Investigation of the Relationship between Surface Features, Protein Adsorption, and Osteoimmunomodulation: The Case of a Chemically Treated Titanium Alloy.

This paper deals with the combined effects of immune response and osseointegration because of the lack of comprehensive studies on this topic. An antibacterial Ti surface was considered because of the high risk of infection for titanium bone implants. A chemically treated Ti6Al4 V alloy [Ti64(Sr-Ag)] with a microporous and Sr-Ag doped surface was compared to a polished version (Ti64) regarding protein adsorption (albumin and fibronectin) and osteoimmunomodulation. Characterization via fluorescence microscopy and zeta potential showed a continuous fibronectin layer on Ti64(Sr-Ag), even with preadsorbed albumin, while it remained filamentous on Ti64. Macrophages (differentiated from THP-1 monocytes) were cultured on both surfaces, with viability and cytokine release analyzed. Differently from Ti64, Ti64(Sr-Ag) promoted early anti-inflammatory responses and significant downregulation of VEGF. Ti64(Sr-Ag) also enhanced human bone marrow mesenchymal cell differentiation toward osteoblasts, when a macrophage-conditioned medium was used, influencing ALP production. Surface properties in relation to protein adsorption and osteoimmunomodulation were discussed.

The mechanical and chemical stability of the interfaces in bioactive materials: The substrate-bioactive surface layer and hydroxyapatite-bioactive surface layer interfaces.

Bioactive materials should maintain their properties during implantation and for long time in contact with physiological fluids and tissues. In the present research, five different bioactive materials (a bioactive glass and four different chemically treated bioactive titanium surfaces) have been studied and compared in terms of mechanical stability of the surface bioactive layer-substrate interface, their long term bioactivity, the type of hydroxyapatite matured and the stability of the hydroxyapatite-surface bioactive layer interface. Numerous physical and chemical analyses (such as Raman spectroscopy, macro and micro scratch tests, soaking in SBF, Field Emission Scanning Electron Microscopy equipped with Energy Dispersive Spectroscopy (SEM-EDS), zeta potential measurements and Fourier Transformed Infra-Red spectroscopy (FTIR) with chemical imaging) were used. Scratch measurements evidenced differences among the metallic surfaces concerning the mechanical stability of the surface bioactive layer-substrate interface. All the surfaces, despite of different kinetics of bioactivity, are covered by a bone like carbonate-hydroxyapatite with B-type substitution after 28 days of soaking in SBF. However, the stability of the apatite layer is not the same for all the materials: dissolution occurs at pH around 4 (close to inflammation condition) in a more pronounced way for the surfaces with faster bioactivity together with detachment of the surface bioactive layer. A protocol of characterization is here suggested to predict the implant-bone interface stability.

Bioactive materials: In vitro investigation of different mechanisms of hydroxyapatite precipitation.

Bioactive materials, able to induce hydroxyapatite precipitation in contact with body fluids, are of great interest for their bone bonding capacity. . The aim of this paper is to compare bioactive materials with different surface features to verify the mechanisms of action and the relationship with kinetics and type of precipitated hydroxyapatite over time. Four different surface treatments for Ti/Ti6Al4V alloy and a bioactive glass were selected and a different mechanism of bioactivity is supposed for each of them. Apart from the conventional techniques (FESEM, XPS and EDX), less common characterizations (zeta potential measurements on solid surfaces and FTIR chemical imaging) were applied. The results suggest that the OH groups on the surface have several effects: the total number of the OH groups mainly affects hydrophilicity of surfaces, while the isoelectric points, surface charge and ions attraction mainly depend on OH acidic/basic strength. Kinetics of hydroxyapatite precipitation is faster when it involves a mechanism of ion exchange while it is slower when it is due to electrostatic effects . The electrostatic effect cooperates with ion exchange and it speeds up kinetics of hydroxyapatite precipitation. Different bioactive surfaces are able to differently induce precipitation of type A and B of hydroxyapatite, as well as different degrees of crystallinity and carbonation. STATEMENT OF SIGNIFICANCE: The bone is made of a ceramic phase (a specific type of hydroxyapatite), a network of collagen fibers and the biological tissue. A strong bond of an orthopedic or dental implant with the bone is achieved by bioactive materials where precipitation and growth of hydroxyapatite occurs on the implant surface starting from the ions in the physiological fluids. Several bioactive materials are already known and used, but their mechanism of action is not completely known and the type of precipitated hydroxyapatite not fully investigated. In this work, bioactive titanium and bioglass surfaces are compared through conventional and innovative methodologies. Different mechanisms of bioactivity are identified, with different kinetics and the materials are able to induce precipitation of different types of hydroxyapatite, with different degree of crystallinity and carbonation.

