Bone marrow stromal cells interaction with titanium; Effects of composition and surface modification

Abstract

Inflammation and implant loosening are major concerns when using titanium implants for hard tissue engineering applications. Surface modification is one of the promising tools to enhance tissue-material integration in metallic implants. Here, we used anodization technique to modify the surface of commercially pure titanium (CP-Ti) and titanium alloy (Ti-6Al-4V) samples. Our results show that electrolyte composition, anodization time and voltage dictated the formation of well-organized nanotubes. Although electrolyte containing HF in water resulted in nanotube formation on Ti, the presence of NH4F and ethylene glycol was necessary for successful nanotube formation on Ti-6Al-4V. Upon examination of the interaction of bone marrow stromal cells (BMSCs) with the modified samples, we found that Ti-6Al-4V without nanotubes induced cell proliferation and cluster of differentiation 40 ligand (CD40L) expression which facilitates B-cell activation to promote early bone healing. However, the expression of glioma associated protein 2 (GLI2), which regulates CD40L, was reduced in Ti-6Al-4V and the presence of nanotubes further reduced its expression. The inflammatory cytokine interleukin-6 (IL-6) expression was reduced by nanotube presence on Ti. These results suggest that Ti-6Al-4V with nanotubes may be suitable implants because they have no effect on BMSC growth and inflammation.

Fig 1. Effect of anodization parameters on surface morphology.

Anodization was performed with five different conditions and their effect on nanotubes formation were observed on Cp-Ti (a-e) and Ti-6Al-4V (f-j) using a FESEM. Uniform nanotubes were observed on Ti (a&b) and Ti-6Al-4V (i). Other anodization conditions resulted in nonuniform distribution and/or distortion in structure of nanotubes (c, d, f, g, h) and formation of nanograss (e and j).

Fig 2. Elemental analysis of Ti substrates.

The EDS elemental analysis of (a) Ti-NT and (b) Ti-6Al-4V-NT showing presence of only Ti and O in Ti-NT, while Ti, Al, V and O are found in Ti-6Al-4V.

Fig 3. Surface roughness of Ti substrates.

Surface roughness of the Ti substrates (a) Ti (b) Ti-NT (c) Ti-6Al-4V (d) Ti-6Al-4V-NT, was determined by an optical profiler. Shows increase in roughness of samples after anodization.

Fig 4. Anodization increases hydrophilicity.

Contact angle of (a) Ti (b) Ti-NT (c) Ti-6Al-4V (d) Ti-6Al-4V-NT. Contact angles in anodized samples (b and d) are significantly less that untreated samples showing the effective role of anodization on increasing hydrophilicity.

Fig 5. Cellular morphology on Ti substrates.

a) Saka cells were grown on Ti discs as indicated in materials and methods followed by examination of cellular morphology using FESEM. b) Saka cells were allowed to adhere to Ti discs for 6 hours followed by investigation of cell adhesion by XTT assay.

Fig 6. Effect of composition and surface morphology on growth of BMSCs and inflammation.

a) Saka cells were allowed to adhere on material as indicated in methods for 3 days followed by XTT assay to determine cell proliferation. qRT-PCR for the inflammatory markers b) GLI2, c) CD40L and d) IL-6. E) Saka cells were allowed to adhere A similar experiment was performed to determine the expression of differentiation markers alkaline phosphatase 1 (ALP-1), Collagen A-1 (COLA-1) and osteocalcin (OCN) on the different substrates by qRT-PCR. Data are presented as averages of 2 independent experiments, each performed in triplicate and the bars represent means ± SEM.