Abstract
Background/purpose
Fortilin is a multi-functional protein involved in several cellular processes. It has been shown promising potential to be a bioactive molecule that can be incorporated in the dental materials. This study aimed to compare the biocompatibility and mineralization activities of modified glass ionomer cement (Bio-GIC) and Biodentine by direct and indirect method on human dental pulp stem cells (hDPSCs).
Materials and methods
Conventional glass ionomer cement (GIC), Bio-GIC (GIC supplemented with chitosan, tricalcium phosphate, and recombinant fortilin from Fenneropenaeus merguiensis), and Biodentine were examined in this study. Recombinant fortilin was purified and tested for its cytotoxicity by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Human DPSCs were treated with different material eluate for particular time intervals. At given time points, viability of hDPSCs was examined using MTT assay and calcium deposition was assessed by Alizarin red staining assay. Comparisons of the data among groups were analyzed by analysis of variance and Tukey's multiple comparisons.
Results
All test materials demonstrated no cytotoxicity. In addition, Bio-GIC promoted cell proliferation at 72 h. For direct and indirect method, cells treated with Bio-GIC demonstrated significantly higher calcium deposition than other groups (P < 0.05).
Conclusion
Bio-GIC and Biodentine are not cytotoxic to hDPSCs. Bio-GIC demonstrates enhanced calcium deposition comparable to Biodentine. Bio-GIC may be further developed as a bioactive material for dentin regeneration.
Keywords: Fortilin, Cytotoxicity, Mineralization, Dental materials
Introduction
Dental pulp exposure can be generated by several causes including caries progression, cavity preparation, and trauma. To preserve pulp vitality, direct pulp capping or pulpotomy is the treatment of choice. The materials used for direct pulp capping include calcium hydroxide, resin-modified glass ionomer cement, and Biodentine.1 Calcium hydroxide has been considered as the gold standard for direct pulp capping.1,2 It has several disadvantages such as poor sealing ability and induction of porous dentin formation.2 There have been numerous studies focusing on development of bioactive materials that induce mineralized tissue formation. Many studies utilized tricalcium silicate-based cements and demonstrated their good bioactivity. Biodentine is a calcium-silicate material that has been shown several biological activities including capacity to induce proliferation and differentiation of dental pulp stem cells.1 However, Biodentine exhibits low radiopacity as a primary pitfall.1
In deep carious lesion that dental pulp is nearly exposed, indirect pulp capping is usually performed to avoid pulp exposure. Under certain circumstances, the affected dentin remained in the cavity as a minimally invasive approach. Calcium hydroxide and glass ionomer cement (GIC) are the materials of choice for indirect pulp capping.1 GIC is composed of fluoroaluminosilicate glass and polyalkenoic acids.3 In addition to its application as a base/liner, GIC is used for filling, luting, and core build-up. GIC provides several beneficial properties including biocompatibility, fluoride release, and chemical bonding with tooth structure.4
Many studies aimed at development of bioactive GIC with improved biocompatibility and mineralization property. Previous research has shown that resin-modified GIC supplemented wtih chitosan and fortilin demonstrated less cytotoxicity and cytoprotective effect.5 Chitosan is a copolymer derived from deacetylation of chitin, which is a natural polymer found in the exoskeleton of crustaceans. It has been widely used in biomedical applications such as drug delivery and scaffold for tissue engineering.6 An in vitro study demonstrated that chitosan can promote proliferation and osteogenic differentiation of dental pulp cells.7 Fortilin, also known as translationally controlled tumor protein (TCTP), is a highly conserved protein with multiple functions. Fortilin is involved in many fundamental cell activities, including proliferation, maturation, anti-apoptosis, and disease processes.8 It was shown to protect cells from diverse stress conditions.9 Tricalcium phosphate (TCP) is a compound that has been widely used to enhance mineralization in many materials. Culture of human dental pulp stem cells in TCP scaffold showed induction of odontogenic differentiation.10
The aim of this study was to compare the cytotoxicity and mineralization promotion of the modified GIC, which is supplemented with fortilin, chitosan, and TCP compared to Biodentine on human dental pulp stem cells (hDPSCs).
Materials and methods
Human DPSCs isolation and cultivation
Human DPSCs were obtained from freshly extracted third molars without caries of adults aged from 18 to 25 years at Faculty of Dentistry, Prince of Songkla University. The study protocol was approved by the Research Ethics Committee (code no. EC6401-002) and all subjects gave informed consent. The isolation of pulp cells was performed by enzymatic digestion and the isolated cells were cultured as previously described.11 The cells between the 2nd and 5th passages were used in all experiments for this study.