Nanogrooves and keratin nanofibers on titanium surfaces aimed at driving gingival fibroblasts alignment and proliferation without increasing bacterial adhesion.

Periimplantitis and epithelial downgrowth are nowadays the main conditions associated to transmucosal dental implants. Gingival fibroblasts can play an important role in periimplantitis because they are the promoters of the inflammatory process and eventual tissue homeostasis and destruction. Moreover, the related inflammatory state is commonly driven also to counteract bacteria implants colonization. In the present research, a new technology based on mechanically produced nanogrooves (0.1-0.2µm) and keratin nanofibers deposited by electrospinning has been proposed in order to obtain titanium surfaces able to drive gingival fibroblasts alignment and proliferation without increasing bacterial adhesion. The prepared surfaces have been characterized for their morphology (FESEM), chemical composition (FTIR, XPS), surface charge (zeta potential) and wettability (contact angle). Afterwards, their performances in terms of cells (human primary gingival fibroblasts) and bacteria (Staphylococcus aureus) adhesion were compared to mirror-like polished titanium surfaces. Results revealed that gingival fibroblasts viability was not negatively affected by the applied surface roughness or by keratin nanofibers. On the opposite, cells adhesion and spread were strongly influenced by surface roughness revealing a significant cell orientation along the produced nanogrooves. However, the keratin influence was clearly predominant with respect to surface topography, thus leading to increased cells proliferation on the surfaces with nanofibers, disregarding the presence of the surfaces grooves. Moreover, nor nanogrooves nor keratin nanofibers increase bacterial biofilm adhesion in comparison with mirror polished surfaces. Thus, the present research represents a promising innovative strategy and technology for a surface modification finalized to match the main requirements for transmucosal dental implants.

How do wettability, zeta potential and hydroxylation degree affect the biological response of biomaterials?

It is well known that composition, electric charge, wettability and roughness of implant surfaces have great influence on their interaction with the biological fluids and tissues, but systematic studies of different materials in the same experimental conditions are still lacking in the scientific literature. The aim of this research is to investigate the correlations between some surface characteristics (wettability, zeta potential and hydroxylation degree) and the biological response (protein adsorption, blood wettability, cell and bacterial adhesion) to some model biomaterials. The resulting knowledge can be applied for the development of future innovative surfaces for implantable biomaterials. Roughness was not considered as a variable because it is a widely explored feature: smooth surfaces prepared by a controlled protocol were compared in order to have no roughness effects. Three oxides (ZrO2, Al2O3, SiO2), three metals (316LSS steel, Ti, Nb) and two polymers (corona treated polystyrene for cell culture and untreated polystyrene for bacteria culture), widely used for biomedical applications, were considered. The surfaces were characterized by contact profilometry, SEM-EDS, XPS, FTIR, zeta potential and wettability with different fluids. Protein adsorption, blood wettability, bacterial and cell adhesion were evaluated in order to investigate the correlations between the surface physiochemical properties and biological responses. From a methodological standpoint, XPS and electrokinetic measurements emerged as the more suitable techniques respectively for the evaluation of hydroxylation degree and surface charge/isoelectric point. Moreover, determination of wettability by blood appeared a specific and crucial test, the results of which are not easily predictable by using other type of tests. Hydroxylation degree resulted correlated to the wettability by water, but not directly to surface charge. Wetting tests with different media showed the possibility to highlight some differences among look-alike materials. A dependence of protein absorption on hydroxylation degree, charge and wettability was evidenced and its maximum was registered for surfaces with low wettability in both water based and protein containing media and a moderate surface charge. As far as bacterial adhesion is concerned, no effect of surface charge or protein adsorption was evidenced, while the presence of a high acid component of the surface energy appeared significant. Finally, the combination of hydroxylation degree, wettability, surface charge and energy (polar component) emerged as a key parameter for cell adhesion and viability.

The response of osteoblastic MC3T3-E1 cells to micro- and nano-textured, hydrophilic and bioactive titanium surfaces.

The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation.

Antibacterial titanium surfaces for medical implants.

