Effects of a micro/nano rough strontium-loaded surface on osseointegration

Abstract

We developed a hierarchical hybrid micro/nanorough strontium-loaded Ti (MNT-Sr) surface fabricated through hydrofluoric acid etching followed by magnetron sputtering and evaluated the effects of this surface on osseointegration. Samples with a smooth Ti (ST) surface, micro Ti (MT) surface treated with hydrofluoric acid etching, and strontium-loaded nano Ti (NT-Sr) surface treated with SrTiO3 target deposited via magnetron sputtering technique were investigated in parallel for comparison. The results showed that MNT-Sr surfaces were prepared successfully and with high interface bonding strength. Moreover, slow Sr release could be detected when the MNT-Sr and NT-Sr samples were immersed in phosphate-buffered saline. In in vitro experiments, the MNT-Sr surface significantly improved the proliferation and differentiation of osteoblasts compared with the other three groups. Twelve weeks after the four different surface implants were inserted into the distal femurs of 40 rats, the bone-implant contact in the ST, MT, NT-Sr, and MNT-Sr groups were 39.70%±6.00%, 57.60%±7.79%, 46.10%±5.51%, and 70.38%±8.61%, respectively. In terms of the mineral apposition ratio, the MNT-Sr group increased by 129%, 58%, and 25% compared with the values of the ST, MT, and NT-Sr groups, respectively. Moreover, the maximal pullout force in the MNT-Sr group was 1.12-, 0.31-, and 0.69-fold higher than the values of the ST, MT, and NT-Sr groups, respectively. These results suggested that the MNT-Sr surface has a synergistic effect of hierarchical micro/nano-topography and strontium for enhanced osseointegration, and it may be a promising option for clinical use. Compared with the MT surface, the NT-Sr surface significantly improved the differentiation of osteoblasts in vitro. In the in vivo animal experiment, the MT surface significantly enhanced the bone-implant contact and maximal pullout force than the NT-Sr surface.

Keywords: implant; micro/nanorough; surface modification.

Figure 1

Images of the titanium surface from gross observation, SEM, and AFM (n=8). Abbreviations: AFM, atomic force microscopy; MNT-sr, micro/nanorough strontium-loaded Ti; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; SEM, scanning electron microscopy; ST, smooth titanium surface.

Figure 2

Cross-sectioning of (A) NT-Sr and (B) MNT-Sr samples from SEM images (n=8). Abbreviations: MNT-Sr, micro/nanorough strontium-loaded Ti; NT-Sr, nano strontium-containing titanium surface; SEM, scanning electron microscopy.

Figure 3

XRD patterns acquired from samples with four different surfaces (n=8). Abbreviations: MT, micro titanium surface; MNT-Sr, micro/nanorough strontium-loaded Ti; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface; XRD, X-ray diffraction.

Figure 4

XPS survey spectra collected from samples with four different surfaces (n=8). Abbreviations: MT, micro titanium surface; MNT-Sr, micro/nanorough strontium-loaded Ti; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface; XPS, X-ray photoelectron spectroscopy.

Figure 5

Sr release of the two Sr-loaded samples. Notes: (A) Noncumulative Sr release time profiles from NT-Sr and MNT-Sr into PBS (n=5 at each time point). (B) Total Sr content of NT-Sr and MNT-Sr samples (n=5). Abbreviations: MNT-Sr, micro/nano strontium-containing titanium surface; NT-Sr, nano strontium-containing titanium surface; PBS, phosphate-buffered saline.

Figure 6

Osteoblast attachment on the four different samples. Notes: (A) Images of osteoblast attachment after 30, 60, and 120 minutes of incubation. The cells were stained with DAPI. n=6 at each time point. (B) Initial number of adherent cells measured by counting cells stained with DAPI. *P<0.05 and **P<0.01 compared with ST; ^#P<0.05 and ^##P<0.01 compared with MT. Abbreviations: MNT-Sr, micro/nano strontium-containing titanium surface; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface.

Figure 7

Osteoblast proliferation after incubation for 1, 4, and 7 days. Notes: **P<0.01 compared with ST; ^#P<0.05 and ^##P<0.01 compared with MT; and ^††P<0.01 compared with NT-Sr. n=6 at each time point. Abbreviations: MNT-Sr, micro/nano strontium-containing titanium surface; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface.

Figure 8

SEM images of osteoblast adhesion on the four surfaces after 3 days. Abbreviations: MNT-Sr, micro/nanorough strontium-loaded Ti; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; SEM, scanning electron microscopy; ST, smooth titanium surface.

Figure 9

Representative CLSM images of cells stained with DAPI to show the nuclei (blue) and FITC to show the actin filaments (green). Notes: (A) ST; (B) MT; (C) NT-Sr; and (D) MNT-Sr. Abbreviations: CLSM, confocal laser scanning microscope; FITC, fluorescein isothiocyanate; MNT-Sr, micro/nanorough strontium-loaded Ti; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface.

Figure 10

ALP activity for different groups cultured for 7 days. Notes: *P<0.05 and **P<0.01 compared with ST; ^##P<0.01 compared with MT; and ^††P<0.01 compared with NT-Sr. n=6. Abbreviations: MNT-Sr, micro/nanorough strontium-loaded Ti; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface; TCP, tissue culture plate.

Figure 11

Micro-CT scanning, double-fluorescence labeling observation, and methylene blue/acid fuchsin staining and histological analysis for evaluating osseointegration around the four different surface implants. Abbreviations: CT, computed tomography; MNT-Sr, micro/nanorough strontium-loaded Ti; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; ST, smooth titanium surface.

Figure 12

Micro-CT parameters for evaluating trabecular ROI. Notes: (A) BV/TV of the four groups; (B) trabecular number (Tb.N) of the four groups; (C) trabecular thickness (Tb.Th) of the four groups; (D) trabecular separation (Tb.Sp) of the four groups. *P<0.05 and **P<0.01 compared with ST; ^#P<0.05 and ^##P<0.01 compared with MT; and ^††P<0.01 compared with NT-Sr. n=10. Abbreviations: BV/TV, bone volume/total volume; CT, computed tomography; MNT-Sr, micro/nanorough strontium-loaded Ti; MT, micro titanium surface; NT-Sr, nano strontium-containing titanium surface; ROI, region of interest; ST, smooth titanium surface; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.