Scientific report on the effect of discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells

2021-11-24 03:22:01 By : Mr. Kent Syu

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Scientific Reports Volume 5, Article Number: 17177 (2015) Cite this article

The anti-tarnishing calcium aluminosilicate cement was developed to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic bone cement. This study tested the effect of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the different cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) Impact. Aging period. Use assays to detect plasma membrane integrity, cytosolic enzyme leakage, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity, and intracellular DNA content to check cell viability and proliferation. The results of the six tests showed that Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after 24 hours of curing, and the cytotoxicity of Quick-Set2 was relatively lower than that of WMTA at this stage. After two aging cycles, the cytotoxicity characteristics of the two hydraulic cements were not significantly different, and the cytotoxicity of the two hydraulic cements was much less than that of the positive control (zinc oxide-eugenol cement). Based on these results, it is conceivable that after the outward diffusion and removal of its cytotoxic components, it is more likely to reveal any potential beneficial effects of the anti-tarnishing calcium aluminosilicate cement on the osteogenesis of differentiated hDPSC.

Calcium silicate cement has been widely used to treat non-surgical and surgical pulp diseases, including vital pulp therapy1. Similar to the ordinary Portland cement used in the construction industry, these cements contain lime and silica as the main oxides in the form of tricalcium and dicalcium silicate. There are also small amounts of tricalcium aluminate, calcium sulfate, and calcium aluminate ferrite (dark phase)2. When reacting with water, these hydraulic cements produce amorphous calcium silicate hydrate and crystalline calcium hydroxide as the main hydration phase that binds unreacted mineral particles together to form an agglomerated structure. Calcium silicate cement designed for biomedical purposes has biocompatibility, bioactivity, and has clinically acceptable sealing properties and the ability to induce the formation of repairable hard tissues2. Their limitations include poor handling characteristics, long coagulation time, scouring during coagulation, minimal adhesion to root canal dentin, and relatively high solubility in humid environments2. Although some of these undesirable properties have been addressed in recent formulations4, none of the currently available cements have solved all of the aforementioned challenges. The main disadvantage of calcium silicate cement is that they cannot be set optimally in an acid environment5. These cements are also susceptible to erosion by acids and calcium chelating flushing agents, because the calcium hydroxide phase is quickly dissolved by these agents, thereby increasing the porosity of the cured cement6.

Calcium aluminate cement was developed in the late 19th century as an alternative to calcium silicate-based cement6. The emergence of these cements stems from the motivation to develop cements that can resist acid attack and bio-corrosion by acid produced by acid-producing bacteria7. Although calcium aluminate cements are also called hydraulic cements, they differ from calcium silicate cements in the properties of the active phase that causes setting and hardening. Calcium aluminate cement contains lime and alumina as the main oxides, with little or no silica8. The oxides combine to form calcium aluminate as the main active phase, which reacts with water to release calcium and hydroxide ions. This is followed by the precipitation of alumina hydrate and various forms of temperature-dependent calcium aluminate hydrate. After solidification, the alkalinity (pH ~10) of calcium aluminate cement is lower than that of tricalcium silicate cement8. They are more acid resistant because alumina hydrate is stable at pH values ​​as low as ~3-4. The dissolution of hydrated calcium aluminate also results in the formation of additional hydrated alumina. The latter fills the pores and protects the solidified cement from further acid attack6,9.

Because calcium aluminate bone cement has potential resistance to the dissolution of acid-producing bacteria derived from oral plaque biofilm and has potential biological activity, calcium aluminate bone cement has been used as a repair material (Doxadent; Doxa Dental AB , Uppsala, Sweden)10 and used to bond crowns and bridges when combined with dental plaque. Glass ionomer (Ceramir C&B, Dosa Dental AB) 11. Although the clinical performance of cemented cements is satisfactory11, filling materials prepared from calcium aluminate cements show an unacceptable failure rate when used for stress-bearing repairs12. Because calcium aluminate bone cements release carbonated apatite precipitation and stimulate hard tissue regeneration necessary for calcium and hydroxide ions, they are also promoted for endodontics and have the same features as calcium silicate bone cements. Indications.

It was first reported in 199113 that calcium aluminate was added to root canal cement. Subsequently, a set of endodontic cement was developed based on the patented formula of Pandolfelli et al. 14. The composition of the formula (weight percentage): Al2O3 (≥68.0), CaO (≤31.0), SiO2 (0.3–0.8), MgO (0.4–0.5) and Fe2O3 (<0.3), with additional rheological modification Agents and radiopaque additives. Cement contains calcium monoaluminate and calcium dialuminate as the active mineral phase responsible for the hydraulic reaction. Impurities such as Fe2O3 and MgO are reduced to minimize the possibility of tooth blackening and undesirable water-induced swelling. Marketed as EndoBinder (Binerware, São Carlos, SP, Brazil)15, calcium aluminate cement is promoted as calcium silicate dental pulp because of its biocompatibility15, minimal stimulation of inflammatory reactions in animal studies16 and its ability Cement substitutes promote the repair of mineralized tissue in cell culture research17.

Based on the premise that the silicate phase is required for the treatment of dental pulp and periapical tissue during dentin formation and osteogenesis, 18 hybrid aluminosilicate cements have been developed. Two calcium aluminosilicate cement formulations have been tested for potential use in endodontics and they have been designated as Capasio19 and Quick-Set (Primus Consulting, Bradenton, FL, USA) 20,21. In addition to the inclusion of silicate in the cement formulation, these materials differ from Endobinder in that the water-based liquid component does not contain salts for accelerating the setting reaction, but contains proprietary water-soluble polymers and other suspending agents22. The setting time of commercial calcium silicate cement (white ProRoot MTA, Dentsply Tulsa Dental Specialties, Tulsa, OK, USA; commercial calcium silicate cement) is 150 minutes, while the setting time of Capasio (experimental aluminum portland cement) No more than 15 minutes 21.

