2.5 Alkaline Phosphatase Activity Assays

The C2C12 cell line was used to evaluate the ability of core-shell MPs to delivery bioactive BMP-2 after shell degradation, as C2C12 cells produce Alkaline Phosphatase (ALP) in response to BMP-2 [33]. C2C12 cells were cultured at 37°C, 5% CO₂ in DMEM with 4.5 g/L glucose, fetal bovine serum (FBS) (10% for growth media, 1% for assay media; Hyclone), 2 mM L-glutamine, 50 IU Penicillin and 50 µg/mL Streptomycin. For assays, cells were plated at 62,500 cells/cm² in 96 well plates and were incubated for 6 hours to allow adherence before the assay was started.

Microparticles were prepared for pre- or post-fabrication load experiments as described in section 2.4.2 and were sterilized by washing with 70% ethanol for 30 minutes, followed by 3 washes with PBS for 30 minutes each. MPs were centrifuged at 10,000 RCF for 3 minutes and supernatant was removed between each sterilization and wash. For pre-fabrication studies, groups included pre-fabrication loaded and non-loaded degradable core-shell MPs, pre-fabrication loaded and non-loaded core-shell MPs, and a soluble BMP-2 and a no BMP-2 media control (Fig. 4A). For post-fabrication studies, groups included post-fabrication loaded and non-loaded degradable core-shell microparticles, loaded and non-loaded degradable PEG-based MPs, and a soluble BMP-2 and no BMP-2 control (Fig. 6A). All MPs were incubated for 2.5 days prior to being added to cells for pre-degradation. This allowed MP shell degradation to occur during the three day cell assay (while degradation occurs throughout the entire time course, the majority of the PEG-based MP degradation occurred between days 3–6 for these MPs; Supplementary Fig. 1B and 2). All core-shell and heparin MP groups had 0.02 mg of heparin and the degradable PEG-based control group had the same number of MPs as the core-shell group. Cells were cultured for 3 days and then media/MPs were removed.

After media and MP removal, cells were washed twice with PBS. DdH₂O was added to cells for 20 minutes and then cells were subjected to a freeze-thaw cycle. Cells were then scraped from the 96-well plate and cell lysate solution was transferred to 1.7 mL tube. Cell lysate was sonicated for 20 minutes, then subjected to another freeze-thaw cycle. This was repeated once, then samples were centrifuged at 10,000 RCF for 5 minutes and supernatant was used for analysis. For ALP activity, 50 µL sample was combined with 50 µL 1.5M 2-amino-2-methyl-1-propanol (Sigma-Aldrich), 50 µL 10 mM magnesium chloride, and 50 µL 20 mM p-nitrophenol phosphate disodium salt hexahydrate (Sigma-Aldrich). For standards, p-nitrophenol (Sigma-Aldrich) was used. Samples were allowed to incubate for 2 hours and absorbance was read at 405 nm. DNA content was assessed with the CyQUANT Assay following the manufacturer’s instructions and using bacteriophage λ DNA to create a standard curve (ThermoFisher Scientific). The assay was read at excitation/emission of 480/520.