-silicate cements (HCSCs) have got the trust of a large number of dental clinicians who have used them for various pulp-related and endodontic indications despite their limited clinical and first in vitro studies. Currently, the use of these cements for dentine remineralization technology is much closer to practical application.[1] That puts more emphasis on investigating their cellular interactions and biological responses. In particular, to study the freshly mixed and set cements cellular interactions that are closer to clinical situations[2] rather than investigating their cellular interaction within longer time after setting.[3] Previous studies have used different types of living cells such as dental pulp stem cells and dental pulp stromal cell,[4] human orofacial bone mesenchymal stem cells (MSCs),[5] and human bone marrow stem cells.[6] As a result of limited availability of dental stem cells,[7] it is more important to find richer resources of stem cells from which they can be more easily obtained. Human adult bone marrow MSCs are regarded as an easier source which has the ability to differentiate into multilineage cells such as osteoblasts, chondrocytes, and adipocytes.[8] As the cell extraction procedures may be complicated and constricted when dealing with humans, animal-derived marrow MSCs may be regarded as easier way to extract and reliable for differentiation to osteogenic cells as well.[9] Two of the recent and commonly used HCSCs are, first, biodentine (Septodont, St. Maur des Fosses, France), which is an interesting calcium-silicate material with high biocompatibility.[10] However, its bond strength with dentin and in combination with composite resin may be unfavorable due to surface erosion and microleakage that occurs after it is etched with 37% phosphoric acid.[11] The other type is TheraCal (Bisco Inc, Schaumburg, IL, USA), which is a light-curable HCSC formed of Portland cement in a methacrylate resin. It releases calcium ions and sets in few seconds after exposure to light-curing units.[12] Although it was efficiently cytotoxic to human dental pulp cells,[13] a more recent study reported that it had low cytotoxicity in comparison to mineral trioxide aggregate-derived products.[14] They showed antibacterial efficiency against various microorganisms using disc-diffusion evaluations.[15] These HCSCs were reported to be used for pulp-capping purpose. As a result, the present study, in Part I, attempts to evaluate the cellular interaction including cytotoxicity and remineralization ability through the osteogenic capacity of freshly set mix of these materials. In addition, it evaluates their antibacterial effectiveness in a direct contact with the tested bacterial strains. The null hypothesis is that no difference between the two HCSCs within the interaction of cell viability and osteogenic differentiation as well as no difference in their antibacterial effect against Streptococcus mutans.

MATERIALS AND METHODS

Methodology

Materials used in the present study are summarized in Table 1.

Table 1

Materials used in the present study

Material	Composition	Manufacturer
Biodentine	Powder: Tricalcium silicate, calcium carbonate, and zirconium oxide, iron Liquid: Water, calcium chloride (accelerator), and a hydrosoluble polymer (modified polycarboxylate) that serves as a water-reducing agent	Septodont, St. Maur des Fossis, France Batch No. B16479
Theracal LC	Light-curing single paste: Resin (Bis-GMA and PEGD) modified calcium silicate, filled with CaO, calcium silicate particles (Type III Portland cement), Sr glass, fumed silica, barium sulfate, and barium zirconate	Bisco Inc, Schaumburg, IL, USA Batch No. H-33014P

Bis-GMA: Bisphenol A-glycidyl methacrylate, PEGD: Polyethylene glycol dimethacrylate

Cell proliferation assay

Materials' cytotoxicity was assisted using cell counting proliferation assay after three intervals (24, 48, and 72 h).[14] For the assay, 24 disc-shaped specimens of both HCSCs (12 per each material) using a split Teflon mold 10-mm diameter and 1-mm thickness were prepared. MSCs were obtained from Medical Experimental Research Centre. They were harvested from Sprague-Dawley female rat bones marrow, subcultured till reaching 70%–80% confluence, and cells of passages 3–4 were used for the experiment. The proliferating cell counting was done by a hemocytometer after cells' trypsinization with trypan blue dye and using a phase-contrast-inverted microscope (Olympus America Inc., Pennsylvania USA). The cell viability evaluation depends on the concept that viable cells do not take up impermeable dyes (trypan blue); however, dead cells are permeable and take up the dye so that viable cell count can be verified. Freshly mixed and prepared disc-shaped specimens were used directly after their setting. They were disinfected using 1% antibiotics (penicillin and streptomycin)/antimycotic solution for 2 h then washed two times with phosphate-buffered saline (PBS). Six-well plates were used to incubate a large number of (approximately 150,000) cells/well in direct contact with material specimens plus a control group (MSCs without tested materials). Material cell incubation was done in Roswell Park Memorial Institute medium with 10% fetal bovine serum (FBS) (Invitrogen Cat. No. 12440046) in addition to 1% penicillin, 1% streptomycin, and 2 mM glutamine (Lonza, Basel, Switzerland) under laminar air-flow, vertical Class II biosafety cabinet, and 37°C and CO2 cell culture incubator for 72 h.

