Mc3t3 e1

A mouse osteoblast cell line, MC3T3-E1, obtained from Thermo Fisher Scientific (Rockford, IL, USA) was maintained in a regular α-MEM medium plus 10% fetal calf serum (FCS). Fluorescein isothiocyanate (FITC)-labeled and rabbit antibodies against paxillin were purchased from Thermo Fisher Scientific. Primary rabbit antibodies against phosphor-FAK (pFAK; Y397), β-catenin, BMP2, COL1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Boston, MA, USA). FITC-conjugated goat anti-rabbit (ZF-0314) secondary antibody specific for primary antibodies were purchased from Thermo Fisher Scientific. The Prolong Gold Antifade Reagent with DAPI was obtained from Life Technologies Inc. (Carlsbad, OTT, Canada). Ascorbic acid, β-glycerol phosphatase and Alizarin red were obtained from Sigma-Aldrich (St. Louis, MO). E. coli toxin cytotoxic necrotizing factor-1 (CNF1), which activates FAK and RhoA [16 (link),17 (link),23 (link)], was obtained from Dr. Harald Genth, Hannover Medical School, Hannover, Germany [49 (link)].

Mouse embryo osteoblast precursor cells (MC3T3-E1) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). The CSDP, metal pins (specification, 0.5 × 5 mm) and discs (diameter, 8 mm) of titanium alloy (Ti6Al4V-AO, Ti6Al4V), were produced by Jiangsu Brightness Medical Devices Co., Ltd. (Jiangsu, China). The Ti6Al4V-AO, which was coated with an oxide modification layer for CSDP construction, was modified from a titanium alloy, Ti6Al4V, by surface anodic oxidation. The standard culture medium was minimum Eagle’s medium alpha (MEM-α, Thermo Fisher, United States) containing 10% fetal bovine serum (Euroclone), 100 U/mL penicillin, and 100 mg/ml streptomycin. The Cell Counting Kit-8 (CCK-8), purchased from DOJINDO, Japan, was used for estimating the viability and proliferation of the MC3T3-E1 cells co-cultured with Ti6Al4V-AO and Ti6Al4V metal discs. The LIVE/DEAD reagent (L7010, Invitrogen), purchased from Thermo Fisher Scientific Inc., was used to quantify the viability of MC3T3-E1 cells co-cultured with Ti6Al4V-AO and Ti6Al4V metal pins.

The MC3T3-E1 cells (cat. # CRL-2593) were used in this study, which was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). MC3T3-E1 cell cultures were maintained in minimum essential medium (MEM, Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc.), and 1% (v/v) penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 °C in a humidified CO₂ incubator. Cells at approximately 80% confluence were passaged by trypsin digestion and expanded through two passages before being used for the study.

The mouse pre-osteoblastic cell line MC3T3-E1 (subclone 4) and mouse myoblast cell line C2C12 were purchased from the ATCC (Rockville, MD). The human osteosarcoma cell lines MG63 and Saos2, and parental heterogeneous MC3T3-E1 were purchased from the RIKEN Cell Bank (Tsukuba, Japan). Human dental pulp (hDPC) cells from three different patients were isolated from healthy teeth extracted for orthodontic purposes with informed consent. The pulp chamber was opened by minimum drilling and pulp tissue was removed from the chamber using a barbed broach. The extracted tissue was placed onto a culture dish plate and cells that were out-growths from the tissue were used as dental pulp cells. MC3T3-E1 cells, both subclone 4 and heterogeneous cells, were maintained in alpha MEM (Life Technologies). 293EBNA, MG63, 3 different kinds of hDPC, and C2C12 cells were maintained in DMEM (Life Technologies). Saos2 cells were maintained in McCoy's medium (Life Technologies). All media were supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies), and 10% FBS, except for McCoy's medium, which was supplemented with 15% FBS. All cells were cultivated at 37°C under humidified 5% CO₂, and 95% air atmospheric conditions.