Protein encapsulation in sol-gel silica for bone reconstruction application

Over the last years, bone repair has increasingly gained in importance. Temporary porous matrices, called scaffolds, are attractive matrices in order to support and control the spatial organization of stem cells intended to be grafted to promote bone reconstruction. Various materials have been proposed for the conception of scaffolds, like ceramics, polymers, and metals. Nevertheless, studies have shown a lack of cell differentiation, bone production, and bone integration at the surface of these materials. The aim of this study is the adjustment of the surface functionalization of hydroxyapatite via sol-gel coating of silica to promote the local sustained delivery of Bone Morphogenic Proteins (BMP). In this optic, the influence of the functional groups present at the surface of silica pores on the release kinetics of a model protein (i.e. Soybean Trypsin Inhibitor, STI) and on its activity have been studied.
The encapsulation of STI was assessed adopting two alternative methods: (1) the impregnation of already synthesized silica gels in the protein solution (i.e. impregnation method), and (2) the direct incorporation of the protein during the gel synthesis (i.e. in situ method). For the impregnation method, tetraethylorthosilicate (TEOS) was used as the main silica precursor and 3-(2-aminoethylamino)propyltrimethoxysilane (EDAS) as nucleating agent in alcoholic medium and under basic conditions. After drying and/or calcination, the gels were impregnated in a STI solution for 3 days. For the in situ method, tetramethylorthosicilate (TMOS) served as silica precursor after hydrolysis under acid conditions. The textural properties were characterized by nitrogen adsorption and mercury porosimetry. The point of zero charge was determined via a method of equilibrium pH at high loading. The release kinetics of the protein and its inhibitory activity were analyzed in vitro on a 3 months period.
For the ex situ method, the release kinetics highlighted a burst observed during the first 24 h of incubation, due to the STI adsorbed at the surface of the SiO2 particles. This burst was followed by a plateau for the calcined samples and by a continuous release for the dried sample. The inhibitory activity decreased after 4 weeks of incubation to a low value (i.e. around 30 %) for the dried sample and was equal to 0 in the case of the calcined samples. These differences could be explained by the difference in surface chemistry of those samples. Indeed, while only hydroxyl groups should be present at the surface of the calcined samples, ethylene diamine groups of the dried sample should provide positive charges at neutral pH able. Regarding the in situ method, a continuous release were observed over the first 24 h followed by a plateau due to the proteins entrapped in closed pores. Interestingly enough, STI released after 4 weeks of incubation was still mostly active (i.e. around 80 %).
Acknowledgements: Rémi Tilkin and Nicolas Régibeau benefit from funding of the Fund for Scientific Research (F.R.S.-FNRS) under a Fund for Research Training in Industry and Agriculture (FRIA) grant. Stéphanie D. Lambert also thanks the F.R.S.-FNRS for her Senior Research Associate position.