Immobilization of Denosumab on Titanium Affects Osteoclastogenesis of Human Peripheral Blood Monocytes.

Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces.

Deposition of phosphate coatings on titanium within scaffold structure.

PURPOSE: Existing knowledge about the appearance, thickness, and chemical composition of phosphate coatings on titanium inside porous structures is insufficient. Such knowledge is important for the design and fabrication of porous implants. METHODS: Metallic scaffolds were fabricated by selective laser melting of 316L stainless steel powder. Phosphate coatings were deposited on Ti sensors placed either outside the scaffolds or in the holes in the scaffolds. The electrochemically-assisted cathodic deposition of phosphate coatings was performed under galvanostatic conditions in an electrolyte containing the calcium and phosphate ions. The phosphate deposits were microscopically investigated; this included the performance of mass weight measurements and chemical analyses of the content of Ca2+ and  24 PO ions after the dissolution of deposits. RESULTS: The thicknesses of the calcium phosphate coatings were about 140 and 200 nm for isolated titanium sensors and 170 and 300 nm for titanium sensors placed inside pores. Deposition of calcium phosphate occurred inside the pores up to 150 mm below the scaffold surface. The deposits were rich in Ca, with a Ca/P ratio ranging from 2 to 2.5. CONCLUSIONS: Calcium phosphate coatings can be successfully deposited on a Ti surface inside a model scaffold. An increase in cathodic current results in an increase in coating thickness. Any decrease in the cathodic current inside the porous structure is slight. The calcium phosphate inside the pores has a much higher Ca/P ratio than that of stoichiometric HAp, likely due to a gradual increase in Ca fraction with distance from the surface.

Electrochemically assisted deposition of hydroxyapatite on Ti6Al4V substrates covered by CVD diamond films - Coating characterization and first cell biological results.

Although titanium and its alloys are widely used as implant material for orthopedic and dental applications they show only limited corrosion stability and osseointegration in different cases. The aim of the presented research was to develop and characterize a novel surface modification system from a thin diamond base layer and a hydroxyapatite (HAp) top coating deposited on the alloy Ti6Al4V widely used for implants in contact with bone. This coating system is expected to improve both the long-term corrosion behavior and the biocompatibility and bioactivity of respective surfaces. The diamond base films were obtained by Microwave Plasma Assisted Chemical Vapor Deposition (MW-PACVD); the HAp coatings were formed in aqueous solutions by electrochemically assisted deposition (ECAD) at varying polarization parameters. Scanning electron microscopy (SEM), Raman microscopy, and electrical conductivity measurements were applied to characterize the generated surface states; the calcium phosphate coatings were additionally chemically analyzed for their composition. The biological properties of the coating system were assessed using hMSC cells analyzing for cell adhesion, proliferation, and osteogenic differentiation. Varying MW-PACVD process conditions resulted in composite coatings containing microcrystalline diamond (MCD/Ti-C), nanocrystalline diamond (NCD), and boron-doped nanocrystalline diamond (B-NCD) with the NCD coatings being dense and homogeneous and the B-NCD coatings showing increased electrical conductivity. The ECAD process resulted in calcium phosphate coatings from stoichiometric and non-stoichiometric HAp. The deposition of HAp on the B-NCD films run at lower cathodic potentials and resulted both in the highest coating mass and the most homogenous appearance. Initial cell biological investigations showed an improved cell adhesion in the order B-NCD>HAp/B-NCD>uncoated substrate. Cell proliferation was improved for both investigated coatings whereas ALP expression was highest for the uncoated substrate.

Detonation nanodiamonds biofunctionalization and immobilization to titanium alloy surfaces as first steps towards medical application.

Due 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.

Biological nano-functionalization of titanium-based biomaterial surfaces: a flexible toolbox.

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.

Oligonucleotide-RGD peptide conjugates for surface modification of titanium implants and improvement of osteoblast adhesion.

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.

Immobilization of oligonucleotides on titanium based materials by partial incorporation in anodic oxide layers.

This paper describes the immobilization of bioactive molecules on titanium based surfaces through a combination of nano-mechanical fixation of nucleic acid anchor strands (ASs) by partial and regioselective incorporation within an anodic oxide layer and their hybridization with complementary strands (CSs) intended to be conjugated to bioactive molecules. We focus on the interaction between the substrate surface and the anchor strands, the integrity of ASs and their hybridization ability. The observed dependence of adsorption on pH suggests that initial interaction of terminally phosphorylated ASs with the substrate surface is mediated by electrostatic interaction. Using ASs labelled with (32)P at different termini, it could be shown that strand breaks occur, which are attributed (i) to the formation of reactive oxygen species during anodic polarization, (ii) the photocatalytic activity of the titanium oxide and (iii) drying effects. Damage to AS could be considerably reduced if the electrolyte contained 5 mol l(-1) ethanol, light was excluded during the experimental procedure, and the number of drying and rewetting steps was minimized. A total surface density of AS of 4.5 pmol cm(-2) was reached and could be hybridized to CS with an efficiency of up to 100%. A non-complementary strand (NS) bound with less than 0.5% of the amount of CS under similar conditions. Therefore, non-specific binding of CS is considered as negligible.

Osteoconductive modifications of Ti-implants in a goat defect model: characterization of bone growth with SR muCT and histology.

In this work the osteoconductive potential of coatings for titanium implants using different extracellular matrix components was evaluated. Cylindrical implants with two defined cavities A and B were coated with collagen type I, type III, or RGD peptide, and placed in the femur of goats together with an uncoated reference state. Bone contact and volume were determined after 5 and 12 weeks implantation, using both histomorphometry and synchrotron radiation micro computed tomography (SR muCT) as the methods complement each other: SR muCT allows for a high precision of bone detection due to the large number of analysed slices per sample, while histology offers a better lateral resolution and the possibility of additionally determining bone contact. Both methods revealed similar tendencies in bone formation for the differently bio-functionalized implants, with the SR muCT data resulting in significant differences. After 5 and 12 weeks, all three coatings showed a significant increase in bone volume over the uncoated reference, with the highest results for the collagen coatings. The coating consisting of just the RGD-sequence to improve cell adhesion showed only a slight improvement compared with the reference material. For uncoated titanium, RGD, and especially collagen type I, the response in cavity A, situated in denser bone, was stronger than in cavity B. Collagen type III, on the other hand, appeared to be the more effective coating in areas of lesser bone density as represented by cavity B. These results indicate that matrix molecules (or combinations thereof) are capable of generating the appropriate signals for the specific microenvironment around implants and can thus accelerate the bone formation process and increase the stability of implants.

Modification of Ti6Al4V surfaces using collagen I, III, and fibronectin. I. Biochemical and morphological characteristics of the adsorbed matrix.

Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). Aiming at load-bearing applications in bone contact, in this study the modification of titanium surfaces with the collagen types I and III in combination with fibronectin was undertaken; fibrillogenesis, fibril morphology and adsorption of type I, III and I/III-cofibrils onto titanium were assessed. Increasing the collagen type III amount resulted in a decrease of fibril diameter, while no significant changes in adsorption could be detected. The amount of fibronectin bound to the heterotypic fibrils depended on fibrillogenesis parameters such as ionic strength or concentration of phosphate, and varied with the percentage of integrated type III collagen.