Symposium SM5 : Surfaces and Interfaces for Biomaterials

Metal-on-metal hip joint prosthesis have decreased in their popularity due to their high failure rates when compared to metal-on-polymer and ceramic-on-ceramic systems. The wear debris released from the bearing surfaces of the implant lead to bone necrosis and resorption. Therefore it is important from both a scientific and medical point of view to understand what is physically happening in such a tribo-system.

The main testing in this work involved a CoCrMo-on-CoCrMo and a CoCrMo-on-alumina tribo-system linearly reciprocating in PBS and diluted Bovine Serum at three different potentials. The potentials used were cathodic (-700 mV vs. SCE), open circuit potential and anodic (100 mV vs. SCE). The total wear obtained at anodic could be further subdivided into: wear only, corrosion only and synergy.

The majority of material loss in a tribo-system that contains a metal is coming from synergy and this is mainly due to the removal and reformation of the passive film. This work highlights the importance of the passive film and the lubrication that is given by the bovine serum.

An attempt was made to harden the surface of the CoCrMo alloy by low temperature carburizing in order to visualize the effect of hardness on the material loss. This has lead to improvements in the metal-on-ceramic system but null or negative effects have been seen in the metal-on-metal system. Since the majority of material loss is occurring because of synergy; hardness plays a very little role and does not contribute to any improvement in metal-on-metal systems.

The static corrosion resistance of the untreated and carburized CoCrMo was also studied using open circuit potential, potentiostatic, potentiodynamic and electrochemical impedance spectroscopy. The elutes of the potentiostatic tests were also tested using a human bone osteosarcoma cell line (Saos-2). In the case of corrosion, improvements haven been seen after the surface modification treatments.

Many problems associated with implants occur at the metal-body interface. To improve the mechanical and chemical stability of this interface and promote bone growth, the implant’s surface is usually coated with a biocompatible thin film. We deposited mixtures of ZrN, ZrC, TiN, TiC and SiC films on Ti implants by a combinatorial pulsed laser deposition technique to investigate their properties function of their structure and composition. The mechanical properties of the films were characterized by nanoindentation, scratch and wear tests. The structural properties were obtained from grazing incidence and symmetrical X-ray diffraction and X-ray reflectivity investigations. The chemical composition was measured by Rutherford backscattering spectrometry. Electrochemical measurements involving corrosion and electrochemical impedance spectroscopy studies were carried out in physiological solutions to investigate the chemical stability of the titanium, bare or covered with the PLD grown films and to compare their performance. The results clearly showed that the deposition of amorphous coatings resulted in more stable surfaces that were less affected by corrosion and exhibited better mechanical properties than bare Ti.

Modifying the nanostructure/chemistry of materials allows to optimize their properties [1]. Our strategy rests on creating nanopatterns that act as surface cues [2,3], affecting cell behavior. Chemical oxidation creates unique topographies [4], becoming a general strategy to improve biocompatibility. Our treatment selectively inhibits fibroblast growth while promoting osteogenic cell activity [5] in vitro. Enhancement of mechano-biocompatibility may occur by coating with spider silk [6, 7]. Improvement of antibacterial properties using laser and plasma strategies will also be discussed [8-11].
[1]F Rosei, J Phys Cond Matt 16, 1373 (2004)
[2]F Variola et al, Small 5, 996 (2009)
[3]F Variola et al, Biomaterials 29, 1285 (2008)
[4]F Vetrone et al, Nanolett 9, 659 (2009)
[5]L Richert et al, Adv Mater 20, 1488 (2008)
[6]C Brown et al, Nanoscale 3, 3805 (2011)
[7]C Brown et al, ACS Nano 6, 1961 (2012)
[8]O Seddiki et al, Appl Surf Sci 308, 275 (2014)
[9] M Cloutier et al, Diam Rel Mater 48, 65 (2014)
[10] L Cardenas et al, Nanoscale 6, 8664 (2014)
[11] M Cloutier et al, Trends in Biotechnology in press (2015

Metals currently used for prosthetic reconstructions (e.g. titanium) enjoy a relatively good success rate, but their performance drops significantly in patients with compromised health status. To address these needs, different nanotechnology-based strategies have been exploited. Among these, the use of bioactive coatings is one of the most promising strategies, since it permits to control surface-cell interactions by beneficial physicochemical cueing. Materials currently used in such coatings range from biocompatible polymers to proteins adlayers. The search is still one for new materials that can be employed to this end, capable of synergistically endowing implantable metals with an enhanced bioactivity and being relatively simple in order to be easily transferable to large scale production in the implant manufacturing industry. For instance, natural polymers (e.g. chitosan) have been explored as a valuable and economical alternative. In this context, we employed a mussel-inspired polymer, polydopamine (PDA), and carried out extensive investigation of its biological in vitro effects as a coating capable of direct physicochemical cueing to adhering cells. Our results show that human derived osteoblastic cells showed a differential activity on samples coated with a thin layer of polydopamine. Fluorescence microscopy permitted to correlate cell structure to the physicochemical features of surfaces, characterized by spectroscopic techniques (Raman and FTIR) and Atomic Force microscopy (AFM). These techniques allowed us to precisely probe the chemical makeup of the surfaces, confirming the presence of a homogeneous PDA layer, as well as their nanotopography, characterized by a granular nanotexture. In addition, the capacity of retaining proteins onto surfaces, a factor that can contribute to explain the observed cellular results, was assessed by mass spectroscopy. Such correlation was fundamental to unveil the cellular mechanisms underlying the observed variations of cell response, and promise to become a fundamental prerequisite for future studies that aim at extending the breath of polydopamine to other applications in bone tissue engineering.

The aim of this paper was to develop a new methodology to coat micron-sized particles with reactive polymer films using Chemical Vapor Deposition (CVD) polymerization. FTIR and XPS data confirm homogenous coating of the entire particle surface. Important variables for Chemical Vapor Deposition will be discussed and include the working pressure and the carrier gas flow rate. After Chemical Vapor Deposition, a surface titration of reactive chemical groups via aldehyde-hydrazide reaction was conducted. It was confirmed that the glassbeads were covered by a homogenous film of a poly[(4-formyl-p-xylylene)-co-p-xylylene] film. We incubated the coated beats in a hydrazide-PEG4-biotin solution, followed by thorough rinsing and reaction with Alexa Flora 647-conjugated streptavidin. Fluorescence images confirm that the particles were fully covered. Similarly, a Huisgen-1,3-dipolar cycloaddition reaction was conducted ton alkinyl-functionalized CVD coatings to further test homogenous coverage. The result also shows that the particles were fully covered.

Bacterial colonization of surfaces can result in adverse effects such as food contamination, deterioration of surface functionality and transmission of diseases. The focus of our research is on the development of antimicrobial coatings that do not release biocides into the environment. Such coatings prevent bacterial colonization either by preventing bacterial adhesion on the surface (anti-adhesive) or by killing the bacterial cells that contact the surface (bactericidal). We have demonstrated that agarose can inhibit bacterial cell adhesion on surfaces while quaternized chitosan is a well-established bactericidal polymer. In order to use these polymers as coatings in practical applications, facile techniques are needed for forming stable coatings with high antimicrobial efficacy. In our presentation, we will describe different techniques for surface functionalization of polymeric and metallic substrates with quaternized chitosan and agarose. These techniques include (i) surface-initiated UV or heat-induced immobilization and crosslinking of acrylated agarose or acrylated quaternized chitosan, (ii) surface-initiated covalent grafting of agarose or quaternized chitosan via the thiol-ol method and (iii) ionic gelation of quaternized chitosan with surface-grafted tripolyphosphate. The physicochemical properties and antimicrobial efficacy of the coatings prepared by these different techniques will be compared. These coatings are not cytotoxic and the best coatings can reduce bacterial colonization by two orders of magnitude. The stability of these coatings under environmental and simulated in vivo conditions will be presented. For each technique, the advantages, limitations, applicability for biomedical devices and for preventing environmental contamination as well as feasibility of scale-up from bench to practical applications will be discussed.

