Biomimetic Properties of Force-Spun PHBV Membranes Functionalised with Collagen as Substrates for Biomedical Application
The fibre membrane surface static contact angles were investigated at
room temperature using a Contact Angle CAM 200 KSV Instrument ,
equipped with Tetha software using the sessile drop method, dispensing 5 μL water droplets (HPLC grade)
directly onto the sample surface.
In vitro biodegradation studies were performed in Phosphate Buffered Saline (PBS) solution, pH 7.4, at 37 °C (VWR, Incu-Line). Differences in mass were measured by subtracting the dry weight after incubation at time points from the initial dry weight. The material was removed from incubation, washed with deionised water, and dried in a vacuum desiccator before weighing.
Metabolic activity was assessed by Presto Blue assay (Life Technologies, Thermo Fisher Scientific, USA). Cell culture media was removed from samples and replaced with 1500 μL of 1:9 v/v Presto Blue to DMEM. Treated samples were incubated at 37 °C, 5% CO2, for 2 h after which the solution was removed and 200 µL from each sample placed into an opaque 96-well plate.
Samples (4-8 mg, aluminium pans) were analysed using a Netzsch Polyma, using Netzsch Proteus software. A heating scan from 20 to 200°C was conducted at a rate of 10 °C min-1 under a nitrogen atmosphere.
The amount of collagen present in coated PHBV fibres and films was quantitatively determined using Sirius Red dye kit. Films and fibrous mats were cut into square samples with a surface of 1 cm2, and then incubated in 1 ml of Sirius Red dye in a cap tube for 30 min, forming a collagen-dye complex following the protocol provided by Sircol. Thereafter, the dye solution was drained and the stained samples were washed twice with cold washing solution, removing the unbound dye. The washing solution was removed along with any fluid from the lip of the Eppendorf tubes using cotton wool buds. The bound dye was dissolved using 250μl of the provided alkali solution to the samples and mixed for 10 min. Then, 200μl of collagen bound-dye solution was transferred into a 96-well plate, and optical density was measured at 555 nm with UV-Vis scanning spectrophotometer (Sunrise, TECAN) using XFluor4 software (TECAN, V4.51). The quantity of collagen bonded to PHBV films and fibres was calculated using a calibration curve prepared with known collagen concentrations. All the measurements were performed in triplicate.
XPS data: X-Ray Photoelectron Spectroscopy (XPS; Theta Probe, Thermo Scientific, East Grinstead, UK), which uses a micro- focused AlKa X-ray source (1486.6 eV), operated with a 400 µm spot size (100 W power). Survey spectra were collected at a pass energy of 200 eV, a step size of 1 eV and a dwell time of 50 ms, with the spectrometer operated in standard (not angle-resolved) lens mode. High resolution regional spectra were collected using a pass energy of 40 eV, a step size of 0.1 eV and a dwell time of 200 ms. High resolution spectra envelopes were obtained by curve fitting synthetic peak components using the software CasaXPS.
Tissue Engineering and Regenerative Therapies Centre Versus Arthritis (Award 21156)