Protein encapsulation in silica by sol-gel process 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. ex situ method), and (2) the direct incorporation of the protein during the gel synthesis (i.e. in situ method). For the ex situ method, tetraethylorthosilicate (TEOS) was used as main silica precursor and 3-(2-aminoethylamino)propyltrimethoxysilane (EDAS), (3-aminopropyl)trimethoxysilane (APTMS) or phenyltrimethoxysilane (PTMS) as nucleating agents 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 nucleating agents were also EDAS, APTMS, or PTMS. 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.
The results of the textural analysis revealed that ex situ gels presented a porous funnel-like structure (micro, meso, and macropores) while in situ gels presented a micro and mesoporous structure. The in vitro release profile of STI has also been shown to be significantly affected by the functionalization strategy. 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 samples. The inhibitory activity decreased after 4 weeks of incubation to a low value (i.e. around 30 %) for the dried samples and was equal to 0 in the case of the calcined samples. These differences could be explained by the sign difference of the charges present at the surface of the pores. Indeed, we can anticipate that the negative charges existing at the surface of the calcined samples should reinforce their interaction with the protein in contrast to the dried samples which bear positive charges at their surface. Accordingly, the entrapment of the protein would be less reversible for the calcined silica with a possible decrease in inhibitory activity. 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.