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.

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.

Summary Statement

The problem facing this research is to promote rapid osteointegration of titanium implants and to minimise the risks of infections by the functionalization with different agents, each designed for a specific action. A patented process gives a multifunctional titanium surface.

Aims: The aim of the research is the functionalization of biosurfaces by anchoring on them biomolecules involved in the process of osteointegration (cellular adhesion, proliferation, differentiation, migration, matrix mineralization). Alkaline phosphatase (ALP) was used as model protein, because it is involved in the mineralization processes. The functionalized surfaces are biomimetic, because they show the biological signals triggering new tissue generation. A rapid osseointegration are the final goal and a good response and fast healing of bad quality bones is one of the main issues. The devices of interest for the research are dental or orthopaedic implants and substitutes of small bones.

Methods: Bioactive glasses of various compositions were employed as substrates. Bioactive glasses, when in contact with biological fluids, stimulate the precipitation of a hydroxyapatite layer on their surfaces, which in turn promotes effective osteointegration of the implant. Since bioactive glasses are prone to hydroxylation, they could be successfully functionalized and grafted by biomolecules. So the biomimetic materials considered will be bioactive both from a physicochemical (osteoconduction and apatite precipitation) and from a biochemical (osteoinduction) point of view. The research was focused first of all on the methods for developing active sites on the substrates. In the case of bioactive glasses the surface must be cleaned of any contaminants and the reactive hydroxyls activated.

Results: The immobilization of ALP was performed both with and without spacer molecules and a comparison among the different techniques will be presented. XPS was used for the analysis of the immobilized enzyme on titanium and bioglasses and specific signals for its identification were set. After the addition of the specific substrate, the ALP activity was evaluated by UV-VIS spectroscopy.

Conclusions: ALP was successfully grafted on the surface of bioactive glasses with and without the use of an intermediate layer of spacer molecules. The presence of ALP was determined on all the samples, as well as its enzymatic activity. Further analyses are necessary to evaluate the opportunity of using a spacer molecule. Cell adhesion and proliferation tests are in progress.

Aims: A serious problem in orthopedic surgery is the development of infections. The realization of antibacterial and biocompatible/bioactive surfaces represents a challenge. In this study antibacterial behavior has been conferred to surfaces of glasses and glass-caramics, with different degrees of bioactivity, by the introduction of silver through ion exchange.

Methods: Materials have been studied both in bulk form, and as coatings. All samples have been analyzed by means of XRD, SEM and EDS before and after the treatment. Coatings’ roughness, porosity and adhesion resistance have been also analyzed. In vitro reactivity and silver release were carried out soaking samples in SBF. Samples have been analyzed by means of SEM/ EDS and XRD; silver has been quantified in solution by GFAAS. Cellular tests have been performed in order to evaluate materials biocompatibility before and after the treatment. Antibacterial behavior has been tested against S.Aureus.

Results: Characterization analyses show that glassy or crystalline structure and morphology are maintained after the ion-exchange. As well the coating adhesion resistance is higher then the limit provided by ISO standard for hydroxyapatite coatings. GFAAS analysis determined that silver is gradually released in solution. Cellular tests demonstrate that biocompatibility is generally maintained after treatment but it is closely connected to the amount of silver released. Microbiological tests show antibacterial behavior for silver-doped samples.

Conclusions: Ion-exchange technique permits the introduction of controlled silver amount without modifying materials’ structural and morphological properties. Comparing cellular and microbiological tests it is possible to design process parameters to confer, antibacterial properties but not cytotoxic behavior.