Structural Characterization and Osseointegrative Properties of Pulsed Laser-Deposited Fluorinated Hydroxyapatite Films on Nano-Zirconia for Implant Applications

Abstract

Standard zirconia implants used in restoration still present problems related to inertness and long-term stability. Various physicochemical approaches have been used to modify the implant surfaces to improve early and late bone-to-implant integration; however, no ideal surface modification has been reported. This study used pulsed laser deposition to deposit a fluorinated hydroxyapatite (FHA) film on a zirconia implant to create a biologically active surface. The film prepared was uniform, dense, and crack-free, and exhibited granular surface droplets; it also presented excellent mechanical strength and favorable biological behavior. The FHA-coated implant was implanted on the femur of Sprague-Dawley rats, and various tests and analyses were performed. Results show that the in vitro initial cell activity on the FHA-coated samples was enhanced. In addition, higher alkaline phosphatase activity and cell mineralization were detected in cells cultured on the FHA-coated groups. Further, the newly formed bone volume of the FHA-coated group was higher than that of the bare micro-adjusted composite nano-zirconia (NANOZR) group. Therefore, the FHA film facilitated osseointegration and may improve the long-term survival rates of dental implants, and could become part of a new treatment technology for implant surfaces, promoting further optimization of NANOZR implant materials.

Keywords: fluorinated hydroxyapatite; implant; nano-zirconia; osseointegration; pulsed laser deposition.

Figure 1

(a–c) Gross appearance of the NANOZR and fluoridated hydroxyapatite (FHA)-coated NANOZR Materials; (d,e) Scanning electron micrographs of the NANOZR and FHA-coated NANOZR disks; (f–h) Scanning electron micrographs of the FHA-coated screws; Partial magnifications of the orange rectangular area are displayed in the lower panel. (i–k) Scanning probe micrographs of the NANOZR and FHA-coated NANOZR surfaces. Roughness values (Ra) of the NANOZR and FHA-coated NANOZR disks, *** p < 0.001.

Figure 2

(a) EDS image of the FHA-coated NANOZR disks for elemental composition and weight percentage diagram; Elemental mapping (Ca, P, Zr, O, F,) of the FHA-coated NANOZR materials. (b) Wide scan of the XPS spectrum of NANOZR and FHA-coated NANOZR disks. High-resolution spectra for (c) C 1 s, (d) O 1 s, (e) P 2p, (f) Ca 2p, and (g) F 1s.

Figure 3

(a) Fourier transformed infrared spectra of FHA-coated NANOZR disks; (b) XRD patterns of NANOZR and FHA-coated NANOZR disks heat-treated at 450 °C for 10 h; (c) Contact angle of NANOZR and FHA-coated NANOZR disks; (d) Stress-displacement curves of the FHA-coated NANOZR disks.

Figure 4

Morphological analysis of rBMMSCs attached to NANOZR (a,b) and FHA-coated NANOZR (c,d) disks after culturing for 24 h. Actin filaments (red) were labeled with Alex Fluor 594-phalloidin and nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole; Cell viability of rBMMSCs attached to NANOZR and FHA-coated NANOZR (e) disks after culturing for 24 h, * p < 0.05. SEM analysis of the morphology of rBMMSCs attached to NANOZR (f–h), and FHA-coated NANOZR (i–n) disks after culturing for 24 h; Pictures at lower magnification (i–k) show the morphology of single cells.; Pictures at higher magnification (l–n) show the detailed interaction the cell with the FHA coating. (marked with the orange rectangular area).

Figure 5

(a–d) Quantitative real-time (qRT)-PCR analysis of osteogenesis-related gene expression in NANOZR and FHA-coated NANOZR; (e) Alkaline phosphatase activity in NANOZR and FHA-coated NANOZR; (f) Calcium deposition in NANOZR and FHA-coated NANOZR, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

(a,b) Transverse reconstructed microcomputed tomography of the NANOZR and FHA-coated NANOZR implants after 8 weeks; (c,d) Reconstructed three-dimensional micro-CT images of bone tissues around the NANOZR and FHA-coated implants; (e–h) Bone volume to total volume ratio (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) around the NANOZR and FHA-coated NANOZR implants after 8 weeks. (i–l) Histological sections of bone tissues around the NANOZR and FHA-coated implants; (m,n) Bone area ratio (BA) and bone implant contact (BIC) around the NANOZR and FHA-coated implants, * p < 0.05, *** p < 0.001.