Mc3t3 cells

MC3T3 cells (ATCC, Manassas, VA) were cultured in 10 ml of 10% fetal bovine serum (FBS) (FisherScientific, Pittsburgh, PA) and 1% penicillin/streptomycin (FisherScientific, Pittsburgh, PA) media. The cells were passaged every three days and no more than fifteen times. Then 20,000 cells were dispensed into 30 wells on a single 48-well plate. Each well also received 1 ml of phenol-free media and MOFs or control treatment. Pre-made MOFs were utilized to treat cells in 18 wells. Three types of MOFs were tested: Ca, Sr, and Ca-Sr. Initially Mg was also used as a MOF treatment however, it was decided to no longer utilize Mg because it did not aid in generating osteoblasts or proliferating cells. Of the remaining 12 wells, 6 were treated with 1 ml osteogenic media (100 nM of dexamethasone (FisherScientific, Pittsburgh, PA), 10 mM of glycerophosphate (FisherScientific, Pittsburgh, PA), and 50 µM of ascorbic acid (FisherScientific, Pittsburgh, PA)) to represent the positive control. The final 6 wells were not treated beyond 1 ml of phenol free media to indicate the negative control. On day 7, the sample underwent an MTT cell proliferation assay to ensure that the cells were metabolically active and alive after having been treated with MOFs. The samples were also simultaneously tested for bone differentiation via alkaline phosphatase assay after three and seven days.

Mouse fibroblast cell line NIH3T3 (ATCC) was cultured in reduced sodium bicarbonate content (1.5 g per liter) Dulbecco’s modified Eagle’s medium with (DMEM) supplemented with l-glutamate (2 mM), 10% bovine calf serum, and 1% penicillin–streptomycin. Mouse C2C12 myoblasts (ATCC) were cultured in DMEM with 20% FBS and 1% penicillin–streptomycin, and committed into mature myoblastic cells using DMEM supplemented with 2% horse serum and 1% penicillin–streptomycin^{32 (link),33 (link)}. Mouse chondrocytes were cultured in minimum essential medium alpha (MEMα) with nucleosides, ascorbic acid, glutamate, sodium pyruvate supplemented with 10% FBS and 1% penicillin–streptomycin. Mouse MC3T3 cells (ATCC) were cultured in MEMα with nucleosides and l-glutamine without ascorbic acid and supplemented with 10% FBS and 1% penicillin–streptomycin. To commit MC3T3 into mature osteoblasts, MC3T3 media was supplemented with 10 nM dexamethasone, 50 µg per ml ascorbic acid and 10 mM β-glycerophosphate^{27 (link),54 (link)}. Lineage committed progenitor cells, referred here as pre-osteoblasts and pre-myoblasts, were also included in the study to mimic the osteogenic and myogenic regeneration profile in the adult tissue^{27 (link),28 (link)}.

Take 4 mL of α-MEM complete medium to the centrifuge tube and preincubate at 37°C for 10 min. The cryopreserved MC3T3 cells (ATCC, USA) removed from the liquid nitrogen were quickly shaken in a 37°C water bath and reshaken quickly. The cells were transferred to the preheated medium and the supernatant was centrifuged. The appropriate amount of α-MEM was completely cultured at 37°C 5% CO₂ incubator. On the next day, the fresh medium was replaced and incubated at 37°C in a 5% CO₂ incubator. Cells were used in experiments 3 to 5 generations.