Crl 11372

The osteoblastic cell line hFOB 1.19 (ATCC® CRL11372™, ATCC, USA)^{77 (link)} was cultured using DMEM/F12 (Dulbecco’s Modified Eagle Medium α) (Gibco 10565018) conditioned media supplemented with 10% (V/V) foetal bovine serum (FBS) (Eurobio CVFSVF00-01). Cells were cultured at 37 °C in 5% CO₂ in a 10 cm diameter petri dish and trypsinized using 0.05% Trypsin–EDTA (Gibco 25300-054). After sterilization with 70% (w/v) ethanol for 30 min and UV irradiation for 1 h, the filaments and printed scaffolds were dried at room temperature and then placed in contact with hFOB cells for 5 days.
Cell viability was analyzed using MTT assay carried out by incubating 100 µL of 0.5 mg/mL of MTT solution on the cells for 3 h. Purple coloured formazan crystals were dissolved using 100 µL of DMSO (BDH Prolab 23486.297) and the absorbance was recorded at 560 nm using Multiskan plat reader (thermos, USA).

The osteoblast-like mouse cell line MC3T3-E1 was maintained in minimum essential medium alpha (α-MEM) medium containing 10% fetal bovine serum (FBS) [11 (link)]. For monolayer cultures, the cells were inoculated into 75-cm flasks at a density of 30,000–50,000/cm². From 24 h after the start of culturing, the medium was renewed every third day with α-MEM containing 10% inactivated FBS and supplemented with 2 mM l-ascorbic acid 2-phosphate and 4 mM β-glycerophospahte to induce osteoblastic differentiation.
hFOB1.19 cells were received from ATCC (ATCC^® Number: CRL-11372™) temperature-sensitive SV40 large T antigen gene was transfected into this cell, it is expressed at 34 °C and its expression stops at 39 °C. Therefore, in growth culture, 10% of inactivated FBS and 1 g/L of glucose were added to the DMEM/F12 GlutaMax culture solution and cultured at 34 °C. In the differentiation culture, l-ascorbic acid 50 µg/mL, vitamin D 3 × 10⁻⁸ M, vitamin K 3 × 10⁻⁸ M was added to the growth medium and incubated at 39 °C. The culture broth was changed almost every 3 days.
In the monolayer culture, gene expression analysis was performed after differentiation culture for 10 days after cell seeding.

The osteoblast cell lines MC3T3 and hFOB1.19 were purchased from American Type Culture Collection (ATCC, CRL-2593, and CRL11372) and cultured as per the manufacturer’s protocol. Dexamethasone (Sigma-Aldrich, D1756, St. Louis, MO, USA) and Betamethasone (Sigma-Aldrich, B7005, St. Louis, MO, USA) treatments were performed as described in the experiments. Lentiviral vectors containing CMV-aurora A and CMV-glucocorticoid receptor were all purchased from GeneCopeia^TM (Rockville, MD, USA) and applied to the cells as per the manufacturer’s protocols.

Five OS and three OB cell lines were used in this study. The OS cell lines CRL‐1543, CRL‐1427, CRL‐2098, HTB‐85, and HTB‐96 and the OB cell line CRL‐11372 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The OB cell line C‐12720 was purchased from PromoCell (Heidelberg, Germany), and the OB cell line HO‐f‐4610 was purchased from SanBio (Uden, The Netherlands). HTB‐96 and HTB‐85 were cultured in McCoy's 5a medium modified. CRL‐1427 and CRL‐1543 were cultured in Eagle's minimum essential medium. CRL‐2098 was cultured in RPMI‐1640. All OS cell lines were supplemented with 40 mg·mL⁻¹ streptomycin, 240 mg·mL⁻¹ penicillin (400 000 U·mL⁻¹), and 10% fetal bovine serum, except for HTB‐85, which was supplemented with 15% fetal bovine serum. CRL‐11372 was cultured in 1 : 1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium without phenol red, supplemented with 0.3 mg·mL⁻¹ G‐418 and 10% fetal bovine serum. C‐12720 was cultured in OB growth medium + supplement mix (PromoCell). HO‐f was cultured in OB medium (ScienCell Research Laboratories, Carlsbad, CA, USA), and the flasks were coated with poly‐L‐lysine solution bioreagent 0.01% (Sigma Aldrich, St Louis, MO, USA).
All eight cell lines were cultured in a humidified atmosphere of 5% CO₂ at 37 °C, except for CRL‐11372, which was cultured at 34 °C.

Human bone tissue-derived osteoblasts (CRL-11372) [American Type Culture Collection (ATCC), Manassas, VA, USA] were maintained in the logarithmic phase of growth in a 1:1 mixture of Ham’s F12 medium/Dulbecco’s modified Eagle’s medium (Gibco-BRL, Grand Island, NY, USA), with 2.5 mM l-glutamine (without phenol red), 0.3 mg/ml G418, and supplemented with heat-inactivated 10% fetal bovine serum (Gibco-BRL) at 34°C in a 5% CO₂–95% air-humidified incubator. Human Jurkat T-lymphocyte leukemia cell lines (TIB-152; ATCC) were maintained in the logarithmic phase of growth in RPMI-1640 medium (Gibco-BRL) supplemented with heat-inactivated 10% fetal bovine serum (Gibco-BRL) at 37°C in a 5% CO₂–95% air-humidified incubator.