Novel Nano-Engineered Titanium Surface for Direct Connective Tissue Attachment
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CitationChen, Chia-Yu. 2019. Novel Nano-Engineered Titanium Surface for Direct Connective Tissue Attachment. Doctoral dissertation, Harvard School of Dental Medicine.
One of the greatest differences between natural teeth and dental implants lies in the attachment apparatus. Collagen fiber bundles connect the tooth to the gingiva in the form of connective tissue attachment which inserts perpendicularly to the root surface. On the contrary, bundles of connective tissue fibers run parallel to the implant surface at the transmucosal level which make for a vulnerable seal.
The goal of the present study is to develop a nano-engineered titanium surface with perpendicularly attached collagen that stimulate platelet activation for the restoration of periodontium-like connective tissue around dental implants.
Materials & Methods:
The titanium surface modification is executed in two stages. First, TiO2 nanotube array is fabricated via anodization. Diameters and depths of TiO2 nanotubes are controlled by applied voltage and duration. Subsequently, an electrophoretic fusion (EPF) method is applied to fuse type I collagen (Col-I) into nanotube arrays on the titanium surface. Surface morphology of the collagen-modified titanium surface was observed using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM).
Next, platelet-rich plasma (PRP) is prepared and samples (tests and control) were incubated with PRP for 30 minutes, one hour and 3 hours on a see-saw table at 37 °C. At the end of the incubation period, the samples were rinsed and fixed for observation under SEM while supernatants were subjected to enzyme-linked immunosorbent assay (ELISA).
To verify the integration of fibroblast-produced Col-I to the nanoengineered surface, samples (tests and control) were co-cultured with fibroblasts using a cell culture insert with mesh size of 3 µm. At the end of the incubation period, samples were either dried in serial alcohol dehydration for SEM observation or stained with a FITC-conjugated anti-Col-I antibody for confocal fluorescence microscopy.
In another culture system, fibroblasts between passaged 10 ~20 were seeded directly onto sample (test and control) surfaces. 50 mg/mL of L-Ascorbic acid was supplemented every other day for 8 days. At the end of the incubation period, samples were fixed with 4% PFA for 20 minutes and store in phosphate buffered solution (PBS). The samples were subjected to observation with SEM, multiphoton laser scanning microscopy and RAMAN spectroscopy.
SEM with EDX observation revealed that a uniform array of nanotubes measuring 67 nm in diameter was obtained by anodization at 30 volts for 3 hours. Electrophoresis of type-I collagen through native PAGE gel into Ti nanotube surface resulted in Col-I depositions in a perpendicular fashion. SEM images showed a uniform array of perpendicular collagen fibrils deposited into and around the nanotubes while the presence of an amide C=O absorption peak at 1550 cm-1 in the spectrum was confirmed with FTIR. The perpendicularly attached Col-I to the nanotubes demonstrated a significantly more robust binding (compared to other methods of Col-I deposition), resistant to high-power sonication.
When platelet-rich plasma (PRP) was applied onto titanium, platelets aggregated on perpendicular collagen-fused nanotube surface while there were none or few for pure titanium surfaces at an early time point. Furthermore, confocal fluorescence microscopy revealed an increased amount of collagen on the modified Ti surfaces in a co-culture model with fibroblasts, confirming that fibroblast-derived Col-I fused with the engineered Col-I.
Polarized Raman spectroscopy revealed perpendicularly oriented collagen fibers extruding out from the nano-engineered collagen on nanotube-titanium surface.
In this study, we hypothesized that our modified TiO2 surface with perpendicular collagen-fused nanotube array would facilitate biomimetic restoration of peri-implant soft and hard tissue. The key innovation is the orientation of the fused Col-I. The nanotubes support the perpendicular insertion of Col-I monomers and these monomer projections, in terms, serve as the priming site for activation of in vivo healing process.
In this report, we have shown that the nano-engineered surface with perpendicular collagen coating promoted fibroblasts attachment and induced platelet activation. An in vitro culture model revealed that fibroblasts secreted collagen fibrils attached to the engineered surface in a perpendicular fashion. We will move toward in vitro 3D culture model as well as in vivo mice animal model to further investigate the establishment of a direct connective tissue attachment to the modified titanium surface.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:42080596