Immunophenotype analysis by flow cytometry
The immunophenotypic characteristics of hDPSCs were evaluated in accordance with the International Society for Cellular Therapy protocols.12 Human DPSCs suspension (n = 4) at density of 1 x 107 cells/mL were incubated with FITC-conjugated with anti-human CD34, CD45, CD73, CD90, CD105, and IgG1 (Beckman coulter, Marseille, France) in the dark at 25 °C for 20 min. The samples were subsequently washed and analyzed by flow cytometer (Beckman Coulter, Life sciences, IN, USA) using CytExpert software.
Expression and purification of a recombinant fortilin from Fenneropenaeus merguiensis (Fm-Fortilin)
The Fm-Fortilin gene derived from F. merguiensis was cloned and expressed in Escherichia coli (E. coli) strain BL21 (DE3) following the method previously described.13 Briefly, E. coli strain BL21 (DE3) harboring pET29a-Fm-Fortilin was inoculated into 10 mL of Luria–Bertani (LB) medium containing 30 μg/mL kanamycin. After incubation for 16–18 h, the culture was grown in 500 mL of LB medium containing kanamycin until the cell density reached optical density (OD) at 600 nm = 0.6. The culture was subsequently induced by 0.4 mM IPTG (isopropyl β-D-thiogalactopyronositol). After 24 h, the cells were collected and resuspended in lysis buffer. After placed on ice for 30 min, the cells were sonicated 10 times for 30 s and centrifuged at 10,000×g at 4 °C for 20 min. The protein was purified by the AKTA start protein purification system (Merck Darmstadt, Germany) using chromatography HiTrap-QFF column (GE Healthcare, Barcelona, Spain). The total concentration of purified protein was measured using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA).
Specimen preparation
The materials tested in this study were GC (GC Gold Label Luting & Lining Cement, Tokyo, Japan; batch no. 1811151) and Biodentine (Septodont, St. Maur des Fosses, France; batch no. B25581). For cell viability test and Alizarin red staining assay, the disk specimens were fabricated using split-ring Teflon molds (5 mm diameter and 1 mm thickness). The specimens were prepared according to the manufacturer's instruction. Fm-Fortilin was added during mixing the powder and the liquid. Specimens were kept at 37 °C for 1 h. For Biodentine, five drops of liquid phase were added into powder phase in the capsule before mixing with amalgamator at 4000 rpm for 30 s. All specimens were prepared with aseptic technique in a laminar flow hood.
In this study, specimens were divided into three groups according to different formulations. The modified GIC was designated as Bio-GIC. The compositions of each test material are presented in Table 1.
Table 1.
Compositions of the powder and liquid of test materials.
| Group of specimens | Composition |
|
|---|---|---|
| Powder (%w/w) | Liquid | |
| GIC | Fluoro-aluminosilicate glass (95%) and polyacrylic acid (5%) | Polyacrylic acid and distilled water |
| Bio-GIC | Fluoro-aluminosilicate glass, polyacrylic acid (79.95%), chitosan (15%), BSA (5%), and TCP (0.05%). | Polyacrylic acid, distilled water, and 1 μg Fm-Fortilin |
| Biodentine | Tricalcium silicate, zirconium oxide, calcium oxide, calcium carbonate, and colorings | Calcium chloride and polycarboxylate |
Cytotoxicity effect of Fm-Fortilin on hDPSCs
The effect of Fm-Fortilin on hDPSCs viability was investigated by 3-(4,5-dimethylthaiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Human DPSCs at a density of 1 x 104 cells/well were cultured in 96-well plates at 37 °C in a 5% CO2 humidified incubator. After 24 h, the cells were treated with various concentrations of Fm-Fortilin (ranging from 1 ng/mL to 20 μg/mL) for 24 and 72 h. At given time points, 200 μL of fresh medium containing 10 mM HEPES and 50 μL of MTT solution was replaced to each well and then incubated at 37 °C in the dark for 4 h. The formazan crystals were dissolved in 200 μL of DMSO and 25 μL of Sorensen's glycine buffer (0.1 M glycine and 0.1 M NaCl, pH 10.5). The OD was read at 570 nm by a microplate reader (Biochrom, Holliston, MA, USA). The data was presented as the percentage of cell viability as follows:
| Percentage of cell viability = [OD570 treated cells]/[OD570 control cells] × 100. |
Cytotoxicity of test materials
The viability of hDPSCs treated with different material eluates was investigated using the MTT assay. The disk specimen was immersed in 1 mL of α-minimum essential medium (α-MEM). The eluate was collected at 24, 48, and 72 h (n = 4 for each time point) and the material was transferred to a new α-MEM. The eluates were then filtered with a 0.45 μm membrane and diluted with 2× supplemented α-MEM. Human DPSCs were seeded at a density of 5 × 103 cells/well in 96-well plates. After 24 h, the cells were treated with 1× diluted eluate for 24 h. The MTT assay was performed as described above.
