Incorporation of proteins and enzymes at different stages of the preparation of calcium phosphate coatings on a degradable substrate by a biomimetic methodology

In this work, the possibility of incorporating proteins into calcium phosphate (Ca-P) coatings, prepared on the surface of starch polymeric biomaterials by means of a biomimetic route, was investigated. The morphology, chemical composition and crystallinity of Ca-P coatings was assessed and related to the incorporation of the studied biomolecules. For that, bovine serum albumin (BSA) and α-amylase were added in concentrations of 1 mg/ml to simulated body fluid (SBF) solutions, being both added at the nucleation or growth stages of the biomimetic coating process. A biodegradable blend of corn starch/ethylene vinyl alcohol (SEVA-C) was used as substrate and bioactive glass (45S5 Bioglass®) was used as the nucleating agent. The obtained Ca-P coatings were characterised by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy using an attenuated reflectance device (FTIR-ATR) and thin-film X-ray diffraction (TF-XRD). Additionally, to evaluate the activity of the incorporated enzyme and the stability of the Ca-P films, coated samples were immersed in an SBF solution for different periods of time. The enzyme activity was measured and the morphology of the coating examined by SEM. The results obtained showed that the presence of protein molecules, at the nucleation or growth stages, lead to the formation of a dense Ca-P film presenting different morphologies that were different of the selected coating conditions. FTIR-ATR analysis detected the presence of carbonate and phosphate groups on the Ca-P layer, indicating the formation of a coating similar to the mineral component of vertebrates bone tissue. When proteins were added, amide I and amide II bands, characteristic groups of protein molecules, were also detected, revealing the efficient incorporation of these biomolecules into the Ca-P coatings.

Ca-P coatings, with α-amylase incorporated at the nucleation stage, showed no degradation of the film after incubation in SBF for 28 days. The release of increasing concentration of reducing sugars with degradation time revealed that α-amylase was efficiently incorporated in the coating remaining active throughout the coating preparation. This can be a strategy that will allow, in addition of conferring osteoconductive properties to biodegradable polymers, also simultaneously tailoring their degradation kinetics.