2,3 In the case of anchorage-dependent cells, protein adsorption studies involving cell-adhesive proteins such as fibronectin (FN) or laminin are particularly useful to predict and explain cell response to biomaterials, such as cell adhesion and cytoskeletal reorganization, as well as other cellular events triggered by else integrin signaling. Although protein adsorption onto biomaterial surfaces has been assessed using a wide range of techniques, the quantification of protein adsorption onto porous scaffolds is still a challenging and controversial issue, namely due to the difficulty in distinguishing adsorbed from non-adsorbed protein present in the inner part of the scaffolds. With the emergence of tissue engineering and the increasing need of three-dimensional (3-D) porous scaffolds, protocols for the quantitative assessment of protein adsorption to porous scaffolds need to be established.

Table 1 provides an overview of the main methodologies that have been used for the quantitative analysis of protein adsorption to porous biomaterials. Their main advantages and limitations are briefly described. Table 1. Methodologies used for the quantitative analysis of protein adsorption in porous biomaterials Radiolabelling is considered the gold standard of the methodologies available to follow protein adsorption, as it is a straightforward and sensitive quantitative technique. Iodine radioisotopes are usually used, as iodine readily binds to the tyrosine residues of proteins and the signals emitted are directly proportional to protein amount.

2 125I-radiolabelling can be used to quantify protein adsorption from single protein solutions or from complex mixtures of proteins, such as serum-containing media and blood plasma.2 The retention, desorption, as well as the exchangeability of the adsorbed protein by other proteins can also be easily determined, which makes radiolabelling a powerful tool to provide insights into protein adsorption phenomena occurring at the interface of biomaterials with the biological milieu.4,5 This sensitive technique has been widely explored to follow protein adsorption onto polymeric surfaces.6,7 Nevertheless, only a few authors have used this technique to measure protein adsorption onto porous scaffolds.

In an attempt to develop a pre-vascularized scaffold for use in cell-based regenerative therapies, we have shown AV-951 that the endothelialisation of chitosan porous scaffolds could be successfully achieved by prior incubation of the porous matrices in a 40 ��g/mL FN solution. In that study physiadsorption of FN was shown to be effective in promoting endothelial (EC) adhesion and proliferation on CH scaffolds with retention of the EC phenotype and angiogenic ability.8 However, the effectiveness of the FN treatment was found to be dependent on the degree of acetylation (DA) of CH, a parameter influencing directly CH susceptibility to enzymatic degradation, in vivo.