Bacterial contamination is a critical problem in different fields (ranging from everyday life to space missions, and from medicine to biosensing). Specifically, in the case of medical implants, foreign materials are preferential sites for bacterial adhesion and microbial contamination, which can lead to the development of prosthetic infections. These problems can in turn lead to the necessity of a prolonged antibiotic therapy (which can last for years) and eventually to the removal of the device, with a consequent significant increase in the hospitalization times and costs, together with a stressful, painful and critical situation for the patient. Commercially pure titanium and its alloys are the most commonly used materials for permanent implants in contact with bone, and the prevention of infections on their surface is therefore a crucial challenge for orthopaedic and dental surgeons. The problem of the bacterial contamination of medical implants is briefly described in the first part of the present review. Then the most important inorganic antibacterial agents (Ag, Cu and Zn) are described, and this is followed by a review of the reported attempts of their introduction onto the surface of Ti-based substrates.

Effects of sterilization and storage on the properties of ALP-grafted biomaterials for prosthetic and bone tissue engineering applications.

Grafting of the biomaterial surfaces with biomolecules is nowadays a challenging research field for prosthetic and bone tissue engineering applications. On the other hand, very few research works investigate the effect of the sterilization processes on the properties of functionalized biomaterials. In this study, the effects of different sterilization techniques (e.g. gamma and electron beam irradiation, ethylene oxide) on the enzymatic activity of bioactive glasses and Ti6Al4V grafted with alkaline phosphatase (ALP) have been analyzed. Sterility maintenance and in vitro bioactivity of the sterilized surfaces have also been investigated. Finally the effect of packaging and storage conditions has been considered.

Characterization of Co-Cr-Mo alloys after a thermal treatment for high wear resistance.

The cobalt-chromium-molybdenum alloys are characterized by a high resistance to wear and corrosion, as well as good mechanical properties, allowing their use in the substitution of hip and knee joints. Five alloys were used as substrates for a coating deposition by a thermal treatment in molten salts, as reported elsewhere, in order to form a tantalum-rich coating on the sample surface, able to improve the biocompatibility and wear resistance of the materials. However, the temperature (970°C), reached during this process, is considered critical for the phase transformation of the Co-based alloys. The aim of this work is the evaluation of the temperature effects on the structure, microstructure, mechanical and tribological properties of the considered substrates, after the removal of the coating by polishing. The substrates are characterized through X-ray diffraction (XRD), scanning electron microscopy with energy dispersion spectrometry (SEM-EDS) and profilometry. The mechanical behavior is evaluated by the macro- and micro-hardness and bending tests, whereas the tribological properties are analyzed through a ball on disc test. A comparison between the as-received alloys and thermal treated substrates is reported. The biocompatibility feature is not reported in this work. The substrate crystalline structure changed during the heat treatment, inducing the formation of the hexagonal cobalt phase and the decrement of the cubic one. This crystallographic modification does not seem to influence the tribological behavior of the substrates. On the contrary, it affects the strength and ductility of the substrates.

Surface modification of Ti-6Al-4 V alloy for biomineralization and specific biological response: part II, alkaline phosphatase grafting.

Titanium and its alloys are the most widespread materials for the realization of orthopaedic and dental implants due to their good mechanical properties and biocompatibility. Surface functionalization of biomaterials aimed to improve and quicken implant integration and tissue regeneration is an active research field. The opportunity to confer biological activity (ability to directly stimulate cells with proper biological signals) to the Ti6Al4 V alloy, previously modified to be bioactive from the inorganic point of view (apatite precipitation), was explored in this research work. The alkaline phosphatase (ALP) enzyme was grafted to metal surface via tresyl chloride activation, maintaining its activity. A synergistic effect between biological functionalization and inorganic bioactivity was observed.

Surface modification of Ti-6Al-4V alloy for biomineralization and specific biological response: Part I, inorganic modification.

Titanium and its alloys represent the gold standard for orthopaedic and dental prosthetic devices, because of their good mechanical properties and biocompatibility. Recent research has been focused on surface treatments designed to promote their rapid osteointegration also in case of poor bone quality. A new surface treatment has been investigated in this research work, in order to improve tissue integration of titanium based implants. The surface treatment is able to induce a bioactive behaviour, without the introduction of a coating, and preserving mechanical properties of Ti6Al4V substrates (fatigue resistance). The application of the proposed technique results in a complex surface topography, characterized by the combination of a micro-roughness and a nanotexture, which can be coupled with the conventional macro-roughness induced by blasting. Modified metallic surfaces are rich in hydroxyls groups: this feature is extremely important for inorganic bioactivity (in vitro and in vivo apatite precipitation) and also for further functionalization procedures (grafting of biomolecules). Modified Ti6Al4V induced hydroxyapatite precipitation after 15 days soaking in simulated body fluid (SBF). The process was optimised in order to not induce cracks or damages on the surface. The surface oxide layer presents high scratch resistance.

