2.1

Materials

Biodentine, TheraCal LC and MTA Angelus (Angelus Dental Solutions, Londrina, PR, Brazil) were evaluated. The compositions of these calcium silicate-based materials are shown in Table 1 . Biodentine and MTA Angelus were mixed with deionized water or the liquid supplied, using the liquid/powder ratio recommended by the respective manufacturer. These materials were placed in pre-sterilized Teflon molds (5-mm diameter and 3-mm thick), covered with pre-sterilized Mylar sheets, and allowed to set in a 100% humidity chamber for 24 h. TheraCal LC was supplied by the manufacturer in pre-mixed syringes and required no preparation before use. The resin-based calcium silicate cement was dispensed in increments into the Teflon mold and polymerized with a light-emitting diode-type light-curing unit for 20 s per increment. The last increment was covered with a pre-sterilized Mylar sheet prior to light-curing to prevent the formation of an oxygen inhibition layer. Untreated cells were used as the negative control. Disks of similar dimensions to the test cements were prepared with zinc oxide-eugenol cement (Intermediate Restorative Material [IRM], Dentsply Caulk, Milford, DE, USA; one drop of liquid to one level scoop of powder) and used as the positive control. The set materials were sterilized with ultraviolet light for 4 h prior to testing.

2.2

Cell culture

Previously characterized hDPSCs with CD90 ⁺ /CD105 ⁺ /CD34 ⁻ /CD45 ⁻ immunophenotype were employed in the present study. The cells were previously obtained from young healthy patients (18–25 years old; 10 teeth from 6 patients) according to a protocol approved by the Ethics Committee of the Fourth Military Medical University, Xi’an, China. The dental pulp of each tooth was minced and digested in a solution containing 3 mg/mL type I collagenase and 4 mg/mL dispase (Gibco, BRL, Gaitherburg, MD, USA) at 37 °C for 2 h. Single-cell suspensions were then obtained by passing the cells through a 70-mm strainer (BD Falcon, Franklin Lakes, NJ, USA) and cultured in control medium in 5% CO ₂ at 37 °C. The control medium consisted of α-modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin and 100 μg/mL streptomycin. The medium was changed every 3 days.

In colony-forming test, the majority of the hDPSCs retained their spindle-shaped morphology, consistent with other mesenchymal stem cell populations . The multipotency of those hDPSCs had been established using chrondrogenic, adipogenic and osteogenic culturing conditions (Niu, unpublished results). The hDPSCs were seeded in 24-well plates at a density of 10 ⁴ cells/cm ² . The hDPSCs were cultured in complete growth medium consisting of α-modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma–Aldrich, St. Louis, MO, USA) in 5% CO ₂ at 37 °C. The growth medium was changed every 3 days; passages 2–4 hDPSCs were used for the present study.

2.3

Cell viability

2.3.1

XTT assay

In pulpal revascularization protocols, materials are placed in close proximity to the blood clot from which hDPSCs are derived. Cytotoxic components derived from the set calcium silicate cements can readily diffuse into the blood clot. Thus, both materials and eluents were evaluated separately, using a previously established cyclic regime .

An XTT Cell Viability Assay Kit (Biotium Inc., Hayward, CA, USA) was used to determine cell viability, based on the ability of mitochondrial enzymes in metabolically active cells to reduce the yellow tetrazolium derivative 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) to form a water-soluble orange-colored formazan product. The activities of those enzymes are inactivated shortly after cell death; hence, the assay enables determination of live cell numbers using spectrophotometric absorbance quantification at 475 nm. The viability of untreated hDPSCs was used as the negative control, the mean absorbance of which was adjusted to 100% for comparison with the relative dehydrogenase activities of the other groups ( N = 12).

The XTT assay was performed on hDPSCs that had been directly exposed to test cements and on eluents derived from those materials. A weekly cycle consisted of direct evaluation of the toxicity of the set cement disks over the plated cells for 3 days, and indirect evaluation of the effect of eluents derived from the set cements on the plated cells. Eluent collection was achieved by immersing cement disks in complete growth medium for 4 days after direct evaluation of the cement disks. During the first part of each weekly cycle, cement and control disks were placed individually in Transwell inserts with a 3-μm pore size (BD Falcon, Franklin Lakes, NJ, USA) to prevent their direct contact with the hDPSCs. After the inserts were placed over the plated cells, an additional 2 mL of complete growth medium was added to each well to ensure that the level of the culture medium was above the sides of the Transwell insert. The disks were exposed to the plated cells for 3 days without further change in culture medium before testing for mitochondrial dehydrogenase activity. During the second part of each weekly cycle, each disk was retrieved and incubated at 37 °C with complete growth medium (1 disk/2 mL) for 4 days to collect the eluent from the set cement before using the same disk for the next cycle. For each disk, the same growth medium was used for eluent collection throughout the entire testing period. This recurrent cyclic aging protocol was continued until the material disks were rendered noncytotoxic (i.e. >85% of the mean dehydrogenase activity exhibited by the untreated control).

