A linear poly(d/l-lactide-co-l-lactide) (50:50) copolymer (Mw = 102,000) was prepared according to a previously established protocol [30, 31]. Briefly, appropriate amounts of l-lactide, d/l-lactide (Boehringer KG, Ingelheim, Germany) and the starter molecule 1,8-octanediol (Sigma–Aldrich Chemie GmbH, Munich, Germany) were placed into a reaction flask and the mixture was heated under an inert nitrogen gas atmosphere to 130°C. After complete melting, stannous 2-ethylhexanoate (0.05 wt% based on total lactide content; Sigma–Aldrich), which had been dissolved in dry toluene, was added. The reaction mixture was stirred until it became solid. After cooling to room temperature, the polymer was dissolved in dichloromethane (Sigma–Aldrich), precipitated in methanol and dried in vacuum. Preliminary cytotoxicity experiments using standard 3T3 mouse embryonic fibroblasts (LGC Standards GmbH, Wesel, Germany) indicated the cytocompatibility of the poly(d/l-lactide-co-l-lactide) material.
Electrospun fibrous matrices
Tetrahydrofuran, acetone, dichloromethane, hexafluoroisopropyl alcohol and chloroform (all Sigma–Aldrich) were tested for their suitability to dissolve poly(d/l-lactide-l-lactide) for its subsequent use for electrospinning. We determined that a 10% (w/w) solution was essential to spin the copolymer under the conditions described: A homogeneous solution was prepared by slow stirring 1.0 g of the copolymer in 6.1 ml of the solvent at room temperature for 3 h using a magnetic stirrer at 250 rpm. The obtained clear and viscous solution was directly transferred into a 5 ml plastic syringe, which was connected to a 0.40 × 25 mm-gauge blunt ended stainless-steel needle (= spinning nuzzle). The copolymer solution was then exposed to the electrospinning process using a custom designed electrospinning apparatus consisting of an adjustable high-voltage power supply with a limited current of 200 μA (ESV-100; Ingenieurbüro G. Fuhrmann, Leverkusen, Germany). The glass surface of a mirror (20 × 20 cm2; glass thickness 3 mm) was selected as collector plate for collecting the electrospun fibers. The needle and the metallic side of the mirror were connected to the ESV-100. The syringe was mounted vertically against the collector and the sample solution was fed at a constant rate through the syringe to the needle tip. The distance between the needle tip and the mirror was maintained at 20 cm. The voltage applied to the needle was adjusted to 15 kV. The flow rate of the solution was controlled between 0.5 to 0.7 ml h−1. Fiber diameters were determined by software analysis of microscopic images (Image-Pro Plus 5.0, Media Cybernetics, Inc., Silver Spring, MD, USA).
In vitro degradation assays
To determine the in vitro degradation of the electrospun poly(d/l-lactide-l-lactide), we used 80 mg of the electrospun material and the same amount of smooth, solid and non-porous poly(d/l-lactide-l-lactide)-composed controls. These controls had a thickness of approximately 50–60 μm and were generated using the same material (poly(d/l-lactide-l-lactide)–chloroform (50:50) solution) as used for the electrospinning. All samples were exposed to 5 ml of simulated body fluid (SBF) medium of pH 7.4 in a thermostatic incubator at 37°C over 18 weeks. Every week 1 ml of the SBF was withdrawn and the SBF in the vessel was replenished up to 5 ml. The degree of degradation was monitored by the l-lactate sample concentration using an enzymatic l-lactate assay (R-Biopharm, Darmstadt, Germany) according to the manufacturer’s protocol.
In order to confirm that all solvent was evaporated from the electrospun poly(d/l-lactide-l-lactide), we performed thermogravimetric analyses at 20–200°C with a heating rate of 20°C/min at room temperature as described previously  using a STA 449C Jupiter (Netzsch-Gerätebau GmbH, Selb, Germany).
Primary cell isolation and culture
Primary human fibroblasts were isolated from foreskin by enzymatic treatment. Dermal and epidermal layers were separated by a 16 h digest in a 2 U/ml dispase solution in phosphate-buffered saline (PBS; Invitrogen GmbH, Darmstadt, Germany) at 4°C. The dermal layer was cut and treated for 45 min with 0.45 U/ml collagenase solution (Invitrogen). Cells were seeded in T-25 culture flasks in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen). The medium was changed every 2–3 days. Fibroblasts were used at passage 2–5.
Cell seeding of the electrospun matrix
After confirming that all solvent was removed from the electrospun material, the matrices were cut to 1.5 × 1.5 cm2. All samples were then sterilized using 70% ethanol solution for 20 min, followed by two washing steps with PBS. For cell seeding experiments, the electrospun matrices were placed in a petri dish with a 3.2 cm diameter. On each electrospun scaffold, 250,000 fibroblasts were seeded in DMEM and cultured for a maximum of 5 days at 37°C.
Cell proliferation assays
After a culture time of 17 h and 5 days, proliferation was measured using the WST-1 assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, the WST-1 reagent is a water-soluble tetrazolium salt that can be used for cell proliferation or cell viability assays. The rate of WST-1 cleavage by mitochondrial dehydrogenases correlates with the number of viable cells in the culture. Therefore the assay primarily detects total enzymatic activities that are able to reduce the WST-1 substrate. Accordingly, the values obtained using this assay are a direct measure of cell number and proliferation, provided that cytotoxic effects and reductions in metabolism can be excluded. Fibroblasts that had been cultured on 3.2 cm diameter tissue culture polystyrene (TCPS) petri dishes served as control. The absorbance of the samples was measured at a wavelength of 450 nm employing a microtiter plate reader (Model 550; Bio-Rad Laboratories GmbH, München, Germany). All data are displayed in arbitrary units (a.u.) ± SD.
Live/dead staining of cells
To assess cytocompatibility, fibroblasts were cultured for 5 days on the electrospun scaffolds, followed by live/dead staining for 3 min using a solution consisting of 0.005% (w/v) fluorescein diacetate (FDA, Sigma–Aldrich) (stock solution: 0.5% (w/v, in acetone), 0.009% (w/v) propidium iodide (PI, Sigma–Aldrich) (stock solution: 0.1% (w/v) in PBS) in PBS. Cells were immediately visualized by fluorescence microscopy using a Zeiss Axiovert 200 M ApoTome equipped with the appropriate filter sets detecting FITC and rhodamine (Carl Zeiss Microimaging GmbH, Jena, Germany). Images were processed with Adobe Photoshop CS3.3 (Adobe Systems Inc., San Jose, CA, USA).
Scanning electron microscopy
All samples were fixed using 2% glutaraldehyde (Sigma–Aldrich) for 45 min at room temperature, dehydrated with a series of graded alcohols and air-dried overnight. The dried samples were sputter coated with platinum and scanning electron microscopic (SEM) micrographs were taken with a Zeiss Leo 1520 VP (Carl Zeiss).
Fiber diameters in the electrospun scaffolds were measured using scanning electron micrographs. The average fiber diameter was determined from the measurements taken as described before in more detail .
Statistical significance was assessed by Student’s t test. P-values less than 0.05 were defined as statistically significant.