Establishment of perpendicular protrusion of type I collagen on TiO2 nanotube surface as a priming site of peri-implant connective fibers

Abstract

Natural teeth are supported by connective tissue collagen fibers that insert perpendicularly in the tooth cementum. Perpendicular insertion plays an important role in the maintenance of the junction between the oral epithelium and the periodontal connective tissue. Most titanium dental implant surfaces have no micro or macro structure to support perpendicularly oriented collagen attachment. Without this tight biologic seal to resist bacterial invasion and epithelial downgrowth, progressive bone loss in peri-implantitis is seen around dental implants. The purpose of this study was to establish the perpendicularly oriented collagen attachment to titanium oxide nanotube (TNT), and to assess its binding stability. TNT was prepared on the titanium-surface by anodization. Scanning electron microscopy (SEM) showed a regularly aligned TNT with an average 67 nm-diameter when anodized at 30 V for 3 h. Subsequently, collagen type I (CoI) was electrophoretically fused to anodic TNT in native polyacrylamide gel system where negatively charged CoI-C term was perpendicularly navigated to TNT. SEM and atomic force microscopy (AFM) were used to analyze CoI on the TiO 2 and TNT surface. Several tens of nanometers of CoI protrusion were recorded by AFM. These protrusions may be long enough to be priming sites for cell-secreted CoI. CoI laid parallel to the titanium surface when fused by a chemical linker. Binding resistance of CoI against drastic ultrasonication was measured by Fourier-transform infrared spectroscopy attenuated total reflection (FTIR-ATR). The electrophoretically fused CoI in the titanium nanotube (TNT–CoI EPF ) showed the significantly greatest binding resistance than the other groups (P < 0.01, a 1-way ANOVA and Tukey HSD post hoc test). Furthermore, TNT–CoI EPF surface rejected epithelial cell stretching and epithelial sheet formation. Chemically linked horizontal CoI on titanium oxide (TiO 2 ) facilitated epithelial cell stretching and sheet formation.

Biological efficacy of perpendicular type-I collagen protruded from TiO2-nanotubes

“… 31 Our previous published data with epithelial cells as well as current result with fibroblasts attachment assay somewhat corroborate previous research. 15 Although no differences in fibroblasts cell numbers were observed at the 3-h mark, our result showed a statistically significant higher number of fibroblasts on the NT-EPF surface in the 24-h fibroblasts attachment assay. This was likely due to the high surface hydrophilicity.…”

“…Adapted with permission from previous published study by our group. 15 b Representative images of water contact angle on different surfaces. c Average contact angle measurements …”

“…[12][13][14] We have also reported the successful establishment of a robust perpendicular Col-I protrusions on TiO 2 nanotube (NT) surface that impeded epithelial downgrowth. 15 TiO 2 NT created by electrochemical anodization has become increasingly popular due to its simplicity and low cost. 16 Many investigators have demonstrated the potential of TiO 2 NT to direct osteogenic differentiation of mesenchymal stem cells and promote osteoblast cell growth.…”

“… 12 – 14 We have also reported the successful establishment of a robust perpendicular Col-I protrusions on TiO 2 nanotube (NT) surface that impeded epithelial downgrowth. 15 …”

“…a SEM images of NT-CL and NT-EPF surfaces, original magnification ×100 000. Adapted with permission from previous published study by our group 15. b Representative images of water contact angle on different surfaces.…”