Introduction: Titanium based alloys as well as ceramic materials are widely used for implant applications usually showing a good biocompatibility. For patients with risk factors (e.g. smokers) or with systemic diseases (e.g. osteoporosis) these implants however often fail to heal because of insufficient bone formation. Our groups have recently developed a modular immobilization system using oligodesoxynucleotide (ODN) strands[1],[2]. The system has been successfully studied for titanium both in vitro[3],[4] and in vivo[5].
This contribution will present recent work (i) to study effects of ODN strand design and immobilization conditions on the hybridization ability of immobilized anchor strands (AS), (ii) to maintain the activity of the AS under γ sterilization conditions, and (iii) to extent the applicability of the system to arbitrary biomaterials.
Materials and Methods: Coin shaped samples from c.p. titanium (Ti), titanium zirconium (TiZr), zirconium oxide (ZrO2), and Bone Ceramic® (Straumann) were used as substrate materials. ODN (60 mer) used as AS contained either one or two hybridization sequences (HS) (5´-A AAC CCG TCA ATC AAG TCT ACA CTG-3´). In case of one HS the ODN were 5´-terminal elongated by homologous spacer (A30, T30, C30 and G30). Specific substrate binding peptide sequences (SSBPS) (length 8 to 20 amino acids) were selected based on literature. Surface interaction analysis combined radioactive and fluorescence labelling, surface plasmon resonance, and conjugation to biotin in combination with the horseradish peroxidase-streptavidin assay.
Results and Discussion: Using strands with only one HS in combination with a spacer allowed to analyze the impact of the spacer sequence on strand stability as well as hybridization efficiency. High contour length resulted in an improved protection of the HS from unfavourable interactions with the surface or damaging attacks by reactive oxygen species. Samples with immobilized AS packed under standard lab atmosphere suffer a complete loss of hybridization ability after γ sterilization. Using a combination of defined sample environment (composition of gas atmosphere, humidity) in combination with scavenger ODN strands however allowed for a preservation of ~70% of hybridization ability as compared to not sterilized samples after standard γ sterilization conditions of 25 kGray. Using SSBPS conjugated to the 5´-terminus of AS allowed for stable binding to all investigated substrate materials. Hybridization with CS conjugated to biologically active molecules has been used for successful presentation of the N-terminal fragment 1-34 of parathyroid hormone (PTH).
Conclusion: The investigations allowed for an improved understanding of the hybridization behavior of ODN immobilized on substrate surfaces. The developed packaging and handling technology maintains the hybridization ability of immobilized ODN under γ sterilization conditions. The SSBPS approach allows for a successful application of the immobilization system to arbitrary biomaterial surfaces.
The authors acknowledge financial support by the DFG (SCHA570/13-1) and ITI (Research Grant No. 680_2010)
References:
[1] Beutner R. et al. J R Soc Interface, 7(2010), 93-105.
[2] Beutner R. et al. Biomaterials 30(2009)14, 2774-81.
[3] Schliephake H. et al. European Cells and Materials 23(2012) 161-69.
[4] Schliephake H. et al. Biomaterials, 33(2012), 1315-22.
[5] Wölfle J.V. et al. PLOS ONE 9(2014)1, e86151.