The prerequisite for hydraulic cements designed for dental applications is the addition of radiopaque additives. This makes it possible to identify cement by radiography. Bismuth oxide is the most commonly used radiopaque filler in calcium silicate, calcium aluminate and calcium aluminosilicate cements. Because of its high atomic number, it has high opacity to X-rays. However, clinical studies have shown that when using calcium silicate cement without calcium aluminate (ie white mineral trioxide aggregate) for endodontic treatment, teeth discolor 23,24. Calcium silicate cements containing bismuth oxide as radiopaque filler without calcium aluminoferrite can occur after exposure to sodium hypochlorite 25, 26, chlorhexidine gluconate 26 and contact with tooth structure 27, blood 28 or formaldehyde 29 Discoloration from white to gray, dark brown or black. Although the discoloration caused by bismuth oxide does not affect the stability or radiopacity of the cured cement, when these cements are used for crown repair in the aesthetic area, The color change is disturbing. Experimental tricalcium silicate 30 and calcium aluminate cement 31 were added with alternative oxides with different radiopacity (such as zirconia). In order to avoid the problem of tooth discoloration in calcium aluminosilicate cement, an experimental anti-tarnishing calcium aluminosilicate cement was prepared by substituting tantalum oxide instead of bismuth oxide radiation transparent agent (Quick-Set2; Primus Consulting, Bradenton, FL, USA) ). In addition, free alumina was removed to increase the percentage of water phase.

During pulpal treatment, the hydraulic cement that closely adheres to the dental pulp and peri-root tissue must be biocompatible to accelerate the production of restorative dentin of dental pulp stem cells. Therefore, the purpose of this study is to examine the effect of experimental anti-tarnishing calcium aluminosilicate cement on the viability and proliferation of human dental pulp stem cells (hDPSC) before differentiation. Although hDPSCs are pluripotent and have the ability to differentiate into chondrocytes, adipocytes, and osteoblasts, the health of primitive stem cells is a prerequisite for these events32,33. The null hypothesis of the test is that when the experimental calcium aluminosilicate bone cement and calcium silicate pulp cement are placed next to undifferentiated hDPSC, it is caused by the experimental calcium aluminosilicate bone cement and calcium silicate pulp cement. There is no difference in all aspects of cytotoxicity.

Two hydraulic cements were tested: Quick-Set2 and white ProRoot MTA (WMTA), the latter is a calcium silicate cement containing bismuth oxide. For each cement, mix the powder with proprietary hydrogel or deionized water using a liquid-to-powder ratio of 0.3 according to the respective manufacturer's instructions. Place the mixed material in a pre-sterilized Teflon mold (5 mm in diameter and 3 mm in thickness), cover it with pre-sterilized polyester film, and place it in a 100% humidity chamber for 24 hours. Similar-sized discs were made of an intermediate repair material (IRM; Dentsply Caulk, Milford, DE, USA), a zinc oxide-eugenol cement, and was designated as a positive control. For the negative control, hDPSC (described below) was not exposed to any material. Before the test, all materials were sterilized with ultraviolet light for 4 hours.

Two discs made of each hydraulic cement were used to check the potential of the cured material to resist discoloration. Each disc was incubated in 5 mL of one of the following solutions at 37 °C for 7 days: deionized water, 2% chlorhexidine gluconate (Clorox HealthcareTM, Oakland, CA, USA), 8.25% sodium hypochlorite (Clorox® Germicide Bleach, Clorox HealthcareTM) and 10% neutral buffered formaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA). The materials and solutions are kept in the dark during the incubation. After 7 days, retrieve the material tray, rinse with deionized water, air dry and take pictures.

Human dental pulp stem cells were used in this cell culture study. According to the protocol approved by the Ethics Committee of the Fourth Military Medical University, pulp tissue was obtained from non-carious third molars extracted from young healthy patients (18-25 years old). All subjects obtained informed consent. The pulp was chopped and digested in a solution containing 3 mg/ml type I collagenase and 4 mg/ml dispase (Gibco BRL, Gaithersburg, Maryland, USA) at 37°C for 2 hours. By passing the cells through a 70 mm filter (BD Falcon, Franklin Lakes, NJ, USA) and adding 10% fetal bovine serum (Gibco) growth medium (α-modified Eagle medium (Gibco)), 100 units/mL Penicillin and 100 mg/mL streptomycin) in 5% CO2, 37 °C. To identify hDPSC, cultured cells were incubated with fluorescent dye-conjugated monoclonal antibodies for different clusters of differentiation (CD) cell surface molecular markers, including anti-CD29, anti-CD34, anti-CD44, anti-CD45, anti-CD90 and anti- CD105 (EMD Millipore Corp., Billerica, MA, USA) and sorted 32, 33 using a flow cytometer (Elite ESP, Beckman Coulter, Fullerton, CA, USA). In order to confirm the specificity of the primary antibody that matches the host species of the primary antibody to the non-specific mouse IgM isotype control (λ monoclonal MOPC-104E, Abcam, Cambridge, MA, USA), the primary antibody was used instead. CD90/CD105/CD45-/CD34- hDPSCs were sorted, collected and expanded. The hDPSCs sorted from the third to the sixth generation were used in subsequent experiments.

