Ogm bulletkit

In this work, the Normal Human Osteoblasts cells (NHOst, Lonza, USA) were used to assess the membranes’ cytocompatibility. Prior to cell seeding, cells were expanded in osteoblast growth medium OGM BulletKit (Lonza, USA) containing 10% FBS, 0.1% ascorbic acid and 0.1% GA-1000 (Gentamicin Sulfate and Amphotericin-B) at 37 °C in a humidified incubator with 5% CO₂. The cultured medium was renewed every 3 days.
The cell culture experiment was carried out with three types of electrospun membranes: (1) PCL, (2) PCL-A2, (3) PCL-AnZn5. The selected materials were cut into disks (round samples matching the size of wells of 48-well culture plate), sterilized by soaking in 70% ethanol for 30 min and by exposure to UV light for 30 min (each side) and then washed with sterile phosphate buffered saline (PBS, HyClone, USA). The membranes were placed at the bottom of 48-well culture plates and seeded with cells at a cell of concentration 1.5 × 10⁴ cells/mL/well. An empty polystyrene well served as a positive control (TCPS). NHOst cells were cultured on the materials for 7, 14, and 21 days in complete osteoblast growth medium OGM supplemented with differentiation kit SingleQuots (Lonza, USA), containing hydrocortisone-21-hemisuccinate and β-glycerophosphate.

Normal human osteoblasts (CC-2538, LONZA, Basel, Switzerland) were maintained in the OGM Bullet Kit (CC-3207, LONZA). Human mesenchymal stem cells from umbilical cord matrix (C-12971, Promo Cell, Heidelberg, Germany) were maintained in mesenchymal stem cell growth medium (C-28010, Promo Cell). Normal human dermal fibroblasts (KF-4109, KURABO, Osaka, Japan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (044-29765, Wako Pure Chemical Industries, Osaka, Japan) containing 10% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan) and 1% penicillin-streptomycin (168-23191, Wako Pure Chemical Industries). The cells were maintained at 37 °C with 5% CO₂ and were used within 4 to 6 passages.