The use of polymer brushes for the design of advanced functional substrates has shown a rapidly growing interest in recent years^{1, 2}. A particular attention has been paid towards the elaboration of brushes made up of stimuli responsive polymers, in order to design smart substrates whose properties can be altered depending on some external parameters³ (pH, temperature, light, ionic strength etc). This has fostered research focusing on the use of responsive brushes for controlling the interactions between a surface and biological objects and has given rise to a breadth of biology-relevant applications⁴. Most of these applications rely on the conformational change of these tethered polymeric chains. It is therefore of major concern to understand how the conformation of a brush depends on its molecular brush parameters and on the applied stimulus. This calls for the development of experimental techniques that can efficiently screen the effects of molecular parameters of a brush on its structure and its target properties.

Herein, we describe an original set up based on the well known Reflection interference Contrast Microscopy (RICM) that allows us to perform space and time-resolved measurements of the spectral reflectivity of brush-coated surfaces over visible spectrum. We thereby investigate, the thermal response of Poly(N-isopropylacrylamide) (PNIPAM) brushes over a range of surface densities and chain lengths. We show that Reflectivity measurements yield quantitative information about the conformational changes of the brushes when the temperature is varied across the Local Critical Solution Temperature of the polymer and allow access to the density profile of the brush. Our results are in qualitative agreement with similar studies performed using Neutron Reflectivity⁵ and Ellipsometry⁶ and provide one of the very few evidences for the phenomenon of vertical phase separation within the brush as expected theoretically. We investigate the response of PNIPAM brushes upon cycling the temperature, and clarify the physical origin of their hysteretic behavior. We finally show that RICM may come as an interesting alternative technique in estimating the molecular parameters of the “grafted from” polymer brushes, which is crucial for the rational design of smart polymer coatings in actuation, gating, or sensing applications

References
1. O. Azaaroni et al. J. Polym. Sci., Part A: Poly. Chem. 2012, 50, 3225-3258
2. A. Olivier et al. Prog. Polym. Sci. 2012, 37,157-181
3. T. Chen et al. Prog. Polym. Sci. 2010, 35,94-112
4. M. Krishnamoorthy et al. et al. Chem. Rev. 2014, 114,10976-11026
5. H Yim et al. Macromoleules 2004, 37,1994-1997
6. E.S. Kooij et al. J.Phys.Chem. B 2012, 116, 9261-9268

Surface Plasmon Resonance (SPR) has been used already for a few decades for label-free detection and characterization of biochemical kinetics and affinities of many different types of analytes. However, the physical phenomenon is not limited to biochemistry, but is applicable to other nanoscale characterization of thin films [1]. Multi-parametric surface plasmon resonance (MP-SPR) is a new approach to the physical phenomenon, which utilizes full SPR angular spectral measurement and among other things allows new type of thin film characterization methods to be used.
MP-SPR can be used to characterize ultrathin nanoscale films for both thickness and refractive index with two methods. It is possible to characterize the films either by measuring them in two different media with high RI difference, such as air and water [1,2], or by measuring the films with two or more wavelengths of light [2,3]. The method effectiveness has been extensively demonstrated using diffetrent ultrathin films systems [2-4].
Stearic acid (SA) LB films showed approximately 2.5±0.2 nm thickness with both 2M and 3W methods, and linear increment with increasing layer number. Similarly the polyelectrolyte multilayer consisting of polystyrenesulfonate and polyallylaminehydrochloride (PSS:PAH) was characterized to grow in 3-3.5 nm steps per layer pair. Lipid bilayers spread from vesicles showed typical thickness of approximately 5 nm. The refractive index of all studied thin films correlated well to earlier literature values. Ex-situ depositions of nanofibrillated cellulose (NFC) by spin coating were characterized by the 2W method to form 14 ±3.4 nm layers. These films were also characterized for their swelling behaviour, which correlated well with QCM based swelling characterization.
Both of the methods are in good agreement with reference methods used in the studies such as QCM, and with previous literature values obtained with different measurement methods. Thus the MP-SPR based characterization methods for ultrathin films appear to be effective, especially for applications requiring both measurement of the film properties and interactions of different compounds with the film.


References

[1]Albers, Vikholm-Lundin, Chapter4 in Nano-Bio-Sensing, Springer2010
[2]Granqvist et al., Langmuir 2013,29(27),2013,8561-8571
[3]Granqvist et al., Langmuir 2014,30(10),2799–2809
[4]Kontturi et al., J.Mater.Chem.A, 2013,1,13655-13663

The major aim of the present study is to develop and explore the potential of large surface area electrospun polymer nanofabric as a carrier for controlled and sustained release, in particular to hydrophobic drugs. Gelatin (type A), FDA approved natural polymer was electrospun in a mixture of solvent (20% acetic acid in water) to yield long, continuous and uniform fibers with average diameter ~ 200 nm. Piperine was chosen as a model hydrophobic drug in this study. As gelatin is dissolved within no time in aqueous medium, we crosslinked electrospun gelatin nanofibers using saturated GTA vapor to increase the water resistive properties. For controlled release over a period of 12 h, we devised several strategies to vary the crosslinking conditions and accordingly understand their effect on drug release mechanism. One of such successful efforts was based on deposition of multiple layers of electrospun fabric by sandwiching drug loaded gelatin nanofibers with only gelatin nanofibers from both sides. Not only the layer by layer deposition, we also crosslinked the different layer in the same sequential way. Sequential crosslinking using GTA vapor in different layers of the fabric, helped in uniform crosslinking throughout the thickness compared to crosslinking after final deposition in the form of single layer. Effect of different crosslinking strategies was investigated in terms of surface morphology and drug stability. Finally, in vitro release study was performed maintaining the physiological conditions mimicking GI tract to analyze the effect of crosslinking on the drug release profile. The in vitro studies concluded that the controlled drug release can be achieved by tuning the thickness of individual fabric layer followed by their sequential crosslinking due to increased diffusional barrier for drug release. Interestingly, we also found that only 6 min exposure to saturated GTA vapor is sufficient to provide the required drug release in contrast to up to 24 h as reported in literature. This finding also addresses the toxicity problem associated with the use of GTA as a cross-linker.

Mastering the composition and structure of surfaces and interfaces, on top of specific bulk characteristic properties, plays a key role into designing new biomaterials ; this implies use or develop deposition/synthesis tools for biocompatible coatings, and use or develop the most adequate advanced charaterization tools for their qualification.
X-ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) are, without any doubt, amongst the best known and most frequently used techniques for surface and interface characterization. Their advantages and disadvantagies will be reviewed, taking one type of biosurface as a motivating guideline.
Coronarian metallic stents do need an external biocompatibilization coating to reduce restenosis, promoting endothelialization and hemocompatibility. In this work, two routes have been explored : (1) TiOxCy layers with in depth (x,y) gradient composition have been tested . RF vacuum plasma treatment has been used to deposit this conformal inorganic coat, to accommodate for the mechanical stress caused by the stent deployment. (2) molecular organic coatings, containing fibronectin and phosphorylcholine have been prepared by wet chemistry, combining adsorption and covalent grafting. Critical evaluation of these inorganic and organic layers using biological tests will be presented.


This work has been made possible as bilateral Wallonie-Bruxelles International and Québec-Wallonie-Bruxelles joint projects ; it involved many coworkers whose respective contribution is gratefully acknowledged : C. Noel, M. Diallo, A. Batan, and L. Houssiau at the University of Namur - LISE; V. Montano-Machado, F. Lewis, P. Chevallier, S. Turgeon, and D. Mantovani at Laval University -LBB

Implantation of any medical prosthetics in human is the result of an expectation that significant improvement in quality of their lives could be achieved. Longevity of those implants is also an important factor related to quality of life as well as the cost to health care system. Such medical devices in addition to satisfying bio- and haemo-compatibility issues must also possess the specific mechanical, electrical or tribological properties in order to respond to specific medical needs of a patient. Thus, selecting materials that could simultaneously satisfy a multiple of physical, chemical, electrical, biological and/or tribological properties constitutes a significant challenge to the biomaterial engineering community.

Introduction of any foreign object in human body triggers interaction of biological cells with the foreign material. These interactions initially take place at the interface of the material and living cell, and the chemical and morphological properties of the surface impacts the outcome of such interactions. There are several organic, inorganic and composite materials that are fair candidates as biocompatible materials. However, their ability to respond to all requirements simultaneously is rare. As a result, the advanced surface engineering techniques become one of the most important tools for designing high quality medical implants, devices or tools. The surface engineering could take various forms, including deposition of thin film coatings, or near surface implantation of materials to form a composite layer that is only several monolayers thick, among others.

