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.

A comparative investigation was undertaken on 1-2mm sized granules of two forms of synthetic bone graft substitute (SBG) with identical pore structure but varied bulk chemistry, stoichiometric hydroxyapatite (HA) and silicate substituted (0.8wt% Si) hydroxyapatite (SA), to assess the influence of SBG chemistry on the relative affinity of an osteogenic growth factor (GF), recombinant human bone morphogenetic protein-2 (rhBMP-2). A previously described novel fluorescent probe, fluoresceinthioureidoaminocaproic acid (FTCA), was covalently attached to rhBMP-2 to give FTCA-rhBMP-2 and facilitate the quantitative monitoring of GF uptake and release from the two chemistries of SBG. The relative affinity of rhBMP-2 for the HA and SA granules was assessed at a physiologically relevant concentration of 300ngmL-1 from three (increasingly complex) environments; phosphate buffered saline (PBS), minimum Eagles' medium (MEM) and serum supplemented MEM (SCEM) in order to closely mimic clinical bone repair procedures. The results demonstrated that rhBMP-2 affinity to SBGs was highly sensitive to both SBG chemistry and the composition of the local environment. Under the most physiologically relevant competitive conditions of SCEM, rhBMP-2 showed greater affinity to SA (P<0.05) such that 50% of the rhBMP-2 in solution was adsorbed to the SA granules after only 15min, as compared to 30% adsorbed to the HA granules. Subsequent investigation of the desorption of adsorbed GF from the SBGs demonstrated that a significantly higher percentage of the adsorbed rhBMP-2 was desorbed from HA as compared to SA granules. Together, these observations suggested that at physiologically relevant concentrations and conditions, rhBMP-2 has a greater affinity to silicate-substituted hydroxyapatite as compared to stoichiometric hydroxyapatite, which may in part explain the enhanced osteoconductivity and reported osteoinductivity for silicate-substituted hydroxyapatite based SBGs.

A low cost synthesis method for functionalised iron oxide nanoparticles for magnetic hyperthermia from readily available materials.

The synthesis of iron oxide nanocrystals from reagents taken from high street sources using thermal decomposition of an iron-fatty acid precursor in a high boiling point solvent in the presence of surfactants is presented. The nanocrystals were characterised using a variety of techniques including: electron microscopy, X-ray dispersive spectroscopy, infrared spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and magnetometry. Thermogravimetric analysis (TGA) is also used to compare the decomposition behaviour of iron oleate and iron palmitate, our nanoparticle precursors. The nanoparticles also exhibit shape anisotropy when prepared under optimum conditions. We show that these nanoparticles have potential in magnetic hyperthermia after transfer to aqueous media via an amphiphilic polymer.

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.

Development of novel fluorescent probes for the analysis of protein interactions under physiological conditions with medical devices.

In this article, a method to analyze protein adsorption on porous, clinically relevant samples under physiologically relevant conditions is described. The use of fluorescent probes was identified as a methodology that would facilitate analysis under a range of conditions including fully competitive conditions where a protein of interest may be labeled in isolation and then allowed to compete with unlabeled proteins on samples that require no specialized surface pretreatment. As a first step, this article describes the covalent labeling of isolated bovine serum albumin (BSA) with fluorescent fluoresceinthioureidoaminocaproic acid, FTCA, giving FTCA-BSA. The fluorescence intensity of FTCA-BSA was then used to monitor the adsorption and desorption of the protein under noncompetitive conditions with two forms of hydroxyapatite discs (silicate-substituted, SA and stoichiometric, HA) in phosphate-buffered saline (PBS) and minimum essential Eagles' medium (MEM). Noncompetitive conditions were used to facilitate the validation of the technique in which data obtained from these experiments were corroborated against data obtained using an established total protein assay method (Quant-IT kit, Invitrogen). These experiments demonstrated that the FTCA-BSA probe had several advantages including a greater sensitivity at lower concentrations and a considerably longer lifetime. The results also demonstrated that the interaction of BSA with SA and HA was also highly temperature- and media-dependent. Under the most physiologically relevant conditions of MEM at 37 °C, BSA was more readily adsorbed to SA with significant differences between biomaterials, but no differences were observed during the desorption process. The use of this method to analyze adsorption under competitive conditions will be the subject of further investigations.