For indirect evaluation of the eluents, each eluent concentrate collected after completion of the aging cycles was diluted with fresh complete growth medium to 1:1, 1:10, and 1:100 of its original concentration to achieve a final volume of 2 mL ( N = 12). Each diluted, eluent-containing growth medium was then used as the respective culture medium for freshly plated hDPSCs. The XTT assay was performed in the same manner as previously described; absorbance was determined using a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA).

2.3.2

Flow cytometry

Flow cytometry was employed with differential staining for sorting and counting of individual cells within a cohort of hDPSCs that expressed changes in plasma membrane permeability induced by the toxicity of the tested materials. The hDPSCs were plated in 6-well plates at a density of 10 ⁵ cells/cm ² and incubated at 37 °C in a humidified 5% CO ₂ atmosphere for 24 h. The material disks placed in Transwell inserts as previously described. After 3 days of exposure to the materials, the hDPSCs were detached from the culture wells using 0.25% trypsin/2.21 mM ethyelenediaminetetraacetic acid (EDTA) (Mediatech, Inc., Corning, Manassas, VA, USA) and re-suspended in binding buffer derived from the Apoptosis and Necrosis Quantification Kit (Biotium). The hDPSCs were stained with fluorescein isothiocyanate–annexin V ( λ _abs / λ _em : 492/514 nm) and ethidium homodimer-III ( λ _abs / λ _em : 528/617 nm) as the respective fluorescence stains for cytoplasmic membrane phospholipids and nucleic acids ( N = 3). During apoptosis, phosphatidylserine is translocated from the inner to the outer surface of the cell for phagocytic cell recognition . Annexin V labeled with fluorescein identifies apoptotic cells by binding to phosphatidylserine exposed on the outer leaflet of the cytoplasmic membrane, resulting in the expression of green fluorescence within the cytoplasm. Ethidium homodimer III is a highly positive-charged nucleic acid probe impermeable to live cells or apoptotic cells, but stains necrotic cells with red fluorescence. The stained cells were incubated in the dark for 15 min and subjected to fluorescence-activated cell sorting (MACSQuant Analyzer, Miltenyi Biotec Inc., Auburn, CA, USA) to determine the percentage distribution of viable, early apoptotic, late apoptotic, and necrotic cells. Experiments were performed in triplicates.

2.4

Cell Differentiation and mineralization

2.4.1

Gene expression

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression of target markers of osteogenic differentiation among the test groups. The set cements were first rendered non-cytotoxic by aging in culture medium for 5 weeks. The hDPSCs were cultured in complete growth medium at a density of 10 ⁴ cells/cm ² to ∼80% confluency. The culture medium replaced by osteogenic differentiation medium, consisting of the original complete growth medium supplemented with 50 mg/mL ascorbic acid, 10 mmol/L β-glycerophosphate and 100 nmol/L dexamethasone (Sigma–Aldrich). The established hDPSCs were exposed to Transwells containing the 3 aged calcium silicate cements (MTA Angelus, Biodentine and TheraCal LC) for 7 days, with the osteogenic differentiation medium changed every 3 days.

The total RNA present in the hDPSCs ( N = 3) from each group was isolated and purified using QIAshredder and RNeasy kit (Qiagen, Valencia, CA, USA). Quality control of the extracted RNA was performed with 2 μL samples by measuring their UV absorbance (NanoDrop ND-1000 UV–Vis spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantity of extracted RNA was determined from the RNA absorbance at 260 nm; RNA purity was determined from the ratio of the absorbance (A@260 nm/A@280 nm and A@260/A@230 > 1.8 and <2.0). Equal amounts of total RNA (0.1 mg RNA/mL) were reverse-transcribed into single-stranded complementary DNA (cDNA) using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific Inc.) in a 7300 real-time PCR system (Applied Biosystems). Reverse transcription was performed using thermocycling conditions recommended by the manufacturer: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min). The generated cDNA was stored at −20 °C until commencement of qRT-PCR.

