Bernd Schwenzer

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.

Characterization of different carbon nanotubes for the development of a mucoadhesive drug delivery system for intravesical treatment of bladder cancer

In order to increase the effectiveness of therapeutics for bladder carcinoma (BCa) treatment, alternative strategies for intravesical applications are needed. The use of carbon nanotubes (CNTs) as basis for a multifunctional drug transporter is a promising possibility to combine traditional chemotherapeutics with innovative therapeutic agents such as antisense oligodeoxynucleotides or small interfering RNA. In the current study four CNT types varying in length and diameter (CNT-1, CNT-2, CNT-3, CNT-4) were synthesized and then characterized with different spectroscopic techniques. Compared to the pristine CNT-1 and CNT-3, the shortened CNT-2 and CNT-4 exhibited more defects and lower aspect ratios. To analyze their mucoadhesive properties, CNTs were exposed to mouse bladders ex vivo by using Franz diffusion cells. All four tested CNT types were able to adhere to the urothelium with a mean covering area of 5-10%. In vitro studies on UM-UC-3 and EJ28 BCa cells were conducted to evaluate the toxic potential of these CNTs. Viability and cytotoxicity assays revealed that the shortened CNT-2 and CNT-4 induced stronger inhibitory effects on BCa cells than CNT-1 and CNT-3. In conclusion, CNT-1 and CNT-3 showed the most promising properties for further optimization of a multifunctional drug transporter.

Tissue specific expression and serum levels of human tissue factor in patients with urological cancer

Human tissue factor (TF) is involved in tumor angiogenesis and metastasis. However, little is known about the distribution of TF in urological cancer. In this study we investigated the TF expression in tumor tissue and autologous non-malignant tissue as well as in serum of patients with renal cell carcinoma (RCC), bladder cancer, and prostate cancer (PCa). To study the distribution of TF in tumor tissue and in the surrounding non-malignant tissue, we measured TF protein by ELISA in tissue specimens obtained intraoperatively from 18 RCC, seven bladder cancer and six PCa patients. Differences in TF expression were found between tumor tissue and nonmalignant tissue for the three tumor types at the protein level (in the order RCC < bladder cancer < PCa). In all but one of the 18 RCC patients (94 %) higher TF protein level was observed in non-malignant tissue as compared to the tumor tissue. In addition, the relative TF mRNA expression analyzed by a quantitative RT-PCR assay in the same RCC tissue sample pairs was higher in 78% of non-malignant tissues in comparison to the tumor tissue specimens. Moreover, using enzyme linked immunosorbent assay the TF protein content was measured in serum samples of 66 patients with bladder cancer, 75 RCC patients and 157 PCa patients, and was compared with the TF serum level of 92 healthy volunteers. Whereas no differences were detected between normal volunteers and patients with PCa or RCC, patients with bladder cancer showed a significantly increased level of serum TF (P=0.0076). However, no causal association between TF levels in serum and TF content in tissue extracts for all three tumor types of urological tumors was found. Our results suggest that TF in non-malignant renal tissues was expressed at a higher level compared to the supposed de novo TF expression in RCC tissue specimens. This indicates a tumor-associated induction of TF expression in the TF-negative RCC progenitor cells. The increased serum TF levels are alike the reported higher urinary TF levels found in bladder cancer patients. The potential clinical relevance of this finding should be further elucidated.

Effect of oligonucleotide mediated immobilization of bone morphogenic proteins on titanium surfaces.

The aim of the present study was to test the hypothesis that oligonucleotides can be used for anchorage and slow release of osteogenic growth factors such as BMP to enhance the osteogenic activity of a titanium implant surface. Strands of 60-mer non-coding DNA oligonucleotides (ODN) were bound to an acid-etched sandblasted cp Ti-surface by nanomechanical fixation using anodic polarization. RhBMP2 that had been conjugated to complementary strands of DNA oligonucleotides was then bound to the anchored ODN strands by hybridization. Binding studies showed a higher binding capacity compared to non-conjugated BMP2. Long term release experiments demonstrated a continuous release from all surfaces that was lowest for the conjugated BMP2 bound to the ODN anchor strands. Proliferation of human bone marrow stroma cells (hBMSC) was significantly increased on these surfaces. Immunofluorescence showed that hBMSC grown on surfaces coated with specifically bound conjugated BMP2 developed significantly higher numbers of focal adhesion points and exhibited significantly higher levels of transcription of osteogenic markers alkaline phosphatase and osteopontin at early intervals. Biological activity (induction of alkaline phosphatase) of conjugated BMP2 released from the surface was comparable to released non-conjugated BMP2, indicating that conjugation did not negatively affect the activity of the released molecules. In conclusion the present study has shown that BMP2 conjugated to ODN strands and hybridized to complementary ODN strands anchored to a titanium surface has led to slow growth factor release and can enhance the osteogenic activity of the titanium surface.

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.

Improved anchorage of Ti6Al4V orthopaedic bone implants through oligonucleotide mediated immobilization of BMP-2 in osteoporotic rats.

The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo.

