Effects of Estradiol on the Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells 2

Materials and Methods

 

Chemical and reagent

Cell culture media (alpha modified Eagle medium) and fetal bovine serum (FBS) were purchased from GIBCO (Gibco-BRL, USA). β-estradiol, β-glycerophosphate, L-ascorbic acid, MTS, PBS and Alizarin Red S were purchased from Sigma (USA). The primary antibodies used were β-actin (Santa Cruz, USA), anti-DMP-1(Biovision, USA) and anti-DSPP (Novus, USA). The BrdU cell proliferation assay kit was purchased from Calbiochem (CA). ALP activity asssy kit was purchased from Biovision.

 

Cell culture

 The HDPCs lines were immortalized by transfection with the telomerase catalytic subunit hTERT gene (Kitagawa et al., 2007). Cells were cultured in α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified atmosphere of 5 % CO₂at 37℃. For differentiation and mineralization experiments, cells were cultured in differentiation inductive media including 100 μM/L ascorbic acid and 10 mM/L β-glycerophosphate.

 

Cell viability assay

Cell viability was determined using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- caarboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). HDPCs were incubated in 48 well plates at a humidified atmosphere of 5 % CO at 37℃ for 1, 3, 5 and 7 days. β-estradiol was dissolved in DMSO and sterilized through 0.2 μm filter. Optical density was spectro-photometrically measured at 405 nm using ELISA reader.

 

Cell proliferation assay

Cells were plated in 96 well plate and treated with different concentrations of β-estradiol (0.1-10 μM) for 7 days. Proliferation was assessed by ELISA reader using the BrdU proliferation kit according to the manufactures instructions. Briefly, cells were labeled and incubated for 24 h. After fixation and denaturation processes, BrdU antibody was applied. Cells was washed 3 times and incubated with peroxidase goat anti-mouse IgG HRP conjugate. Subquently, cells reacted with a substrate for 15 min and added with stop solution and then measured at 540 nm in a microplate reader.

 

Reverse transcription-PCR

At 7 days of incubation, human dental pulp cells were harvested. For extraction of total RNA, cells were homogenized with in Trizol reagent. For synthesis of cDNA, 2 μg of total RNA and 1㎕ oligo-dT were mixed with 20㎕ RNase-free water, and then incubated at 42℃ for 1h and 70℃ for 10 min. PCR products were generated in PCR buffer containing 10 pmoles of each primer using PCR-premix kit (Genet Bio). The following primer pairs were used : for ER-α, 5′-GGAGACGGACCAAAGCCACT-3′-(sense), 5′-TTCCCAACAGAAGA CAGAAGATGAGATG-3′ (antisense), for ER-β, 5′-GCTGAACACCGTGACCGATGCT-3′ (sense), 5′-CCCGTGATGGAGGACTTGC-3′(antisense), for ALP, 5′-CCCAAAGGCTTCT TCTTG-3′ (sense), 5′-CTGGTAGTTGTTGTGAGC-3′(antisense), for BSP, 5′-TGGAGATG ACAGTCAGAAG-3′ (sense), 5′-GTACTGGTGCCGTTTATG-3′ (antisense), for DSPP, 5′-CAGTGATGAATCTAATGG-3′ (sense), 5′-CTGATTTGCTGCTGTCTGAC-3′ (antisense), for DMP1, 5′-CAGGAGCACAGGAAAAGGAG-3′ (sense), 5′-CTGGTGGTATCTTGGG CAC T-3′ (antisense).

The amplified products were analyzed on 1.5% agarose gels containing SYBR™(safe DNA gel stain) and visualized by UVP Transilluminator/Polaroid camera System (UVP Laboratories, CA). RT-PCR was performed with primers for GAPDH, a housekeeping gene, as a control. The following primer pairs for GAPDH were used : 5’AGTCACGGATTT GGTCGT -3’(sense) and 5’-ACAAGCTTCCCGTTCTCAG -3′(antisense).

 

ALP activity assay

Briefly, 1x10 pulp cells/well in 12 well plates were exposed to different concentrations of β-estradiol (0.1-10μM) for 7 days. ALP activity in cell lysates were evaluated by determining the amount of p-nitrophenol production using ALP activity assay kits. To measure intracellular ALP, washed cells were lysated in the assay buffer, centrifuged to remove insoluble material at 13000 x g for 3 min. Added samples into 96 well plate, bring with assay buffer and added 5 mM pNPP solution. After the reaction was incubated for 60 min at room temperature and protected from light, the stop solution was added and then measured O.D. at 405 nm in a micro plate reader.

 

Alizarin red-S stain

After 2 weeks of differentiation induction, cells were rinsed with phosphate buffered saline and fixed in ice-cold 70% ethanol for 1 hr at room temperature. Subsequently, the cells and the matrix were stained with 40 mmol/L Alizarin red-S (pH 4.2) for 15 min at room temperature, washed extensively five times with deionized water and once with PBS for 15 min at ambient temperature in order to remove nonspecifically bound stain. Bound dye was solubilized in 10% cetylpyridinium chloride and quantitated spectrophotometrically at 562 nm.

 

Western blotting

Cells were washed twice with PBS and proteins solubilized in a lysis buffer (1% NP-40, 500 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Benzamid, 1㎕/㎖ Trypsin inhibitor) containing a cocktail of protease inhibitor (Complete, Germany). Lysates were incubated for 30 min at 4℃, centrifuged at 11,000 xg for 20 min at 4℃ and protein concentrations were determined by BSA protein assay (Pierce, IL). Protein extracts (50-100㎍) were boiled for 5 min with SDS-sample buffer and then subjected to electrophoresis on 10% polyacrylamide gel. Proteins were electroblotted onto nitrocellulose membrane and blocked with 5% skim milk (Becton Dickinson, USA) in tris-buffered saline-0.1% Tween 20 (TBS-T). Primary antibodies of rabbit polyclonal anti-DMP-1 and DSPP were applied. Blots were subsequently washed three times in TBS-T for 5 min and incubated with specific peroxidase-coupled secondary antibodies (Sigma, USA) for 1 hr. Bound antibodies were visualized using an enhanced chemiluminescent detection system (Amersham Pharmacia Biotech, UK).