Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells

Abstract

Background and methods: Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells.

Results: The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells.

Conclusion: This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.

Keywords: adhesion; bone marrow mesenchymal stem cells; micro/nanotexture; nanorod; osteogenic differentiation; titanium surface.

Figure 1

Scanning electron microscopic images of two titanium surfaces at various magnification. Abbreviations: Ti, titanium; HP, hydrogen peroxide.

Figure 2

X-ray diffraction patterns for various titanium surfaces. Abbreviations: Ti, titanium; HP, hydrogen peroxide.

Figure 3

Full x-ray photoelectron spectra of titanium-acid and titanium-acid-hydrogen peroxide surfaces. Abbreviations: Ti, titanium; HP, hydrogen peroxide.

Figure 4

Real-time polymerase chain reaction. (A) After 14 days’ incubation, expression of adhesion-related genes (fibronectin, integrin α1, and integrin β1) and osteogenic differentiation-related genes (Runx-2, ALP, OPN, and OCN) was measured by real-time polymerase chain reaction. (B) Reaction products of reverse transcriptase PCR were further examined by 2.0% agarose gel electrophoresis. Abbreviations: Ti, titanium; HP, hydrogen peroxide; PCR, polymerase chain reaction; ALP, alkaline phosphatase; OPN, osteopontin; OCN, osteocalcin.

Figure 5

Cell adhesion assay. (A) Cell nuclei stained with DAPI were imaged by confocal laser scanning microscopy for the two groups at one, 4, and 24 hours after seeding. (B) Statistical results for adhesive cell numbers. (C) Expression of integrin β1 was detected by confocal laser scanning microscopy. Notes: Green color, actin cytoskeleton of bone marrow mesenchymal stem cells stained with FITC-phalloidin; red color, integrin β1 of bone marrow mesenchymal stem cells stained with DyLight 549; blue color, nuclei of bone marrow mesenchymal stem cells stained with DAPI. *P < 0.05 versus titanium-acid group. Abbreviations: Ti, titanium; HP, hydrogen peroxide.

Figure 6

Analysis of cell metabolism. Note: *P < 0.05, when compared with the titanium-acid group. Abbreviations: Ti, titanium; HP, hydrogen peroxide.

Figure 7

Alkaline phosphatase activity and calcium deposition assay. (A) After 14 days culture, bone marrow mesenchymal stem cells on the various titanium surfaces were stained with an alkaline phosphatase kit and Alizarin Red S solution. (B) Alkaline phosphatase activity of bone marrow mesenchymal stem cells was measured with quantitative assay. (C) Calcium deposition status of cells was assayed using colorimetrically quantitative analysis. Note: *P < 0.05 versus titanium-acid group. Abbreviations: Ti, titanium; HP, hydrogen peroxide; ALP, alkaline phosphatase; ARS, Alizarin Red S.

Figure 8

Detection of osteopontin/osteocalcin immunofluorescence. Notes: After a 14-day incubation period in Dulbecco’s modified Eagle’s medium, osteopontin and osteocalcin immunofluorescence results were observed by confocal laser scanning microscope. Green, actin cytoskeleton; blue, nuclei; red, osteopontin/osteocalcin positive area; and yellow, merged color of green and red. Scale bar 100 μm.