Cell line and cell culture
OCCM-30 cells were obtained by isolating tooth root surface cells from transgenic mice containing a SV40 large T-antigen under control of an OCN promoter, and were characterized as highly differentiated cementoblasts [18, 19]. OCCM-30 were maintained in standard culture medium containing DMEM (Gibco, Life Technologies, USA) supplemented with penicillin (100 U/ mL), streptomycin (100 mg/mL) and 10% fetal bovine serum (FBS) (Gibco), at 5% CO2 atmosphere conditions and 37°C. All the assays described below were performed in triplicate in two independent experiments.
Cell proliferation and viability assays
To address the question of whether CHIR99021 (Sigma Aldrich, St. Louis, MO) affected cell proliferation and viability, a dose-response assay was performed. OCCM-30 were seeded at 1.5 x104 cells/well in 48-well culture plates in standard culture medium. After 24 h, culture medium was replaced by DMEM supplemented with 2% FBS (and penicillin, streptomycin, and L-glutamine) with or without CHIR99021 at 2.5, 5 and 10 µM. The number of viable and non-viable cells were obtained at days 1, 2 and 4 by the trypan blue method.
MTT assay
In order to determine the effect of CHIR99021 (GSK-3 inhibitor) on OCCM-30 metabolic activity the MTT assay was performed at days 1, 2 and 4 after treatment. OCCM-30 cells were seeded in 48-well plates at 2x104 cells/well and incubated (37°C, 5% CO2, 95% humidity) for 24 h in standard culture medium. Thereafter, growth medium was replaced by DMEM with 5% FBS (and penicillin, streptomycin, and L-glutamine) with or without CHIR99021 at 2.5, 5 and 10 µM. Cell metabolic activity was determined by the MTT assay according to the manufacturer's instructions. Briefly, culture medium was replaced by 900 µL of DMEM supplemented with 100 µL of MTT (5 mg/mL) (Life Technologies) at 37°C, and 5% CO2 and incubated for 4 h, under dark conditions. Subsequently, dimethyl sulfoxide (Sigma-Aldrich) was used to dissolve the formazan crystals, and the solution’s optical density was read at 570 nm (VersaMax; Molecular Devices).
Gene expression analysis
In order to determine whether GSK-3 inhibition led to an increased activity of the canonical Wnt signaling, transcripts levels for Axin2 were assessed in OCCM-30 cells treated or not with CHIR99021(2.5, 5 and 10 µM). OCCM-30 cells were seeded in 12-well plates at 1x105 cells/well and kept in standard culture medium until they reached 80% confluence, and culture medium was replaced by DMEM supplemented with 2% FBS (and penicillin, streptomycin, and L-glutamine) with or without CHIR99021. Total RNA was isolated after 30 minutes, 1, 4, 6 and 12 h using the Qiagen RNeasy Micro kit (Valencia, CA, USA). In addition, complementary DNA (cDNA) synthesis and quantitative polymerase chain reaction were performed on the Veriti™ 96-Well Fast Thermal Cycler (Applied Biosystems, USA) using intron-spanning primers to assess the expression of Axin2 (5’-AAGAAGGAGACCGGTCACAG-3’/5’-GGTCCTGGGTAAATGGGTGA-3’). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as housekeeping gene (5’-TGAGCAAGAGAGGCCCTATC-3’/5’-AGGCCCCTCCTGTTATTATG-3’). In addition, the effect of CHIR99021 on the mRNA levels of bone markers, including alkaline phosphatase (Alp) (5’-ATCGGAACAACCTGACTGACCCTT-3’/5’-ACCCTCATGATGTCCGTGGTCAAT-3’), runt-related transcription factor 2 (Runx-2) (5’-ATGATGACACTGCCACCTCTGAC-3’/5’-ACTGCCTGGGGTCTGAAAAAGG-3’), osteocalcin (Ocn) (5’-TGAACAGACTCCGGCG-3’/5’-GATACCATAGATGCGTTTG-3’) and osterix (Osx) (5’-GATGGCGTCCTCTCTGCTT-3’/5’-CGTATGGCTTCTTTGTGCCT-3’), was also assessed by qPCR.
Mineral nodule formation
In order to determine the impact of GSK-3 inhibition on mineral nodule formation in vitro, OCCM-30 were seeded at 2x104 cells/well in 12-well culture plates and incubated under differentiation or control conditions in an atmosphere of 5% CO2 and 37°C for 5, 8 and 12 days. Differentiation medium was composed by DMEM plus antibiotics (penicillin/streptomycin) (10 µg/mL) with 100 nM dexamethasone (Gibco, Life Technologies), 10% FBS, 0.05 mM L-ascorbic acid and 10 mM β-glycerophosphate disodium salt hydrate whereas the control medium was DMEM with 10% FBS and antibiotics. CHIR99021 was added to both, differentiation and control culture medium at 2.5, 5 and 10 µM, and mineral nodules assessed by Xylenol orange (XO) staining as previously described [21].
Statistical analysis
Intra and intergroup analyses were performed using one-way ANOVA followed by the Tukey test in order to determine the effect of GSK-3 inhibition on mitochondrial activity, proliferation rate, gene expression and mineral nodule formation. Significance levels were set at 5%.