Test the pluripotency of hDPSCs using cartilage formation, adipogenesis, and osteogenic culture conditions34. For chondrogenic differentiation, culture 1.0 × 106 hDPSCs until they reach 60-70% confluence. The cells were centrifuged at 800 rpm for 6 minutes to form a pellet. After culturing in complete growth medium for 24 hours, the pellet was cultured in cartilage induction medium, which was composed of high glucose Dulbecco's Modified containing 0.1 mM dexamethasone and 50 mg/mL L-ascorbic acid-2-phosphate. Eagle medium (Gibco) composition (Sigma-Aldrich, St. Louis, MO, USA), 40 mg/mL L-proline (Sigma-Aldrich), 1% insulin transfer selenium (ITS premix; 100X; Sigma -Aldrich), 15% fetal bovine serum, 10 ng/mL transforming growth factor-β1 (R&D Systems, Minneapolis, MN, USA) and 2% Antibiotic-Antimycotic (Life Technologies, Thermo Scientific, Carlsbad, CA, USA). Four weeks after the induction of chondrogenesis, the specimens were fixed with 4% paraformaldehyde, stained with Alcian blue (Lifeline Cell Technology, Frederick, MD, USA) and examined by light microscopy (Leica DM 2500, Wetzlar, Germany). For adipogenic differentiation, 3 × 105 hDPSC was supplemented with 0.5 mM methylisobutylxanthine, 0.5 mM hydrocortisone and 60 μM indomethacin (all from Sigma-Aldrich) and 2% antibiotic-antifungal Cultured in the fat-inducing medium of the drug for 4 weeks. The cultured cells were fixed with 4% polyoxymethylene, stained with 0.3% oil red O solution (Sigma-Aldrich) and examined by an optical microscope. For osteogenic differentiation, 3 × 105 hDPSC was supplemented with 100 nM dexamethasone, 0.2 mM ascorbic acid-2-phosphate and 10 mM β-glycerophosphate (all from Sigma-Aldrich) and 2% antibiotic-antifungal Cultivate in induction medium. After culturing in osteogenic medium for 4 weeks, the cells were fixed with 4% paraformaldehyde and stained with 1% Alizarin Red S for optical microscopic examination of mineralized nodules.

Since the cytotoxic components present in hydraulic cement can diffuse out of the material, the cyclic aging protocol was used to evaluate the material's effect on stem cell viability and proliferation35. The weekly cycle includes assessing the toxicity of the material after 3 days in the vicinity of the stem cells. The newly mixed hydraulic cement pan has been cured for 24 hours for the first cycle. After the first test cycle, the cement pan was retrieved and re-immersed in sterile deionized water for 4 days to allow the potentially toxic components to diffuse out of the pan. Use the same disk for testing in the next weekly cycle. Three cycles are used for the evaluation described in the subsequent sections.

Flow cytometry and differential staining techniques are used to classify and count individual cells in a group of hDPSCs, which express changes in plasma membrane permeability caused by the toxicity of the material. HDPSC was seeded in a 6-well plate at a density of 105 cells/cm2, and cultured in a humidified atmosphere of 5% CO2 at 37°C for 24 hours. As mentioned earlier, the material was tested in three cycles. For each cycle, the cement and positive control discs were individually placed in a Transwell insert (BD Falcon, Franklin Lakes, NJ, USA) with a pore size of 3 μm to prevent the sample from directly contacting the cells. After placing the insert on the plated cells, add an additional 2 mL of complete growth medium to each well to ensure that the level of the medium is higher than the side of the Transwell insert. Expose the disc to the plated cells for 3 days without further changes to the medium. The negative control uses the same procedure, except that no material is placed in the Transwell insert.

After contacting the material, the stem cells were separated from the culture wells with trypsin and resuspended in the apoptosis and necrosis quantification kit (Biotium Inc, Hayward, CA, USA) at a concentration of 2 × 106 cells/mL. In binding buffer. Cells are stained with FITC-Annexin V (AnV; λabs/λem = 492/514 nm) and ethidium homodimer III (Etd-III; λabs/λem = 528/617 nm) as cytoplasmic membrane phospholipids And nucleic acid fluorescent dyes, respectively. During apoptosis, phosphatidylserine is transferred from the inner surface of the cell to the outer surface for phagocyte recognition36. The human anticoagulant Annexin V is a 35 kDa, Ca2-dependent phospholipid binding protein with high affinity for phosphatidylserine. Annexin V labeled with fluorescein (FITC) can recognize apoptotic cells by binding to phosphatidylserine exposed on the outer leaflets of the cell plasma membrane, resulting in the expression of green fluorescence in the cytoplasm. Necrosis is usually caused by severe cell damage, leading to loss of nuclear membrane integrity. Ethidium homodimer III is a highly positively charged nucleic acid probe. It is impermeable to living or apoptotic cells, but it will stain necrotic cells with red fluorescence. The stained hDPSCs were sorted with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) to determine health (AnV/Etd-III negative), early apoptosis (AnV positive, Etd-III negative), and late apoptosis. The percentage distribution of death is apoptotic (AnV/Etd-III positive) and necrotic cells (AnV negative, Etd-III positive). The experiment was carried out in six copies.

Fluorescence microscopy is used to record fluorescence associated with changes in hDPSCs membrane permeability. Spread the cells on a glass cover slip at a density of 400 cells/cm2. Place the cell-covered coverslip in a 6-well plate for culture, and allow the stem cells to establish for 24 hours. The material was tested 24 hours after mixing (first cycle) and after 2 weeks of aging (third cycle). After being exposed to the material for 3 days, hDPSCs were triple-stained with AnV (green fluorescence), Etd-III (red fluorescence) and Hoechst 33342, which is a blue fluorescent bisbenzimide nucleic acid counterstain that can penetrate cells It emits blue fluorescence (λabs/λem = 350/461 nm) when combined with double-stranded DNA in living and non-living cells. The stained hDPSC was examined with a fluorescence microscope (Axioplan 2, Carl Zeiss, Oberkochen, Germany).