For qRT-PCR, alkaline phosphatase ( ALP ), osteocalcin (OCN), bone sialoprotein (BSP), runt-related transcription factor 2 ( Runx2 ), dentin sialophosphoprotein ( DSPP ) and dentin matrix protein-1 ( DMP-1 ) were used as target markers of osteogenic/dentiogenic differentiation . Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as the housekeeping gene. Untreated hDPSCs grown in osteogenic differentiation medium was assigned as the negative control. The cDNA (100 ng each) was mixed with Taqman ^® PCR Universal Master Mix II (Invitrogen, Thermo Fisher Scientific Inc.) and added in 20 μL aliquots to 96-well sequence-specific TaqMan ^® array standard plates preloaded with the designated primers and housekeeping gene ( ALP : Hs01029144_m1; OCN : Hs01587814_g1; BSP : Hs00173720_m1; Runx2 : Hs00231692_m1; DSPP : Hs00171962_m1; DMP-1 : Hs01009391_g1; GADPH : Hs99999905_m1). The plate was loaded in the thermocycler and run in regular cycles. Each cycle consisted of 50 °C for 2 min, 95 °C for 10 min, 40 cycles of denaturing/annealing at 95 °C for 15 s and 60 °C for 1 min. Experiments were conducted in triplicate. The comparative threshold cycle method (ΔΔ C _T ) was employed for relative quantification of gene expression; the expression level of each target cDNA marker was normalized to the GAPDH cDNA endogenous control. Data output was expressed as fold changes of mRNA expression levels, given by 2 ^−ΔΔCt .

2.4.2

Alkaline phosphatase enzyme activity

Cells cultured in osteogenic differentiation medium were exposed to the 4 aged materials for 14 days ( N = 12). Intracellular ALP activity was evaluated using the Quantichrom ALP assay kit (Bioassay Systems, Hayward, CA, USA). Colorimetric determination was based on hydrolysis of p -nitrophenyl phosphate by ALP into inorganic phosphate and p -nitrophenol, a yellow-colored product. After 14 days of exposure, the cement disks and their respective Transwell inserts were retrieved. The cells were washed with PBS and lysed with 0.2% Triton X-100 for 20 min. Assay buffer and p -nitrophenyl phosphate were added to a clear-bottomed 96-well plate (150 μL/well). Following addition of the cell lysate, the optical density of p -nitrophenol at t = 0 min was recorded at 405 nm using the Synergy microplate reader. The samples were shielded from direct light at room temperature for 4 min prior to recording the optical density of p -nitrophenol at t = 4 min. The ALP activity of the cell lysate (in IU/L = μmol/L min) was calculated from the difference in optimal density readings.

2.4.3

Extracellular mineralization

Cells cultured in osteogenic differentiation medium were exposed to the 4 aged materials for 21 days ( N = 6). The hDPSCs were evaluated for calcium production by rinsing three times with phosphate-buffered saline. The cells were then fixed in 10% formaldehyde and stained with Alizarin red S (40 mmol/L, pH 4.2), a dye with affinity to calcium salts. Images of the specimens were taken for qualitative evaluation. Stained calcium deposits were incubated in 10% acetic acid for 30 min and neutralized with 10% ammonium hydroxide . The supernatant was pipetted into a clear-bottom 96-well plate and the absorbance of the supernatant was determined at λ = 550 nm. The concentration of dye extracted from the specimens (mg/mL) was determined according to a linear regression equation derived from a calibration curve that correlated absorbance with known Alizarin red S concentrations. Because one mole of Alizarin red S binds to two moles of calcium to produce an Alizarin Red S-calcium complex , the extent of calcium deposition was expressed as molar equivalent of calcium.

Because Alizarin red S is not specific for calcium or alkali earth metals and can also react with a wide variety of cations , hDPSCs in various groups before and after addition of the osteogenic differentiation medium were examined by transmission electron microscopy (TEM) to confirm the formation of extracellular calcium deposits. The hDPSCs cultured in complete growth medium were detached by incubating in trypsin-EDTA for 2 min. Pooled dislodged cells were centrifuged, fixed with Karnovsky’s fixative, post-fixed in 1% osmium tetroxide, dehydrated in an ascending ethanol series (30–100%), transferred to propylene oxide, and embedded in epoxy resin. Sections prepared by ultramicrotomy (90 nm thick) were stained with uranyl acetate and Reynold lead citrate. Stained sections were examined using a JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV. After completion of the mineralization assay, hDPSCs from unstained wells were similarly processed and examined without further staining to identify the status of mineralization within the extracellular matrix.