Amongst various surface engineering techniques, plasma-based techniques offer the most versatile approach, since they allow surface modifications with a level of precision that approaches monolayer accuracy. Other advantages include their ability to allow independent control of thermal effects to heat sensitive materials. An important group of heat sensitive biomaterials are polymers, which could be synthesized using pulsed plasma assisted techniques. The main advantage of pulsed plasma is that it produces significant quantities of ions, electrons and radicals, which are required for the process of deposition or surface modification, at low average energy cost.

In this presentation we will give a brief highlight of the technologies that are readily available for application to surface engineering of biomaterials and we will present some examples of their applications.

Microfluidic devices have become increasingly attractive for sundry biological applications due to their reduced size and high surface area-to-volume ratio, enabling fast analysis, short reaction times and potential for patterning. Robust application of microfluidics to biology requires surfaces that can be hydrophilic, patterned, optically transparent and charged. Currently, the most commonly used microfluidics material is polydimethylsiloxane (PDMS). Despite its several advantageous properties, PDMS surfaces are extremely hydrophobic. In order to overcome this issue, a new family of thermoplastic polymers such as COC (Cyclic Olefin Copolymers), NOA (Norland Optical Adhesive) and THV (Dyneon) were investigated using low and atmospheric pressure plasma processes. The major concern is the stability of such coatings.

Introduction. Plasma immersion ion implantation (PIII) is a plasma modification technique that can be used to tune the surface properties of metallic alloys. In particular, it can be used for alloys used in biomedical applications, such as stainless steel, Co-based alloys, Ti-based shaped memory alloys and Ti alloys. For L605, a Co-Cr-Ni-W alloy, the release of Cr and Ni in the blood stream can be a health concern; for this reason, a surface treatment can effectively accommodate surface deformation and act as a barrier, reducing the adverse events following endovascular procedures as restenosis and thrombosis. The present work deals with the structural, mechanical and chemical characterization of an oxygen plasma implanted Co alloy surface. The correlation between the properties of the electropolished substrate and the plasma treated samples were investigated for antenna power, P, and implantation time, d. The characterization of the surface condition was performed by different techniques and the interaction of endothelial cells with the modified surfaces was assessed.
Materials and methods. L605 flat samples were electropolished implanted with a pulse duration in the range of 50-100 µs and a frequency in the range 10-40 Hz. X-Ray photoelectric spectroscopy (XPS) and scanning electron microscopy (SEM) were used to assess surface chemistry and morphology. Roughness was studied by atomic force microscopy (AFM). The viability of human endothelial cells was evaluated by resazurin test.
Results and discussion. Oxygen implantation led to the formation of a Co-oxygen rich phase on the surface of the material; the presence of Si found by XPS analysis is due to quartz window sputtering. Increasing doses cause the increase of Co in the topmost layer; the same effect was found for increasing antenna power.
The oxide surface layer created by the implantation process showed a different chemical composition, structure and thickness compared to the layer formed after electropolished. In fact, the latter was thinner and richer in Ni and Cr than the implanted one, composed only by a thicker oxide Co and Si layer. Both AFM and SEM did not evidence a relevant change of the surface topography, even if electron microscopy micrographies showed the absence of grain boundaries after implantation, due to amorphization of the surface. Scratch test evidenced the formation of slip lines, both for the EP condition and the implanted ones. A critical load of ∼5 N was found for the thicker layer. Nanoindentation measurements showed a hardness of around 4.5 GPa, increasing with increasing oxygen dose. Endothelial cell investigations in L605 treated samples show the highest cell viability for higher doses implantation.
Conclusions. Oxygen PIII effectively modified the chemistry and the structure of the surface layer. Increasing both implantation doses and antenna power increase the surface Co oxide, improving the biological response.

The use of bone implantable devices has become a viable treatment for patients suffering from bone fractures, joint degradation, osteoporosis, and bone cancer. In 2010, there were more than one million knee and hip replacement surgeries undertaken in USA alone. Zirconium-based alloys are promising materials for orthopedic prostheses due to their low toxicity, superb corrosion resistivity, and favourable mechanical properties. The sub-optimal biocompatibility of bare metal surfaces, however, often leads to adverse foreign body responses, inflammation, or infection requiring additional medical interventions that increase costs and discomfort for patients. The integration of such bio-implantable devices with local host tissues can be strongly improved by a plasma polymerized acetylene and nitrogen (PPAN) coating that covalently immobilizes bio-active molecules. The stability of the plasma polymerized layer in body fluids is critically important, and the coating must resist failure even when scratched. In this study, we present a novel approach for the fabrication of chemically and mechanically robust films through plasma polymerization of PPAN on biased zirconium substrates. A custom-made plasma polymerization system consisting of a radio frequency (RF) electrode and a pulsed voltage source was utilized for plasma polymer deposition. The precursor gas composition, pulsed bias voltage, and RF input power were varied to achieve optimized deposition conditions. The surface chemistry of PPAN was analysed using X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS), while atomic force microscopy (AFM) was applied to evaluate the morphology of coatings at early stages of growth. The chemical and mechanical stability of the coatings in a simulated body fluid was examined by incubation of samples in Tyrode’s solution at 37^oC for durations of 1 week to 2 months. As evidenced by both XPS data and SEM observations, the PPAN film resisted failure, and no delamination, cracking, or buckling was observed after scratching and subsequent incubation in Tyrode’s solution. XPS results revealed that excellent zirconium-PPAN adhesion is linked to the formation of metallic carbide and carbonate bonds, induced by ion implantation, at early stages of film growth. Such atomic interfacial mixing also resulted in the formation of a continuous smooth film near the substrate as suggested by AFM and ToF-SIMS results. Deposition of PANN using this technique holds great promise for the fabrication of robust bioactive surfaces on other carbide-forming early transition metals such as titanium and niobium.

Artificial corneas are viable alternatives for patients that need to replace damaged corneas but are unsuitable candidates for corneal transplants. The main issues that lead to failure of implants are poor integration into the patient’s eye and high risk for bacterial infection. Current proposals address both issues with chemical modification of the surface. However, this approach provides only a short-term solution. Our solution is to incorporate nanotopography on the surface of the artificial cornea. There has been evidence that natural nanoscale topography has significant effects on cell behavior. Nanotopography can be applied to material surfaces and maintain material properties and surface chemistry of materials. We have applied nanotopography on a surface that will be incorporated into the artificial cornea with nanoimprint lithography, an industrially scalable process. By pressing a patterned mold directly into the material surfaces under heat and pressure, we have fabricated well-defined nanostructures. We have three design criteria for the artificial cornea: it needs to be antibacterial, optically transparent, and capable of controlling adhesion of corneal cells. We have been able to fulfill these requirements in the design that I will describe. We found that nanopillars (100-500 nm range) decrease adhesion of E. coli while killing those that come in contact with the pillars (Dickson, Liang, et al. 2015). Structures in this size range do not scatter light so patterned surfaces maintain their optical transparency. Finally, we observed fewer fibroblasts adhering to nanopillared surfaces while more cells adhered to nanolined surfaces compared to a flat surface. Fibroblasts exhibited increased cell motility on nanopillars. This was further validated by observing the presence of higher order aggregates of paxillin in focal adhesions of cells on nanopillars compared to those of cells on flat or nanolines. These findings indicate that we can use nanopatterns for selective control of cell adhesion to improve the implant’s integration with the host eye. Ongoing work seeks to transfer such nanofeatures to quasi-three-dimensional (curved) surfaces of the corneal prosthesis prototype for in vivo testing.