Cells with impaired plasma membrane integrity release lactate dehydrogenase (LDH) into the surrounding medium. This cytoplasmic enzyme catalyzes the conversion of lactic acid to pyruvate by reducing NAD (the oxidized form of nicotinamide adenine dinucleotide) to NADH. The loss of intracellular LDH and its release into the culture medium are biomarkers of irreversible cell membrane damage induced by material toxicity. The quantification of LDH activity used PierceTM LDH Cytotoxicity Detection Kit (Thermo Scientific). In short, hDPSCs were seeded in a 24-well plate at a density of 104 cells/cm2 and exposed to cement discs derived from three aging cycles. For each cycle, the discs were exposed to plated cells for 3 days with no further changes in the medium. At the specified time, the LDH released into the culture medium is transferred to a new plate and mixed with the reaction mixture. After incubating for 30 minutes, stop the reaction by adding stop solution. A microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA) was used to measure the absorbance at 490 nm and 680 nm. Determine the LDH activity by subtracting the 680 nm background absorbance from the 490 nm absorbance. For the control, additional hDPSC was used to test the spontaneous LDH activity in sterile ultrapure water, and the maximum LDH activity of the cells was tested by exposing the cells to 10X lysis buffer. The experiment was carried out in six copies. Use the following formula to calculate the percentage of cytotoxicity: [(material-mediated LDH activity-spontaneous LDH activity)/(maximum LDH activity-spontaneous LDH activity)] × 100.

Caspase-3 is a member of the aspartic protease family that requires cysteine ​​and plays a vital role in mediating intracellular events related to apoptosis, including chromatin condensation, DNA fragmentation, and cellular Bubble 38. Caspase-3 colorimetric assay kit (Sigma-Aldrich) was used to determine the caspase-3 activity of hDPSCs exposed to different cements. In short, after exposing hDPSC (105 cells/cm2) to materials from three aging cycles for 3 days each, they were lysed with lysis buffer for 15 minutes. The cell lysate was reacted with p-nitroaniline (pNA)-conjugated peptide substrate for 90 minutes. Hydrolysis of the peptide substrate by caspase-3 present in cell lysates results in the release of p-NA, the absorbance of which is recorded at 405 nm. The concentration of released p-NA is determined by a calibration curve prepared with different concentrations of p-NA standards. The experiment was carried out in six copies.

Redox homeostasis is dynamically regulated in cells because there is a narrow concentration range to control whether reactive oxygen species (ROS) induce toxicity or act as the second messenger of redox signaling in cell proliferation, differentiation, apoptosis, or autophagy39 . Oxidative stress reflects the imbalance between the production of ROS and the ability of cells to detoxify active intermediates and repair damage. Excessive ROS production can cause toxic effects through the production of peroxides and free radicals. These peroxides and free radicals can damage cellular components such as proteins, lipids, and DNA. Therefore, the evaluation of intracellular ROS formation provides another perspective for evaluating the response of cells to hydraulic cement. CellROX® Orange oxidative stress reagent (Life Technologies, Thermo Fisher) was used to detect intracellular ROS in hDPSC. After exposing the cells (105 cells/cm2) to the test materials from the three aging stages for 3 days each, they were separated, centrifuged and resuspended in 1% phosphate buffered saline. CellROX® Orange (a fluorescent redox cytoplasmic stain; λabs/λem = 545/565 nm) was added to the cells at a final concentration of 5 μM and incubated at 37 °C for 30 minutes. The FACSCalibur flow cytometer is used to detect the percentage of ROS positive cells for each material/aging time. The experiment was carried out in six copies. Spread additional cells on a cover glass, double stain with CellROX® Orange and Hoechst 33342, and check with a fluorescence microscope (Axioplan 2) to qualitatively assess the intracellular ROS distribution.

Use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the mitochondrial activity of hDPSCs after exposure to materials from three aging cycles . This assay is based on the basic principle that dead cells cannot metabolize tetrazolium salt by mitochondrial dehydrogenase involved in the citric acid cycle and electron transport chain to measure cell metabolic activity. hDPSCs were seeded in a 24-well plate at a density of 104 cells/cm2 and incubated for 24 hours. The assay was performed by incubating hDPSCs with MTT-succinate solution for 60 minutes, and then fixing the cells with Tris-formalin. Use dimethyl sulfoxide-NaOH to dissolve the purple MTT formazan produced in the cells in situ. The optical density of formazan was measured at 562 nm. Subtract the optical density of the blank dimethyl sulfoxide-NaOH from all wells. The formazan content in each well was calculated as a percentage of the average value of the unexposed hDPSC negative control.

CyQUANT cell proliferation detection kit (Life Technologies, Thermo Fisher) was used to examine the effect of test materials on hDPSC proliferation. The measurement uses a fluorescence method to indicate the number of cells, based on the fluorescence displayed by the binding of CyQUANT GR cyanine dye to cell nucleic acid. In short, hDPSC was seeded in a 24-well plate (104 cells/cm2), cultured for 24 hours and exposed to materials from three aging cycles, each for 3 days. After removing the material, expose the cells to CyQUANT GR dye/cell lysis buffer for 5 minutes. Measure the absorbance of the cell lysate at λabs/λem = 480/530 nm using a fluorescence microplate reader (FL600, BioTek Instruments, Winooski, VT, USA). Calculate the DNA concentration (in ng/μL) using a pre-established standard curve, which correlates the fluorescence intensity with the known DNA concentration.