2.5

Statistical analyses

Data from the IRM positive control was excluded from all statistical analyses to increase the robustness of the respective analysis. For XTT assay, results derived from weekly cycles were analyzed using two-factor repeated measures analysis of variance (ANOVA) and post-doc Holm–Sidak multiple comparison procedures to examine the effects of calcium silicate cements and aging cycle on hDPSC viability. Similar tests were used for analyzing the effect of eluent dilution and aging cycle on cell viability. For flow cytometry, one-factor ANOVA and Holm–Sidak multiple comparison procedures were used to examined the effects of calcium silicate cements on the percentage of vital, non-apoptotic, non-necrotic hDPSCs. For qRT-PCR, the effect of materials on expression of each osteogenic marker was analyzed separately by comparing with the GADPH endogenous control, using a one-factor ANOVA and Holm–Sidak multiple comparison procedures. The results obtained from ALP enzymatic activity and Alizarin red S staining were separately analyzed using one-factor ANOVA and Holm–Sidak multiple comparison procedures. For all statistical analyses, parametric versions of these tests were used after evaluation of the normality (Shapiro–Wilk test) and equal variance assumptions (modified Levene test) of the individual data tests. If those assumptions were violated, the data were nonlinearly transformed to satisfy those assumptions prior to the use of parametric testing methods. Except for qRT-PCR, statistical significances for all analyses were set at alpha = 0.05. For qRT-PCR, because the chance of finding one or more significant differences in testing six hypotheses on osteogenic markers with the same samples (type I error) is 26.49%, the Dunn–Šidák correction was used for controlling the familywise error rate. For each osteogenic marker, the critical value (alpha) was reduced to 0.0085, so that test results with P < 0.0085 were only considered significantly different from the GADPH endogenous control.

2.1

Materials

Biodentine, TheraCal LC and MTA Angelus (Angelus Dental Solutions, Londrina, PR, Brazil) were evaluated. The compositions of these calcium silicate-based materials are shown in Table 1 . Biodentine and MTA Angelus were mixed with deionized water or the liquid supplied, using the liquid/powder ratio recommended by the respective manufacturer. These materials were placed in pre-sterilized Teflon molds (5-mm diameter and 3-mm thick), covered with pre-sterilized Mylar sheets, and allowed to set in a 100% humidity chamber for 24 h. TheraCal LC was supplied by the manufacturer in pre-mixed syringes and required no preparation before use. The resin-based calcium silicate cement was dispensed in increments into the Teflon mold and polymerized with a light-emitting diode-type light-curing unit for 20 s per increment. The last increment was covered with a pre-sterilized Mylar sheet prior to light-curing to prevent the formation of an oxygen inhibition layer. Untreated cells were used as the negative control. Disks of similar dimensions to the test cements were prepared with zinc oxide-eugenol cement (Intermediate Restorative Material [IRM], Dentsply Caulk, Milford, DE, USA; one drop of liquid to one level scoop of powder) and used as the positive control. The set materials were sterilized with ultraviolet light for 4 h prior to testing.

2.2

Cell culture

Previously characterized hDPSCs with CD90 ⁺ /CD105 ⁺ /CD34 ⁻ /CD45 ⁻ immunophenotype were employed in the present study. The cells were previously obtained from young healthy patients (18–25 years old; 10 teeth from 6 patients) according to a protocol approved by the Ethics Committee of the Fourth Military Medical University, Xi’an, China. The dental pulp of each tooth was minced and digested in a solution containing 3 mg/mL type I collagenase and 4 mg/mL dispase (Gibco, BRL, Gaitherburg, MD, USA) at 37 °C for 2 h. Single-cell suspensions were then obtained by passing the cells through a 70-mm strainer (BD Falcon, Franklin Lakes, NJ, USA) and cultured in control medium in 5% CO ₂ at 37 °C. The control medium consisted of α-modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin and 100 μg/mL streptomycin. The medium was changed every 3 days.

In colony-forming test, the majority of the hDPSCs retained their spindle-shaped morphology, consistent with other mesenchymal stem cell populations . The multipotency of those hDPSCs had been established using chrondrogenic, adipogenic and osteogenic culturing conditions (Niu, unpublished results). The hDPSCs were seeded in 24-well plates at a density of 10 ⁴ cells/cm ² . The hDPSCs were cultured in complete growth medium consisting of α-modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma–Aldrich, St. Louis, MO, USA) in 5% CO ₂ at 37 °C. The growth medium was changed every 3 days; passages 2–4 hDPSCs were used for the present study.

2.3

Cell viability

2.3.1

XTT assay

In pulpal revascularization protocols, materials are placed in close proximity to the blood clot from which hDPSCs are derived. Cytotoxic components derived from the set calcium silicate cements can readily diffuse into the blood clot. Thus, both materials and eluents were evaluated separately, using a previously established cyclic regime .

An XTT Cell Viability Assay Kit (Biotium Inc., Hayward, CA, USA) was used to determine cell viability, based on the ability of mitochondrial enzymes in metabolically active cells to reduce the yellow tetrazolium derivative 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) to form a water-soluble orange-colored formazan product. The activities of those enzymes are inactivated shortly after cell death; hence, the assay enables determination of live cell numbers using spectrophotometric absorbance quantification at 475 nm. The viability of untreated hDPSCs was used as the negative control, the mean absorbance of which was adjusted to 100% for comparison with the relative dehydrogenase activities of the other groups ( N = 12).

The XTT assay was performed on hDPSCs that had been directly exposed to test cements and on eluents derived from those materials. A weekly cycle consisted of direct evaluation of the toxicity of the set cement disks over the plated cells for 3 days, and indirect evaluation of the effect of eluents derived from the set cements on the plated cells. Eluent collection was achieved by immersing cement disks in complete growth medium for 4 days after direct evaluation of the cement disks. During the first part of each weekly cycle, cement and control disks were placed individually in Transwell inserts with a 3-μm pore size (BD Falcon, Franklin Lakes, NJ, USA) to prevent their direct contact with the hDPSCs. After the inserts were placed over the plated cells, an additional 2 mL of complete growth medium was added to each well to ensure that the level of the culture medium was above the sides of the Transwell insert. The disks were exposed to the plated cells for 3 days without further change in culture medium before testing for mitochondrial dehydrogenase activity. During the second part of each weekly cycle, each disk was retrieved and incubated at 37 °C with complete growth medium (1 disk/2 mL) for 4 days to collect the eluent from the set cement before using the same disk for the next cycle. For each disk, the same growth medium was used for eluent collection throughout the entire testing period. This recurrent cyclic aging protocol was continued until the material disks were rendered noncytotoxic (i.e. >85% of the mean dehydrogenase activity exhibited by the untreated control).

For indirect evaluation of the eluents, each eluent concentrate collected after completion of the aging cycles was diluted with fresh complete growth medium to 1:1, 1:10, and 1:100 of its original concentration to achieve a final volume of 2 mL ( N = 12). Each diluted, eluent-containing growth medium was then used as the respective culture medium for freshly plated hDPSCs. The XTT assay was performed in the same manner as previously described; absorbance was determined using a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA).

2.3.2

Flow cytometry

Flow cytometry was employed with differential staining for sorting and counting of individual cells within a cohort of hDPSCs that expressed changes in plasma membrane permeability induced by the toxicity of the tested materials. The hDPSCs were plated in 6-well plates at a density of 10 ⁵ cells/cm ² and incubated at 37 °C in a humidified 5% CO ₂ atmosphere for 24 h. The material disks placed in Transwell inserts as previously described. After 3 days of exposure to the materials, the hDPSCs were detached from the culture wells using 0.25% trypsin/2.21 mM ethyelenediaminetetraacetic acid (EDTA) (Mediatech, Inc., Corning, Manassas, VA, USA) and re-suspended in binding buffer derived from the Apoptosis and Necrosis Quantification Kit (Biotium). The hDPSCs were stained with fluorescein isothiocyanate–annexin V ( λ _abs / λ _em : 492/514 nm) and ethidium homodimer-III ( λ _abs / λ _em : 528/617 nm) as the respective fluorescence stains for cytoplasmic membrane phospholipids and nucleic acids ( N = 3). During apoptosis, phosphatidylserine is translocated from the inner to the outer surface of the cell for phagocytic cell recognition . Annexin V labeled with fluorescein identifies apoptotic cells by binding to phosphatidylserine exposed on the outer leaflet of the cytoplasmic membrane, resulting in the expression of green fluorescence within the cytoplasm. Ethidium homodimer III is a highly positive-charged nucleic acid probe impermeable to live cells or apoptotic cells, but stains necrotic cells with red fluorescence. The stained cells were incubated in the dark for 15 min and subjected to fluorescence-activated cell sorting (MACSQuant Analyzer, Miltenyi Biotec Inc., Auburn, CA, USA) to determine the percentage distribution of viable, early apoptotic, late apoptotic, and necrotic cells. Experiments were performed in triplicates.

