Chemically Modified Nanoporous Titanium Oxide for Biomedical Applications
Zusammenfassung der Projektergebnisse
The paradigm in the design of biomimetic materials such as bone or tooth implants, vascular stents, or cardiac tissue is to generate three-dimensional scaffolds which support essential cell functions and mimic biomechanical properties of the host tissue, while avoiding toxic reactions and advert immune responses. Current efforts are directed towards maximizing biocompatibility of the implant material, which means to optimize cell adhesiveness and to support physiological cell reactions such as spreading, proliferation, migration and differentiation. Major strategies to improve cell adhesiveness of implant biomaterial include surface roughening, etching or modifying by physical and chemical methods, and/or coating with adhesive proteins of the extracellular matrix such as collagens, fibronectin, laminins. In addition, evidence is accumulating that not only the surface chemistry of the biomaterial, but also the surface topography at nanoscale is a critical parameter for cellular recognition of biomaterials. Recently we have developed a technique to generate geometrically defined titanium dioxide surface patterns by anodic oxidation of titanium in the presence of HF/H3PO4 which results in vertically aligned TiO2 nanotubes of defined diameters between 15 and 100 nm, depending on the anodization potential. We demonstrated that bone marrow stem cells (MSC) respond to titanium chips covered with TiO2 nanotubes in a size-dependent manner, with a maximum of cell adhesion, proliferation, and migration rates on 15 nm diameter nanotubes. This observation prompted us to further investigate whether the size-dependence of cellular responses to the surface geometry applies also to other cell types possibly involved in biomaterial-host tissue interactions, mostly osteoblasts, endothelial cells and hematopoetic stem cells, and whether the responses depend on the surface chemistry and crystalline status of the titanium dioxide surface. Furthermore, it remained to be resolved whether other cell functions such as cell differentiation and apoptosis, the programmed cell death, is dependent on the nanotubular surface geometry, and whether cell interactions with the titanium dioxide nanotube surface are mediated by integrins, in a manner similar to cell interactions with the extracellular matrix. Besides its function as structural scaffold and substrate for cell adhesion, a physiologically important role of the extracellular matrix is its ability to store and sequester growth factors. For example, based on their high affinity for FGFs (Fibroblast growth factors), PDGF (Platelet derived growth factor) or Indian hedgehog, heparin sulfate proteoglycans control the intercellular transport and presentation of these factors to cell surface receptors; similarly, collagens, fibrillin and decorin bind TGF-β and BMPs (Bone morphogenic proteins) and thus are involved in storage and sequestering these growth factors. For this reason, one of the goals of this project was therefore to endow biomaterial with the ability to store growth factors, either by covalent linking growth factors to the surface or inner lumen of TiO2 nanotubes, or by coating nanotubular surfaces with collagens, fibronectin and laminin. In a first series of in vitro studies using titanium chips coated with TiO2 nanotubes of 6 different diameters between 15 and 100 nm, we were able to show that differentiation of MSC to osteoblasts was highest on surfaces with a 15 nm spacing, while nanotubes of 70-100 nm diameter induced cell death. Other cell types involved in bone regeneration such as osteoblasts, osteoclasts and endothelial cells showed the same maximum of cell responses at 15 nm. This size-dependent cell behavior did not change whether TiO2 nanotubes were prepared in the amorphous or in the crystalline (anatase) form, but it was lost when the surface wettability was impaired by coating with a hydrophobic substance. The dominance of the nanoscale surface geometry over surface chemistry was also demonstrated by analyzing the cellular reactions to zirconium dioxide nanotubes which can be prepared in a similar nanotubular structure as TiO2 nanotubes by anodic oxidation. Similar to TiO2 nanotubes, cell adhesion and spreading on nanotubular ZrO2 chips were enhanced on nanotube diameters of 15–30 nm. A clue to the size-controlled cell responses was provided by studies on focal contact formation, cytoskeletal arrangements and integrin localization of cells adhering toTiO2 nanotubes of different diameter. The 15 nm spacing supported a maximum of microfilament formation, paxillin phosphorylation and β1 integrin clustering, indicating a maximum of focal contact formation and integrin activation. Almost all cellular activities regulated by the extracellular matrix are mediated by integrins, which are clustered and activated by the ECM. Since the size of the extracellular domains of the transmembrane integrin receptors is about 10 nm, we postulate that 15 nm spacing may be a universal geometric constant of surface topography supporting cell adhesion and differentiation. The strong support of osteoblast and osteoclast differentiation by nanotubular TiO2 initiated a collaborative project with Prof. Neukam and Prof. Schlegel at the Dept. of Oro-Maxillary Surgery on the effect of nanotubular coating of dental titanium implants. Further studies on the use of TiO2 nanotubes in the generation of novel biomaterials to be used as coatings on bone and teeth implants are expected in the next years. In addition, we intend to pursue basic cell - and molecular biological studies on the finding of the 15 nm nanoscale range as universal parameter for supporting cell functions by inducing integrin clustering.
Projektbezogene Publikationen (Auswahl)
- Improved attachment of mesenchymal stem cells on super-hydrophobic TiO2 nanotubes. Acta Biomater. 2008 Sep;4(5):1576-82. Epub 2008 Apr 25
Bauer S, Park J, von der Mark K, Schmuki P
(Siehe online unter https://doi.org/10.1016/j.actbio.2008.04.004) - Another look at "Stem cell fate dictated solely by altered nanotube dimension". Proc Natl Acad Sci USA. 2009 Jun 16;106(24):E60; Epub 2009 Jun 8
von der Mark K, Bauer S, Park J, Schmuki P
(Siehe online unter https://dx.doi.org/10.1073%2Fpnas.0903663106) - Narrow window in nanoscale dependent activation of endothelial cell growth and differentiation on TiO2 nanotube surfaces. Nano Lett. 2009 Sep;9(9):3157-64
Park J, Bauer S, Schmuki P, von der Mark K
(Siehe online unter https://doi.org/10.1021/nl9013502) - Size selective behavior of mesenchymal stem cells on ZrO2 and TiO2 nanotube arrays. Integr Biol (Camb). 2009 Sep;1(8-9):525-32. Epub 2009 Jun 19
Bauer S, Park J, Faltenbacher J, Berger S, von der Mark K, Schmuki P
(Siehe online unter https://doi.org/10.1039/B908196H) - TiO2 nanotube surfaces: 15 nm-an optimal length scale of surface topography for cell adhesion and differentiation. Small. 2009 Mar;5(6):666-71
Park J, Bauer S, Schlegel KA, Neukam FW, von der Mark K, Schmuki P
(Siehe online unter https://doi.org/10.1002/smll.200801476) - Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix (Review). Cell Tissue Res. 2010 Jan;339(1):131-53. Epub 2009 Nov 7
von der Mark K, Park J, Bauer S, Schmuki P
(Siehe online unter https://doi.org/10.1007/s00441-009-0896-5) - Covalent functionalization of TiO2 nanotube arrays with EGF and BMP-2 for modified behavior towards mesenchymal stem cells. Integr Biol (Camb). 2011 Sep;3(9):927-36. Epub 2011 Aug 10
Bauer S, Park J, Pittrof A, Song YY, von der Mark K, Schmuki P
(Siehe online unter https://doi.org/10.1039/c0ib00155d) - Synergistic Control of Mesenchymal Stem Cell Differentiation by Nanoscale Surface Geometry and Immobilized Growth Factors on TiO(2) Nanotubes. Small. 2011 Nov 18
Park J, Bauer S, Pittrof A, Killian MS, Schmuki P, von der Mark K
(Siehe online unter https://doi.org/10.1002/smll.201100790)