Two-factor repeated measures analysis of variance (ANOVA) was used to analyze the data obtained from each measurement separately to examine the influence of "material" and "aging cycle" and the interaction of these two factors on the studied parameters. Since the IRM positive control group was included to identify the discriminatory potential of each assay, data from this group was excluded to increase the robustness of the statistical analysis. Use the Holm-Sidak program for post-mortem comparison. First evaluate the normality (Shapiro-Wilk test) and equal variance (modified Levene test) assumptions for each data set. When these assumptions are violated, before using the parameter statistical procedure, the corresponding data set is non-linearly transformed to meet these assumptions. The statistical significance of all tests is set to α = 0.05.

The color change of the set hydraulic cement after incubation in different solutions (Figure 1) is indirect evidence that the material may cause tooth discoloration. After being immersed in deionized water, the color of the white ProRoot MTA (WMTA) remains stable. Although no spectrophotometric method was used, after WMTA was incubated in other solutions, the color change was strong enough to be clearly seen; the sample was gray when immersed in 2% chlorhexidine, and dark brown when immersed in 8.25% sodium hypochlorite, 10% After being immersed in formaldehyde, it turns black. These color changes are similar to those reported in the literature for calcium silicate cement using bismuth oxide as a radiopaque agent25,26,29. Sodium hypochlorite and chlorhexidine are commonly used irrigation agents in root canal treatment. Although there are no signs of formaldehyde in clinical practice, the WMTA root-tip fillings performed in animal studies turned black after being fixed with formaldehyde during tissue section preparation (Primus, unpublished results). In contrast, Quick-Set2 is less likely to change color after samples are incubated in different solutions. The results indicate that tantalum oxide may be a better substitute than bismuth oxide, as a color-stable radioopaque agent, suitable for hydraulic cement designed for clinical operations involving the coronal surface of teeth.

After incubating the cured material in deionized water (DI), 2% chlorhexidine (CHX), 8.25% sodium hypochlorite (NaOCl) and 10% formaldehyde (FADH) for 7 days, the color of white ProRoot MTA (WMTA) and Quick-Set2 Variety.

The European cosmetics industry has banned the use of animal substitutes for toxicological testing since March 201342. Prior to this implementation, the National Academy of Sciences published a consensus report in 2007 entitled "Toxicity Testing in the 21st Century: Vision and Strategy"43. The report envisages a paradigm shift in future toxicity testing from the current qualitative endpoint. Expensive and lengthy in vivo animal testing has been transformed into an in vitro toxicity assay on human cells or cell lines using a set of quantitative parameter toxicity pathway assays.44 This report supports the use of human stem cells cultured in vitro as a key target for future toxicology testing Cells 45, 46. Dental pulp stem cells are one of five types of stem/progenitor cells derived from dental tissues, with mesenchymal stem cell-like properties, including the ability to regenerate hard tissues of teeth.47. In the current work, undifferentiated hDPSC is used , Because when hydraulic cement is placed on exposed human dental pulp during direct pulp capping or pulpectomy, these cells may be involved. Although in previous hydraulic cement cytotoxicity studies, almost only non-human Cell lines, but human cells can predict the human body’s response to chemicals more accurately than animal cells. Due to species differences, humans and animals have different responses to chemical exposure. The consistency rate of using non-rodent models is about 63%. The consistency rate of using rodent models to predict human toxicity is only 43%. These relatively low consistency indicate the need to use human cells for toxicology testing; another advantage of using human cells is that they may reveal human toxicity The susceptibility factor 46.

Consistent with other mesenchymal stem cell populations, most hDPSCs showed strong expression of mesenchymal surface molecular markers (CD29-98.9%, CD44-98.5%, CD90-99.5% and CD105-96.4%). hDPSC also showed weak expression of hematopoietic system-derived cell surface markers (CD34–0.9% and CD45–0.9%) (Figure 2A). Weak staining of the mouse IgM isotype control antibody confirmed the specificity of primary antibody binding (Figure 2A). The pluripotency of hDPSCs was confirmed by observation that these cells may develop into chondrocytes and form chondrocyte-like extracellular matrix after chondrogenesis is induced (Figure 2B). After induction of adipogenesis, intracellular lipid vacuoles can be identified in differentiated adipocytes (Figure 2C). After hDPSCs were cultured in osteogenic differentiation medium, mineralized nodules stained with Alizarin Red S appeared in the extracellular environment (Figure 2D).

The immunophenotype and pluripotency characteristics of hDPSCs.

(A) Immunophenotyping of hDPSC using FITC dye-conjugated antibody to identify different clusters of differentiation (CD) cell surface molecules. FITC dye-conjugated mouse IgM antibody was used as an isotype control. The weak staining of the isotype IgM control antibody indicates the binding specificity of the CD antibody. (B) Chondrocyte extracellular matrix (stained with Alcian blue) formed by differentiated cells after incubation in chondrogenic medium. (C) Intracellular lipid vacuoles (stained with oil red O) found in differentiated cells after incubation in lipogenic medium. (D) Mineralized nodules formed by differentiated cells after cultured in osteogenic medium (stained with Alizarin Red S). For (B–D) bar = 50 μm.