2.4

Cell Differentiation and mineralization

2.4.1

Gene expression

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression of target markers of osteogenic differentiation among the test groups. The set cements were first rendered non-cytotoxic by aging in culture medium for 5 weeks. The hDPSCs were cultured in complete growth medium at a density of 10 ⁴ cells/cm ² to ∼80% confluency. The culture medium replaced by osteogenic differentiation medium, consisting of the original complete growth medium supplemented with 50 mg/mL ascorbic acid, 10 mmol/L β-glycerophosphate and 100 nmol/L dexamethasone (Sigma–Aldrich). The established hDPSCs were exposed to Transwells containing the 3 aged calcium silicate cements (MTA Angelus, Biodentine and TheraCal LC) for 7 days, with the osteogenic differentiation medium changed every 3 days.

The total RNA present in the hDPSCs ( N = 3) from each group was isolated and purified using QIAshredder and RNeasy kit (Qiagen, Valencia, CA, USA). Quality control of the extracted RNA was performed with 2 μL samples by measuring their UV absorbance (NanoDrop ND-1000 UV–Vis spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantity of extracted RNA was determined from the RNA absorbance at 260 nm; RNA purity was determined from the ratio of the absorbance (A@260 nm/A@280 nm and A@260/A@230 > 1.8 and <2.0). Equal amounts of total RNA (0.1 mg RNA/mL) were reverse-transcribed into single-stranded complementary DNA (cDNA) using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific Inc.) in a 7300 real-time PCR system (Applied Biosystems). Reverse transcription was performed using thermocycling conditions recommended by the manufacturer: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min). The generated cDNA was stored at −20 °C until commencement of qRT-PCR.

For qRT-PCR, alkaline phosphatase ( ALP ), osteocalcin (OCN), bone sialoprotein (BSP), runt-related transcription factor 2 ( Runx2 ), dentin sialophosphoprotein ( DSPP ) and dentin matrix protein-1 ( DMP-1 ) were used as target markers of osteogenic/dentiogenic differentiation . Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as the housekeeping gene. Untreated hDPSCs grown in osteogenic differentiation medium was assigned as the negative control. The cDNA (100 ng each) was mixed with Taqman ^® PCR Universal Master Mix II (Invitrogen, Thermo Fisher Scientific Inc.) and added in 20 μL aliquots to 96-well sequence-specific TaqMan ^® array standard plates preloaded with the designated primers and housekeeping gene ( ALP : Hs01029144_m1; OCN : Hs01587814_g1; BSP : Hs00173720_m1; Runx2 : Hs00231692_m1; DSPP : Hs00171962_m1; DMP-1 : Hs01009391_g1; GADPH : Hs99999905_m1). The plate was loaded in the thermocycler and run in regular cycles. Each cycle consisted of 50 °C for 2 min, 95 °C for 10 min, 40 cycles of denaturing/annealing at 95 °C for 15 s and 60 °C for 1 min. Experiments were conducted in triplicate. The comparative threshold cycle method (ΔΔ C _T ) was employed for relative quantification of gene expression; the expression level of each target cDNA marker was normalized to the GAPDH cDNA endogenous control. Data output was expressed as fold changes of mRNA expression levels, given by 2 ^−ΔΔCt .

