Mg 63 cells

Polycaprolactone (PCL, Mn 70,000), 2-aminoethyl methacrylate hydrochloride, and fluorescamine were purchased from Sigma (Saint Louis, MO, USA). Methanol and tertahydrofuran (THF) were purchased from Duksan Chemical (Seoul, Korea). N,N-dimethyl formamide (DMF) was purchased from Showa Chemical (Japan). E. coli-derived recombinant human bone morphogenetic protein-2 (BMP-2) was donated by Cowellmedi (Busan, Korea). Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin-streptomycin were purchased from Gibco-BRL (Rockville, MD, USA). The cell counting kit-8 (CCK-8) was purchased from Dojindo (Tokyo, Japan). The BMP-2 ELISA kit was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). All chemicals and solvents were used without further purification. MG-63 cells (human osteosarcoma cell line) were purchased from Korea Cell Line Bank (KCLB number 21427, Seoul, Korea).

MG-63 cells were obtained from Korea Cell Line Bank (No. 21427, Seoul, Korea). In this study, we used MG-63 cells because they are commonly used to evaluate osteogenic evaluation of bioceramices, bioactive drugs, peptides, and proteins in/on various scaffolds.
MG-63 cells were carefully seeded at a density of 1 × 10⁵ cells/mL on 10 mg of PMSs, aminated-PMSs, BCP-M-PMSs, or BCP-IM-PMSs in 24-well tissue culture plates. Cells were cultured in DMEM supplemented with 10% FBS, 50 μg/mL ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerophosphate with 100 U/mL penicillin and 100 μg/mL streptomycin for 10 days. At predetermined time intervals (3, 7, and 10 days), each sample was rinsed with PBS, and 1× RIPA buffer was added for cell lysis. To remove cell debris, cell lysates were centrifuged at 13,500 rpm for 1 min. After centrifugation, p-nitrophenyl phosphate solution was added to the supernatants. The reactions were incubated for 30 min at 37 °C and then stopped with 1 M NaOH. Based on the standard of p-nitrophenyl phosphate solution, the ALP activity was determined by converting p-nitrophenyl phosphate to p-nitrophenol. ALP activity was evaluated using a Flash Multimode Reader to measure the absorbance at 405 nm.

Poly (d,l-lactic-co-glycolic acid) (PLGA, 50:50, molecular weight: 30,000–60,000), poly vinyl alcohol (PVA, molecular weight: 13,000–23,000, 98% hydrolyzed), dichloromethane (DCM), gelatin from porcine skin, ascorbic acid, dexamethasone, and β-glycerophosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin-streptomycin were obtained from Gibco BRL (Rockville, MD, USA). Cell counting kit-8 (CCK-8) reagents were obtained from Dojindo, Inc. (Kumamoto, Japan). Biphasic calcium phosphate (BCP; Hydroxyapatite = 60%, Tricalcium phosphate = 40%) nanoparticles were kindly donated by Ossgen Corporation (Gyeongbuk, Korea). MG-63 cells (human osteosarcoma cell line) were obtained from the Korea Cell Line Bank (KCLB No. 21427, Seoul, Korea). All chemical reagents were of the purest analytical grade available.