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Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_5 | Pages 25 - 25
1 Apr 2019
Cazzola M Ferraris S Stella B Orlygsson G Ng CH Cempura G Scolaro C Prenesti E Yamaguchi S Pezzotti G Cochis A Rimondini L Spriano S
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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.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 105 - 105
1 Jan 2017
Cazzola M Ferraris S Bertone E Prenesti E Corazzari I Cochis A Rimondini L Spriano S Vernè E
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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.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_9 | Pages 150 - 150
1 May 2016
Porporati A Spriano S Ferraris S Rimondini L Cochis A
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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.

Objectives

To verify the capability of ceramic bearings to reduce bacteria biofilm adhesion by means of their surface chemico-physical properties.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 86 - 86
1 Jul 2014
Spriano S Ferraris S Miola M
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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.

Introduction

A patented process of surface modification is described. It gives a multifunctional surface with a multiscale roughness (micro and nano topography), that is excellent for osteoblast adhesion and differentiation. It has a high degree of hydroxylation, that is relevant for inorganic bioactivity (apatite-HA precipitation) and it is ready for a functionalization with biological factors. A direct grafting of ALP has been obtained. Moreover, the growth of an antibacterial agent within the surface oxide layer can be useful in order to combine the osteoinduction ability to antimicrobial effects. The selection of an inorganic agent (metal nanoparticles) has the advantage to avoid an eventual development of antibiotic resistance by bacteria.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 265 - 266
1 May 2009
Spriano S Vernè E Guala C Faga M Eitel F
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Aims: The aim of this research activity is the synthesis and characterization of surface modified cobalt alloys, in order to obtain a biocompatible material presenting at the same time good wear performances, low metal ion release and low toxicity. It is of interest for hip and knee joints with metal-on-metal or metal-on-UHMWPE contacts. In the first case the main aim is to enhance the biocompatibily of the surface and to reduce the amount and the toxicity of the metal debris. In the second case the main aim is to reduce the amount of polymeric debris. The surface chemical composition of several implant cobalt alloys (as cast and forged alloys, low and high carbon alloys) was modified, producing relevant tantalum enrichment. Tantalum was chosen because of its low toxicity and high corrosion resistance. The employed process is of interest because it is low-cost and it does not involve the formation of a brittle ceramic coating.

Methods: The chemical composition and morphology (SEM-EDS), roughness (prophilometry), crystallographic structure (x-ray diffraction analysis) and wettability (contact angle) of the surface were characterized. The mechanical behavior of the modified materials was investigated by nanoindentations, scratch tests (Revetest), friction measurements and wear tests (pin-on-disc).

Results: A surface enrichment with tantalum was obtained through a thickness of several microns and the interface between the substrate and the modified layer is continuous and crack free. The maximum tantalum content on the surface is about 90 wt%. Scratch tests showed that the modified layer is well adherent to the substrate through a diffusion layer and its first detachment appeared at 55 N without any brittle or catastrophic event. The modified layer presented a higher wettability than the untreated one, with a contact angle of about 48° respect to 82°. The elastic modulus of the treated surface layer (nanoindentation tests) is 352 GPa, while it is 281 GPa in the untreated alloy and its hardness is 17 GPa while it is 8.6 GPa in the untreated one. The surface presents also a better wear resistance (0.7·10−6 respect to 5.7 10−6 mm3/ Nm) and lower friction coefficients (0.24 respect to 0.34) in the Me-on-Me contact, according to its higher hardness and wettability.

Conclusions: A surface with high tantalum content can be obtained by a low-cost process. It shows good mechanical properties (high elastic modulus, high hardness value, low friction coefficient, high wear resistance) and chemical properties (high wettability, good corrosion resistance). So it is of interest for joints presenting good biocompatibility and great longevity.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 265 - 265
1 May 2009
Vernè E Ferraris S Spriano S Brovarone CV Bianchi C Morra MM Cassinelli C
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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.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_I | Pages 60 - 60
1 Mar 2005
Spriano S Bronzoni M Vernè E
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Aims: The main objective of the research was to investigate alternative processes, respect to hydroxylapatite plasma spray coating, in order to obtain metallic bio-materials presenting good osteointegration ability. An innovative process consisting of mechanical and thermochemical treatments was tested and a surface and mechanical characterization performed on treated samples.

Methods: The material investigated was the Ti-6Al-7Nb alloy. The surface modification process consists of grit blasting, passivation, alkali etching and thermal treatment performed in air or in vacuum. Crystallographic structure was investigated by XRD and TEM. Surface morphology and composition were assessed by SEM, EDS and AES analysis. Bioactivity was tested by soaking in standard SBF solution. Metal ion release measurements were performed by GFAA-ICP technique on withdrawn solution after soaking samples in SBF. Scratch and fatigue tests were performed as mechanical characterization of the material.

Results: The alkali etching strongly modifies the surface morphology of titanium and its alloys producing a microporous layer and a drastic increment in surface wettability. The use of previous passivation treatment modifies the surface crystallographic structure, forms a graded interface between the surface and the substrate, enhances the surface layer adhesion and scratch resistance, increases the corrosion resistance of the material and causes a low metal ion release. The use of a vacuum atmosphere during heat treatment inhibits rutile formation and scratch tests evidenced low damage on it. During soaking in SBF the formation of a reaction layer and of precipitated crystals containing Ca and P was detected on the treated samples. The precipitate morphology resembles that of apatite. The fatigue strength was 260 MPa for the treated series, while it was 460 MPa in the case of the grit blasted series without any additional treatment and therefore significantly higher.

Conclusions: It can be concluded that the surface of treated samples shows chemical, structural and morphological modifications. The passivation pre-treatment causes the formation of different crystallographic phases and of a smoother interface with the substrate. The treated samples evidenced a quite low metal ion release and interacted with SBF solution, showing a moderate bioactivity. The disadvantage of this process is the decrease in fatigue strength. This aspect suggests that when surface etching and modifications are performed with the aim of enhancing metal osteointegration ability, a careful investigation of their influence on the fatigue resistance must be performed.