Acetone

Pellet cultures (2 with EMF treatment and 2 without treatment for each patient) were washed in phosphate buffered saline (PBS) pH 7.4, incubated in 5% sucrose in PBS (21 °C, 15 min), dry embedded in Tissue-Tek (Sakura, Zoeterwoude, Netherlands) and frozen at −20 °C. Serial cryosections (8 μm) were prepared by mounting pellet cultures on SuperFrost glass slides (Menzel-Gläser, Braunschweig, Germany). Cryosections were fixed in acetone (AppliChem, Darmstadt, Germany) and dried at room temperature (RT). Serial sections were stained in triplicate with 0.75% safranin-O (Fluka, Buchs, Switzerland) and 0.02% fast green (Chroma, Münster, Germany).

Prior to immunofluorescent staining, the sections were firstly fixed with acetone (Applichem) for 15 minutes. Then 5% bovine serum albumin (BSA) (Sigma–Aldrich) was applied to block non-specific binding sites. Thereafter, primary antibody of hBMP-2 (rabbit anti-human) (Abcam) was added and incubated overnight at 4 °C. For negative control, the primary antibody was omitted. After intensive washing, sections were treated with 0.1% Triton X-100 solution (Sigma–Aldrich). The secondary antibody (goat anti-rabbit) (Jackson ImmunoResearch) was added and incubated for 30 minutes at RT. Thereafter, the nuclei were stained with Hoechst 33342 (Life Technologies). The slides were mounted with Fluoromount W (SERVA Electrophoresis), and allowed to dry in darkness at 4 °C. Microscopy was performed with the Zeiss Axioskop 40 equipped with AxioCam MRc 5 (Zeiss) and FluoArc (Zeiss). Images were obtained using Axio Vision, Rel. 4.9 software (Zeiss). Constant exposure time was assured for all the samples so that fluorescence intensity could be compared.

All materials used were of reagent grade and from commercial sources. Iron (III) sulphate hydrate, iron (II) sulphate heptahydrate (ACS, 99+%), and citric acid (99+%) were purchased from Alfa Aesar (Haverhill, MA, USA). Acetone (AppliChem GmbH, Darmstadt, Germany), ethanol absolute (Carlo Erba Reagents GmbH, Emmendingen, Germany), NH₄OH (aq) 25% (Honeywell Fluka, Charlotte, NC, USA), and HCl 1 M (Honeywell Riedl-de-Haën, Charlotte, NC, USA) were used as received. Polyethylene oxide (PEO; Mw, 400,000 g/mol) was from Sigma-Aldrich, Co. (St. Louis, MO, USA), and poloxamer 188 (P188; Lutrol F68) was from BASF (Ludwigshafen, Germany). The water used was purified by reverse osmosis.

The chemicals and solutions used for tissue fixation, histology, and immunohistochemistry were purchased from Applichem (acetone, catalogue number A1582; ethylenediaminetetra-acetic acid, catalogue number A3553), Carl Roth (Alizarin red S or Alizarin sulphonic acid sodium salt; catalogue number 0348.2; ethanol, catalogue number K9285; formaldehyde solution 37%, catalogue number 4979.1; glacial acetic acid, catalogue number 3738.4; hydrogen peroxide, catalogue number 9681.4; molybdatophosphoric acid hydrate, catalogue number 4440.3; nuclear fast red aluminum sulfate solution, catalogue number N069.1; Orange G, catalogue number 0318.2; Roticlear, catalogue number A538.1; Weigert's hematoxylin solution A and B, catalogue numbers X906.1 and X907.1; xylene, catalogue number 9713.5), Dako (antibody diluent, background reducing, S302283–2), Sigma-Aldrich (acid fuchsin, catalogue number F8129; citric acid monohydrate, catalogue number C1909; ponceau xylidine, catalogue number P2395; potassium hexacyanoferrate, catalogue number P3289; saturated picric acid solution, catalogue number P6744; and zinc formaline, catalogue number Z2902–3), Vector Laboratories (AEC Peroxidase Substrate Kit, catalogue number SK-4200; Vectastain Elite ABC-HRP kit, catalogue number PK-6100) or Waldeck GmbH (azophloxine, catalogue number 1B-103; light green SF, catalogue number 1B-211R).

Unless stated otherwise, the materials were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany), and the volume concentration of a solution is expressed as % v/v. PVDF (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) with a molecular weight of ~530 kDa in pellet form and P(VDF-TrFE) 70/30 mol% (Piezotech Arkema Group, Pierre-Benite, France) with a molecular weight of ~695 kDa in powder form were dissolved in a mixed solution of two solvents; N,N-dimethylformamide (DMF, Fluka Analytical, Seelze, Germany) and acetone (AppliChem GmbH, Darmstadt, Germany) in a weight ratio of 6:4. For the identification of favourable conditions for producing the scaffolds, concentrations of 10, 15, and 20 wt% PVDF and 10, 15, and 20 wt% P(VDF-TrFE) were prepared at 50 °C and stored at room temperature overnight prior to electrospinning. As a non-piezoelectric material, 17 wt% polycaprolactone (PCL, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in pellet form with a molecular weight of ~80 kDa was dissolved in 2,2,2-Trifluorethanol (TFE, abcr GmbH, Karlsruhe, Germany) for the scaffold production via electrospinning.

For 24 h, hADSC-seeded scaffold constructs were fixed in 4% PFA (Thermo Fischer Scientific) at 4 °C, then embedded in 1% agarose solution (Sakura, Tokyo, Japan). All the scaffolds were decalcified in 15% EDTA solution (DCS Innovative, Hamburg, Germany) for 3 weeks, then embedded in paraffin. Scaffolds were cut at 10 µm with a Leica RM2255 microtome (Leica). Tissue sections were fixed with acetone (Applichem, Darmstadt, Germany) for 15 min, blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich) for 30 min and permeabilized with 0.1% Triton X-100 solution (Sigma-Aldrich) in PBS (5 min). Samples were incubated first with primary antibody for 1 h at 4 °C and then with 1:500 diluted secondary antibody for 30 min at room temperature. The primary antibody was omitted for negative control. The primary antibodies (OCN, OPN, Scl) were purchased from R&D Systems (Wiesbaden, Germany) and the secondary antibody conjugated with Alexa Fluor 488 was obtained from Thermo Fischer Scientific. Cell nuclei were stained with Hoechst 33,342 (Sigma-Aldrich ) and then air-dried in darkness at 4 °C. The staining results were examined under a Zeiss Axioskop 40 (Zeiss, Jena, Germany).