Abstract
Introduction
The annual incidence of fractures in the UK is almost 4%. Bone grafting procedures and segmental bone transport have been employed for bone tissue regeneration. However, their limited availability, donor site morbidity and increased cost mean that there is still a large requirement for alternative methods and there is considerable research into regeneration using bone morphogenetic proteins (BMPs). The aims of this study are to synthesise and combine BMP-2 with a novel nanocomposite and study its release.
Materials and Methods
BMP-2 was synthesised using an E. coli expression system and purified. C2C12 cells were used to test its bioactivity using an alkaline phosphatase (ALP) assay. The modified solution evaporation method was used to fabricate 30% a-TCP/PLGA nanocomposite and it was characterized using SEM, TEM, TGA, XRD, EDX and particle size analysis. The release pattern of adsorbed BMP-2 was studied using an ELISA assay.
Results
SEM suggests that there was a homogeneous distribution of a-TCP nanoparticles within the PLGA matrix. The concentration of BMP-2 adsorbed onto a-TCP/PLGA nanocomposites directly correlated with the incubation concentration of BMP-2. Approximately 10-15% of BMP-2 was adsorbed on to the discs, up to an incubation concentration of 25 μg/ml. At a higher incubation concentration (50 μg/ml), however, only 4% of the BMP-2 appears to have been adsorbed. The ALP activity shows that the BMP-2 was bioactive and successfully adsorbed onto the surface of the a-TCP/PLGA nanocomposite. A burst release pattern of BMP-2 was observed over 24h, being maximal at 2 h.
Discussion
Increasing incubation concentrations of BMP-2 resulted in an increase of detected adsorbed BMP-2 on the discs, however this was not observed at the highest incubation concentration (50 μg/ml). As adsorption of BMP-2 onto the ground surface of the a-TCP/PLGA nanocomposite occurs primarily through electrostatic interactions between cationic BMP-2 and anionic a-TCP, this might reflect saturation in adsorption secondary to saturation of surface anionic a-TCP by BMP-2, or heterogeneity of the discs' content and/or surface area.
Adsorbed BMP-2 was shown to have bioactivity which significantly increased with increasing incubation concentrations of BMP-2 and suggests this nanocomposite could have osteoinductive potential in-vivo.
The burst pattern of BMP-2 release has been shown previously from BMP adsorbed onto mPCL/collagen/HA composite and this significantly increased the bone formation of critical-sized defects. Whilst a more sustained release profile of BMP-2 is generally considered desirable, this nanocomposite of a-TCP/PLGA has been shown to possess some osteoconductive and weak osteoinductive properties itself (unpublished). The addition of BMP-2 to the nanocomposite by adsorption results in an early burst release, which can promote the differentiation of mesenchymal cells into osteoblasts. The proliferation of these might then be sustained by the nanocomposite itself, without the need for sustained delivery of BMP-2.
Conclusions
Bioactive BMP-2 was synthesised and combined with a-TCP/PLGA nanocomposite, producing a biodegradable and osteoinductive material which has potential for use in bone regeneration
