In order to improve fast osseointegration, to modulate inflammatory response and to avoid biofilm formation, several attempts of surface modifications of titanium alloy in term of surface topography and chemistry have been performed over years, but this is still an open issue.

In our research work, a patented chemical treatment was developed and tailored to improve fast osseointegration and to allow further surface functionalization in order to get a multifunctional surface.

After the chemical treatment, Ti6Al4V shows a micro and nano-textured surface oxide layer with high density of hydroxyls groups, as summarized Figure 1: it is able to induce apatite precipitation (during soaking in Simulated Body Fluid), high wettability by blood, specific protein adsorption, positive osteoblast response and surface mechanical resistance to implantation friction.

Hydroxyl groups exposed by the treated surface also allow binding natural biomolecules such as polyphenols, which can further improve the rate and quality of osseointegration by adding anti-inflammatory, antibacterial and antitumoral effects suitable for implants in critical situations. Polyphenols have the further added value of being a low cost and eco-sustainable product, extractable from byproducts of wine and food industry.

On the chemically treated and functionalized samples, the surface characterization was performed using Folin&Ciocalteu test, fluorescence microscopy and XPS analysis in order to check the presence and activity of the grafted biomolecules (polyphenols from red grape pomace and green tea leaves). Cell tests were performed with Kusa A-1 cells highlighting the ability of polyphenols to improve osteoblasts differentiation and deposition of mineralized extracellular matrix.

Surface functionalization can also be performed with chitin derived biomolecules to reduce inflammation.

With the purpose of obtaining the antibacterial effect, during the chemical treatment a silver precursor can also be added to obtain in situ reduced silver nanoparticles embedded in the nano-structured oxide layer. The samples containing nanoparticles on the surface were characterized by means of TEM and FESEM observation highlighting the presence of well distributed and small-sized nanoparticles on the surface and through the thickness of the oxide layer. A long-lasting release in water was observed up to 14 days and antibacterial tests on Staphylococcus aureus showed the ability of the surface to reduce bacteria viability avoiding biofilm formation.

The results showed that the patented chemical treatment can improve the response of osteoblasts to titanium alloy implants, but is also a promising way to obtain multifunctional surfaces with antibacterial, antioxidant, anti-inflammatory and antitumoral properties that can be the future of orthopedic implants.

Orthopedic metallic medical devices are essential in the treatment of a wide range of skeletal diseases and disabilities. However, they are often related with surgery complications due to acute prosthetic joint infections (PJI) causing devastating complications. Gallium (Ga) antibacterial activity has been recently demonstrated: in aqueous solutions, Ga ionize in a trivalent form Ga³⁺ that can replace Fe³⁺ in bacterial metabolism thus leading to bacteria death. However, it is not yet clear whether such effect is typical to Ga³⁺ release, and how this would affect longer term performance. Here we investigated Ga addition into titanium alloys using metallurgical methods. The study has confirmed that metallurgical addition of gallium even in small amounts (1–2% wt.) to titanium alloys have highly efficient antibacterial function without any visible cytostatic or cytotoxic effects. The presence of gallium within the metal matrix might ensure that antibacterial effect will persist for a long time towards multi-drug resistant S. aureus, which might not be possible if gallium or other metal are only in thin degradable coatings or similar formulations. A 5-logs decrease in CFU number was detected for alloys with 2% Ga and more after 72 h (alamar blue and CFU count assays). The alloys also show to be in vitro cytocompatible with both mature U2OS osteoblasts and progenitor pre-osteoblasts hFOB. Since gallium is metallurgically analogous to aluminium in titanium alloys, it might be used without affecting other alloy properties.

Among plant derived molecules, polyphenols have antioxidant, anticancer and antibacterial ability [1,2]. Moreover, they can stimulate osteoblast differentiation and promote apoptosis of tumoral cells [3–4]. It's thus possible combine the properties of these molecules with those of bioactive materials trough surface functionalization.

A silica-based bioactive glass and chemically treated bioactive Ti6Al4V were used as substrates while gallic acid and polyphenols extracted from green tea or red grape skin as biomolecules for functionalization. The surface functionalization procedure was optimized in order to maximize the grafting and investigated by means of the Folin&Ciocalteu method and X-Ray Photoelectron Spectroscopy (XPS) analyses. The in vitrobioactivity was studied by means of Field Emission Scanning Electron Microscopy (FESEM) and Fourier Transform Infrared Spectroscopy (FTIR) after soaking in simulated body fluid (SBF).