Alizarin red staining (ARS) assay
The effect of test materials on mineralization of hDPSCs was assessed by direct and indirect methods. In the direct method, hDPSCs were seeded in a 6-well plate at a density of 5.0 x 103 cells/well. After 24 h, the medium was replaced with inductive medium, which composed of 0.05 mM ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone, and 100 mg/mL antibiotic-antimycotic in α-MEM supplemented with 10% FBS. The specimens were then placed in contact directly with the cells (n = 4). For the Transwell method, hDPSCs at a density of 3.0 x 103 cells/well were seeded on the lower part of the 12-well Transwell plate with pore size of 3.0 μm. After 24 h, the culture medium was replaced with inductive medium and each specimen was placed on the upper part of the Transwell plate (n = 4). The medium was refreshed every 3 days. After 14, 21, and 28 days, the cells were stained with 40 mM Alizarin red and incubated for 30 min. For quantification, the Alizarin red was solubilized with 500 μL of 0.1 g/mL cetylpyridinium chloride (CPC) in 10 mM Na2HPO4 pH 7.0. The absorbance was measured with a microplate reader at OD 550 nm.
Statistical analysis
Data were presented as mean ± standard deviation (SD). One-way repeated ANOVA with Tukey's multiple comparisons was used to determine the differences between the experimental groups. Two-way repeated ANOVA and Tukey's multiple comparisons were used to analyze the results of ARS staining assay. The statistical significance level was defined as P < 0.05.
Results
Characterization of hDPSCs
Cells derived from dental pulp exhibited a fibroblast-like morphology (Fig. 1A).
Figure 1.
Morphology and flow analysis of hDPSCs. (A) hDPSCs formed a monolayer of fibroblast-like cells, scale bar = 200 μm. (B) Expression of MSC surface markers. (C) Expression of hematopoietic stem cell surface markers.
Flow cytometry analysis revealed that the cells demonstrated positive expression for mesenchymal stem cell (MSC) markers; CD73, CD90, and CD105 (Fig. 1B) and negative expression for CD34 and CD45, which are hematopoietic stem cell markers (Fig. 1C).
Effect of Fm-Fortilin on hDPSCs viability
Different concentrations of Fm-Fortilin (1 ng/mL-1 μg/mL) were not cytotoxic to hDPSCs after exposure for 24 and 72 h (Fig. 2). At 24 h, Fm-Fortilin at concentrations of 10 μg/mL and 20 μg/mL had significantly lower cell viability than the control group (P < 0.05). On the contrary, all concentrations of Fm-Fortilin exhibited no cytotoxicity and appeared to promote cell proliferation at 72 h. Furthermore, Fm-Fortilin at concentration of 20 μg/mL showed significantly higher cell viability (P < 0.05) than the control group. Fm-Fortilin at concentration of 1 ng/mL was the lowest concentration that had no cytotoxicity and promoted cell proliferation. Therefore, this concentration was selected to perform further experiments.
Figure 2.
Effect of various concentrations of Fm-Fortilin on hDPSCs viability. The results were represented as mean ± SD. ∗ indicates significant difference compared to control (P < 0.05). One-way ANOVA with Tukey's multiple comparisons.
Cytotoxicity of test materials
All test materials demonstrated no cytotoxicity at all time points investigated (Fig. 3). Furthermore, the Bio-GIC group showed significantly higher cell viability at 72 h (P < 0.05).
Figure 3.
Effect of different test materials on hDPSCs viability. The data were presented as mean ± SD. ∗ indicates significant difference compared to control. One-way ANOVA with Tukey's multiple comparisons.
Effect of calcium deposition of different materials on hDPSCs
For the direct method, the level of calcium deposit of hDPSCs treated with Bio-GIC and Biodentine was significantly higher than GIC (P < 0.05) (Fig. 4A). In addition, the calcium deposit in the Bio-GIC group markedly increased on days 21 and 28 compared to day 14 (P < 0.05), whereas the Biodentine group showed comparable level on days 21 and 28.
Figure 4.
ARS assay (A) Direct method and (B) Transwell method. The data were presented as mean ± SD. Different letter indicates significant difference between groups (two-way ANOVA with Tukey's multiple comparisons). ∗ indicates significant difference within a group at each time point (one-way ANOVA with Tukey's multiple comparisons).