Non-equilibrium atmospheric pressure plasmas offer an astounding versatility relying on a wide range of physical principles and source architectures for their generation and sustainment. Spanning from dielectric and resistive barrier discharges to plasma guns, from jet-to-jet coupled sources to the more energetic meso-plasmas, these plasmas are being explored for an ever increasing number of applications, including decontamination, medical therapies and surface modification of materials. The talk will touch three subjects that put together plasma sources and processes for surface modification of biological materials in plasma medicine and antibacterial applications. Low power atmospheric pressure inductively coupled thermal plasma sources integrated with a quenching device (cold ICP) for the efficient production of biologically active agents have been recently developed for potential biomedical applications. To evaluate the antibacterial activity and possible associated cytotoxicity of cold ICP treatment on both prokaryotic and eukaryotic cells, an in-vitro model realistically representing a typical wound environment has been defined. Bacteria typical of wound infections (S. aureus and E. coli) adherent eukaryotic cells, Kidney Epithelial Cells (Vero) and Lung Fibroblasts (HEL 299), were separately plasma treated (direct treatment) in physiological solution. The role of the liquid medium was also investigated, comparing such results with those obtained by adding plasma treated liquid to prokaryotic and eukaryotic cells (indirect treatment). UV radiation and nitric oxide species produced at the biointerphase, as well as the pH of plasma treated saline solution, were measured. An important challenge for endodontic treatments is represented by the complete decontamination of tooth root canals, for which fully efficient methods do not yet exist. A study of the antibacterial effects of a handheld PG source, properly designed for endodontic applications, was carried out, with the aim of determining an efficient procedure for the decontamination of tooth root canals. Indeed, since biological substrates are never completely dry, the decontamination potential of Plasma Gun devices was quantitatively analyzed on E. faecalis suspensions and also used used to decontaminate tooth root canals in realistic in vitro models with: A) a dry-direct treatment of the contaminated root canal; B) a direct treatment of the root canal filled with 50 µl of bacterial suspension; C) an indirect treatment of contaminated root canal with 100 µl of water activated by a PG treatment. Results were quantitatively evaluated by means the bacterial load reduction (LogR) with the final aim of designing the best plasma assisted process for root canal decontamination. Finally some preliminary results on the beneficial modification induced by Plasma Gun treatment on the adhesion properties between materials typically used in the dental field will presented.

In my talk, I will present recent developments from our group on various biomaterial coatings that are facilitated by plasma deposition. These include antibacterial coatings, drug release platforms and cell guidance/capture surfaces.

Undesired bacterial adhesion and subsequent colonisation of medical devices is a substantial medical problem causing complex and sometime fatal infections. We have developed various strategies for generation of antibacterial coatings that can be applied to medical service surfaces. These involve means such as surface silver nanoparticles, antibiotics, nitric oxide, quaternary ammonium compounds (QACs) or simply coatings that have intrinsic low fouling properties. All these coatings are facilitated by plasma deposition, a technique that provides functional films applicable to the surface of any type of material. Important for applications, we not only extensively test our coating for their antibacterial efficacy but also assess their potential cytotoxicity to mammalian cell and inflammatory consequences.

My talk will also outline our research on advanced plasma polymer based coatings capable of directing cellular behaviour. We often use surface gradients which are powerful tools for studying and guiding cellular responses such as adhesion, proliferation, migration, differentiation, etc. We have developed a range of surface gradients such as of chemistry, bound ligand and protein densities, nanomechanics and nanotopography. We have used these gradients to interrogate the behaviour of various cell types, including stem cells. In my talk, I will focus on recent work where we used gradients of nanoparticles density to study the influence of surface nanotopography of cell behaviour. Our data demonstrates that surface nanotopography strongly affects the adhesion of fibroblasts and osteoblasts, and alters the level of expression of pro-inflammatory cytokines from microphages and neutrophils. I will present a strategy for developing gradients of surface elastic modulus. Our studies on the adhesion and proliferation of primary human fibroblasts show that these cells prefer stiffer surfaces. I will also present our surfaces that are capable of driving the differentiation of stem cells via surface chemistry, nanotopography or density of signalling molecules.

I will also briefly present drug delivery and release platforms that we have developed including a method for solvent free encapsulation of drug particles. A recently developed device for selective cancer cell capture for complex liquids will also be presented.

Selected references:
Ramiasa et al Chem Commun 51 (20) 4279-4282 (2015)
Cavallaro et al Chem Commun 51 (10), 1838-1841 (2015)
Delalat et al Adv Funct Mater 25 (18) 2737-2744 (2015)
Taheri et al Biomaterials 35 (16)4601–4609 (2014)
Hopp et al Biomaterials 34 (21) 5070–5077 (2013)
K. Vasilev et al Nano Letters 10 (1), 202–207 (2010)

Nosocomial infections continue to be a major public health concern in hospitals and healthcare units worldwide. Since infections are primarily caused by bacterial colonization of biomedical surfaces, antibacterial coatings have emerged as a primary component of the global mitigation strategy of bacterial pathogens. Still, far too little attention has been paid to the microbial contamination in the inanimate environment, despite mounting evidence linking contaminated environmental surfaces to nosocomial infections. Therefore, the development of antibacterial surfaces for near-patient clinical areas, acting in conjunction with cleaning and disinfection procedures, are needed to reduce the spread of nosocomial infections. The suitability of engineered materials for such task typically depends on their stability and general ability to withstand harsh operating conditions. Plasma technologies have been proposed as interesting options since they offer the possibility to deposit strongly adherent and cohesive coatings with a tunable set of properties on various substrates.
Herein, we present the development of silver-containing diamond-like carbon (Ag-DLC) nanocomposite coatings as highly stable antibacterial surfaces. A hybrid plasma enhanced chemical vapor deposition – physical vapor deposition (PECVD-PVD) system was developed to deposit Ag-DLC coatings in a one-step process. X-Ray photoelectron spectroscopy and Atomic force microscopy analyses showed the integration of silver as nanosized clusters within the film. Silver clustering and dispersion as well as the coating’s morphology were found to be primarily influenced by the ions energy during plasma deposition. Direct contact antibacterial tests revealed that Ag-DLC coatings possessed a strong antibacterial activity against E. Coli, which could be directly tuned by controlling the amount of incorporated silver. The mechanical properties and distribution of silver within the coating were strongly correlated with the depositing ion energy and could be optimized to provide a coating with high hardness and wear-resistance. The resulting coatings could withstand up to 1000 cycles of mechanical wear with no apparent delamination, major loss of integrity or antibacterial properties.

Introduction:
Hydrogenated amorphous carbon (a-C:H) films have a good biocompatibility as anti-thrombosis, anti-bacterial, histocompatibility, and/or cytocompatibility. In order to make best performances, characteristics of a-C:H film surface should be modified and carefully selected for each application. In our previous work, relationship between a-C:H film surface condition and NIH 3T3 cell proliferation was investigated and it was observed that C=O bond of a-C:H film surface strongly depends on cell adhesion with regardless deposition techniques. Additionally, it was observed that oxygen plasma treatment of a-C:H films was widely effective for cell adhesion. In this study, oxygen plasma treatment were carried out for different type of typical a-C:H films and influence on the film properties was investigated.

Materials and Methods:
In this experiment, several types of typical a-C:H films were used for estimation of cell adhesion control. The a-C:H films were deposited by CVD and PVD method technique. After the films deposition, surfaces of the a-C:H film were treated by oxygen plasma (r.f. power at 100 W, O2 gas at 10 Pa). In cell culture, NIH-3T3 cells were spread and cell adhesion response was studied on the oxygen plasma treated a-C:H films surface. The a-C:H films were examined with regard to their surface conditions using X-ray Photoelectron Spectroscopy (XPS). Furthermore, the film structure was examined by Fourier Transport Infrared Spectroscopy (FT-IR) and Raman Spectroscopy (Raman).

Results and discussion:
C=O bond ratio of the a-C:H film surfaces were changed by oxygen plasma treatment, respectively. The result shows that by the oxygen plasma treated a-C:H films, their cell adhesion could be changed with C=O bond ratio and film structures. It means that the different type a-C:H film surfaces with regardless deposition methods were modified to quality of uniformity surface conditions for cell adhesion by oxygen plasma treatment. Oxygen plasma treatment is useful for control of cell adhesion on various a-C:H films.

