Monoclonal anti vinculin fitc conjugate

At the end of each experiment, cells were washed with prewarmed phosphate buffered saline (PBS), pH 7.4, and fixed with 3.7% paraformaldehyde at room temperature, washed, and then permeabilized with 0.1% Triton x-100 (Sigma, St Louis, MI, USA). After washing, cells were preincubated with 1% bovine serum albumin (Sigma) in PBS for 20 min prior to cell immunolabelling for actin cytoskeleton with rhodamine phalloidin (Sigma) and monoclonal anti-vinculin FITC conjugate (Sigma). After 20 min. TiO₂/PLGA-10, TiO₂/PLGA-3, TiO₂/PLGA-100 samples were rinsed with prewarmed PBS prior to mounting with Vectashield ® (Vector, Burlingame, CA, USA).

Chitosan (CS) (low molecular weight 50,000–190,000 Da, 75–85% deacetylated, CAS 9012-76-4) was purchased from Sigma-Aldrich (San Louis, MO, USA). Tetraethylortosilicate (TEOS, 99%, CAS 78-10-4,) was supplied by Alfa Aesar (Haverhill, MA, USA). Hydrochloric acid (37%, Pharma grade, CAS 7647-01-0) was obtained from Alfa Aesar. Absolute ethanol (99.5%, CAS 64-17-5) was purchased 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, methanol rhodamine-phalloidin, and monoclonal anti-vinculin FITC conjugate were all purchased from Sigma-Aldrich. “Hard Set Vectashield with DAPI^® (Vector, Burlingame, CA, USA)”.

At the end of each experiment, cells were washed twice with prewarmed phosphate-buffered saline (PBS), pH of 7.4, and fixed with 3.7% paraformaldehyde at room temperature, washed, and then permeabilized with 0.1% Triton x-100. After washing, cells were preincubated with 1% bovine serum albumin (Sigma) in PBS for 20 min prior to cell immunolabeling for actin cytoskeleton with rhodamine-phalloidin (Sigma) and monoclonal anti-vinculin FITC conjugate (Sigma). After 20 min, SCS8 aerogels and control samples were rinsed with prewarmed PBS prior to mounting with Hard Set Vectashield with DAPI^® (Vector, Burlingame, CA, USA). The measurement of FA (Focal Adhesion) size and location was conducted for at least 10 cells on each substrate.

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).