For the transwell method, hDPSCs treated with Bio-GIC demonstrated significantly higher calcium deposit than other groups (P < 0.05) (Fig. 4B). The level of calcium deposit in the Bio-GIC group significantly increased on each time interval. In contrast, the calcium deposit in the Biodentine group showed highest level on day 14 and significantly decreased on days 21 and 28. In addition, the calcium deposit in the Biodentine group was not statistically different from those in the GIC group.
Discussion
The induction of dentin regeneration is one of the important properties of materials used for direct and indirect pulp capping. Stem cells derived from dental pulp tissue are classified as mesenchymal stem cells. In this study, the morphology of cells isolated from dental pulp was spindle-shaped. The identification of cell surface markers revealed that the cells exhibited mesenchymal stem cell markers (CD73, CD90, and CD105) and did not display hematopoietic markers (CD34 and CD45).
In the present study, fortilin is a recombinant protein that has potential to function as a bioactive molecule incorporated in the dental materials. Fortilin is a multifunctional protein that has an anti-apoptotic function and promotes cell proliferation.14 Fm-Fortilin at various concentrations was not cytotoxic to hDPSCs. Moreover, it promoted cell proliferation after treatment for 72 h, which is consistent with the previous study.11
During dental restoration, residual components released from restorative materials may diffuse into the pulp tissue underneath. In this study the eluate from the test material was used to investigate the toxicity and the proliferative effect on hDPSCs. According to ISO standard, cell viability should be evaluated on three time intervals (24, 48, and 72 h).15 The results revealed that all test material eluate were not cytotoxic to hDPSCs at all times investigated. Basically, the essential components of GIC powder contains metal ions and glass particles such as silica, which are neither toxic nor irritate to living cells.16 Although the mechanism of GIC setting is an acid–base reaction, the powder/liquid ratio is well proportioned, thus rendering low residual acid release. The proliferative effect of Bio-GIC is in agreement with previous study that reported higher proliferation of osteoblasts treated with GIC supplemented with Pmer-TCTP and chitosan.17 Chitosan was shown to enhance proliferation of dental pulp cells.7 The proliferative effect of Bio-GIC may be attributed to the addition of chitosan and Fm-Fortilin at suitable ratio.
There have been many studies on the effect of Biodentine on cell viability, proliferation, and differentiation.18 Biodentine has been developed to improve physical and biological properties for many years. It has wide range of applications and can be used as a dentine substitute.18 In addition to its biocompatibility, Biodentine was shown to promote proliferation, migration, and adhesion of hDPSCs.19 A clinical study reported that Biodentine induced dentin bridge formation with well-organized odontoblast-like cells.20 In the present study, we examined the calcium deposition of hDPSCs via direct and indirect culture with different test materials for 14, 21, and 28 days. For direct method, the GIC and Bio-GIC groups enhanced the mineral deposit in a similar pattern, whereas Biodentine showed the highest calcium deposition on days 21 and 28. Furthermore, Biodentine appeared to promote mineralization at earlier stage than other groups. The possible explanation is that the setting mechanism of Biodentine is a hydration reaction that provides by-products such as calcium hydroxide.21 In addition, Biodentine demonstrated high amount of calcium ion release, which subsequently enhance mineralization in hDPSCs.21,22 Therefore, Biodentine can rapidly induce mineralization than other test materials. Bio-GIC demonstrated the highest calcium deposition on day 28 in both direct and indirect methods. These findings are consistent with previous studies that reported mineralization enhanced by materials supplemented with chitosan and fortilin.5,17 In addition to these two bioactive components, TCP has been shown to be involved in promoting cell mineralization as well. Previous study reported that β-TCP reduced inflammatory cytokines releases and foster osteogenic differentiation of bone marrow stem cells (BMSCs) and osteosarcoma cells (SaOS-2).23 The induction of mineral formation is probably due to combinatorial effect of fortilin, chitosan, and TCP.
The condition in transwell-based model was designed to partially mimic the in vivo condition, in which the substances from the materials leach into the surrounding tissue. However, this model cannot fully simulate the microenvironment in pathological condition. There are numerous factors involved in the cell–biomaterial interaction including inflammatory response, endogenous growth factors, and vascularization. Further studies may investigate on molecular events involved in cell-modified GIC interaction and capability to promote mineralization in animal model.
Within the limitation of this in vitro study, Bio-GIC is not cytotoxic and can induce cell proliferation and mineralization comparable to Biodentine. This modified GIC showed promising potential to develop as an alternative biomaterial for dentin regeneration.
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by the Program Management Unit for Competitiveness (PMUC), Eagle Dream, Co., Ltd., Postgraduate unit, Faculty of Dentistry, Prince of Songkla University, as well as Graduate School, Prince of Songkla University. The authors would like to thank Professor Peter A. Leggat, James Cook University, for critical reading and editing this manuscript.
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