Atmospheric pressure non-equilibrium plasmas, or cold atmospheric plasmas (CAP) due to their characteristically low temperature (possibly lower than 40°C), are extremely versatile sources of reactive species, UV radiation, radicals and electrons, offering innovative means to induce chemical reactions and material modification. While the use of low pressure plasmas for these aims is well assessed, CAPs potentialities are still largely unexplored, notwithstanding their advantages of cost and ease of use.
In this presentation, different applications of CAPs in the field of surface modification of materials will be introduced and discussed, such as surface functionalization to affect cell response, plasma polymerization of polyacrilic acid (pPAA) for thin film deposition, plasma co-deposition of pPAA and Ag nanoparticles for the production of nanostructured AgNPs/pPAA thin films.
In particular, a simple and straightforward process for introducing COOH groups in a polymeric surface, relying on the use of a scalable non-equilibrium atmospheric pressure plasma source, is presented for the case of electrospun poly(L-lactic acid) (PLLA) non-woven mats. Thermo-mechanical properties, morphology, hydrophilicity, and water uptake of the scaffolds after plasma treatment are shown and compared with the behavior of pristine mats. The amount of carboxyl functional groups on the scaffold surface is evaluated using fluorescein isothiocyanate conjugation and microdensitometry. The effect of plasma treatment on mouse embryonic fibroblast morphology is assessed through image analysis.
Furthermore, atmospheric pressure plasma-polymerization of acrylic acid for the deposition of pPAA thin films having a high retention of carboxyl groups will be discussed. Chemical characteristics of the deposited coatings, evaluated by means of attenuated total reflectance-Fourier transform infrared and X-ray photoelectron spectroscopies will be shown.
Finally, a recently developed single step process for the deposition of nanocomposite AgNPs/pPAA coatings will be presented. Through SEM and ATR-FTIR analysis, the deposition of a micrometric pPAA coating on the polyethylene (PE) film used as substrate is assessed; AgNPs embedded in the polymeric matrix are visible in SEM pictures and their presence is confirmed by XPS and EDS analysis. XPS also highlights a high retention of carboxylic groups in the pPAA chemical structure and the surface oxidation of AgNPs. Preliminary results of the antibacterial activity of the codeposited coatings will be shown as well.
The different plasma sources required to enable these applications will also be detailed, and a few techniques for their characterization, holding the keys for process understanding and optimization, will be shown and results discussed.

The ability to modify the contact angle of water on silicon has applications ranging from thermal management of electronics to miniaturized biomedical and microfluidic devices. That the contact angle for bulk silicon can be increased or decreased through surface treatments as well as micro- or nano-structuring is well known. Such modification processes must be simple and robust for practical applications such as DNA printing and gold nanoparticle adhesion. The motivation for this work is to explore simpler oxygen plasma treatments that can both expand the overall range of contact angles and ensure long-term behavior. Through simple variation in the thickness of poly(dimethylsiloxane) (PDMS) and oxygen plasma treatment, we were able to create both hydrophobic and superhydrophobic surface on the same sample for adhesion of both DNA and other biomaterials.

Here, we report ~30° variation in superhydrophobic contact angles on silicon that is stable after ~3 months of hydrophobic recovery using a relatively simple plasma treatment on silicon nanowires coated with few nanometers of PDMS. The variation in contact angle arises from choosing nanowires of different lengths and is approximately five times more than that observed previously due to clustering effects alone.

We characterize the surfaces using a combination of X-ray photoelectron spectroscopy (XPS), contact angle measurements, and ellipsometry. Taken together with contact angles available from similar treatment on bulk silicon, it is possible to non-lithographically create regions of diverse contact angles, from ~50° to ~149°.

The cell behaviors such as adhesion, proliferation, and differentiation are greatly affected by cell-material interface [1]. It is well known that the surface topographies and chemistry of micro- or nano-patterned polymer brushes acting as cell culture substrates are important for both the initial cell adhesion and the followed cell response [2, 3]. However, the nanopatterned polymer brushes for cell orientation control have not been study. In this work, the parallel dip-pen nanodisplacement lithography (p-DNL), a scanning probe-based lithography, is used to serially fabricate two-component 3D polymer brush with micro- or nano-structures for cell adhesion and orientation study.
Poly (N-isopropylacrylamide) (PNIPAAm) brushes with a thickness of 10 nm act as the polymer resist layer to modify the Au-glass substrates. Atomic Force Microscopy tip arrays (55, 000 tips per square centimeter) pre-inked with initiator-bearing thiol molecules can cleave the PNIPAAm brushes away from the substrate at the tip-substrate contact domain and the initiator molecules simultaneously self-assemble onto the bare areas, achieving the “nanodisplacement” process. Poly (glyceryl methacrylate) (PGMA) brushes are then grown from the patterned initiators via surface initiated atom transfer radical polymerization. Therefore, the 3D PGMA brush patterns on PNIPAAm brush-coated Au-glass substrate are successfully prepared. The PGMA patterns including line arrays, dot arrays and gradients. The pattern areas are 1 cm². The height and width of 90-μm long PGMA lines are 80 nm and 230 nm, respectively. The polymer resist layer on the substrate make the PGMA patterns less lateral collapse and more tightness. Then gelatin is bonded to the PGMA line arrays and the Hela cells are well aligned along the gelatin/PGMA nanolines.
[1] Viswanathan P, Themistou E, Ngamkham K, Reilly GC, Armes SP, Battaglia G. Biomacromolecules 2014; 16: 66-75.
[2] Théry M. Journal of Cell Science 2010; 123: 4201-13.
[3] Yu Q, Johnson L M, López G P. Advanced Functional Materials, 2014, 24: 3751-3759.

We present a method of modeling nanoparticle (NP) hydrophobicity using coarse grained molecular dynamics (CG MD) simulations for water/solid and water/oil/solid systems, and we apply this to modeling the interaction of lipids with nanoparticles. To model at a coarse-grained level the wettability or hydrophobicity of a given material, we choose the Martini coarse-grained force field, and determine the contact angles of polarizable Martini water residing on flat regular surfaces composed of various Martini bead types (C1, C2, etc.), where each surface is composed of a single bead type in each of three crystallographic symmetries (FCC, BCC, and HCP). While this method lumps together atoms (such as one cerium and two oxygens of CeO₂) into a single CG bead, we can still capture the overall hydrophobicity of the actual material by choosing the Martini bead type that gives the best fit of the contact angle to that of the actual material, as determined by either experimental or by all-atom simulations. For five different Martini bead types, the macroscopic contact angle is obtained by extrapolating the microscopic contact angles of droplets of eight different sizes (containing N_w = 3224 to 22978 water molecules) to infinite droplet size. For each droplet, the contact angle was computed from a best fit of a circular curve to the droplet interface extrapolated to the first layer of the surface. We then examine how small nanoparticles of differing wettability interact with Martini dipalmitoyl phosphotidylcholine (DPPC) lipids, which show transitions from tails coating the nanoparticle to hemimicelle coating to water-wet NPs, as the hydrophobicity of the NP increases. The results are relevant to developing a taxonomy describing the nanotoxicity of nanoparticles in the lung.

Lenses in laparoscopes, arthroscopes, and laryngoscopes fog during closed body surgery, due to bodily fluids, gases, humidity and the difference between body and operating room temperatures [1,2]. Fogging obscures surgeon’s vision, so scopes must be repeatedly removed, cleaned, and reinserted for surgery to continue, increasing surgery duration, along with infection risks and scarring due to air exposure from repeated scope removal and reinsertion [1,2]. Methods to address fogging introduce other complications: alcohol-based coatings are too acidic for contact with tissue and quickly evaporate, and many require heating or re-application every 5 to 20 minutes [2]. Our anti-fog coating is a super-hydrophilic, bio-compatible, pH neutral (7.2-7.4) material called VitreOx™ [4]. It overcomes these shortfalls and can be used wet or dry, without using alcohol or heat. Once applied as a wet film, it easily espouses a lens' surface and edges, and dries within seconds into a permanent coating. When dry, it retains the high transparency of wet films while remaining super-hydrophilic, whether used within a minute or after years of application, on silica and polymers.
Nucleation and growth theory applied to condensing films predict three mechanisms for condensation: 2-D (resulting in a flat transparent film), 3-D (resulting in fogging), or mixed [3]. On a hydrophobic surface, such as an optical lens, condensation occurs via spherical 3-D droplets, as predicted by the Volmer-Weber model [3]. Such droplets scatter light in all directions due to their high curvature air/water interface, leading to opaque translucent films commonly called “fog”. Applying VitreOx™ on lenses which are typically hydrophobic renders them super-hydrophilic. Condensation changes, as predicted by the 2-D Frank-Van der Merwe growth model [3], into flat, highly transparent 2-D condensed films. Such 2-D films do not scatter light nor distort optics.
In vitro and in vivo animal studies of VitreOx™ were conducted to measure performance and duration of anti-fog effectiveness and bio-compatibility. Bare lenses, lenses treated with competing anti-fog coatings, and heating were compared side-by side. In vitro testing spanned continuously from 3 to 72 hours and over a 3 years range show that anti-fog effectiveness and optical clarity are continuously maintained for at least 3 years, until removed. In vivo exploratory gastro-endoscopies were conducted with VitreOx^TM on Yucatan swines for 90 minutes without fogging or reapplication, while all other methods lasted at most 35 minutes without fogging. No adverse reaction was observed on the swines and their tissues at the time of surgery and in the 14 months following endoscopy.

