Introduction: Implant failure due to bacterial infection is a major concern, in particular for transcutaneous implants (like dental implants, catheters or external fixators). Great effort has been put into developing surface treatments preventing the early adhesion of bacteria but at the same time improving the behavior of the host cells to shift the outcome of the “race to the surface” towards stable soft or hard tissue attachment. A major disadvantage of approaches based on antimicrobial coatings consists in the reduced efficiency once the compounds are released. Hence, recent attention has shifted toward exploiting the impact of surface topography and its potential to prevent or change microbial adhesion[1]. The idea of our approach consists in the implementation of nanostructures (nanotubes or nanorods) onto microscopically flat, electropolished surfaces. It is expected that starting from a polished surface the bacterial adhesion can be markedly reduced while the nanofeatures are intended to stimulate the differentiation of bone forming cells and would eventually favor bone formation over bacterial adhesion.
Materials and Methods: Three different surface types (sandblasted and acid etched – SBAE, electropolished - EPOL and electropolished and anodized – EPOLAN) were compared with respect to the adhesion behavior of bacteria and adhesion, proliferation and differentiation of human bone marrow derived stromal cell (hMSC) from adult donors.
EPOLAN samples were first electropolished. then etched in a HF/ H3PO4 mixture and subsequently anodized in a C2H4(OH) based solution containing NH4F at 80 V for 900 s. The average diameter of the formed nanotubes was ~68 nm.
For analysis of bacterial adhesion and biofilm formation of Streptococcus sanguinis ɣ-sterilized samples were pre-coated with human saliva. After 24 h static cultivation the attached cells were detected using fluorescence in situ hybridization (FISH).
For in vitro cell culture proliferation was evaluated by analysis of cell numbers (via Lactate dehydrogenase (LDH) activity as well as via DNA). As markers for bone differentiation alkaline phosphatase (ALP) as well as deposited mineral was analyzed. Scanning electron microscopy and immunostaining was performed to evaluate cell morphology.
Results and Discussion: The results of the bacterial adhesion demonstrate that in comparison to the current gold standard for dental implants (SBAE) the biofilm formation could be reduced from ~ 60% to 16 – 18% for both flat surfaces (EPOL and EPOLAN). The reduced biofilm formation is obviously based on the roughness in the micrometre scale. The additional implementation of nanostructures for EPOLAN did not affect the bacterial adhesion. For both electropolished surfaces the cells spread well developing numerous focal contacts and prolonged proliferation was observed in comparison to SBAE surfaces. The implemented nanostructures provoked a further positive impact on differentiation in terms of ALP activity and mineralization. Analysis of gene expression profiles is still in progress.
Conclusions: The results show that the implementation of nanostructures into microscopically flat surfaces is an efficient modification to reduce bacterial adhesion due to its microroughness while at the same time bone cell differentiation is supported.
The authors acknowledge financial support by the EC [EC SMP-2013- 606624 nanoTi].
References:
[1] Rizzello L et al., Nanomedicine 8(5), 807–821, 2013