Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect

  Objective  To explore the preparation method of copper (Cu)-doped hydroxyapatite (HA) microspheres loaded with vancomycin (Van), and evaluate their antibacterial and osteogenic effects in vitro.

  Methods  The Cu doped HA microspheres (Cu-HA) with molar doping ratios of 1%, 5%, 10%, and 20% were prepared by hydrothermal synthesis. The microscopic morphology changes were observe with scanning electron microscope. X-ray diffractometer (XRD) was used to study the phase composition and analyze the crystallinity of the sample. Cu-HA with a molar doping ratio of 10% was selected for analysis of the elemental composition of the sample with energy dispersive X-ray spectroscopy (EDS), and was then coated with polydopamine (PDA) as the medium to prepare Cu-HA-PDA. XRD and Fourier infrared spectrometer were used to examine the coating effect of the sample. Van was load on Cu-HA-PDA to prepare Cu-HA-PDA-Van. HA, Cu-HA, HA-PDA, and Cu-HA-PDA-Van were added to α medium at 10 mg/mL to prepare different groups of extract solutions.The main components of the extract solutions were examined, and the Van concentration was checked. We examined the toxic effect of material extract solutions on osteogenic precursor cells and the proliferation and differentiation of bone marrow mesenchymal stem cells, and checked the expression of osteocalcin (OCN), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP), the osteogenic related genes. Sterilized HA, Cu-HA, HA-PDA, Cu-HA-PDA, Cu-HA-DPA-Van microsphere materials were prepared, and the colony counting method was used to evaluate the antibacterial effect of the materials for Staphylococcus aureus.

  Results  Various types of Cu-doped HA (Cu-HA) were successfully synthesized. As the proportion of Cu increased, the morphology gradually changed from being strip or belt-shaped to a uniform spherical shape. Cu-HA of 10% molar doping ratio showed a clearly microspherical shape and a petal-like porous micro-nano morphology on the surface. EDS and XRD analyses showed that the main structure of the material was still made up of hydroxyapatite crystals and Cu was successfully doped with HA. The infrared spectrometer showed that the PDA was successfully coated on the surface of the material. Examination of the main components of the extract solution once again verified that the Cu element had successfully entered and replaced part of the Ca element in the HA. The 10 mg/mL Cu-HA-PDA-Van extract solution contained 0.27 mg/mL of Van. In vitro cell experiments and bone-formation-related gene testing showed that Cu-HA-Van had good biological activity and promoted bone differentiation. The minimum inhibitory concentration (MIC) of Cu-HA-PDA-Van microspheres was 16 μg/mL. Compared with Cu-HA, HA-PDA and pure HA, Cu-HA-Van microspheres had significant and long-lasting antibacterial effects.

  Conclusion  Cu element was used to control the microscopic morphology of HA, and the Cu-HA-PDA-Van microspheres prepared by successfully coating of PDA and loading of Van had good antibacterial properties and biological activity.