[1] Eaton AM et al (1995). Ophthalmology. 102(5):733-736
[2] Lawrentschuk N et al (2010). Journal of Endourology. 24(6):905-913
[3] Bauer, E. (1958).” Zeitschrift für Kristallographie. 110(1-6):372-394
[4] US Patents Filed N. Herbots, C.F. Watson, et al.

Orthopedic implants in the market to bone fracture fixation applications present a great limitation due to the mechanical properties incompatibility with the natural bone, mainly about the Young’s modulus, which is related to the amount of load supported by the material in a mechanical stress. This research aims to develop a new material consisting of a thermoplastic polymer matrix of poly(3-hydroxybutirate) (PHB) loaded with a nanodiamons (ND), vising the confection of orthopedic devices (screws/pins bone fixation) that have the closest human bone mechanical properties.
The results were related to study the effect of the addition of ND in the PHB matrix on the mechanical based on bending, compression and microhardness tests according to ASTM D790, D695 and E384 standards, respectively. The evaluation of the viscoelastic properties and a study on the effect of the addition of ND were made by dynamic mechanical analysis (DMA). In addition, in vitro tests was performed to evaluate the cytotoxicity and the induction of inflammatory response by the biocomposite on the fibroblasts and macrophages cell cultures.

Time-lapse videos of growing biofilms were analyzed using a background subtraction method, which removed camouflaging effects from the heterogeneous field of view to reveal evidence of streamer formation from optically dense biofilm segments. In addition, quantitative measurements of biofilm velocity and optical density, combined with mathematical modeling, demonstrated that streamer formation occurred from mature, high-viscosity biofilms. We propose a streamer formation mechanism by sudden partial detachment, as opposed to continuous elongation as observed in other microfluidic studies. Additionally, streamer formation occurred in straight microchannels, as opposed to serpentine or pseudo-porous channels, as previously reported. Finally, other new analytical tools for monitoring physiochemical propeties of biofilms in microfluidic channels will be presented, including electrochemical measurements and pH imaging.

Dialysis is the process by which an artificial kidney device removes waste and excess water from a patient. The problem with this paradigm is that anything other than the patient’s own body will cause the removal of antigens and proteins, even if the material is biocompatible. Due to the unreactive nature of polytetrafluoroethylene(PTFE), the focus of this project is to determine if less proteins will attach to the new PTFE material during dialysis rather than the traditional material polyvinylchloride (PVC), therefore resulting in minimal protein and antibody loss for the patient. The materials were used side by side and then the protein concentrations were tested using UV Vis spectrophotometry. The objective of using this new polymer PTFE is thought to be able to replace PVC as the current material for the tubing for dialysis machines. Results showed there was a nine percent drop in protein concentration from the PVC over period of time representing a dialysis treatment, conversely this protein was retained by using PTFE.

Silk fibroin from Bombyx mori silkworms is used in diverse applications in materials science due to its high availability, low cost, and rich polymer chemistry. Although silk can be processed into various material classes that are suitable for biological material applications, such as tissue engineering,¹ further enhancement of its properties can be achieved through facile chemical modifications of the amino acid side chains. Such modifications greatly diversify the properties of silk in a minimal number of synthetic steps.

One method to retain the biocompatibility of silk, while enhancing its optical properties is functionalization with azobenzene, yielding a material called AzoSilk.² Tyrosine, an amino acid contained in silk can be converted into azobenzene via traditional diazonium coupling under aqueous conditions. In this study, families of AzoSilk materials have been synthesized by varying the structure of aniline used in the reaction. Subsequently, Azosilk solutions are plated onto plasma-treated petri dishes, and allowed to dry slowly over 5 days. This produced a family of light-responsive water-resistant films for study that can be swelled with water.

Upon examining re-hydrated AzoSilk films under a two-photon microscope, we discovered the appearance of persistent fluorescent patterns within the irradiated areas of the films. The written patterns can be easily visualized by observing the fluorescence emitted at 550 nm, excited by 800 nm. In this microlithographic process, out-of-plane expansion of the film causes micrometre-sized blisters to form near the surface. Micro-blisters were characterized using underwater AFM, and exhibited a 10-fold decrease in modulus. The induced radius of curvature associated with blister formation, together with significant photo-softening are expected to be valuable material characteristics for guided cell growth.³

In order to assess cytotoxicity of hydrated AzoSilk films, they were plated with Chinese hamster ovary cells, and tested with a standard live/dead assay over 3 hours. Sodium sulfanilate-based AzoSilk exhibited the best cell viability(90%+ alive) due to its highly charged surface. Having identified the best surface for cell viability, we currently study the effect of this surface on chick embryo neurons. We notice that surfaces with high acidity provide the best surfaces, due to their high positive charge and thus good adhesion.

To summarize, photo-induced patterning in AzoSilk materials provides optically tunable, biocompatible surfaces that can be photo-softened by laser light irradiation. The patterning of Azosilk films can be controlled through the two-photon process and yields promising surfaces to guide cell growth by providing a tunable radius of curvature of photo-induced features.

References:
1. Kaplan, D.L et al. Nat. Prot. 6, 2011, 1612.
2. Murphy, A.R. et al. Biomaterials, 29, 2008, 2829.
3. Lucido, A.L. et al J. Neuro. 29, 2009, 12466.

Calcium phosphates such as hydroxyapatite have a wide range of applications both in bone graft applications and as coating for the improved integration of implants. Hydroxyapatite is a brittle material with a hexagonally close packed structure, and understanding the failure modes of this crystal at the nanoscale will have significant impact in the application of this biomaterial in medical research. Moreover, understanding the fracture mechanics of hydroxyapatite crystal has a significant effect in its contributions to bone’s mechanical properties. The chemistry of hydroxyapatite is complex, with a series of substitutions possible, giving rise to various impure or calcium deficient apatites.
Here we investigate how the substitution of carbonate groups changes the mechanical properties of hydroxyapatite crystal. In this study, we use density functional theory to find the fracture toughness and unstable stacking fault energy of hydroxyapatite’s basal and prismatic planes, both before and after the carbonate substitution. Using these values we predict changes in hydroxyapatite’s fracture toughness and propensity for slip. Our results indicate that there is an overall decrease in the fracture toughness of hydroxyapatite due to carbonate substitution. Further we find that the unstable stacking fault energy of hydroxyapatite decreases with carbonate substitution, resulting in a greater potential for slip in the basal and prismatic planes. These results imply a loss in hardness for hydroxyapatite after undergoing carbonate substitution.

Nano porous anodic alumina is a widely studied material that is used for corrosion protection of aluminum surfaces or as dielectric material in microelectronics applications. For more than 40 years porous alumina has been the subject of investigations. It exhibits a homogeneous morphology of parallel pores which grow perpendicular to the surface with a narrow distribution of diameters and interpore spacings, the size of which can easily be controlled between 20 and 400 nm. Monodomain porous alumina templates with very high aspect ratios can also be synthesized by using lithographic preparation. The combination of self-assembly and lithography allows the preparation of porous alumina templates with various configurations of pore arrangement that are not accessible by other state-of-the-art methods.
Because of the above mentioned unique properties, nano porous alumina can be used in a wide range of applications, such as filters, as platforms for muliti-functional sensors, for fuels cells, and especially as templates for the fabrication of nanometer-scale composites, such as nanotubes or nanowires by electrochemical deposition or by using polymer melts.
Those polymer nanowires and nanotubes are investigated as novel biomimetic materials for anti-fouling surfaces, self-cleaning surfaces or stongly adhesive properties.

Hereby we report the study and development of graphene-based flexible cutaneous electrodes that are suitable for short- and long-term skin contact for physiological recordings. Nitrogen-doped graphene layers were grown on copper foils by Hot Filament Chemical Vapor Deposition (HFCVD) technique and transferred onto polyimide substrate (Kapton) that contain gold patterned electrodes integrated to PEDOT:PSS (conductive polymer). The performance of these electrodes were assessed at 1 kHz. The graphene electrodes show lower impedance in comparison to Ag/Al-based electrodes, which can be useful for large amounts of medical data that involve long time monitoring periods. The study will demonstrate that nitrogen-doped graphene remains stable, inert, and suitable for wearable medical devices intended for long-term electrophysiological recordings.

