L ascorbic acid 2 phosphate sesquimagnesium salt hydrate asc

Scaffolds were cut, clamped into 11 mm diameter cell crowns and afterwards sterilized by autoclavation. Hdf were seeded on top in a concentration of 30,000 cells per square centimeter. Cell culture medium consisted of dulbecco’s modified eagle medium (DMEM, Gibco^® Life Technologies, Carlsbad, CA, USA), containing 10% Fetal Calf Serum (FCS, LOT: BS 210601.5, Bio and SELL GmbH, Feucht, Germany), 1% penicillin/streptomycin (pen/strep, PAA, Coelbe, Germany) and either 100 or 500 µmol L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Asc) (Sigma-Aldrich, Schnelldorf, Germany) for proliferation and ECM synthesis stimulation. Medium was renewed three times a week. Cultivation lasted four weeks under standard cell culture conditions (37 °C, with an amount of 5% CO₂ and a relative humidity of 95%). The complete decellularization process was conducted under sterile conditions. Therefore, specimens were rinsed thrice with phosphate buffered saline (PBS^-) (Sigma-Aldrich) and incubated in decellularization solution containing 22.5 g/L sodium desoxycholate (Sigma-Aldrich, Schnelldorf, Germany) in deionized (DI) water. The procedure was repeated on the following day. Specimens were stored in pen/strep containing PBS- until further usage.