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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. 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. 91-B, Issue SUPP_II | Pages 266 - 266
1 May 2009
Ferraris S Miola M Robotti P Bianchi G Di Nunzio S Vernè E
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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.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_I | Pages 67 - 68
1 Mar 2005
Bretcanu O Vernè E
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Aims: The purpose of this work is the preparation and characterisation of bioactive ferrimagnetic biomaterials. These materials can form a stable bond to the bone and can be heated by the application of an external alternating magnetic field, so they are good candidates for non-invasive hyperthermic treatment of solid bone tumours.

Methods: The investigated materials are glass-ceramics belonging to the system SiO2-CaO-Na2O-P2O5-FeO-Fe2O3. They can be obtained by different methods, such as melting of traditional raw materials (oxides, carbonates or phosphates), thermal treatment of wet-chemistry derived precursors, or sintering. In the first two methods, different amounts of magnetite can crystallize inside the amorphous phase during cooling from the processing temperature to r.c., leading to a glass-ceramic. In the sintering method, glass powders and magnetite particles are intimately mixed and successively thermally treated, so that a composite material is obtained. A complete characterization was performed in terms of morphology and microstructure (SEM, TEM, XRD, DTA), bioactivity (soaking in SBF), magnetic properties (hysteresis loss) and calorimetric measurements (specific power loss).

Results: Depending on the synthesis process it is possible to obtain both dense and macroporous devices (glass-ceramic or composites up to some centimeters size) as well as glass-ceramic micrometric particles. The magnetite crystals inside the amorphous phase are nanometric or submicrometric, depending on the synthesis method. The glass-ceramics have a bioactive behaviour, since hydroxyapatite grows on their surface after few days of soaking in Simulated Body Fluid. The hysteresis loss and the specific power loss are compatible with the temperature required for hyperthermic treatments of neoplastic tissues.

Conclusions: Innovative magnetic biomaterials have been designed and synthesized by a careful optimization of the composition and processing parameters. Different synthesis methods can be used to prepare these biomaterials, in function of the tissue characteristics and magnetic field conditions. Due to the possibility of producing very small devices, these materials can be implanted by non-invasive surgical techniques, and since they are bio-compatible, can be let inside the body for a long period, being subjected to multiple heating cycles. Due to their bioactivity they could be proposed as an alternative for the treatment of bone tumors after surgical resection


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.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_I | Pages 65 - 65
1 Mar 2005
Brovarone CV Vernè E Bosetti M Cannas M
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Aims: The aim of this research work was the realization of an inorganic bioactive scaffold for bone regeneration. This biomaterial should be macroporous, in order to allow the bone in-growth, and bioactive aiming to promote the bone regeneration and healing.

Methods: The macroporous biomaterial was prepared by consolidation of a suspension of starch and SiO2-CaO-Na2O-MgO glass powders. Starch powders were used as both pore former and consolidation agent. Starch-glass green bodies were prepared by uniaxial pressing and, after drying, they were heated to remove the organic phase and to sinter the inorganic one. The sintered scaffolds were characterized by X-Ray diffraction, scanning electron microscopy and mercury intrusion porosimetry. The scaffolds bioactivity was evaluated soaking the samples in a simulated body fluid for periods up to 4 weeks. On the most representative samples, in vitro tests of adhesion and proliferation were performed using human primary osteoblast-like cells.

Results: The obtained scaffolds showed an interconnected macroporosity of 50–100 B5m and a satisfactory degree of sintering. The sintering treatment induced the nucleation and growth of Na2Ca2(SiO2)3 crystals which is a phase that possess a very high bioactivity index. By soaking the scaffolds in SBF for period up to 1 month, an extensive precipitation of hydroxylapatite, with the typical globular morphology, occurred both inside and outside the pores. The adhesion and proliferation tests showed a remarkable spreading of the osteoblasts on the scaffold surface and thus a good biological response.

Conclusions: Scaffolds with interconnected porosity were successfully obtained. The pores are highly interconnected and homogenously distributed in the samples. The chosen thermal treatment and the use of starch powders led to a final macroporous glass-ceramic structure. The obtained scaffolds showed a very high in vitro bioactivity with precipitation of HAp. Moreover, preliminary biological tests, showed a satisfactory cellular interaction with the proposed biomaterials. For the above-mentioned reasons, the starch consolidation method, the optimized processing parameters and the tailored glass composition can be used to produce scaffolds suitable for bone substitutions and tissue engineering.