Cells exposed to toxic substances may lead to multiple cell fates. Depending on the level of toxicity, cells may undergo necrosis, thereby losing membrane integrity and dying due to cell lysis. Alternatively, the cell can activate a genetic program that controls cell death (apoptosis). They can also prevent active growth and cell division (decrease cell viability and proliferation). In addition, cytotoxic substances may reduce the production of antioxidants in cells or increase the production of ROS in mitochondria, thereby increasing the level of oxidative stress in cells, thereby adversely affecting cell proliferation. The results of the four cell viability assays are shown in Figure 3. Figure 3A shows the percentage of healthy cells with intact plasma membrane present in the hDPSCs complex after exposure to different materials that have been aged for 3 cycles. Two-factor repeated-measure analysis of variance compared hDPSCs exposed to two hydraulic cements with unexposed hDPSCs, indicating the type of material (P <0.001), the aging period (P <0.001), and the interaction of these two factors (P <0.001) Both have a significant impact on the percentage of healthy hDPSCs that are impermeable to the two fluorescent staining of cell plasma membrane phospholipids and nucleic acids. Post-mortem pairwise comparisons (only the comparisons with significant differences are described) show that for the "aging cycle" factor within the material, the number of healthy cells in the first cycle of Quick-Set2 is lower than the number in the next two cycles. For WMTA, the number of healthy hDPSCs in each cycle is higher than that in subsequent cycles, and the order is: the first cycle <the second cycle <the third cycle. For the "material" factor in the first cycle, the number of unexposed healthy hDPSC is higher than the number exposed to Quick-Set2 or WMTA, while the number of healthy cells in Quick-Set2 is higher than that of WMTA. For the factor "material" in the second cycle, the number of healthy unexposed hDPSC is higher than Quick-Set2 or WMTA. For the "material" factor in the third cycle, only a significant difference in the number of healthy cells was observed between unexposed hDPSC and hDPSC exposed to Quick-Set2.

Cell viability test results of hDPSCs exposed to materials from 3 aging cycles.

(A) The membrane integrity of hDPSCs after cells are stained with FITC-Annexin V and Ethidium homodimer III. The graph shows the percentage of healthy hDPSCs that are not stained with annexin V and ethidium homodimer III. (B) Leakage of lactate dehydrogenase in hDPSCs, these enzymes have damaged the permeability of the plasma membrane. (C) The Caspase-3 activity of hDPSCs is used as an indicator of cell apoptosis. (D) The expression of reactive oxygen species in hDPSCs is used as an indicator of intracellular oxidative stress. Statistical analysis was performed only on hDPSC exposed to two hydraulic cements and unexposed hDPSC (negative control). For the factor "material" in each chart, the groups marked with the same code (numbers in the first cycle, capital letters in the second cycle and lowercase letters in the third cycle) have no significant difference (P> 0.05) . For the "aging cycle" factor in each chart, there is no significant difference in the cycle of connecting the same hydraulic cement to the horizontal bar (P> 0.05). For unexposed hDPSC, there was no difference in activity between 3 cycles (P> 0.05; horizontal bars are not shown).

The images obtained by fluorescence microscopy are complementary to the results of flow cytometry (Figure 4a). The hDPSCs that were not exposed during the first or third cycle were mainly healthy and showed blue fluorescent nuclei with few signs of apoptosis or necrosis. Most of the cells exposed to the IRM positive control are apoptotic or necrotic, and the prevalence of green fluorescent cytoplasm can be attributed to apoptosis. Sometimes, the cells show part of the red fluorescent cytoplasm (caused by the leaching of nucleic acid components) or pink nuclei (blue and red fluorescence combined), which is a characteristic of necrosis or cell death caused by apoptosis. Stem cells exposed to Quick-Set2 or WMTA in the first cycle are mainly healthy; however, cells with green fluorescent cytoplasm can be observed. Starting from the third cycle, after hDPSCs were exposed to Quick-Set2 or WMTA, the number of apoptotic cells showing green fluorescent cytoplasm was significantly reduced.

Fluorescence microscopy assessment of plasma membrane integrity and oxidative stress after hDPSCs were exposed to materials from the first and third aging cycles.

(A) Triple staining with Hoechst 33342 (blue fluorescent nuclear counterstain), ethidium homodimer III (red fluorescent non-important DNA dye) and FITC-Annexin V (green fluorescent phosphatidylserine bound cytoplasmic dye) Fluorescence microscope image of hDPSC. Healthy cell nuclei are stained blue. Apoptotic cells show green cytoplasm and blue nuclei. Necrotic cells have red or pink nuclei. Dead cells developed from apoptotic cell populations are stained green, red, and blue. Bar = 25 μm. (B) Fluorescence microscope image of hDPSC double stained with CellROX® Orange and Hoechst 33342. The cytoplasm of cells exhibiting oxidative stress is stained orange. Bar = 50 μm.

Unlike complex life systems, cultured eukaryotic cells that undergo apoptosis will eventually die due to secondary necrosis49. Since the characteristics of apoptosis and necrosis in cell culture overlap, it is necessary to perform two or more tests to confirm that cell death occurs through apoptosis. This prompted the examination of hDPSCs for LDH leakage and caspase-3 activity in this study. For LDH leakage (Figure 3B), the type of material (P <0.001), the aging cycle (P <0.001) and the interaction of these two factors (P <0.001) all have a significant impact on the cytosolic enzyme leakage of hDPSCs For the pairwise comparison of the "aging cycle" factors within the material (only the comparisons with significant differences are described), after each aging cycle of the two hydraulic cements, the LDH leakage from hDPSC continues to decrease. Regarding the "material" factor in the first aging cycle, the LDH leakage of the unexposed hDPSC is lower than that of the exposed two hydraulic cements; the LDH leakage of Quick-Set2 is also lower than that of WMTA. Regarding the “material” factor in the second and third cycles, the LDH leakage of unexposed hDPSC is still lower than that of the two hydraulic cements.

For caspase-3 activity (Figure 3C), the type of material (P <0.001), the aging cycle (P <.001) and the interaction of these two factors (P <0.001) all have a significant impact on the production of this enzyme . hDPSCs are enzymes in the early stages of apoptosis. For the pairwise comparison of the "aging cycle" factors in the materials (only the comparisons with significant differences are described), the caspase-3 activity of hDPSCs continues to decrease after each aging cycle of the two hydraulic cements. For the "material" factor in the first and second cycles, the caspase-3 activity of unexposed hDPSCs was lower than that of the two hydraulic cements; the enzyme activity in the cells exposed to Quick-Set was also lower than that of WMTA. For the "material" factor in the third cycle, the caspase-3 activity of unexposed hDPSC was still significantly lower than that of hDPSC exposed to Quick-Set2 or WMTA.

