Bioactive and biodegradable silica biomaterial for bone regeneration

Abstract

Biosilica, a biocompatible, natural inorganic polymer that is formed by an enzymatic, silicatein-mediated reaction in siliceous sponges to build up their inorganic skeleton, has been shown to be morphogenetically active and to induce mineralization of human osteoblast-like cells (SaOS-2) in vitro. In the present study, we prepared beads (microspheres) by encapsulation of β-tricalcium phosphate [β-TCP], either alone (control) or supplemented with silica or silicatein, into the biodegradable copolymer poly(d,l-lactide-co-glycolide) [PLGA]. Under the conditions used, ≈5% β-TCP, ≈9% silica, and 0.32μg/mg of silicatein were entrapped into the PLGA microspheres (diameter≈800μm). Determination of the biocompatibility of the β-TCP microspheres, supplemented with silica or silicatein, revealed no toxicity in the MTT based cell viability assay using SaOS-2 cells. The adherence of SaOS-2 cells to the surface of silica-containing microspheres was higher than for microspheres, containing only β-TCP. In addition, the silica-containing β-TCP microspheres and even more pronounced, a 1:1 mixture of microspheres containing β-TCP and silica, and β-TCP and silicatein, were found to strongly enhance the mineral deposition by SaOS-2 cells. Using these microspheres, first animal experiments with silica/biosilica were performed in female, adult New Zealand White rabbits to study the effect of the inorganic polymer on bone regeneration in vivo. The microspheres were implanted into 5mm thick holes, drilled into the femur of the animals, applying a bilateral comparison study design (3 test groups with 4-8 animals each). The control implant on one of the two hind legs contained microspheres with only β-TCP, while the test implant on the corresponding leg consisted either of microspheres containing β-TCP and silica, or a 1:1 mixture of microspheres, supplemented with β-TCP and silica, and β-TCP and silicatein. The results revealed that tissue/bone sections of silica containing implants and implants, composed of a 1:1 mixture of silica-containing microspheres and silicatein-containing microspheres, show an enhanced regeneration of bone tissue around the microspheres, compared to the control implants containing only β-TCP. The formation of new bone induced by the microspheres is also evident from measurements of the stiffness/reduced Young's modulus of the regenerated bone tissue. The reduced Young's modulus of the regenerating bone tissue around the implants was markedly higher for the silica-containing microspheres (1.1MPa), and even more for the 1:1 mixture of the silica- and silicatein-containing microspheres (1.4MPa), compared to the β-TCP microsphere controls (0.4MPa). We propose that based on their morphogenetic activity on bone-forming cells in vitro and the results of the animal experiments presented here, silica/biosilica-based scaffolds are promising materials for bone repair/regeneration.

Keywords: Animal experiments; Biosilica; Bone metabolism; Microspheres; Silicatein; β-Tri-calcium phosphate.