Alkaline phosphatase activity and alizarin red staining

The ability of the tested cements discs (24 specimens) to induce mineralized tissue formation through MSCs differentiation was evaluated through alkaline phosphatase (ALP) activity. Before being seeded with the cells, prepared discs of the tested materials were placed in a six-well culture plate and disinfected by the same way for cell proliferation assay. After disinfection, they were washed twice with PBS and finally prewet with culture media. Afterward, specimens received a number of 3 × 104 MSCs in each 1 mL of osteogenic media with composition of Dulbecco's Modified Eagle's medium, 10% FBS, 1% penicillin, 1% streptomycin, and 2 mM glutamine (Lonza, Basel, Switzerland), 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, Germany). A negative control cell group with the previous media and without the tested materials was also used. The cells were allowed to be in a direct contact with the material and incubated for 8 h before observation. The culture medium and the osteogenic supplements were refreshed every 2 days. After culturing for 12 days, cells were washed with PBS and fixed in 10% (v/v) formaldehyde (Sigma, USA). After 15 min, 2% alizarin red solution at pH 4.1 was added to each well.[16] The cells were incubated at room temperature for 20 min and then washed four times with distilled water for 5 min. Digital pictures of the proliferated cells at the surrounding area of the specimens were taken using an inverted light microscope (Olympus America Inc., Pennsylvania USA). Images were taken with magnifications ×4 and ×10.

For ALP activity, fixed cells within the wells were trypsinized then resuspended in 100–200 μL of the lysis buffer, and centrifuged for 15 min at 2000 ×g. The ALP activity was assayed utilizing the conversion of a colorless p-nitrophenyl phosphate to a colored p-nitrophenol according to the manufacturer's protocol (Sigma, St. Louis, MO, USA). The color change was measured spectrophotometrically at 405 nm (Spectra III, TECAN, Austria), and the ALP levels were normalized to the total protein content of cells measured by bicinchoninic acid assay kit (Sigma, St. Louis, MO, USA) according to the company's guidelines. The results were expressed in μU (micro unit) of yellow p-nitrophenol per μg of protein.

Antibacterial activity with direct contact test

The concept of direct contact test depends on measuring the turbidity associated with bacterial growth in 96-well microtiter plates.[17] S. mutans (ATCC 25175) was cultured in 5 ml of brain-heart infusion broth (BHI) (Oxoid, England) and incubated overnight at 37°C. Before testing, bacterial culture was centrifuged at 3000 rpm for 10 min, and cells were resuspended in fresh media to an optical density (OD) at 650 nm (OD650) of 0.5. Then, the suspension was 10-fold serially diluted and plated on BHI agar to determine colony-forming units (CFU). In a 96-well microtiter plate (Nunc, Maxisorp, Denmark), each row was divided into three sets (first, middle, and last) each of four adjacent wells. At the time of testing, “Biodentine” capsules were mixed according to manufacturer's instructions; sidewalls of the first and the last sets were coated evenly with an equal amount of tested materials while holding the plate vertically. The visible light-emitting diode curing unit Lite Q LD-107 (Monitex Industrial Co., LTD, New Taipei, Taiwan) was used to cure the Theracal LC according to manufacturer's instructions. Then, 10 μl of the bacterial suspension (106 CFU) was placed on each well of the first set in each row and incubated for 1 h at 37°C while the plate is still in a vertical position to ensure the direct contact between the bacteria and the tested materials. After that, 245 μl of BHI broth was added to each well of the first set and mixed well. Then, 15 μl was transferred from the wells of the first set to the adjacent middle set of four wells containing 215 μl of fresh BHI broth, respectively. The last set received 230 μl of uninoculated BHI broth and served as negative control. The positive control was included in a separate row in the same microtiter plate, which consisted of two sets of uncoated wells, that were treated the same as the first and middle experimental sets. The bacterial outgrowth was estimated after direct contact with the tested material on the basis of the changes in the readings of OD650, which were recorded by the spectrophotometer (ELISA microtiter plate reader model 608, Bio-Rad) every 2 h for 18 h at 37°C. The negative control wells values were considered as the baseline as they were subtracted from the respective experimental data. A regression line was calculated using the ascending linear portion of the curves for each well.

