Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 µgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

Microspheres of alginate encapsulated minocycline-loaded nanocrystalline carbonated hydroxyapatite: therapeutic potential and effects on bone regeneration.

Background and objective: Tetracycline and its derivatives, combined with calcium phosphates, have been proposed as a delivery system to control inflammatory processes and chronic infections. The objective of this study was to evaluate the microspheres of alginate encapsulated minocycline-loaded nanocrystalline carbonated hydroxyapatite (CHAMINO) as a biomimetic device to carry out target-controlled drug delivery for alveolar bone repair. Methods: CHAMINO microspheres were implanted in a rat central incisor socket after 7 and 42 days. New bone was formed in both groups between 7 and 42 days of implantation. However, the bone growth was significantly higher for the CHAMINO microspheres. Results: The minocycline (MINO) loading capacity of the nanocrystaline carbonated hydroxyapatite (CHA) nanoparticles was 25.1±2.2 µg MINO/mg CHA for adsorption over 24 hrs. The alginate microspheres containing minocycline-loaded CHA were biologically active and inhibited the Enterococcus faecalis culture growth for up to seven days of the MINO release. An osteoblastic cell viability assay based on the resazurin reduction was conducted after the cells were exposed to the CHAMINO powder and CHAMINO microspheres. Thus, it was found that the alginate extracts encapsulated the minocycline-loaded CHA microspheres and did not affect the osteoblastic cell viability, while the minocycline-doped CHA powder reduced the cell viability by 90%. Conclusion: This study concluded that the alginate microspheres encapsulating the minocycline-loaded nanocrystalline carbonated hydroxyapatite exhibited combined antibacterial activity against Enterococcus faecalis with cytocompatibility and osteoconduction properties. The significant improvement in the new bone formation after 42 days of implantation suggests that the CHAMINO microsphere has potential in clinical applications of bone regeneration.