A novel approach to enhance protein adsorption and cell proliferation on hydroxyapatite: citric acid treatment

Abstract

Hydroxyapatite (HA) is a promising carrier for delivery of therapeutic proteins, especially bone morphogenetic proteins (BMPs), due to the affinity between the amino and carboxyl groups of proteins and the calcium and phosphate of HA. The main challenge of employing HA as a protein carrier is the burst release profile of protein. In this study, we developed a strategy to improve the protein loading capacity of HA and to sustain protein release from HA by immobilizing COO⁻ groups onto the HA surface. HA particles were precipitated in presence of different amounts of citric acid (CA). The physico-chemical properties (crystallinity, surface area and surface charge) of CA-treated HA (CA–HA) were significantly changed compared to those of the unmodified HA. CA–HA demonstrated significantly higher affinity towards lysozyme, in which the lysozyme adsorption increased by increasing the concentration of CA during the HA preparation. Using the optimized parameters obtained from lysozyme adsorption, the results demonstrated that functionalizing HA with CA significantly increased the amount of BMP-2 loaded onto HA and prolonged its release profile. The in vitro results showed that the CA–HA is not toxic and indeed citric ions on HA shift the surface charge towards negative value, which promoted osteoblast-like cell proliferation on HA.