2.4.2

Alkaline phosphatase enzyme activity

Cells cultured in osteogenic differentiation medium were exposed to the 4 aged materials for 14 days ( N = 12). Intracellular ALP activity was evaluated using the Quantichrom ALP assay kit (Bioassay Systems, Hayward, CA, USA). Colorimetric determination was based on hydrolysis of p -nitrophenyl phosphate by ALP into inorganic phosphate and p -nitrophenol, a yellow-colored product. After 14 days of exposure, the cement disks and their respective Transwell inserts were retrieved. The cells were washed with PBS and lysed with 0.2% Triton X-100 for 20 min. Assay buffer and p -nitrophenyl phosphate were added to a clear-bottomed 96-well plate (150 μL/well). Following addition of the cell lysate, the optical density of p -nitrophenol at t = 0 min was recorded at 405 nm using the Synergy microplate reader. The samples were shielded from direct light at room temperature for 4 min prior to recording the optical density of p -nitrophenol at t = 4 min. The ALP activity of the cell lysate (in IU/L = μmol/L min) was calculated from the difference in optimal density readings.

2.4.3

Extracellular mineralization

Cells cultured in osteogenic differentiation medium were exposed to the 4 aged materials for 21 days ( N = 6). The hDPSCs were evaluated for calcium production by rinsing three times with phosphate-buffered saline. The cells were then fixed in 10% formaldehyde and stained with Alizarin red S (40 mmol/L, pH 4.2), a dye with affinity to calcium salts. Images of the specimens were taken for qualitative evaluation. Stained calcium deposits were incubated in 10% acetic acid for 30 min and neutralized with 10% ammonium hydroxide . The supernatant was pipetted into a clear-bottom 96-well plate and the absorbance of the supernatant was determined at λ = 550 nm. The concentration of dye extracted from the specimens (mg/mL) was determined according to a linear regression equation derived from a calibration curve that correlated absorbance with known Alizarin red S concentrations. Because one mole of Alizarin red S binds to two moles of calcium to produce an Alizarin Red S-calcium complex , the extent of calcium deposition was expressed as molar equivalent of calcium.

Because Alizarin red S is not specific for calcium or alkali earth metals and can also react with a wide variety of cations , hDPSCs in various groups before and after addition of the osteogenic differentiation medium were examined by transmission electron microscopy (TEM) to confirm the formation of extracellular calcium deposits. The hDPSCs cultured in complete growth medium were detached by incubating in trypsin-EDTA for 2 min. Pooled dislodged cells were centrifuged, fixed with Karnovsky’s fixative, post-fixed in 1% osmium tetroxide, dehydrated in an ascending ethanol series (30–100%), transferred to propylene oxide, and embedded in epoxy resin. Sections prepared by ultramicrotomy (90 nm thick) were stained with uranyl acetate and Reynold lead citrate. Stained sections were examined using a JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV. After completion of the mineralization assay, hDPSCs from unstained wells were similarly processed and examined without further staining to identify the status of mineralization within the extracellular matrix.

2.5

Statistical analyses

Data from the IRM positive control was excluded from all statistical analyses to increase the robustness of the respective analysis. For XTT assay, results derived from weekly cycles were analyzed using two-factor repeated measures analysis of variance (ANOVA) and post-doc Holm–Sidak multiple comparison procedures to examine the effects of calcium silicate cements and aging cycle on hDPSC viability. Similar tests were used for analyzing the effect of eluent dilution and aging cycle on cell viability. For flow cytometry, one-factor ANOVA and Holm–Sidak multiple comparison procedures were used to examined the effects of calcium silicate cements on the percentage of vital, non-apoptotic, non-necrotic hDPSCs. For qRT-PCR, the effect of materials on expression of each osteogenic marker was analyzed separately by comparing with the GADPH endogenous control, using a one-factor ANOVA and Holm–Sidak multiple comparison procedures. The results obtained from ALP enzymatic activity and Alizarin red S staining were separately analyzed using one-factor ANOVA and Holm–Sidak multiple comparison procedures. For all statistical analyses, parametric versions of these tests were used after evaluation of the normality (Shapiro–Wilk test) and equal variance assumptions (modified Levene test) of the individual data tests. If those assumptions were violated, the data were nonlinearly transformed to satisfy those assumptions prior to the use of parametric testing methods. Except for qRT-PCR, statistical significances for all analyses were set at alpha = 0.05. For qRT-PCR, because the chance of finding one or more significant differences in testing six hypotheses on osteogenic markers with the same samples (type I error) is 26.49%, the Dunn–Šidák correction was used for controlling the familywise error rate. For each osteogenic marker, the critical value (alpha) was reduced to 0.0085, so that test results with P < 0.0085 were only considered significantly different from the GADPH endogenous control.