Uv vis spectroscopy

Porphysomes were synthesized following a protocol reported previously (29 (link)). The lipid film of porphysomes consists of 55 mol % porphyrin-lipid (pyropheophorbide-lipid), 40 mol % cholesterol (Avanti Polar Lipids, Alabaster, AL, USA) and 5 mol % distearoyl-sn-glycero-3- phosphoethanolamine-N-methoxy(polyetheneglycol) (PEG2000-DSPE, Avanti). Lipid films were dried under a gentle stream of nitrogen gas, followed by 1 h under vacuum. Each lipid film was then rehydrated with PBS buffer (150 mM, pH 7.5) at a concentration of 10 mg/mL and extruded through a polycarbonate membrane (pore size = 100 nm) 10 times. The porphyrin concentration was determined by UV/Vis spectroscopy (Varian Inc., Palo Alto, CA, USA). Porphysomes were kept sterile and stored at 4 °C prior to use.

The surface wettability was characterized by measuring the static water contact angle, which was obtained from a DSA 100 drop shape analysis system (KRÜSS) with a computer-controlled liquid dispensing system. One μL DI water droplets were deposited onto substrate surfaces, and the water contact angles were measured within 10 s through the analysis of photographic images. The cross-linking kinetics of BPMPC coating was investigated by a UV—vis spectroscopy (Varian) with 254 nm UV light. The thickness of the spin-coated polymer layer on the silicon substrates and CarboSil substrates were measured by a M-2000 V Spectroscopic Ellipsometer (J.A Woollam co., INC.) with a white light source at three incident angles (65°, 70°, and 75°). The thickness of the modified layer was measured and calculated using a Cauchy layer model. Infrared spectroscopy studies of polymer coated films were done using a Thermo-Nicolet model 6700 spectrometer equipped with a variable angle grazing angle attenuated total reflection (GATR-ATR) accessory (Harrick Scientific).

The osteoblast cell viability was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) assay after 1 and 2 days of culture. Samples were moved to a new 24-well plate and 100 μL of MTT (Sigma, St. Louis, MO) solution was added on top of each sample, followed by the addition of 900 μL of cell media. After 2 hours of incubation at 34 °C, the solution was removed from each well and 600 μL of solubilizer solution (composed of 10 % Triton X-100, 0.1N HCl and isopropanol) was added to each sample to dissolve formazan crystals. Formazan is a purple colored compound produced after reduction of MTT by cellular enzymes. 100 μL of this solution was transferred to 96-well plate and read by UV–Vis spectroscopy (BioTek) at 570 nm.

Firstly, vitamin C (Sigma Aldrich, MO) is dissolved in DI water at two different concentrations of 4 and 8 mg/ml. Then, 50 μl of drug solution is loaded on both sides of TCP scaffolds, so that each sample contains either 400 or 800 μg of vitamin C. From now on, scaffolds loaded with 400 and 800 μg of vitamin C will be mentioned in this paper as 400C-TCP and 800C-TCP, respectively. After drug loading, the scaffolds are kept in the dark at room temperature for drying. 5% PCL (M_w=14000, Sigma Aldrich, MO) solution is prepared by dissolving in acetone. 50 μl of PCL solution is added dropwise to both sides of the scaffolds.
To study the in vitro release kinetics, a phosphate buffer and an acetate buffer with pH values of 7.4 and 5.0, respectively, are prepared. Three scaffolds from each group of composition (pure TCP, 400C-TCP and 800C-TCP) with and without PCL are kept in separate vials containing 4 ml of phosphate or acetate buffer. Vials are kept under continuous shaking at 150 rpm at 37 °C. Release media are collected after 1.5, 3, 6, 12, 24, 48, 96, and 144 h, and replaced with fresh buffer solution. Finally, 200 μl of release media from each sample are pipetted to a 96-well plate. To determine the concentration of the released drug, the absorbance of all the scaffolds are measured at 260 nm wavelength using the UV–Vis spectroscopy (BioTek).