Statistical analysis

Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA) for Windows. One-way analysis of variance (ANOVA) was conducted to assess ALP activity and bacterial growth curve slopes of both hydraulic calcium-silicate dental cements. Two-way ANOVA was used to analyze viable cellular counts after the three intervals. Before that, data were assessed for normality using Shapiro–Wilk test. Post hoc Tukey's test was used afterward. Statistical significance was determined at P ≤ 0.05. Results were expressed as mean ± standard deviation.

RESULTS

Cell proliferation number assay

Factorial ANOVA showed that there was a significant impact (P ≤ 0.05) of variables, material groups, and time intervals, on the number of viable MSCs after the three intervals of counting. The highest mean values of viable MSCs were for biodentine (277,350 ± 7000) after 72 h of incubation. On the other hand, Theracal showed a statistically significant decrease in the number of viable MSCs (119,000 ± 8000) than the control group after 72 h of incubation denoting cytotoxic effect in direct contact with the cells. Figure 1a shows comparison among all groups mean values after 24, 48, and 72 h of incubation. Post hoc Tukey's test results are represented as letters for each column.

Figure 1

Bar charts showing a comparison for (a) mesenchymal stem cells proliferating No. among all groups after periods of 24, 48, and 72 h of incubation, (b) alkaline phosphatase activity μU/μg protein) after a period of incubation for 12 days. Values are expressed as means ± standard deviations (as error bars) in addition to mesenchymal stem cells (negative control). The mean values were statistically significant at P≤ 0.05. The letters are for Tukey's test

Alkaline phosphatase activity evaluation

After a period of 12 days of incubation with MSCs in osteogenic media, the results showed a statistically significant difference between tested groups (P ≤ 0.05). Biodentine showed a statistically significant higher ALP values (209.26 ± 11.17 μU/μg protein) than Theracal, which indicates a higher ability to form mineralized structure. The bar chart in Figure 1b shows a comparison of ALP activity of all tested groups after the complete period of incubation with MSCs. Inverted light microscope images reveal difference in MSCs morphology and mineral deposits formation among groups [Figure 2a–c]. Control group displayed irregular cells and evident mineralized nodule whereas Theracal group disclosed ill-defined cells with no obvious mineral deposits. Biodentine showed clustered cells with apparent minute mineral deposits.

Figure 2

Inverted light microscope images showing flatter and irregular shape of mesenchymal stem cells and the appearance of calcium deposits: (a) Control, (b) Theracal, and (c) Biodentine, after incubation of 12 days in osteogenic media

Antibacterial activity with direct contact test

The bacterial growth curves of both tested HCSCs and positive control group, after total period of incubation with bacteria, are shown in Figure 3. The curves are representative of the average of OD values for four wells of each tested group over the 18 h. There was a statistically significant difference between the control groups with the two tested cements. Biodentine showed the lowest mean value for the growth curve slope (0.005 ± 0.0005) with no statistical significant difference in comparison to the curve slope mean value of Theracal [Table 2].

Figure 3

Streptococcus mutans growth curves of different groups after being incubated in direct contact with the tested pulp-capping materials for 18 h

Table 2

Means and standard deviations of bacterial growth rate indicated by the slope values of linear portion of growth curves

Group	Mean value (slope of linear potion of curve)	F	P
Positive control	0.037±0.0025A	473.191	≤0.05
Biodentine	0.003±0.0005B
Theracal	0.005±0.0005B

P≤0.05 means there is a statistically significant difference, and the letters are for Tukey’s test. SD: Standard deviation

