Surface Immobilization of TiO2 Nanotubes with Bone Morphogenetic Protein-2 Synergistically Enhances Initial Preosteoblast Adhesion and Osseointegration

Abstract

Although titanium (Ti) alloys have been widely used as implant materials, the bioinertness of pristine Ti impairs their bioactivity and early osseointegration. In the present work, we prepared TiO₂ nanotubes (TNT) layer on the titanium (Ti) surface by anodic oxidation. The anodized surface was functionalized with human bone morphogenetic protein-2 coating to form the hBMP-2/TNT surface. The release behavior of hBMP-2 on the hBMP-2/TNT surface displayed a controlled and sustained pattern, compared to that on the hBMP-2/Ti surface, which showed a rapid release. In vitro cellular activity tests demonstrated that both TNT and hBMP-2/Ti surfaces, particularly the hBMP-2/TNT surface, enhanced adhesion, proliferation, and differentiation of osteoblast cells. Increased cell adhesion, improved cytoskeleton organization, and immunofluorescence staining of vinculin were observed on the modified surfaces. The TNT, hBMP-2/Ti, and hBMP-2/TNT surfaces, especially the hBMP-2/TNT surface, further displayed an upregulated gene expression of adhesion and osteogenic markers vinculin, collagen type 1, osteopontin, and osteocalcin, compared to the pristine Ti surface. In vivo experiments using a rat model demonstrated that the TNT and hBMP-2/Ti surfaces, in particular the hBMP-2/TNT surface, improved osseointegration and showed a superior bone bonding ability compared to Ti. Our study revealed a synergistic role played by TiO₂ nanotubes nanotopography and hBMP-2 in promoting initial osteoblast adhesion, proliferation, differentiation, and osseointegration, thus suggesting a promising method for better modifying the implant surface.

Figure 1

SEM images of Ti (a), TNT (b), hBMP-2/Ti (d), and hBMP-2/TNT (e) samples observed at 20,000 × and 50,000 ×(magnified insets). Figure 1(c) is the fracture surface of 45 degree tilting sample of the nanotube microstructure; Figure 1(f) is the magnified SEM picture of Figure 1(e)'s inset (100,000 ×).

Figure 2

XPS spectra of Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT samples.

Figure 3

(a) Images of contact angles on Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT samples. (b) Summary of water contact angle degrees on different surfaces. Values are mean ± SD, n=3; ∗, #, and @ indicate p < 0.05 compared with Ti, TNT, and hBMP-2/Ti, respectively.

Figure 4

Release behavior of hBMP-2 from hBMP-2/Ti and hBMP-2/TNT surfaces. Samples in PBS (pH 7.4) were monitored for 21 days (values are mean ± SD, n=3).

Figure 5

Osteoblast adhesion measured by counting cells stained with DAPI under a fluorescence microscope after 4h of incubation. (a): representative images of Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT samples. (b): summary of adherent cell numbers on different surfaces. Values are mean ± SD, n=3; ∗, #, and @ indicate p < 0.05 compared with Ti, TNT, and hBMP-2/Ti, respectively. Scale bar, 100 μm.

Figure 6

CLSM images of cells cultured on different substrates for 4 h. 1: actin filaments (red), 2: immunofluorescence for vinculin (green), 3: DAPI staining of nucleus (blue), and 4: a merged image of 1, 2, and 3. Scale bar, 100 μm.

Figure 7

Proliferation of MC3T3-E1 cells on Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT substrates incubated for 1, 4, and 7 days. Values are mean ± SD, n=3; ∗, #, and @ indicate p < 0.05 compared with Ti, TNT, and hBMP-2/Ti, respectively.

Figure 8

ALP activity of cells on Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT substrates cultured for 7 and 14 days. Values are mean ± SD, n=3; ∗, #, and @ indicate p < 0.05 compared with Ti, TNT, and hBMP-2/Ti, respectively.

Figure 9

Real-time PCR results representing the MC3T3-E1 cell gene expression levels on the Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT substrates. Values are mean ± SD, n=3; ∗, #, and @ indicate p < 0.05 compared with Ti, TNT, and hBMP-2/Ti, respectively.

Figure 10

(a) Hematoxylin and eosin (H&E) staining images at the bone-implant interfaces of Ti, TNT, hBMP-2/Ti, and hBMP-2/TNT surfaces after 4-week implantation. CNT: connective tissue; B: bone; scale bar, 200 μm. (b) Push-out tests results. Values are mean ± SD, n=3; ∗, #, and @ indicate p < 0.05 compared with Ti, TNT, and hBMP-2/Ti, respectively.