Surface functionalization with bioactive molecules (BAMs) on a nanometre scale is a main field in current biomaterial research. The immobilization of a vast number of substances and molecules, ranging from inorganic calcium phosphate phases up to peptides and proteins, has been investigated throughout recent decades. However, in vitro and in vivo results are heterogeneous. This may be at least partially attributed to the limits of the applied immobilization methods. Therefore, this paper highlights, in the first part, advantages and limits of the currently applied methods for the biological nano-functionalization of titanium-based biomaterial surfaces. The second part describes a new immobilization system recently developed in our groups. It uses the nanomechanical fixation of at least partially single-stranded nucleic acids (NAs) into an anodic titanium oxide layer as an immobilization principle and their hybridization ability for the functionalization of the surface with BAMs conjugated to the respective complementary NA strands.
A new concept for modular biosurface engineering of titanium implants based on the self-assembly of complementary oligonucleotides was biochemically investigated and optimized. This study describes the synthesis and characterization (RP-HPLC and Sakaguchi assay) of oligodeoxyribonucleotide (ODN) conjugates of the hexapeptide GRGDSP containing the RGD sequence as the recognition motif for cellular adhesion receptors (integrins). The peptide was chosen exemplarily as a model molecule, because it is a simple but potent bioactive molecule and relatively well investigated. The conjugation products must fulfill two main requirements: (I) the ability to hybridize and (II) the preservation of biological activity of the RGD peptide for the enhancement of osteoblast adhesion. In the following text, the term "hybridization" is generally used for Watson-Crick base pairing. The ability of the conjugates to hybridize to surface-immobilized complementary ODN was verified by competitive hybridization with radiolabeled ((32)P) complementary strands and by hybridization experiments using a quartz crystal microbalance (QCM). Surface hybridization was further characterized using different adsorption isotherms (e.g., Freundlich and Frumkin types), since the type of isotherm and the derived thermodynamic parameters may reveal characteristic differences between ODN and conjugates thereof. Biological activity of the conjugates was examined in vitro with osteoblasts. The cells were either cultured directly on the ODN-GRGDSP modified titanium implants or used for competition adhesion studies with dissolved ODN-GRGDSP conjugates. All results support the successful establishment of the new surface modification system. Hybridization of RGD peptide-modified nucleic acids to ODN-modified titanium implant materials is thus a promising method for osteoblast attachment in a modular and self-organizing system on implant surfaces.
SummaryDue to their outstanding properties nanodiamonds are a promising nanoscale material in various applications such as microelectronics, polishing, optical monitoring, medicine and biotechnology. Beyond the typical diamond characteristics like extreme hardness or high thermal conductivity, they have additional benefits as intrinsic fluorescence due to lattice defects without photobleaching, obtained during the high pressure high temperature process. Further the carbon surface and its various functional groups in consequence of the synthesis, facilitate additional chemical and biological modification. In this work we present our recent results on chemical modification of the nanodiamond surface with phosphate groups and their electrochemically assisted immobilization on titanium-based materials to increase adhesion at biomaterial surfaces. The starting material is detonation nanodiamond, which exhibits a heterogeneous surface due to the functional groups resulting from the nitrogen-rich explosives and the subsequent purification steps after detonation synthesis. Nanodiamond surfaces are chemically homogenized before proceeding with further functionalization. Suspensions of resulting surface-modified nanodiamonds are applied to the titanium alloy surfaces and the nanodiamonds subsequently fixed by electrochemical immobilization. Titanium and its alloys have been widely used in bone and dental implants for being a metal that is biocompatible with body tissues and able to bind with adjacent bone during healing. In order to improve titanium material properties towards biomedical applications the authors aim to increase adhesion to bone material by incorporating nanodiamonds into the implant surface, namely the anodically grown titanium dioxide layer. Differently functionalized nanodiamonds are characterized by infrared spectroscopy and the modified titanium alloys surfaces by scanning and transmission electron microscopy. The process described shows an adsorption and immobilization of modified nanodiamonds on titanium; where aminosilanized nanodiamonds coupled with O-phosphorylethanolamine show a homogeneous interaction with the titanium substrate.
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