Fabio Variola

Improving biocompatibility of implantable metals by nanoscale modification of surfaces: an overview of strategies, fabrication methods, and challenges.

The human body is an intricate biochemical-mechanical system, with an exceedingly precise hierarchical organization in which all components work together in harmony across a wide range of dimensions. Many fundamental biological processes take place at surfaces and interfaces (e.g., cell-matrix interactions), and these occur on the nanoscale. For this reason, current health-related research is actively following a biomimetic approach in learning how to create new biocompatible materials with nanostructured features. The ultimate aim is to reproduce and enhance the natural nanoscale elements present in the human body and to thereby develop new materials with improved biological activities. Progress in this area requires a multidisciplinary effort at the interface of biology, physics, and chemistry. In this Review, the major techniques that have been adopted to yield novel nanostructured versions of familiar biomaterials, focusing particularly on metals, are presented and the way in which nanometric surface cues can beneficially guide biological processes, exerting influence on cellular behavior, is illustrated.

Nanoscale Oxidative Patterning of Metallic Surfaces to Modulate Cell Activity and Fate

In the field of regenerative medicine, nanoscale physical cuing is clearly becoming a compelling determinant of cell behavior. Developing effective methods for making nanostructured surfaces with well-defined physicochemical properties is thus mandatory for the rational design of functional biomaterials. Here, we demonstrate the versatility of simple chemical oxidative patterning to create unique nanotopographical surfaces that influence the behavior of various cell types, modulate the expression of key determinants of cell activity, and offer the potential of harnessing the power of stem cells. These findings promise to lead to a new generation of improved metal implants with intelligent surfaces that can control biological response at the site of healing.

Self-assembled monolayer of alkanephosphoric acid on nanotextured Ti

Surface modification of titanium and its alloys is of great importance for their practical application as biomedical implants. We have studied and compared assembly of dodecylphosphoric acid on commercial polished and on nanostructured titanium disks. The latter were produced by chemical etching that created nanoscale pits of typical size of about 20 nm. Enhanced hydrophobicity and high molecular density were obtained after functionalization of the nanotextured substrate. Aging tests showed a lifetime of the organic films of about one month in phosphate buffer. The samples were characterized by means of infrared spectroscopy, contact angle measurements, ellipsometry, and atomic force and scanning tunneling microscopies.

Colocation and role of polyphosphates and alkaline phosphatase in apatite biomineralization of elasmobranch tesserae.

Elasmobranchs (e.g. sharks and rays), like all fishes, grow continuously throughout life. Unlike other vertebrates, their skeletons are primarily cartilaginous, comprising a hyaline cartilage-like core, stiffened by a thin outer array of mineralized, abutting and interconnected tiles called tesserae. Tesserae bear active mineralization fronts at all margins and the tesseral layer is thin enough to section without decalcifying, making this a tractable but largely unexamined system for investigating controlled apatite mineralization, while also offering a potential analog for endochondral ossification. The chemical mechanism for tesserae mineralization has not been described, but has been previously attributed to spherical precursors, and alkaline phosphatase (ALP) activity. Here, we use a variety of techniques to elucidate the involvement of phosphorus-containing precursors in the formation of tesserae at their mineralization fronts. Using Raman spectroscopy, fluorescence microscopy and histological methods, we demonstrate that ALP activity is located with inorganic phosphate polymers (polyP) at the tessera-uncalcified cartilage interface, suggesting a potential mechanism for regulated mineralization: inorganic phosphate (Pi) can be cleaved from polyP by ALP, thus making Pi locally available for apatite biomineralization. The application of exogenous ALP to tissue cross-sections resulted in the disappearance of polyP and the appearance of Pi in uncalcified cartilage adjacent to mineralization fronts. We propose that elasmobranch skeletal cells control apatite biomineralization by biochemically controlling polyP and ALP production, placement and activity. Previous identification of polyP and ALP shown previously in mammalian calcifying cartilage supports the hypothesis that this mechanism may be a general regulating feature in the mineralization of vertebrate skeletons.

Nanoporous titanium surfaces for sustained elution of proteins and antibiotics.