Surface charge and isoelectric point were investigated by means of zeta potential measurements. Free radical scavenging activity evaluation was performed in order to investigate the antioxidant ability of glass samples. Finally, the functionalization selective killing activity towards osteosarcoma cells was in vitroassayed by the metabolic 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test and compared with non-tumoral control bone cells.

The presence of polyphenols on the surfaces was confirmed by XPS analyses by the appearance of characteristic peaks (C-O and C=O bonds) in the carbon and oxygen regions. The Folin&Ciocalteu test demonstrated the presence and activity of polyphenols on all the substrates and evidenced a clear relation between surface reactivity and grafting ability. The bioactivity tests showed the deposition of hydroxyapatite on the functionalized samples and an influence of biomolecules on its amount and shape for glasses. Zeta potential measurements evidenced a shift of the isoelectric point of glass samples after functionalization. A certain antioxidant activity of bare glass has been evidenced and it is improved by the grafting of tea polyphenols. Accordingly, MTT results confirmed polyphenols selective killer activity towards osteosarcoma cells whose viability was significantly decreased in comparison with safe bone cells.

XPS analyses, zeta potential measurements and Folin&Ciocalteu tests showed the presence and the activity of the polyphenols on the surfaces. Bioactivity tests highlighted an improvement of the deposition of hydroxyapatite on the surface of the functionalized glass samples. Certain antioxidant ability has been evidenced for glass samples and was further improved by tea polyphenols. Moreover, a selective toxic activity towards tumor cells was in vitropreliminary confirmed.

In conclusions polyphenols were successfully grafted to the surface of glass and Ti6Al4V samples maintaining their activity. Polyphenols improve in vitro bioactivity, antioxidant and anticancer ability of glass. The surface functionalization seems to be a good way to combine the properties of bioactive materials for bone contact applications with those of polyphenols.

Introduction

According to the Australian registry 2014, periprosthetic joint infection (PJI) is the fourth important reason for revision of a primary total hip arthroplasty (THA). PJI is frequently caused by commensal strains of the skin such as Staphylococcus aureus or Staphylococcus epidermis. Deep infection is depending on many factors, such as implant surface chemical and physical behaviour, device design, host site, surgery and host response. Nevertheless, a lack of knowledge is seen concerning the specific effects of different surfaces on the biological response of different biomaterials. In addition, it is difficult to discriminate the material chemico-physical properties by the topological features, such as surface roughness. Indeed, it has been widely demonstrated that surface composition, electric charge, wettability and roughness of implant surfaces have a strong influence on their interactions with biological fluids and tissues. Therefore, also bearing surface properties can influence the incidence of PJI, just shown recently.

The aim of the work is to develop innovative antibacterial surface modification treatments for titanium capable to limit the bacterial adhesion and proliferation as weel as the biofilm formation while maintaining an high osteointegrative potential. The goal is to contrast the infections which represent a serius complication related to the use of implantable devices.

Aims:. To test the effect of different surface roughness and fluorohydroxyapatite (FHA) coating on osteoblast-like cell (MG63) viability, proliferation, differentiation and synthetic activity, then to compare the various surfaces tested and try to identify an osteoblast parameter that can better explain the different behaviour of the tested surfaces observed in previous in vivo studies.

Methods: The tested materials were made of Ti6Al4V coated with Ti and with Ti plus FHA with different roughness; they can be divided into four groups: low roughness (LR; Ra: 5.9 B5m), low roughness plus FHA coating (LR+FHA; Ra: 5.6 B5m), high roughness (HR; Ra: 22.5 B5m), high roughness plus FHA coating (HR+FHA; Ra: 21.2 B5m). MG63 were cultivated on 6 samples of each group and on polystyrene as control; after 72 hours the proliferation assay (WST-1) was done, alkaline phosphatase activity (ALP) was determined and the synthesis of osteocalcin (OC), type 1 collagen (CICP), transforming growth factor α 1 (TGF-A71), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-a) were measured. Samples of each material were randomly processed for analysis with a scanning electron microscope (SEM).

Results: Cells proliferated on biomaterials more slowly than in the control group (p < 0.0001), the proliferation rate was higher on FHA-coated LR than uncoated HR (p = 0.037). CICP production was positively affected by the LR surface (p = 0.001) as compared to controls, while it was significantly lower (p = 0.0001) in the HR surfaces. Compared to controls, LR and HR surfaces led to enhanced production of TGF-A71, further improved by FHA (FHA-coated LR: p = 0.007; FHA-coated HR p < 0.0001 respectively). ALP, OC, IL-6 levels were not significantly different from the controls

Conclusions: Results suggest that CICP production could be useful in predicting the in vivo osteointegration rate of biocompatible biomaterials observed in previous studies.