Ramón Pérez-Tanoira

In vitro assessment of Staphylococcus epidermidis and Staphylococcus aureus adhesion on TiO2 nanotubes on Ti–6Al–4V alloy

The aim of this study was to evaluate Staphylococcus sp. adhesion to modified surfaces of titanium alloy (Ti-6Al-4V). Specimens of Ti-6Al-4V alloy 6-4 ELI-grade 23 that meets the requirements of ASTM F136 2002A (AMS 2631B class A1) were anodized in a mixture of sulfuric/hydrofluoric acid at 20 V for 5 and 60 min to form nanoporous (NP) and nanotubular (NT) oxide layers with pore diameter of 20 and 100 nm, respectively. The amount of fluorine incorporated in the oxide films from the electrolyte was 6 and 4 wt %, respectively. Bacterial adherence was studied using laboratory strains and six clinical strains each of Staphylococcus aureus and Staphylococcus epidermidis. Lower adherence of laboratory strains was demonstrated on fluoride nanostructured surfaces in comparison with the fluoride-free surfaces. Significant differences between clinical strains and laboratory strains were also found (p < 0.0001, Kruskal-Wallis test) when NP and NT specimens were compared with chemically polished (CP) surfaces. The results of the tests using multiple clinical strains confirmed a decrease in bacterial adherence on F-containing titanium oxide surfaces, suggesting a potential applicability of this surface, with a confirmed added value of decreasing clinical staphylococci adherence, for medical prosthetic devices.

Doped TiO2 anodic layers of enhanced antibacterial properties

Ti-6Al-4V joint replacement implants foster uncemented fixation in orthopaedic surgery. However, bacterial colonization competes with host cells and ultimately may produce implant-related difficult-to-treat infections, justifying the efforts to obtain infection-resistant materials. In a previous work, the authors demonstrated the antibacterial properties of anodic fluoride-TiO2 nanostructured layers on Ti-6Al-4V alloy. In this work, the anodizing bath has been modified in order to grow fluoride-TiO2 barrier layers (FBL). A bacterial adherence protocol, run with reference and six different clinical strains of Staphylococcus aureus and Staphylococcus epidermidis, showed a statistically significant decrease in the percentage of covered surface (p<0.0001, Kruskal-Wallis test) for FBL specimens when compared with non fluoride-containing specimens, i.e. chemically polished Ti-6Al-4V and F-free TiO2 barrier layers. The results obtained on the F-barrier layers allowed discrimination between the effects of the presence of fluoride in the layer and the layer nanostructure on bacterial adhesion.

Influence of the nanostructure of F-doped TiO2 films on osteoblast growth and function

The aim of this study was to evaluate the proliferation and mineralization ability of mouse osteoblastic MC3T3-E1 cells on F-containing TiO2 films with different morphology and nanostructure that previously confirmed antibacterial properties. F-containing TiO2 films were fabricated by anodizing Ti-6Al-4V alloy ELI -grade 23. By using a mixture of H2SO4/HF acid at 20 V for 5 and 60 min, a TiO2 film grows with nanoporous (NP) and nanotubular (NT) features, characterized with a pore diameter of 20 and 100 nm, respectively. Fluoride-TiO2 barrier films (FBL) were produced in 1M NH4H2PO4/0.15M NH4F solution at constant voltage controlled at 20 V for 120 min. The amount of F incorporated in the nanostructured oxide films was 6 at % and of 4 at %, for the NP and NT, respectively, while for the FBL film was 12 at %. MC3T3-E1 cells exhibited different behavior when seeded and grown onto these surfaces. Thus, F-doped TiO2 films with NP structures increased proliferation as well as osteogenic gene expression and the mineralization capacity of these osteoblastic cells. These results confirm that anodizing process is suitable to fabricate multifunctional surfaces on Ti-6Al-4V alloy with improved not only antibacterial but also osteogenic properties useful for bone fixation of prosthetic devices

