Abstract
Introduction
Titanium (Ti) alloys are used as porous bone ingrowth materials on non-cemented knee arthroplasty tibial tray implants. Nano-surface mechanism that increase the osseointegration rate between Ti alloys, and surrounding tissue has been recognized to improve the interface to ultimately allow patients to weight bear on non-cemented arthroplasty implants sooner. Bioactive TiO2 nanotube arrays has been shown to accelerate osseointegration. Ideally, these surfaces would both increase the adhesion of bone to the implant and help to reduction of infection to substitute for antibiotic bone cement. This study examines a combination treatment of both TiO2 nanotubes combined with silver nano-deposition, that simultaneously enhances osseointegration while improving infection resistance, by testing ex vivo implantation stability in an equine cadaver bone followed by in vitro and in vivo analysis to understand the biocompatibility and early stage osseointegration.
Methods
100nm diameter and 300nm length TiO2 nanotubes were formed on a CP titanium surface using anodization method at 20V for 45mins using 1% HF electrolyte. Silver deposition on TiO2 nanotubes were performed using 0.1M AgNO3 solution at 3V for 45s. Figure 1 shows SEM images showing (a) TiO2 nanotubes of 300nm length and (b) nanotubes with silver coating). Ti anodized samples with and without silver nanotubes implanted into an equine cadaver bone in an ex vivo manner to study the stability of nanotubes and the adherence of silver deposition. Silver release study was performed for a period of 14 days in a similar ex vivo manner. Dimensions for implantation samples: 2.5 mm diam. × 15 mm. For cell culture, circular disc samples 12.5mm in diameter and 3 mm in thickness were used to study the bone cell-material interactions using human fetal osteoblast (hFOB) cells. To evaluate the cell proliferation, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay was used. The in vitro cell-materials interaction study was performed for a period of 4 and 7 days. In vivo study was performed using rat distal femur model for a period of 12 weeks with dense Ti samples as control (Sample dimensions: 3mm diam. × 5mm). At the end of 12 weeks, the samples were analyzed for early stage osseointegration using histological analysis and SEM imaging.
Results
No significant changes in the morphology of nanotubes was observed due to the implantation process which signifies the damage resistance these nanotubes can endure during implantation and explantation. Figure 2 shows SEM images of (a) & (b) nanotubes without silver coating before and after implantation and (c) & (d) nanotubes with silver coating before and after implantation respectively. Silver nanocoatings can be observed after implantation which shows the adherence of the antimicrobial nano-coating on the surface of nanotubes. Cumulative release profiles of silver ions after 14 days showed the total release was in the effective range for antimicrobial characteristics and was well below the toxic limit specified for human cells (10 ppm) Figure 3(a) shows cumulative release profile of silver after 14 days. MTT assay and SEM images show good cell proliferation, antimicrobial effect, and increase in cell density after 7 days for samples with nanotubes and silver with no cytotoxic effects and good cell attachment on the samples as shown in Figure 3(b) MTT assay results showing cell densities after 4 and 7 days and Figure 3(c) SEM images showing cell attachment after 4 and 7 days on samples. Histological analysis and SEM images showed osteoid formation around the implant with improved bonding towards the implant and bone showing signs of early stage osseointegration. Figure 4 shows histological and SEM images showing bonding between bone and implant surface for respective samples after 12 weeks.
Conclusions
Mechanically stableTiO2 nanotubes with strongly adhered antimicrobial silver coating were grown on the surface of titanium which were biocompatible and non-toxic. In vitro and in vivo tests indicate improved cell-materials interaction with signs of early stage osseointegration. This nano-surface treatment shows promise towards simultaneously improving early stage osseointegration and providing an infection barrier on bone ingrowth materials.