When the cell’s metabolic pro-oxidant production exceeds its antioxidant capacity, oxidative stress 50 occurs. Approximately 2% of cell oxygen consumption is used by mitochondria to produce ROS 51. When the production of ROS is low, the antioxidant enzymes produced in the cell can deal with the damage of key intracellular biomolecules. Excessive ROS in the form of hydrogen peroxide, superoxide or hydroxyl free radicals will react with cellular DNA, proteins and lipids to produce other free radicals or cytotoxic products that cause cell damage. Figure 4B shows an image of hDPSC stained with CellROX® Orange and Hoechst 33342 nuclear counterstain. Cells with elevated levels of oxidative stress showed orange fluorescence in their cytoplasm, which was most prominent in the IRM positive control group. The graph in Figure 3D represents the percentage of ROS-positive hDPSCs after exposure to the material. It was found that the material type (P <0.001), the aging cycle (P = 0.022) and the interaction of these two factors (P = 0.013) significantly affected the oxidative stress of unexposed hDPSCs or hDPSCs exposed to two hydraulic cements Level from three cycles. Regarding the "aging cycle" factor in the material (only pairwise comparisons with significant differences are described), the oxidative stress level of hDPSCs exposed to WMTA from the first cycle is higher than that of the second and third cycles. For the factor "material" in the first cycle, compared with hDPSC exposed to two hydraulic cements, there are fewer ROS positive cells in unexposed hDPSC. Compared with WMTA, ROS in Quick-Set-2 There are fewer positive cells. For the factor "material" in the second cycle, the percentage of ROS-positive cells in unexposed hDPSC is still lower than hDPSC exposed to two hydraulic cements.

There are four main types of cell proliferation testing: i) Metabolic cell proliferation testing, such as detection based on tetrazolium salt reduction, ii) DN​​A synthetic cell proliferation testing, and iii) detection of cell proliferation markers, such as Ki-67 expressed Proteins in the S, G2, and M phases of the cell cycle and iv) bioluminescence-based adenosine triphosphate detection. In this study, MTT determination and DNA content quantification were used to examine the effect of hydraulic cement on cell proliferation. The CyQUANT cell proliferation detection kit only quantifies the relative number of cells in the cohort based on the total DNA content of the cells, and has nothing to do with cell metabolism, but does not recognize DNA synthesis. The latter involves the incorporation of 3H-thymidine or thymidine analogs such as 5-bromo-2'-deoxyuridine into nascent DNA when these cells are actively proliferating during the S phase of the cell cycle. Although not as accurate as quantitative DNA synthesis, the measurement of cell number with DNA combined with fluorescent dyes represents an indirect indicator of cell proliferation. This is a reasonable alternative method for comparing the cytotoxic effects of different groups with the same initial cell number.

For MTT determination (Figure 5A) and relative DNA content analysis (Figure 5B), a similar trend was observed, that is, both materials were initially relatively cytotoxic; after two additional aging cycles, cement had an effect on cell metabolism and cell metabolism. The number of cytotoxic effects gradually decreases. For any determination, the factors "material" (P <0.001), "aging cycle" (P <0.001) and the interaction of these factors (P <0.001) significantly affect the respective cell proliferation parameters (mitochondrial enzyme activity measured by MTT) And DNA content used to determine cell DNA content). For the pairwise comparison of the factor "aging cycle" in each article (only pairwise comparisons with significant differences are described), hDPSC exposed to Quick-Set2 or WMTA exhibited its own in each of the three aging cycles The gradual increase in cell proliferation parameters. For the "material" factors in the first cycle, the corresponding cell proliferation parameters of unexposed hDPSCs were higher than those of cells exposed to the two hydraulic cements; those exposed to Quick-Set2 hDPSC's expression of corresponding parameters is higher than that of WMTA. For the "material" factor in the second cycle, the cell proliferation parameters in the unexposed hDPSC were higher than those in the cells exposed to the two hydraulic cements. For the factor “material” in the third cycle of the MTT assay (Figure 5A), the mitochondrial enzyme activity in unexposed hDPSCs was higher than that in hDPSCs exposed to WMTA. For the factor "material" in the third cycle of DNA content determination (Figure 5B), the DNA content of unexposed hDPSCs is higher than that of hDPSCs exposed to two hydraulic cements.

The results of cell proliferation assays for hDPSCs after exposure to materials from 3 aging cycles.

(A) MTT determination. The mitochondrial dehydrogenase activity of hDPSCs exposed to different materials was expressed as a percentage relative to the unexposed hDPSCs negative control (100%). (B) Cell DNA content. Only hDPSC exposed to two hydraulic cements and hDPSC (negative control) that were not exposed for 3 aging periods were statistically analyzed. For the factor "material" in each chart, the groups marked with the same code (numbers in the first cycle, capital letters in the second cycle and lowercase letters in the third cycle) have no significant difference (P> 0.05) . For the "aging cycle" factor in each chart, there is no significant difference in the cycle of connecting the same hydraulic cement to the horizontal bar (P> 0.05). For unexposed hDPSC, there was no difference in activity between 3 cycles (P> 0.05; horizontal bars are not shown).

In summary, the results of the six assays with different end-point measurements show that after 24 hours of setting, the cytotoxicity of Quick-Set2 to undifferentiated hDPSCs is relatively lower than that of WMTA. The initial cytotoxicity of WMTA and Quick-Set2 may be due to the high pH of the set cement caused by the diffusion of Ca(OH)2 into the environmental medium. It is observed that Quick-Set2 is initially less cytotoxic to WMTA, which may be due to the low pH value of calcium aluminosilicate cement (~10), while the pH value of the solidified tricalcium silicate cement (~12)2 Higher; pH closer to physiological pH will make the medium less corrosive to hDPSC. The difference in initial cytotoxicity characteristics between the two hydraulic cements warrants rejection of the null hypothesis tested in this study.

