Osteogenic differentiation media

The osteogenic differentiation of MC3T3-E1 cells on the synthesized matrices was determined by ALP (SensoLyte pNPP, AnaSpec, Fremont, CA, USA) activity at days 1, 3, 7, and 14. The assay is based on the conversion of p-nitrophenyl phosphate (pNPP) to p-nitrophenol, which is measured at 405 nm. Briefly, the cells were seeded on the 15 × 15 mm² samples at a density of 3×10⁶ and cultured in osteogenic differentiation media (Lonza, Walkersville, MD, USA) for 14 days. At the predetermined timepoints, the cells were digested using lysis buffer (300 µL) and subjected to three freeze-thaw cycles. The cell suspension was centrifuged for 10 min at 4°C. 50 µL of the supernatant was added to each well of a 96-well plate and allowed to react for 15 min with 50 µL of pNPP substrate solution at 37°C. Standards of 0–300 ng/mL p-nitrophenol were run in parallel. The absorbance of standards and samples were read at 405 nm. Protein concentrations of the sample supernatants were determined using a Pierce™ assay kit (Thermo Scientific, Waltham, MA, USA).

To evaluate osteogenic potential of arterial and venous Flk1 populations, cells were cultured under osteogenic differentiation media (Lonza) for seven days. Calcium deposition and alkaline phosphatase staining were performed. For calcium deposition, cells were washed twice in PBS without magnesium and calcium and fixed in 4% paraformaldehyde for 10 minutes. After two washes of PBS, Alizarin Red staining was performed based on manufacturer’s instructions (American MasterTech Sci.). For alkaline phosphatase staining, cells were washed twice in PBS and fixed for one minute with 4% paraformaldehyde. Wells were washed in PBS and stained with SigmaFast BCIP/NBT following Sigma manufacturer protocol.