Porcine skin gelatine

ECFCs (140,000/ml culture medium per well) were seeded in a 12‐well plate pre‐coated with 1% porcine skin gelatine (Sigma‐Aldrich, St Louis, MO, USA; supplemented with 1% gentamycin (Thermo Fisher Scientific)). After 24 h, the confluent cell monolayer was scratched with a 100 μl pipette tip. Images were taken every 2 h using an inverted microscope (Olympus CKX53). The width of the scratch was measured at the same three vertical positions per image and the mean width was calculated. The width of the scratch (0 h) and the percentage of decrease of the scratch (2, 4 and 6 h) were used for statistical analyses. The assay was conducted in triplicate.

All solutions were prepared with ultrapure water (18.2 MΩ cm of resistivity) obtained from a Milli-Q® IQ 7003 purification device system (Millipore, Bedford, MA, USA). Argon (99.998%) and helium (99.999%) were from Nippon Gases (Madrid, Spain). Mono-elemental 1000 mg L⁻¹ standard of titanium [(NH₄)₂TiF₆] was from PerkinElmer.
Citrate-coated TiO₂ NPs with a primary size of 45 nm (45 nm TiO₂ NPs) stock dispersions were prepared from pristine 45 nm TiO₂ NPs [99.5% purity, mixture of rutile and anatase; nanoparticle sizes of < 100 nm (BET) and < 50 nm (XRD)] from Sigma-Aldrich (Osterode, Germany). The 45-nm TiO₂ NPs were stabilised with trisodium citrate dehydrate aqueous solution reaching a weight ratio of 1:1.5 TiO₂:citrate. The mixture was dispersed for 30 min using an ultrasonic probe (Branson Disintegrator Ultrasonic Mod. 450; with 30 s pulse on/5 s pulse off, and 50% amplitude). The final concentration of citrate-coated TiO₂ NPs was 13.3–15.5 g L⁻¹ depending on the batch. Figure S1 (Electronic Supplementary Information, ESI) shows a typical TEM image from the prepared material.
Other reagents as sodium hydroxide, sodium hydrogen carbonate, hyperpure nitric acid 69% (w/v), and absolute ethanol were from Panreac (Barcelona, Spain). Porcine-skin gelatine, type A, bloom strength 300, and formaldehyde solution (36.5–38% in water) were from Sigma-Aldrich (Osterode, Germany). Glass sample holders were from Labbox (Barcelona, Spain). To avoid metal contamination, all glassware and plastic ware were washed with ultrapure water and kept in 10% (v/v) nitric acid for 48 h, and then rinsed several times with ultrapure water before use.

Potato dextrose agar was purchased from Merck (Warsaw, Poland), glucose test was from Biomaxima (Lublin, Poland), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) sodium salt (ABTS), laccase from Trametes versicolor (EC 1.10.3.2), bovine serum albumin, porcine skin gelatine, sodium azide (NaN₃) and Lowry reagent were from Sigma-Aldrich (Poznan, Poland). Other chemicals were purchased from POCh (Gliwice, Poland). Transglutaminase (mTGase) Activa^®WM was kindly donated by Ajinomoto (Tokyo, Japan). All the chemicals used were of analytical grade.

Isolation of fpEC from placental chorionic vessels was approved by the ethics committee of the Medical University of Graz (29-319 ex 16/17). Written informed consent was obtained. Directly after delivery, primary fpEC were isolated as described by Lang et al. (2008 (link)). Placentas were collected after caesarean sections or spontaneous deliveries. Pieces (3 cm) of arterial chorionic plate vessels were dissected and washed with 1 × Hanks Balanced Salt Solution (HBSS, Thermo Fisher Scientific, MA, USA). Then, arteries were cannulated using a 20 gauge needle (Smiths Medical) and perfused with 20 ml of prewarmed (37 °C) 0.5% Collagenase/Dispase solution (Roche, Mannheim, Germany) for 8 min (flow rate 2.5 ml/min). The released cells were collected in a tube containing 10 ml fetal calf serum (FCS, HyClone, GE Healthcare) and centrifuged for 7 min at 900 rpm at room temperature (RT). Cells were resuspended in 1 ml of the respective media. Only during the first culture outgrowth, FCS was replaced by 10% human pooled and heat inactivated serum (Atlanta Biologicals, Flowery Branch, GA, USA). This was not possible for the media from Gibco and Cell applications which provided all supplements premixed. The cell suspension isolated from one individual vessel was seeded per well in a 12-well culture plate (Corning, NY, USA) pre-coated with 1% porcine skin gelatine (Sigma-Aldrich, St. Louis, MO, USA). Wells were observed daily and medium was changed after 2 days and then twice a week. After reaching confluency, fpEC were transferred into 12.5 cm² culture flasks and human serum was replaced by FCS according to the original media composition. Cells were then further expanded and subjected to immunocytochemical characterization for identity and purity as described below. fpEC isolations were cultured at 12% O₂, 5% CO₂ and 37 °C in a 90% humidified incubator.

Scaffolds were synthesized as previously described [50 (link),51 (link)]. Multiwalled CNTs (from SouthWest NanoTechnologies Inc., Norman, OK, USA) had the following dimensions: outer diameter 10 ± 1 nm, internal diameter 4.5 ± 0.5 nm, lengths 3–6 µm. CNHs (from Carbonium s.r.l., Padova, Italy) presented a dahlia-type shape with a diameter of 60–120 nm. RGO powder (from ACS Material, LLC, Pasadena, CA, USA) had lateral dimensions between 1 and 2 µm, and flakes consisted of few irregularly overlapping layers with many corrugations. Chemical modification and purification of the CNMs was performed through diazonium-based reactions, as previously described [52 (link)], yielding p-methoxyphenyl-functionalized derivatives (CNM-PhOMe) with improved dispersibility. CNM@PLLA blend solutions in CHCl₃ were prepared by adding a dispersion of CNM-PhOMe in chloroform obtained via sonication to a chloroform solution of PLLA (6 wt%) under continuous stirring. Each CNM-PhOMe was dispersed within the various blends at 0.25 and 1 wt% (with respect to polymer content). Scaffold films were obtained through solvent evaporation at 50 °C. Functionalized CNM@PLLA films were characterized as reported [52 (link)].
In experiments with cells, scaffolds were cleaned with 70% ethanol, sterilized under UV radiation for 90 minutes, and incubated for 3 h with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) GlutaMAX™ supplement (Invitrogen, Milan, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Euroclone, Milan, Italy). In the control samples, culture wells were coated with 0.005% gelatine (porcine skin, Sigma-Aldrich, Milan, Italy)/1µg/ml poly-L-lysine (Invitrogen).