Osteoblast growing medium

Tetraethylortosilicate (TEOS, 99%) and hydrochloric acid (HCl) (37%) were purchased from Alfa Aesar (Haverhill, MA, USA). Gelatin (from porcine skin, Type A, gel strength 300) and 3-glycidoxypropyltrimethoxysilane (GPTMS, >98%) were both purchased from Sigma Aldrich (St. Louis, MI, USA). Microsized β-tricalcium phosphate (Captal β-TCP, 2.0 μm average particle size) was purchased from Plasma Biotal Ltd. (Buxton, Derbyshire, UK). Ethanol absolute (99.5%) was purchased from Panreac (Spain). HOB^® human osteoblasts, foetal calf serum and Osteoblast Growing Medium were purchased from Promocell (Heidelberg, Germany). PBS, methanol, Triton x-100, bovine serum albumin, paraformaldehyde, rhodamine phalloidin and monoclonal anti-vinculin FITC conjugate were all acquired from Sigma, (St Louis, MI, USA) and Hard Set Vectashield with DAPI ^® from Vector (Burlingame, CA, USA).

Chitosan (CS; 50,000–190,000 Da; 75–85% deacetylation degree) was supplied by Sigma Aldrich (St. Louis, MI, USA). Tetraethylortosilicate (TEOS, 99%) and hydrochloric acid (37%, Pharma grade) were purchased from Alfa Aesar (Haverhill, MA, USA). Tri-calciumphosphate (TCP, pure, pharma grade) and absolute ethanol (99.5%) were obtained from Panreac (Barcelona, Spain), HOB^® human osteoblasts, fetal calf serum, and Osteoblast Growing Medium (Promocell, Heidelberg, Germany) Paraformaldehyde, PBS, Triton x-100, bovine serum albumin, Metanol, rhodamine phalloidin, and monoclonal anti-vinculin FITC conjugate were all purchased from Sigma Aldrich, (St. Louis, MI, USA) and Hard Set Vectashield with DAPI^® (Vector, Burlingame, CA, USA).

HOB^® human osteoblasts (Promocell, Heidelberg, Germany) were seeded at a density of 5000 cells/cm² and incubated in Osteoblast Growing Medium (Promocell) supplemented to a final concentration of 0.1 ml/ml of foetal calf serum (Promocell) at 37° and 5% CO₂ on test surfaces and immunolabelled after 24, 48 h and 7 days. Growth medium was changed every three days. HOB cells did not exceed ten population doublings. Test surfaces were exposed to U.V. light for 20 min. each side, in a laminar flow chamber under sterile conditions, in order to achieve optimal sterilization, prior to cell seeding. The test groups were as follows: PLGA, TiO₂/PLGA-10, TiO₂/PLGA-3, TiO₂/PLGA-100. At least five samples of each type were seeded and analysed in each experiment.