DISCUSSION

This study aims to comprehensively evaluate cellular interaction and antibacterial efficacy of two famous HCSCs (Biodentine and Theracal) in Part I. A successive study on their remineralizing ability using cell-independent model is in progress (Part II). The MSCs derived from rats bone marrow were used to evaluate materials cellular interaction. They have proved to have osteogenic activity with mineralized nodules formation and expression of collagen I.[9] This type of MSCs is considered of nondental origin which is a limitation as dental stem cells induce stronger osteogenic capability.[18] One of the main principles needed for successful direct pulp-capping procedures is the reliable sealing ability and occluding of exposed area.[19] This will be instantaneous with the capping material and later on with the mineralized dentine bridges formation. In addition, profound antibacterial ability, that minimizes or eliminates bacteria penetrating to the pulp, is crucial for the pulp-capping process. If the pulp-capping procedures are successful, it prevents the need for more invasive, and expensive treatment.[1920] That is why the present study focused on both tested materials' ability to induce mineralized tissue formation through their osteogenic potential and antibacterial effect. In addition, their cytotoxic effect in contact with living stem cells, simulating the condition of direct pulp capping, was evaluated. The null hypothesis was rejected, and the results of the present study showed a significant decrease in the number of viable cells within all intervals (24, 48, and 72 h) when they come into contact with Theracal. These findings agree with what was reported previously for cellular interaction of Theracal.[1314] Theracal composition contains 40%–50% by weight of Portland cement powder and 5%–10% by weight of a hydrophobic resin monomer such as Bis-GMA.[21] The main cause for cytotoxicity may be the leaching out of incompletely polymerized resin monomers which could accumulate over time to reach a cytotoxic level. The resin components can alter the lipid layer within the cell membrane and hence increase cell permeability so that it can intake the impermeable dyes such as trypan blue.[22] As biodentine lack this polymer, it showed significantly high number of viable cells than the control cell group, and despite of its reported high alkaline pH, it did not significantly affect cell proliferation.[1323] Biodentine also showed a significantly higher amount of ALP enzyme activity after 12 days of incubation in an osteogenic media indicating higher osteogenic ability. This is in agreement with previous reports that showed a significant increase of mineralized nodules when used with human dental pulp stem cells.[1323] Another important issue rather the two materials' composition difference is the heat generation occurs during curing of Theracal which was not considered and should be studied in future studies. The direct contact method was used to test the antibacterial efficacy of both materials against S. mutans instead of the disc-diffusion method used for almost all previous studies.[1415] Although the disc-diffusion method allows direct comparison between the tested microorganisms, it has several limitations. Most important of these limitations are related to the diffusion capacity of materials within agar media such as agar gel viscosity and ionic concentration of tested material in relation to the medium. The slopes of the bacterial growth curves of S. mutans showed no statistically significant difference between both tested HCSCs after 18 h as biodentine showed the lowest slope value. This disagrees with what was reported previously[1415] where Theracal had more significant antibacterial activity against S. mutans than biodentine. That may be attributed to, first, the different test method used which eliminated the agar media and allowed the bacterial strains to be in direct contact with tested material hence directly exposed to any ingredients diffusing out of the material. Second, both materials were reported to have high alkaline pH ranging between 11 and 12 during the 1st h after preparation.[24] The former may explain the significant inhibition of both HCSCs for S. mutans growth than the control group. Although Biodentine showed lower curve slope and higher inhibition to bacterial growth, there was no statistically significant difference with Theracal. That may be justified by the hydration process of tricalcium silicate materials which produce calcium-silicate hydrate gel, calcium hydroxide, and unreacted tricalcium silicate.[11] Calcium hydroxide is known to induce both antibacterial and anti-inflammatory properties by acting on the cytoplasmic membrane. They also are considered as highly oxidizing free radicals that show extreme reactivity with many biomolecules. Biodentine was reported to release higher amounts of Ca hydroxide than Theracal as revealed in previous X-ray diffraction results.[25] The Ca ions rarely diffuse away from their site of generation. Consequently, biodentine may have these reactive ions in close contact to it during the hydration period.

CONCLUSION

Based on the results and within the limitations of the present study, Biodentine gave a highly positive effect on both the proliferation of MSCs and ALP activity after incubation in direct contact with the cells than Theracal which revealed significant cytotoxic effect. Both HCSCs had the ability to significantly inhibit the S. mutans growth which is crucial for the exposed pulp situations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.