Characterisation of polyphenol-functionalized biomaterials under physiologically relevant conditions using perfusion bioreactor

Polyphenols are a class of bioactive compounds of natural origin, which are gaining great attention in biomedical applications for their antioxidant, anti-inflammation, and anticancer, properties, as well as for their positive impact on bone health. Therefore, they can be used to functionalize biomaterials, endowing them with multifunctional properties. In this study, green tea polyphenols (TPH) and tannic acid (TA) have been used to functionalize two kinds of biomaterials: porous titanium disk and chitosan hydrogels. Porous titanium offers mechanical strength with low rigidity, minimizing the stress-shielding phenomenon, while its optimized porosity increases the capacity of osseointegration. On the other hand, chitosan is a biocompatible and biodegradable polymer of natural origin, widely used for its ability to form hydrogels with tunable properties. They were investigated as substrate for the functionalization with TPH focusing the attention on the anti-cancer properties. The functionalization was achieved by immersing substrates in polyphenolic solutions, and then are characterized through Folin-Cicalteau test, zeta potential titration curves, FTIR and fluorescence microscopy. The objective of this work was to evaluate the release of these bioactive compounds under physiologically relevant conditions. To achieve this, biomimetic bioreactors were used to provide an accurate and reliable platform which replicates the dynamic physiological conditions. A perfusion bioreactor, set to a superficial velocity of 200 µm/s - corresponding to capillary blood flow - was used to investigate the release behaviour of polyphenols under in-vitro physiological-like conditions. Additionally, the mechanical properties of pure and functionalized hydrogels were evaluated using a bioreactor with dynamic compression. Uv-Vis Spectroscopy was used to monitor the release kinetics. Finally, the biological activity of the release polyphenols was evaluated through and indirect assay, culturing healthy (hMSC) and tumour cells (U2OS, K7M2-wt) with the eluates obtained from the release study.