Alcohols and phenolic compounds are utilized as an alternative means of modifying silicon oxide surfaces. Silane based organic compounds and other compounds have been extensively used for modifying silicon oxide surfaces. Moisture control is challenging, yet vital in silane based surface chemistry. We have successfully developed alternative strategies for surface modification of silicon oxide surfaces using alcohol and phenolic compounds. The strategy includes microwave radiation of silicon oxide substrates immersed in a neat solution of alcohols or phenolic compounds. Silane chemistry requires control over moisture in the environment, while such control is unnecessary for reactions with alcohols or phenolic compounds. Other advantages of using alcohols and phenolic compounds for modifying properties of silicon oxide surfaces include their widespread availability and ease of handling. Also, alcohols and phenolic compounds form a strong covalent bond with the silicon oxide surfaces (i.e. silyl ether), which results in an excellent long-term stability when immersed in organic solvents, acids, and bases. The strategy introduced in this research can be incorporated into conventional manufacturing processes to create functionalized surfaces, such as silicon oxides and other oxides, towards the fabrication of biological sensors and implantable devices.

Self-setting bone pastes can be utilized for minimally invasive surgery and can fit to the complicated shape of bone defects easily; however, presently practically available pastes are hardened via transformation of calcium phosphates into hydroxyapatite which has weak mechanical strength and no or low biodegradability. Therefore, surgeons desire biodegradable/bioresorbable self-setting bone paste. A hydroxyapatite/collagen bone like nanocomposite (HAp/Col) porous body has been clinically applied in Japan as a bioresrobable bone filler, because the HAp/Col is incorporated into bone remodeling process. A self-setting HAp/Col paste could be a good candidate for the next generation bone void filler. In this study, 1.0 or 10 % aqueous solution in volume of (3-glycidoxypropyl)trimethoxysilane (GPTMS), which works as a cross-linker of collagen via amino groups and forms siloxane network by self-condensing after hydrolysis, was mixed with the HAp/Col powder to prepare the HAp/Col biodegradable self-setting pastes, and their mechanical and biological properties were investigated.
The HAp/Col at the hydroxyapatite and collagen mass ratio of 80:20 was synthesized by the simultaneous titration method. The HAp/Col powder, a powder phase of HAp/Col self-setting bone pastes, was prepared by a uniaxial compacting dehydration, freeze-drying, crushing and classifying to 100 μm or less in particle size. Liquid phase of the pastes was 1.0 or 10 % GPTMS aqueous solution 1 hour after mixing, to allow hydrolysis of GPTMS. The powder/liquid (P/L) ratio of the pastes was 1.00 g/cm³, which would be optimal condition for the paste anti-decay. The viscosity of the HAp/Col pastes fabricated was measured by according to follows, a total 2 kg of glass plate and weight were placed on 0.1 cm³ of the cylindrical paste 10 minutes after mixing started, and the spread area was measured at 10 minutes after placing the weight as its viscosity. Biological reactions of the pastes were investigated by the implantation in porcine tibia. Cylindrical defects of 4.0 mm in diameter were formed by surgical drill. The pastes, prepared on site, were then injected into the defects. After 12 weeks, histological sections of the paste and surrounding tissues were observed to evaluate its biodegradability and biocompatibility.
Viscosity of the paste with 10 % GPTMS solution was larger than that 1.0 %. Rapid siloxane network formation in the paste prepared with 10 % GPTMS than 1.0 % would cause increasing in solution viscosity. The results of histological analysis for the HAp/Col-GPTMS pastes will be presented on a podium.

To act as a mechanical barrier in guided bone regeneration (GBR) techniques, the adhesiveness is a required property to the biomaterial-body interface remain stable without biologic rejection until complete bone regeneration. In this work natural rubber (NR) obtained from the Hevea brasiliensis tree composed of poly-isoprene rubber-matrix with Hevein proteins (Hev. b1 and Hev. b3) particles was processed to obtain sustainable membranes. Calcium phosphate (CaP) micro grains with multi-crystalline phases, including Ca₅P₈ (47.8 wt. %), CaCO₃(36.3 wt.%), Ca(PO₄)₃OH (9.5 wt.%) and Ca (6.3 wt.%), obtained by sol-gel, were incorporated in the polymeric matrix to produce a functionalized surface. The structural and vibrational characterization by confocal spectrum-microscopy probing shows the CaP particles trapped by proteins on surfaces. As first finding of the surface properties, the morphology was changed. Thus, all aspects of the wettability were studied in detail to establish a relation with the roughness and free energy. The wettability effects were observed in different dynamic mode by monitoring the contact line and the contact angle. As main result, an induced pinning at interface is observed by two experimental ways. This phenomenon increases the adhesive forces at interface in 60%. In the final, the induced pinning is discussed taking account the possible consequences in the first stage of bone regeneration.

Planar array infrared (PAIR) spectroscopy was used in this study to probe the thermally reversible gelation of solutions of bio-based, biodegradable poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (Nodax) to gain insight into the time-dependent evolution of the gel micro-structure. Analysis of the time-resolved infrared spectra was performed using two dimensional correlation spectroscopy to determine the origin of the gel micro-structure. PAIR spectroscopy was vital in this investigation for its very fast sampling of infrared spectra. Although the fundamental driving force of the gelation is a thermal transition in nature, the development of the gel structure is kinetic, meaning that once the gel solution has cooled, it takes time for the solution to become a gel. The growth of said structure occurs in a rapid, step-wise fashion that can only be observed through a very fast sampling, which traditional Fourier transform infrared spectroscopy (FTIR) cannot adequately achieve. 2D correlation spectroscopy allowed the subtle evolution of spectra during the gelation, which can be difficult to observe, to be analyzed in depth and easily compared.

The concept of bone replacement has been dominant in the development of metallic implants and the biodegradable metals with the potential to stimulate bone formation have recently captured the attention of scientists. Among these metals, magnesium alloy is an attractive biodegradable material with unique set of properties. The corrosion of magnesium is accompanied by hydrogen evolution and a local increase in pH, which impose constraints on many potential biomedical applications. We have overcome these limitations and created a road map to the next generation of metallic biodegradable implant materials with the addition of completely biocompatible elements. Along with the addition of Ca, which is a biocompatible element that plays major in bone formation and remodeling, excellent material properties were achieved through the in-house built special mechanical extrusion machine. The state of the art method to synchronize the corrosion potentials of two constituent phases (Mg + Mg2Ca) with the selective doping of Zn into Mg2Ca was developed to control the corrosion rate. Furthermore, mechanical extrusion broke the connectivity of the Mg2Ca phases, which prevented continuous corrosion and the formation of a galvanic circuit that caused severe corrosion of the Mg-Ca alloy. Animal studies confirmed the large reduction in hydrogen evolution and revealed good tissue compatibility with increased bone deposition around the newly developed Mg alloy implants. Newly developed set of KIST magnesium alloys have the mechanical strength, ability to stimulate bone growth and controlled slow degradation rate to be considered as an ideal candidate for biodegradable implant applications.
With all the extensive in vivo experience using developed magnesium alloy material over the past 3 years, we were able to receive an approval from Korea Food and Drug Administration (KFDA) for the first human clinical trial of orthopedic biodegradable metallic implant devices. Working closely with major hospitals in Korea, we have performed upto 100 cases of small bone fixation screws last year.
This presentation will cover the entire development process of the orthopedic biodegradable implant material and long-term clinical study result of 53 cases.

In recent years, the application of magnesium in biomedical use has been getting more and more attention. Regarded as a promising bio-compatible material, magnesium has both good bio-degradability and sufficient mechanical properties, which allow the applications in not only drug delivery vehicles, but also implants including temporary anchorage devices and artificial joints. However, when serving as an implant, magnesium has a highly active chemical property and tends to suffer a high rate corrosion that is generally accompanied with observable hydrogen evolution^[1]. In order to control the degradation rate to a proper value, surface modification is thus indispensable.

