2.6.1. Isolation and Cell Culture

Cartilage tissue consists of only one cell type, the chondrocyte, embedded in a dense extracellular matrix (ECM). To evaluate the cytotoxicity of the developed biofunctionalized nanofibrous substrates, human articular chondrocytes (hAC) isolated from knee cartilage samples, collected from arthroplasties surgeries biopsies, were used. These cells were chosen as they were isolated from diseased joints cartilage. Samples were collected under the cooperation agreement between Centro Hospitalar do Alto Ave, Guimarães, Portugal, and the 3B's Research Group, after informed donor consent. Briefly, cells were isolated by enzymatic digestion, according to the protocol described previously [46].

Dulbecco's modified Eagle's medium (DMEM, Sigma D5671), containing 10 mM Hepes buffer (Life Technologies, Paisley, UK), L-alanyl-L-glutamine (Sigma), Non Essential Aminoacids (Sigma) 10,000 units/ml penicillin, 10,000 μg/mL streptomycin, and 10% foetal calf serum was the basis for the expansion and differentiation medium used herein (Basic medium). Basic medium was supplemented with 10 ng/mL of bFGF for hAC expansion. Basic medium was further supplemented with 1 mg/mL of insulin and 1 mg/mL of ascorbic acid, when hAC were seeded onto the PCL NFM (differentiation medium). These supplements enhanced extracellular matrix (ECM) deposition by the cultured hACs.

Human articular chondrocytes where used at passage 4. Expansion medium was changed every 2 days until the cells reached a confluence of 90%. The cells were harvested and seeded onto both the activated NFM, as well the NFM with the TNF-α antibody immobilized using the differentiation medium.