Current medically relevant metals for prosthetic reconstructions enjoy a relatively good success rate, but their performance drops significantly in patients with compromised health status, and post-surgical infections still remain an important challenge. To address these problems, different nanotechnology-based strategies have been exploited to create implantable metals with an enhanced bioactivity and antibacterial capacities. Among these, oxidative nanopatterning has emerged as a very effective approach to engender nanoporous surfaces that stimulate and guide the activity of adhering cells. The resulting nanoporosity is also attractive because it offers nanoconfined volumes that can be exploited to load bioactive compounds and modulate their release over time. Such extended elution is needed since a single exposure to growth factors and/or antibiotics, for instance, may not be adequate to further sustain bone regeneration and/or to counteract bacterial colonization. In this article, we assessed the capacities of nanoporous titanium surfaces generated by oxidative nanopatterning to provide controlled and sustained elution of proteins and antibiotic molecules. To this end, we have selected bovine serum albumin (BSA) and vancomycin to reflect commonly used compounds, and investigated their adsorption and elution by Fourier-transform infrared (FT-IR) and ultraviolet–visible (UV-VIS) spectroscopy. Our results demonstrate that while the elution of albumin is not significantly affected by the nanoporosity, in the case of vancomycin, nanoporous surfaces provided an extended release. These findings were successively correlated to the establishment of interactions with the surface and physical-entrapment effects exerted by the nanopores, ultimately highlighting their synergistic contribution to the release profiles and thus their importance in the design of nanostructured eluting platforms for applications in medicine.

Oxidative nanopatterning of titanium generates mesoporous surfaces with antimicrobial properties.

Mesoporous surfaces generated by oxidative nanopatterning have the capacity to selectively regulate cell behavior, but their impact on microorganisms has not yet been explored. The main objective of this study was to test the effects of such surfaces on the adherence of two common bacteria and one yeast strain that are responsible for nosocomial infections in clinical settings and biomedical applications. In addition, because surface characteristics are known to affect bacterial adhesion, we further characterized the physicochemical properties of the mesoporous surfaces. Focused ion beam (FIB) was used to generate ultrathin sections for elemental analysis by energy-dispersive X-ray spectroscopy (EDS), nanobeam electron diffraction (NBED), and high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) imaging. The adherence of Staphylococcus aureus, Escherichia coli and Candida albicans onto titanium disks with mesoporous and polished surfaces was compared. Disks with the two surfaces side-by-side were also used for direct visual comparison. Qualitative and quantitative results from this study indicate that bacterial adhesion is significantly hindered by the mesoporous surface. In addition, we provide evidence that it alters structural parameters of C. albicans that determine its invasiveness potential, suggesting that microorganisms can sense and respond to the mesoporous surface. Our findings demonstrate the efficiency of a simple chemical oxidative treatment in generating nanotextured surfaces with antimicrobial capacity with potential applications in the implant manufacturing industry and hospital setting.

Enhanced Raman scattering in graphene by plasmonic resonant Stokes emission

Abstract Exploiting surface plasmon polaritons to enhance interactions between graphene and light has recently attracted much interest. In particular, nonlinear optical processes in graphene can be dramatically enhanced and controlled by plasmonic nanostructures. This work demonstrates Raman scattering enhancement in graphene based on plasmonic resonant enhancement of the Stokes emission, and compares this mechanism with the conventional Raman enhancement by resonant pump absorption. Arrays of optical nanoantennas with different resonant frequency are utilized to independently identify the effects of each mechanism on Raman scattering in graphene via the measured enhancement factor and its spectral linewidth. We demonstrate that, while both mechanisms offer large enhancement factors (scattering cross-section gains of 160 and 20 for individual nanoantennas, respectively), they affect the graphene Raman spectrum quite differently. Our results provide a benchmark to assess and quantify the role and merit of each mechanism in surface-plasmon-mediated Raman scattering in graphene, and may be employed for design and realization of a variety of graphene optoelectronic devices involving nonlinear optical processes.

Osteogenic Cell Cultures Cannot Utilize Exogenous Sources of Synthetic Polyphosphate for Mineralization

Phosphate is critical for mineralization and deficiencies in the regulation of free phosphate lead to disease. Inorganic polyphosphates (polyPs) may represent a physiological source of phosphate because they can be hydrolyzed by biological phosphatases. To investigate whether exogenous polyP could be utilized for mineral formation, mineralization was evaluated in two osteogenic cell lines, Saos‐2 and MC3T3, expressing different levels of tissue non‐specific alkaline phosphatase (tnALP). The role of tnALP was further explored by lentiviral‐mediated overexpression in MC3T3 cells. When cells were cultured in the presence of three different phosphate sources, there was a strong mineralization response with β‐glycerophosphate (βGP) and orthophosphate (Pi) but none of the cultures sustained mineralization in the presence of polyP (neither chain length 17‐Pi nor 42‐Pi). Even in the presence of mineralizing levels of phosphate, low concentrations of polyP (50 μM) were sufficient to inhibit mineral formation. Energy‐dispersive X‐ray spectroscopy confirmed the presence of apatite‐like mineral deposits in MC3T3 cultures supplemented with βGP, but not in those with polyP. While von Kossa staining was consistent with the presence or absence of mineral, an unusual Alizarin staining was obtained in polyP‐treated MC3T3 cultures. This staining pattern combined with low Ca:P ratios suggests the persistence of Ca‐polyP complexes, even with high residual ALP activity. In conclusion, under standard culture conditions, exogenous polyP does not promote mineral deposition. This is not due to a lack of active ALP, and unless conditions that favor significant processing of polyP are achieved, its mineral inhibitory capacity predominates. J. Cell. Biochem. 115: 2089–2102, 2014.