The initial cytotoxicity exhibited by Quick-Set2 and WMTA was significantly reduced after these cements were aged in deionized water and then exposed to hDPSC. After two aging cycles, the cytotoxicity curves of the two cements were basically similar in all six assays. This phenomenon clearly reflects Paracelsus’ classic toxicology guidelines on the dose/response relationship: "Alle Ding sind Gift und nichts ohn Gift; alein die Dosis macht das ein Ding kein Gift ist Non-toxic; only the dose can make things not poison)"53. The toxicological risk only exists when the cells are exposed to setting hydraulic cement. The conditions that exist in isolated monolayer cell cultures are non-steady because there is no mechanism to eliminate toxic substances in the body. Although isolated monolayer cell cultures are powerful models for toxicity assessment, these models have limitations in their ability to generalize physiological processes and cell characteristics in vivo. A parallel example is the cytotoxicity of borate bioactive glass under conventional static in vitro culture conditions. Borate bioactive glass is toxic to cells due to the release of borate ions. However, toxicity is significantly reduced under dynamic culture conditions, where the culture medium flows continuously to mimic the in vivo reactions of the living host, such as the availability of lymphatic vessels to remove toxic substances. Cytotoxicity is a multi-factorial process involving active transport mechanisms and passive diffusion, cell apoptosis, metabolites and ROS production, the biotransformation of toxic components by intracellular or extracellular enzymes, and the interaction with the immune system. Dynamic accumulation/removal of toxic ingredients. Many of these processes involve cell-cell interactions and cell-extracellular matrix interactions. These important microenvironment-driven determinants of cell behavior are often lost in isolated two-dimensional cell culture models or even three-dimensional culture models55. These limitations may explain the paradox that dental materials considered to be cytotoxic in cell culture can be tolerated in the in vivo environment. Therefore, the results from this study should be interpreted as a risk estimate of the relative rate at which healing or tissue repair occurs in the presence of hydraulic cement. In the future, dental explant organ culture models can be used to check the toxicity of these cements to circumvent some of the limitations associated with the use of isolated cell culture models.

Since hDPSCs have the potential to differentiate into specialized cells capable of producing mineralized tissues, as an extension of this study, it is logical to examine whether the differentiation and osteogenic potential of these stem cells are affected by hydraulic cement. Although it is almost certain that the presence of toxic components in these cements will adversely affect hDPSC differentiation and hard tissue formation, it is necessary to emphasize that the components in dental hydraulic cements (such as silicates) are potential stimuli for the synthesis of type I collagen. Agent and mineralization 57. The beneficial effects of these components are initially masked by the cytotoxicity of the set cement, and will not become apparent until the cement gradually depletes its toxic components. The results of this study indicate that the experimental anti-tarnishing calcium aluminosilicate cement must be aged for at least three cycles to reduce in vitro cytotoxicity before determining its expected enhancement effect on osteogenic differentiation. The research in this direction is orderly.

Within the limits of using a separate monolayer cell culture model, it can be concluded that the experimental anti-tarnish calcium aluminosilicate pulp cement is initially cytotoxic to hDPSC after 24 hours of curing. At this stage, the cytotoxicity characteristics of the improved calcium aluminosilicate cement, as determined using the assay to detect different aspects of cell viability and proliferation, are significantly more advantageous than the calcium silicate cement containing bismuth oxide. After two cycles of aging in deionized water, the cytotoxicity characteristics of the two hydraulic cements are similar, and the cytotoxicity is much lower than that of zinc oxide eugenol-based repair cement. Therefore, a more favorable in vivo tissue reaction is expected to occur. In addition to the cell biocompatibility of the material, the effect of hydraulic cement on the osteogenic differentiation of hDPSCs is also very important. The investigation of these responses is orderly. The results of this study indicate that the potential beneficial effects of anti-tarnishing calcium aluminosilicate dental pulp cement on the osteogenic differentiation and osteogenic potential of hDPSCs are more likely to be revealed by reducing the initial cytotoxicity of the cemented cement through water expression . Ageing.

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National Institute of Oral and Craniofacial Research R44 DE20204-02, National High-Tech Research and Development Plan 2015AA020942, National Natural Science Foundation of China 81400555, 81300898 and 81130078, and IRT1305 Innovation Research Team Project were funded in universities.

Niu Li-na, Watson Devon, and Thames Kyle made similar contributions to this work.

Department of Prosthodontics, State Key Laboratory of Military Stomatology, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi, China

Niu Lina, Jiao Kai, Chen Jihua

Department of Endodontics, Georgia Regent University, Augusta, Georgia, USA

Niu Lina, Devin Watson, Kyle Thames, Brian E. Bergeron and Franklin R. Tay

LECOM School of Dentistry, Bradenton, Florida, USA

Department of Dentistry, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil

Department of Periodontology, Georgia Regent University, Augusta, Georgia, USA

Department of Oral Biology, Georgia Regent University, Augusta, Georgia, USA

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LNN, DW, and KT conducted the experimental and analytical parts of the research and wrote the manuscript. CMP, BEB, KJ and EAB are useful for flow cytometry testing in cell viability determination. CWC, JHC, and DHP provided suggestions for experimental design and edited the manuscript. FRT supervised the project and edited the manuscript. All authors reviewed the manuscript.

The author declares that there are no competing economic interests.

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Niu, Ln., Watson, D., Thames, K. etc. The effect of discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells. Scientific Report 5, 17177 (2015).


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