Enhanced Biological Response of AVS-Functionalized Ti-6Al-4V Alloy through Covalent Immobilization of Collagen

Abstract

This study presents the development of an efficient procedure for covalently immobilizing collagen molecules on AVS-functionalized Ti-6Al-4V samples, and the assessment of the survival and proliferation of cells cultured on these substrates. Activated Vapor Silanization (AVS) is a versatile functionalization technique that allows obtaining a high density of active amine groups on the surface. A procedure is presented to covalently bind collagen to the functional layer using EDC/NHS as cross-linker. The covalently bound collagen proteins are characterized by fluorescence microscopy and atomic force microscopy and their stability is tested. The effect of the cross-linker concentration on the process is assessed. The concentration of the cross-linker is optimized and a reliable cleaning protocol is developed for the removal of the excess of carbodiimide from the samples. The results demonstrate that the covalent immobilization of collagen type I on Ti-6Al-4V substrates, using the optimized protocol, increases the number of viable cells present on the material. Consequently, AVS in combination with the carbodiimide chemistry appears as a robust method for the immobilization of proteins and, for the first time, it is shown that it can be used to enhance the biological response to the material.

Figure 1

Morphology of the MSC cells cultured on bare Ti-6Al-4V, adsorbed collagen on Ti-6Al-4V (Col-ads) and plastic control samples after 4 and 48 hour of seeding. Viable cells are stained green while the dead cells appear as red dots. Scale Bar: 100 μm.

Figure 2

Number of cells obtained by (a) cell counting from micrographs after 4 and 48 hours of seeding, and (b) XTT reaction after 48 hours of seeding for MSC cells on bare Ti-6Al-4V, collagen adsorbed on Ti-6Al-4V (Col-ads) and control samples. *Denotes p < 0.05.

Figure 3

Fluorescence microscopy images of samples incubated with FITC-tagged collagen on (a) bare Ti-6Al-4V incubated with EDC/NHS cross-linker before SDS treatment and (d) after SDS treatment, (b) AVS-functionalized Ti-6Al-4V without EDC/NHS cross-linker before SDS treatment and (e) after SDS treatment, and c) AVS-functionalized Ti-6Al-4V incubated with EDC/NHS cross-linker (Col-imm-f) before SDS treatment and (f) after SDS treatment. Scale Bar 100 μm.

Figure 4

AFM topography images of samples incubated with FITC-tagged collagen on (a) bare Ti-6Al-4V incubated with EDC/NHS cross-linker before SDS treatment and (d) after SDS treatment, (b) AVS-functionalized Ti-6Al-4V without EDC/NHS cross-linker before SDS treatment and (e) after SDS treatment, and (c) AVS-functionalized Ti-6Al-4V incubated with EDC/NHS cross-linker (Col-imm-f) before SDS treatment and (f) after SDS treatment. Scale Bar 5 µm.

Figure 5

Fluorescence microscopy images of functionalized Ti-6Al-4V samples incubated with collagen and different concentrations of EDC/NHS. (a,d) 1X concentration of EDC/NHS (b,e) 1/10X concentration of EDC/NHS (c,f) 1/20X concentration of EDC/NHS. (a,b,c) Scale Bar 1 mm and (d,e,f) Scale Bar: 100 μm.

Figure 6

AFM topography images of functionalized Ti-6Al-4V samples incubated with collagen and different concentration of EDC/NHS at different scan sizes (a,d) 1X concentration of EDC/NHS, (b,e) 1/10X concentration of EDC/NHS, and (c,f) 1/20X concentration of EDC/NHS.

Figure 7

MSC cells adhered on bare Ti-6Al-4V, functionalized Ti-6Al-4V with immobilized collagen containing 1X, 1/10X and 1/20X concentrations of EDC/NHS, and control sample at 4 and 48 hours after seeding. Viable cells are stained green, while the dead cells appear as red dots. Scale Bar 100 μm.

Figure 8

Number of MSC cells obtained by (a) cell counting from micrographs after 4 and 48 hours of seeding and (b) XTT reaction at 48 hours after seeding on bare Ti-6Al-4V, functionalized Ti-6Al-4V with immobilized collagen containing 1X, 1/10X and 1/20X concentrations of EDC/NHS, and control sample. *Denotes p < 0.05.

Figure 9

Fluorescence microscopy images of Phalloidin/Hoechst stained MSC cells adhered on bare Ti-6Al-4V, functionalized Ti-6Al-4V with immobilized collagen containing 1X, 1/10X and 1/20X concentrations of EDC/NHS, and control sample after 4 hours of seeding. Scale Bar 20 μm.

Figure 10

Surface area of MSCs on bare Ti-6Al-4V, functionalized Ti-6Al-4V with immobilized collagen containing 1X, 1/10X and 1/20X concentrations of EDC/NHS, and control sample after 4 hours of seeding. *Denotes p < 0.05 with respect to the control sample.