Progression of osteogenic cell cultures grown on microtopographic titanium coated with calcium phosphate and functionalized with a type I collagen-derived peptide.

BACKGROUND The functionalization of metallic surfaces aims at promoting the cellular response at the biomaterial-tissue interface. This study investigates the effects of the functionalization of titanium (Ti) microtopography with a calcium phosphate (CaP) coating with and without peptide 15 (P-15), a synthetic peptide analog of the cell-binding domain of collagen I, on the in vitro progression of osteogenic cells. METHODS Sandblasting and acid etching (SBAE; control) Ti microtopography was coated with CaP, enabling the loading of two concentrations of P-15: 20 or 200 μg/mL. A machined Ti was also examined. Rat calvarial osteogenic cells were cultured on Ti disks with the surfaces mentioned above for periods up to 21 days (n = 180 per group). RESULTS CaP coating exhibited a submicron-scale needle-shaped structure. Although all surfaces were hydrophobic at time zero, functionalization increased hydrophilicity at equilibrium. Microtopographies exhibited a lower proportion of well-spread cells at 4 hours of culture and cells with long cytoplasmic extensions at day 3; modified SBAE supported higher cell viability and larger extracellular osteopontin (OPN) accumulation. For SBAE and modified SBAE, real-time polymerase chain reaction showed the following results: 1) lower levels for runt-related transcription factor 2 at 7 days and for bone sialoprotein at days 7 and 10 as well as higher OPN levels at days 7 and 10 compared to machined Ti; and 2) higher alkaline phosphatase levels at day 10 compared to day 7. At 14 and 21 days, modified SBAE supported higher proportions of red-dye-stained areas (calcium content). CONCLUSION Addition of a CaP coating to SBAE Ti by itself may affect key events of in vitro osteogenesis, ultimately resulting in enhanced matrix mineralization; additional P-15 functionalization has only limited synergistic effects.

Assessment of the titanium dioxide absorption coefficient by grazing-angle Fourier transform infrared and ellipsometric measurements.

Thin films, either deposited or native (i.e., oxide layers), have been widely exploited in different technological fields (e.g., optics, electronics, and biomedicine) to improve the efficiency of devices and extend their range of applications. Among the different physical/chemical characteristics of thin layers that determine their overall properties and therefore their potential for new applications, thickness has been demonstrated to play a pivotal role in affecting different phenomena; this is particularly significant at nanoscale dimensions, since nanostructured materials often behave very differently from their bulk counterparts. For example, thickness affects the microstructure, morphology, and optoelectronic properties of ZnS, ZnO, and Mo-doped indium oxide thin films, as well as the electrical properties of MOS structures in microelectronic and optical devices. In the biomedical field, the thickness of the superficial TiO2 layer affects the biological response of titanium-based materials. Due to its effects on several optical, physical, and biological events, different techniques have been developed to precisely determine the thickness of thin oxide layers, such as, for example, that of SiO2 9 and TiO2. 10 However, the majority of these methods have limitations that can compromise their applicability to a wide range of samples and materials. In addition, these techniques (e.g., X-ray photoelectron spectroscopy (XPS) sputter profiling) may damage the sample surface and alter its micro and nanometric topographical features. One of the most efficient nondestructive methods to precisely measure the thickness of thin organic and inorganic layers is ellipsometry. Infrared (IR) spectroscopy has found increasing applications in materials science, biology, and nanotechnology due to the possibility of determining the surface chemical composition of thin inorganic and molecular layers. However, to our knowledge, this technique has never been used to determine quantitatively the thickness of inorganic thin films. In this work, we demonstrate that ellipsometry and Fourier transform infrared (FT-IR) spectroscopy can be combined to estimate the absorption coefficient and the thickness of IRactive nanometric oxide layers. In particular, we selected the amorphous TiO2 layer present on the surfaces of bulk titanium (cpTi) and Ti6Al4V resulting from natural passivation and chemical oxidation with H2SO4/H2O2 solutions. 28–33 Results from this study will permit the exclusive use of FT-IR to determine the thickness of amorphous titanium dioxide thin films. More generally, our approach can be potentially applied to investigate a wider variety of materials (IR-active thin films deposited on a reflective substrate), without altering their surface topography.