Investigation of the Peptide Adsorption on ZrO2, TiZr, and TiO2 Surfaces as a Method for Surface Modification

Specific surface binding peptides offer a versatile and interesting possibility for the development of biocompatible implant materials. Therefore, eight peptide sequences were examined in regard to their adsorption on zirconium oxide (ZrO2), titanium zircon (TiZr), and titanium (c.p. Ti). Surface plasmon resonance (SPR) measurements were performed on Ti coated sensor chips to determine the kinetics of the interactions and kinetic rate constants (kon, koff, KD, and Rmax). We also investigated the interactions which are present in our system. Electrostatic and coordinative interactions were found to play a major role in the adsorption process. Four of the eight examined peptide sequences showed a significant adsorption on all investigated materials. Moreover, the two peptides with the highest adsorption could be quantified (up to 370 pmol/cm(2)). For potential biomaterials applications, we proved the stability of the adsorption of selected peptides in cell culture media, under competition with proteins and at body temperature (37 °C), and their biocompatibility via their effects on the adhesion and proliferation of human mesenchymal stem cells (hMSCs). The results qualify the peptides as anchor peptides for the biofunctionalization of implants.

Peptide linkers for the immobilization of bioactive molecules on biphasic calcium phosphate via a modular immobilization system

Herein we describe the use of peptide linkers to establish a nucleic acid-based immobilization system based on biphasic calcium phosphates (BCP), with which different molecules can be immobilized at the same time in defined ratios. It consists of single-stranded oligonucleotides, anchor strands (AS) which are immobilized to the surface and conjugates of complementary strands (CS) and bioactive molecules that bind to the AS via Watson-Crick base pairing. AS immobilization can be achieved on calcium phosphate ceramics using conjugates of AS and peptides that bind specifically to the ceramic. We successfully immobilized three different peptide sequences on BCP. Among them, we identified Stath (DpSpS EEK FLR RIG RFG, phosphoserine) as the most suitable one and further immobilized Stath-AS conjugates on BCP. This immobilized AS was able to hybridize with CS. Unspecific adsorption of oligonucleotides on the BCP surface was negligible. The stability of the system was proven by short term desorption experiments. The amounts of immobilized peptides, oligonucleotides and peptide-AS conjugates were determined by an enzymatic assay using biotin-streptavidin interactions, and were found to reach surface densities that are of therapeutic relevance (0.03 pmol cm(-2)).

Functionalization of titanium implants using a modular system for binding and release of VEGF enhances bone-implant contact in a rodent model

AIMS To test the immobilization of vascular endothelial growth factor (VEGF165 ) on the surface of titanium implants using DNA oligonucleotide (ODN) anchor strands for the ability to enhance periimplant bone formation. MATERIALS AND METHODS DNA oligonucleotides were anchored to the surface of sandblasted acid-etched (SAE) titanium screw implants and were hybridized with complementary strands of ODN conjugated to rhVEGF165 . The implants were tested against blank SAE implants and SAE implants with nano-anchored ODN. The implants were inserted into the tibiae of 36 Sprague-Dawley rats. Primary outcome parameters were bone-implant contact (BIC), amount of new bone formation and periimplant bone density (BD). density after 1, 4 and 13 weeks. Unit of analysis has been the individual implant. RESULTS Implants with rhVEGF165 hybridized to ODN anchor strands exhibited significantly increased average BIC after 1 month compared to blank implants and implants with anchored ODN strands. CONCLUSIONS It is concluded that rhVEGF165 immobilized on the surface of titanium implants through nano-anchored oligonucleotide strands can accelerate BIC of sandblasted and etched titanium implants to a certain extent. The radius of effect of the growth factor appears to be limited to tissue immediately adjacent to the implant surface.

Functionalized carbon nanotubes as transporters for antisense oligodeoxynucleotides

The use of DNA-based therapeutics requires efficient delivery systems to transport the DNA to their place of action within the cell. To accomplish this, we investigated multiwalled carbon nanotubes (pristine MWCNT, p-MWCNT) functionalized with hydroxyl groups via 1,3-dipolar cycloaddition. In this way, we have obtained MWCNT-f-OH with improved stability in aqueous dispersions which is an advantageous property for their use in cellular environments. Afterwards, a carrier strand oligodeoxynucleotide (CS-ODN) was adsorbed to MWCNT-f-OH followed by hybridization with a therapeutic antisense oligodeoxynucleotide (AS-ODN). The amount of adsorbed CS-ODN, as well as the complementary AS-ODN and a non-complementary oligodeoxynucleotide (NS-ODN) as reference, was directly measured by radionuclide labeling of ODNs. We show that subsequent release of AS-ODNs and NS-ODNs was possible for MWCNT-f-OH above the melting temperature of AS-ODNs at 80 °C and under physiological conditions at different pH values at 37 °C. We also show a very low influence of p-MWCNT and MWCNT-f-OH on the cell viability of the bladder carcinoma (BCa) cell line EJ28 and that both MWCNT types were internalized by EJ28. Therefore, MWCNT-f-OH represents a promising carrier able to transport and release AS-ODNs inside cells.