Tantalum-based thin film coatings for wear resistant arthroprostheses.

Cobalt-chromium-molybdenum alloys with high carbon content (HC-CoCrMo) are widely used as materials for arthroprosthesis, in particular in metal-on-metal (MoM) hip joints. In spite of their good wear and corrosion resistance, production of metallic wear particles and metal ion release will occur on a large time-scale. An enhancement of the metal ion level in the patient's blood and urine is often reported in clinical data. Hypersensitivity, inflammatory response and cell necrosis can occur as consequence. So implants on young patients and women on childbearing age are not so widespread. The aim of this research is the realization of a thin film coating in order to improve the biocompatibility of Co-based alloys and to reduce debris production, ion release and citotoxicity. The innovative process consists of a thermal treatment in molten salts, in order to obtain a tantalum enriched thin film coating. Tantalum is chosen because it is considered a biocompatible metal with high corrosion resistance and low ion release. Three HC-CoCrMo alloys, produced by different manufacturing processes, are tested as substrates. The coating is a thin film of TaC or it can be composed by a multilayer of two tantalum carbides and metallic tantalum, depending on the temperature of the treatment and on the carbon content of the substrate. The thin films as well the substrates are characterized from the structural, chemical and morphological point of view. Moreover mechanical behaviour of treated and untreated materials is analyzed by means of nanohardness, scratch and ball-on-disc wear tests. The coating increases the mechanical and tribological properties of HC-CoCrMo.

Characterization of surface modified Ti-6Al-7Nb alloy.

In the last years different types of surface modifications were developed with the aim of improving the osteointegration ability of titanium alloys. The chemical composition, crystallographic structure and morphology of a surface layer can be modified in order to obtain a better interaction between the implant, the cells and the organic fluids. The final goal is to obtain a more efficient bone growth also in critical clinical cases. In the present paper were reported several data about the characterization of the Ti-6Al-7Nb alloy treated by two innovative surface treatments. They consist of blasting, followed by a two step chemical etching and heat treatment performed in air or in vacuum. TEM, XRD and SEM investigations were performed in order to assess the structure and morphology of the modified surfaces. The surface chemical composition was investigated by XPS ad AES analyses. The ability to interact with physiological fluids was tested by immersion of the treated materials in an acellular simulated body fluid (SBF). Metal ion concentration analyses of the fluid and SEM observations of the samples were performed after different times of soaking. The mechanical characterization involved scratch and fatigue tests. The surface of treated samples shows chemical, structural and morphological modifications. The passivation pre-treatment has influence on the surface modification. The treated samples evidenced a quite low metal ion release and interact with SBF solution, showing a moderate bioactivity. A relevant decrease in fatigue strength was observed on modified samples.

New chemical treatment for bioactive titanium alloy with high corrosion resistance.

It was recently claimed that titanium metal and its alloys can bond to the living bone, without being coated by apatite (VPS coatings), but by being chemically and heat-treated. The bioactivity of treated titanium is of interest because of the opportunity to obtain orthopaedic or dental implants presenting, at the same time, high toughness, strength and fatigue resistance as well as bone-bonding ability. The bioactive behaviour of the treated implants is due to the presence of a modified surface, which, during soaking in body fluid, promotes the precipitation of apatite. The apatite formed is strongly bonded to the substrate and promotes living bone bonding. In this work were characterised samples of Ti-6Al-7Nb alloy with surfaces presenting a different chemical and mechanical state. The aim of the research was twofold. The first objective was to characterise chemically and heat-treated samples with different surface topography, in order to define the best conditions for osteogenic integration. The second aim was to assess the corrosion behaviour of the bioactive implants, because they expose a microporous and quite thin modified surface layer. No-treated and passivated samples, with a surface state closed to that nowadays used on implants, were used as reference. The surface structure, morphology, electrochemical behaviour and bioactivity of the different samples were assessed by means of XRD, SEM-EDS, anodic polarizations, open circuit measurements and in-vitro tests. Results evidence that it is possible to modify the surface of the Ti-6Al-7Nb alloy in order to obtain the formation of a bioactive layer and that the substrate roughness influences the characteristics of the surface layer formed. It was also evidenced that the as treated surfaces present inadequate corrosion behaviour, so a new two-step chemical treatment has been developed in order to obtain a bioactive material with good corrosion resistance.

