Use of a fluorescent probe to monitor the enhanced affinity of rh-BMP-2 to silicated-calcium phosphate synthetic bone graft substitutes under competitive conditions

This work was funded by the Engineering and Physical Science Research Council (EPSRC) and the Central Research Fund (CRF) (AR/CRF/B) awarded in 2010

SSNMR confirms silicate ion substitution in the apatitic structure of 0.8wt% Si hydroxyapatite

This study investigates the structural location of silicon present in ‘silicate-substituted’ apatite. It has been hypothesized that silicon is present in the form of silicate groups site-specifically substituted in the hydroxyapatite lattice for phosphate groups, or that there may be an amorphous silicon-rich phase in the vicinity of the grain boundaries. Solid-state nuclear magnetic resonance (SSNMR) spectroscopy was carried out to view the elemental interactions using phosphorus (31P) and silicon (29Si) probes to confirm or refute the presence of silicate groups in relation to phosphate groups in the bulk material structure. The analysis was performed on stoichiometric hydroxyapatite (HA) and silicate-substituted hydroxyapatite (SA) with a nominal silicon content of 0.8wt%, in as-precipitated, calcined (700°C), and sintered (1,250 and 1,300°C, respectively) powder forms. XRD confirmed all forms of powder were phase pure. FTIR confirmed both hydroxyl and phosphate group functionalities in all forms of HA and SA, while silicate group functionality was only observed in all forms of SA. SSNMR using 31P- and 29Si-coupled probes demonstrated that as the crystallinity of the powders increased from the precipitate to the sintered form, the signal associated with the presence of a silicate group in the phosphate environment developed as the crystal structure became more ordered. These results support the hypothesis that in SA containing 0.8wt% silicon, silicate groups are site-specifically substituted in the hydroxyapatite lattice for phosphate groups. This observation may be key to understanding the mechanisms by which the introduction of 0.8wt% silicon enhances bone regeneration in apatitic bone graft substitute materials

Complexation and sequestration of BMP-2 from an ECM mimetic hyaluronan gel for improved bone formation

Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG