PCL is a semicrystalline thermoplastic polyester widely used in biomedical field. This popular biomaterials for tissue engineering scaffold often has suboptimal properties when analyzed for cell attachment and growth, thus, it should be desirable to improve its performance by immobilization of bioactive moieties on its surface.

Moreover, short amino acid sequence such as arginine-glycine-aspartic acid (RGD) has been widely used as recognition motif to mediate cell attachment.

The aim of this work is to set-up a procedure to covalently immobilize RGD-based peptide sequences onto functionalized PCL surface. The procedure consists of three steps: aminolysis of polymeric scaffold, grafting of a tether and peptide immobilization.

The aminolysis of PCL substrates with 1,6-hexanediamine has been investigated and the yield of the reaction evaluated. In particular, the amount of amino group introduced, measured by Ninhidryn assay reached a maximum NH₂ density of ~1*10^-7mol/cm² (30 minutes, 37°C)(fig 1).

Contact angles decrease with increasing time of treatment, ranging from 76° (pure) to 67° (90 min at 37°C). The rates of NH₂ grafting at different temperatures (from 24 to 37°C) ranged from 0.92 to 3.2*10^-8 mol/cm²min, and, an activation energy of 59 kJ/mol have been evaluated.

Moreover, ATR-FTIR spectroscopy characterization evidenced the presence of amine group (3337cm^-1 NH stretch, 1639cm^-1 and 1556cm^-1 -CONHR- absorption) (fig 2). Afterwards, indeed, glutaraldeide solutions were used to graft GRGDY-Rho sequences on scaffold surface after reaction in mild aqueous conditions by reductive amination.

CLSM observations were also preformed on cross-sections of 3D PCL scaffolds demonstrating the capability to control in space and amount the peptide immobilization also in the case of 3D PCL substrate to be used as scaffold for tissue engineering.

Fig 1

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1. Biomimetic hydrogels for tissue reconstruction