The influence of crystallised Fe3O4 on the magnetic properties of coprecipitation-derived ferrimagnetic glass-ceramics.

Ferrimagnetic glass-ceramics are potential candidates for magnetic induction hyperthermia, which is one form of inducing deep-regional hyperthermia, by using a magnetic field. The aim of this work was to analyse the influence of the amount of crystallised magnetite on the magnetic properties of glass-ceramic samples. Thus, two different ferrimagnetic glass-ceramics with the composition of the system Na(2)O-CaO-SiO(2)-P(2)O(5)-FeO-Fe(2)O(3) were prepared by melting at 1500 degrees C for 30 min of the coprecipitation-derived starting products. The X-ray diffraction patterns show the presence of nanometric magnetite crystals in a glassy matrix after cooling from melting temperature. The estimated amount of crystallised magnetite varies between 20 and 45 wt.%, as a function of the chemical composition. The morphology of the crystals was studied by scanning electron micrography and transmission electron micrography. Glass transition temperature and thermal stability were investigated by differential thermal analysis. Magnetic hysteresis cycles were analysed using a vibrating sample magnetometer with a maximum applied field of 17 kOe, at room temperature, in quasi-static conditions. Calorimetric measurements were carried out using a magnetic induction furnace. The power losses estimated from calorimetric measurements under a magnetic field of 40 kA/m and 440 kHz are 65 W/g for the glass-ceramic with lower iron oxides content and 25 W/g for the glass-ceramic with higher iron oxide content.

Fluoroapatite glass-ceramic coatings on alumina: structural, mechanical and biological characterisation.

The aim of this work was to realise bioactive coatings on full density alpha-alumina substrates. An SiO2-CaO-based glass (SC) and an SiO2-Al2O3-P2O5-K2O-CaO-F--based glass-ceramic (SAF) were used for this purpose. Specifically, SAF is a fluoroapatite containing glass-ceramic and previous studies have shown that it is a highly bioactive biomaterial. Furthermore, these fluoroapatite crystals possess a needle-shaped morphology which mimics that of hydroxylapatite found in human hard tissues, particularly in teeth. SAF is a very viscous glass-ceramic and for this reason an intermediate, less viscous, SC layer was interposed in direct contact with alumina aiming to obtain a good coating adhesion. Moreover, this intermediate layer strongly lowers the Al3+ diffusion and thus minimises both compositional changes in the SAF outer layer and the risk of detrimental modifications of the nature of the crystalline phases. A complete characterisation of the coated samples was performed by means of X-ray diffraction, optical and scanning microscopy. Coating adhesion on alumina was tested by comparative shear tests while biocompatibility was investigated on alumina. bulk SAF and on the realised coatings. For this purpose, cytotoxicity, adhesion and proliferation of human osteoblast-like cells were cultured onto the three materials. Results showed that the interposition of the SC layer was successful in allowing a good softening and spreading of the SAF outer layer and in avoiding the crystallisation of undesired crystalline phases maintaining the good bioactive properties of the bulk one. In vitro results on the coatings showed osteoblast-like cell behaviour similar to bulk fluoroapatite glass-ceramic and better respect to alumina substrates, being a promising index of bone material integration and of its in vivo possible applications.

[Precancerous oral lesions. Diagnostic and therapeutic protocol]. / Le precancerosi orali. Protocollo di diagnosi e terapia.

The NRC (National Research Council) task force on head and neck cancer issued this document to help clinicians involved in diagnosing and treating precancerous oral diseases. Chapter 1 includes definition, epidemiology, risk factors (alcohol, smoking, poor oral hygiene), WHO classification, clinical picture of leukoplakias, erythroplasia, lichen planus, as well as their natural history. Chapter 2 points out the clinical signs to be looked for in order to make a proper diagnosis and discusses biopsy techniques. Chapter 3 deals with therapeutic procedures (surgical technique and medical treatment), while Chapter 4 concerns follow-up according to the clinical and histological diagnosis. Finally, some statistical forms are enclosed: Form 1 consists of information regarding educational qualifications, occupation, alcohol consumption, smoking and dietary habits; Form 2 consists of information about previous diseases and the history of the present disease; Form 3 records the signs and symptoms observed for the precancerous lesion; Form 4 records treatment modalities; Form 5 records patient follow-up.