Silk Fibroin Particle Enhances Stability and Sustained Release of Bone Morphogenetic Protein-2

Objectives: Bone morphogenetic protein 2 (BMP-2) is a strong osteoinductive growth factor. Conventional delivery of BMP-2 soaked in absorbable collagen sponge generated many complications such as swelling and ectopic bone formation around applied area. These unwanted problems came from inappropriate high dosage of BMP-2 because of its short half-life and abrupt release from the carrier. The proper carrier and delivery system need to be investigated further.
Methods: Mixture of Silk fibroin solution, polyvinyl alcohol (PVA) and BMP-2 were achieved successful encapsulation of BMP-2 by sonication. Morphology characterization of BMP-2/silk fibroin particle (BMP-2/SF) was analyzed by Scanning electron microscope (SEM) and zeta-sizer. ELISA was used for encapsulation efficiency and in vitro release profile of BMP-2. Cytotoxicity of BMP-2/SF was tested by WST cell viability assay. Bioactivities of encapsulated BMP-2 were assessed by quantitative real time PCR, alkaline phosphatase activity assay in vitro. Four-week old C57BL/6 mouse and eight-week old Sprague Dawley rat were used for intramuscular heterotopic ossification and calvarial critical-size defect model respectively. Specimens were analysed by soft X-ray, micro-computed tomography and histological examination.
Results: Sonication of mixture solution of silk fibroin, PVA and BMP-2 achieved successfully encapsulated of BMP-2 in the particle. Self-assembled BMP-2/SF showed smooth surfaced spherical and average diameter 500-700nm and zeta-potential -20mV particles. Loading capacity of BMP-2 in the particle is around 80%. Releasing profile was sustained over 30 days and reached platau until 40% release. The stability and bioactivity of encapsulated BMP-2 was strongly enhanced even in 10% fetal bovine serum in the media compared with naïve BMP-2 protein. Particle itself does not show cytotoxicity. Encapsulated BMP-2 successfully stimuated ALP activity in vitro and osteogenic potentials in vivo.
Conclusions: These results imply the clinical potential of silk fibroin particle and BMP-2 because of its long-lasting release pattern and enhancement of protein stability.