Bacterial adhesion on biomedical surfaces covered by micrometric silver Islands

A set of Cu-Mn-O and Ag-Cu-Mn-O films were sputter-deposited onto polished Ti-6Al-4V coupons and the microbiological adherence of Staphylococcus sp. was studied in these biomedical surfaces modified by using advanced ternary and quaternary oxides, these latter incorporated micrometric silver islands. Silver is known to have a natural biocidal character and its presence in the surface of Ti-6Al-4V forming large micrometric islands. In principle, predicted to enhance the antimicrobial properties of biomedical surfaces. Microbial adhesion tests were performed using collection strains and six clinical Staphylococcus aureus and Staphylococcus epidermidis strains. The adherence study was performed using a previously published protocol by Kinnari et al. Collection strains and clinical strains showed decreased adherence to modified materials; however, only on the clinical strains were there statistically significant differences between Cu-Mn-O and Ag-Cu-Mn-O containing silver islands. Nanocrystalline silver dissolves and releases both Ag(+) and Ag(0) whereas other silver sources release only Ag+. We can conclude that nanocrystalline silver coating, confirmed by XRD, appears to alter the biological properties of the solution, particularly antimicrobial activity.

Competitive Colonization of Prosthetic Surfaces by Staphylococcus Aureus and Human Cells

Implantation of a biomaterial provides an adhesion substratum both to host cell integration and to contaminating bacteria. We studied simultaneous competitive adhesion of Staphylococcus aureus in serial 1:10 dilutions of 108 colony forming units (CFU)/mL and human osteogenic sarcoma (SaOS-2) or primary osteoblast (hOB) cells, both 1x105 cells/mL, to the surfaces of titanium, polydimethylsiloxane and polystyrene. The bacterial adherence and human cell proliferation, cytotoxicity and production of reactive oxygen species (ROS) were studied using fluorometric (fluorescent microscopy and flow cytometry) and colorimetric methods (MTT, LDH and crystal violet). The bacterial cell viability was also evaluated using the drop plate method. The presence of bacteria resulted in reduced adherence of human cells to the surface of the biomaterials, increased production of ROS, and into increased apoptosis. On the other hand, the presence of either type of human cells was associated with a reduction of bacterial colonization of the biomaterial with Staphylococcus aureus. These results suggest that increasing colonization of the biomaterial surface in vitro by one negatively affects colonization by the other. Host cell integration to an implant surface reduces bacterial contamination, which opens novel opportunities for the design of infection-resistant biomaterials in current implantology and future regenerative medicine.

Bacterial adhesion on biomedical surfaces covered by yttria stabilized zirconia

The aim of this study was to compare the bacterial adhesion of Staphylococcus spp. on Ti–6Al–4V with respect to Ti–6Al–V modified alloys with a set of Cubic yttria stabilized zirconia (YSZ) and Ag-YSZ nanocomposite films. Silver is well known to have a natural biocidal character and its presence in the surface predicted to enhance the antimicrobial properties of biomedical surfaces. Microbial adhesion tests were performed using collection strains and twelve clinical strains of Staphylococcus aureus and Staphylococcus epidermidis. The adherence study was performed using a previously published protocol by Kinnari et al. Both collection strains and clinical isolates have shown lower bacterial adhesion to materials modified with respect to the alloy Ti–6Al–4V and the modification with silver reduced the bacterial adhesion for most of all the strains studied. Moreover the percentage of dead bacteria have been evaluated, demonstrating increased proportion of dead bacteria for the modified surfaces. Nanocrystalline silver dissolves releasing both Ag+ and Ag0 whereas other silver sources release only Ag+. We can conclude that YSZ with nanocrystalline silver coating may lead to diminished postoperative infections and to increased corrosion and scratch resistance of YSZ incorporating alloys Ti–6Al–4V.

Effect of S53P4 bone substitute on staphylococcal adhesion and biofilm formation on other implant materials in normal and hypoxic conditions

To study the effect of bioactive glass bone substitute granules (S53P4) on bacterial adhesion and biofilm formation on other simultaneously used implant materials and the role of the hypoxic conditions to the adhesion. Bacterial and biofilm formation were studied on materials used both in middle ear prostheses and in fracture fixtures (titanium, polytetrafluoroethylene, polydimethylsiloxane and bioactive glass plates) in the presence or absence of S53P4 granules. The experiments were done either in normal atmosphere or in hypoxia simulating atmospheric conditions of middle ear, mastoid cavity and sinuses. We used two collection strains of Staphylococcus aureus and Staphylococcus epidermidis. In the presence of bioglass and hypoxic conditions the adhesion of the planktonic bacterial cells was decreased for most of the materials. The biofilm formation was decreased for S. epidermidis on titanium and polydimethylsiloxane in both atmospheric conditions and on bioglass plates in normoxia. For S. aureus the biofilm formation was decreased on bioglass plates and polytetrafluoroethylene in normoxia. Hypoxia produces a decrease in the biofilm formation only for S. aureus on polytetrafluoroethylene and for S. epidermidis on bioglass plates. However, in none of the cases bioactive glass increased the bacterial or biofilm adhesion. The presence of bioglass in normoxic and hypoxic conditions prevents the bacterial and biofilm adhesion on surfaces of several typical prosthesis materials in vitro. This may lead to diminishing postoperative infections, however, further in vivo studies are needed.

Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the “race for the surface” theory

Objectives Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied. Methods We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis. Results Our results show that clinical strains adhere to the material surface at lower concentrations than collection strains. A destructive effect of bacteria on preosteoblastic cells was also detected, especially with higher concentrations of bacteria. Conclusions The method described herein can be used to evaluate the effect of surface modifications on bacterial adherence more accurately than conventional monoculture studies. Clinical strains behave differently than collection strains with respect to bacterial adherence. Cite this article: M. Martinez-Perez, C. Perez-Jorge, D. Lozano, S. Portal-Nuñez, R. Perez-Tanoira, A. Conde, M. A. Arenas, J. M. Hernandez-Lopez, J. J. de Damborenea, E. Gomez-Barrena, P. Esbrit, J. Esteban. Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the “race for the surface” theory. Bone Joint Res 2017;6:315–322. DOI: 10.1302/2046-3758.65.BJR-2016-0226.R2.

Use of an experimental model to evaluate infection resistance of meshes in abdominal wall surgery

BACKGROUND Staphylococcal species are the most common organisms causing prosthetic mesh infections, however, infections due to rapidly growing mycobacteria are increasing. This study evaluates the resistance of biomaterial for abdominal wall prostheses against the development of postoperative infection in a rat model. MATERIAL AND METHODS In 75 rats, we intramuscularly implanted three different types of prostheses: (1) low-density polypropylene monofilament mesh (PMM), (2) high-density PMM, and (3) a composite prosthesis composed of low-density PMM and a nonporous hydrophilic film. Meshes were inoculated with a suspension containing 108 colony-forming units of Staphylococcus aureus, Staphylococcus epidermidis, Mycobacterium fortuitum, or Mycobacterium abscessus before wound closure. Animals were sacrificed on the eighth day postoperatively for clinical evaluation, and the implants were removed for bacteriologic analyses. RESULTS Prostheses infected with S aureus showed a higher bacterial viability, worse integration, and clinical outcome compared with infection by other bacteria. Composite prostheses showed a higher number of viable colonies of both M fortuitum and Staphylococcus spp., with poorer integration in host tissue. However, when the composite prosthesis was infected with M abscessus, a lower number of viable bacteria were isolated and a better integration was observed compared with infection by other bacteria. CONCLUSIONS Considering M abscessus, a smaller collagen-free contact surface shows better resistance to infection, however, depending on the type of bacteria, prostheses with a large surface, and covered with collagen shows reduced resistance to infection, worse integration, and worse clinical outcome.

Antibacterial properties of biomedical surfaces containing micrometric silver islands

A set of Cu-Mn-O and Ag-Cu-Mn-O films were sputter-deposited onto polished Ti-6Al-4V coupons and the microbiological adherence of Staphylococcus sp. was studied in these biomedical surfaces modified using advanced ternary and quaternary oxides that incorporated micrometric silver islands. The as-deposited ternary and quaternary compounds were amorphous. Upon air annealing the Ag-Cu-Mn-O films, silver-oxygen bonds in the compound destabilize, resulting in the segregation of metallic silver in the form of micrometric layered silver islands with high specific area dispersed at the surface of the remaining oxide. Silver is well known to have a natural biocidal character and its presence in the surface forming large micrometric escalonated islands is, in principle, predicted to enhance the antimicrobial properties of biomedical surfaces. Microbial adhesion tests were performed in triplicates using collection strains of Staphylococcus aureus and Staphylococcus epidermidis. Preliminary results indicate that both strains showed decreased adherence to modified materials, S. epidermidis showed higher adherence these materials than S. aureus, however, there was no statistically significant differences between Cu-Mn-O and Ag-Cu-Mn-O containing silver islands.