In previous studies about magnesium in biomedical applications, many different kinds of surface modification processes have been investigated, including magnesium oxide coatings formed using micro-arc oxidation (MAO) and hydroxyapatite (HA) coatings deposited through electrodeposition or conversion coating process. Nonetheless, these coatings have either no biological activity or comparable biodegradability, and may be not the best solution for biodegradable magnesium alloys. Amongst various potential replacements, one candidate standing out is polyaspartic acid (PAS). PAS is a biodegradable polyaminoacid used in many different applications including corrosion inhibition, and the its effect on both HA deposition and dentin remineralization has also been disclosed in the past few years^[2]. To synthesis PAS, the key precursor polysuccinimide (PSI) is required, yet the preparation is usually complicated and expensive.

In 2012, Jiang et al. presented a new approach to produce a copolymer called poly(succinimide-co-citramide) (PSICA), which has the molecular structure of PSI, through a solvent-free condensation reaction between hexamethylene (HDMA) and citrate ester^[3]. In this present study, the same approach was used to form a PSICA top coating on AZ31 magnesium alloys with or without a citrate intermediate layer. The corrosion properties of polymer-coated AZ31 in both chloride-containing solution and simulated body fluid were investigated using electrochemical impedance spectroscopy (EIS). As shown in the results, the PSICA coating elevated the total impedance, which indicates a slower corrosion rate and decelerated hydrogen evolution. Besides, as the immersion time increased, the change in impedance of coated AZ31 was smaller than that of untreated AZ31, suggesting that the degradation of PSICA coating itself can help maintaining the degradation of magnesium coated underneath in a relatively stable rate.

Reference:
1. G. Song, Corros. Sci., 49(4), 1696 (2007)
2. S.Z. Xu, X.Y. Yang, X.Y. Chen, X.J. Lin, L. Zhang, G.J. Yang, C.Y. Gao, and Z.R. Gou, Biomed. Mater., 6, 035002 (2011)
3. M. Jiang, W. Chen, P. Fang, S.L. Chen, J.X. Bai, Y.F. Huang, M.W. Han, P. Lu, and J. Dong, J. Polym. Sci., Part A: Polym. Chem, 50, 3819 (2012)

Magnesium and its alloys have excellent physical and mechanical properties for a wide range of applications. Their tunable degradability may benefit the use of temporary implants in biomedical applications, avoiding extraction surgery sometimes required with non-biodegradable implants. In order to optimize the use of magnesium alloys for such applications, the rate of their degradation in vivo must be controlled and carefully chosen. The use of functional implant coatings can be a promising way to gain such control. This paper describes the growth, characterization and corrosion analyses of nanoporous alumina (Al₂O₃) functional coatings which can slow down the degradation of Mg in vivo. Alumina thin films were deposited by reactive pulse DC magnetron sputtering. The structural and optical properties of alumina films deposited at different partial oxygen pressures were studied with XRD, FE-SEM, AFM and optical microscopy. A bilayer Mg-Al₂O₃ system was used as a model for evaluating the sustainability of Al₂O₃ thin films on Mg surface. In order to evaluate resorption time of alumina thin films, immersion tests were performed on Mg-Al₂O₃ systems with varying alumina film thickness in the range 10-100 nm. The tests were performed at room temperature with deionized water, phosphate buffered saline, cell adhesion assay media and 0.1 - 2.0 % NaCl solutions in water. The results of these tests indicate that nanosized porosity of non-degradable alumina thin films controls the resorption rate of these corrosion-protective coatings. These coatings may therefore be used to control delay of degradation of fast degrading implants, such as magnesium implants, to benefit the post-surgery recovery process.

Degradable, transient orthopaedic implants have been proposed for years, with the goal to replace permanent biomaterials that are left in the body indefinitely or that have to be removed via surgical procedures. Current resorbable implant designs either degrade too quickly, injuring surrounding tissue while losing necessary mechanical strength before full tissue reconstruction, or degrade too slowly, thereby acting like a permanent implant. One of the biggest issues in metallic resorbability is controlling the ever-changing interface between the degrading implant and newly-grown tissue. The present researchers are investigating the use of degradable biocomposites such as FeMn+Hydroxyapatite to replace more inert implants with nontoxic, transient ones. Microporosity was achieved through the use of NaCl-leaching of bulk FeMn samples, which can be tailored to a wide variety of conditions to alter initial cell attachment and overall degradation rate. Additionally, hydroxyapatite was integrated into the alloy to aid in encouraging bone ingrowth while stabilizing the matrix. A thirty day corrosion study on nine various FeMn-based materials was conducted in vitro. Results will be discussed of the effects of these unique bioactive surfaces on bone marrow stromal cells attachment and overall degradation.

Inspired by their unique biodegradable properties, magnesium and their alloys have received extensive attentions as a candidate material for bone-fixation devices and stents. However, electrons and hydrogen gas generated from the corrosion of such metals have an adverse effect on human body, thus significantly limiting their practical usage in clinical setting. In this study, we developed a novel strategy that utilizes these electrons for the generation of the reactive-oxygen-species (ROS), which is one of the pivotal signal molecules in various physiological events.
In the first section, we presented a new type of electrochemical system using biodegradable metals. Similar to the chemical reaction in the primary battery, electrons generated from the corrosion of the biodegradable magnesium anode were transferred to the nanostructured titanium cathode. The transferred electrons were utilized for converting oxygen molecules near the cathode to ROS molecules by oxygen-reduction-reaction (ORR). Combining spectroscopic and electrochemical analyses, we found that hydrogen peroxide (H₂O₂) can be spontaneously generated by the corrosion of the metal. By precisely tuning the material parameters and discharging time, we could control the released amount of H₂O₂ from 1 uM to 100 uM. Interestingly, the controlled release of H₂O₂ (~ 10 uM ) from our electrochemical system apparently enhanced in-vitro angiogenesis, even in the condition where no growth factor exists. It is noteworthy that our system can support the angiogenesis, which is one of the core prerequisites in tissue regenerative engineering, comparable to positive results with growth factors. Finally, by combining magnesium anode in the titanium bone-fixation devices, we developed integrated type bone-fixation devices that can support angiogenesis.
In the following section, we applied our electrochemical system for stent application. Because the re-coarctation have been one of the most imminent problems in stent-applications, previous efforts have been made on increasing the proliferation of HUVECs and simultaneously decreasing that of smooth muscle cells (SMCs). Combining a small amount of biodegradable metals on the metal stent, we could precisely tune the released amount of H₂O₂, and finally we were successful at finding the optimum H₂O₂ concentration where the proliferation of HUECS increased, while that of smooth muscle cells (SMC) decreased. Additionally, long-term fatigue tests showed that our metal-based system is superior to conventional polymer-metal hybrid one.
Ultimately, we applied corrosion of biocompatible metals to generate ROS near the implantable devices. Conventional devices could be functionalized to improve tissue engineering aspect of it by utilizing our electrochemical system. We believe that our approach can be further extended to versatile biodegradable metal platforms.

Post-surgical site infections are common after medical implant placement. Infections in tissue surrounding an implant can cause patient suffering, medical device failure, and can potentially spread systematically. Post-operational infection associated with orthopedic implants is a critical and escalating problem which demands urgent attention for a decrease of occurrences. Implant related infections can require a patient to undergo additional surgeries following the initial implant placement surgery. Another challenge that exists for implant placement, due to orthopedic injuries, is tissue integration. Each year, there are more than 30,000 revision surgeries partially due to poor orthopedic implant fixation with bone. In order to combat infection, biomaterials and functional coatings used for medical implants are evaluated either for their ability to resist infection (resist bacterial adhesion and biofilm formation) or for their ability for tissue integration (to support tissue cell adhesion and proliferation). No viable clinical technology currently exists to address both these issues simultaneously. Our hypothesis is that laser micro-nano machining can create surface topographies on orthopedic implant surfaces that could provide a platform for simultaneous tissue integration and therapeutic delivery for biofilm prevention. This study explores the role of laser micro-nano machining in creating surface topographies on an orthopedic relevant bio-metal, cobalt chromium (Co-Cr) alloy. Co-Cr alloys are extensively used for orthopedic and dentistry applications. Laser modified Co-Cr samples were compared to other laser modified bio-metals, such as titanium. Co-Cr alloy and Ti were cut into 1cm x 1cm squares and were then modified using a nanosecond pulsed laser. A CoherentTM Avia 355X nanosecond pulsed laser with pulse energy of 95 µJ, spot size of 130 µm, line width of 100 µm, scan rate of 200 mm/min, and repetition rate of 20 kHz was used to raster scan the coupons with an overlap of 23%. A lens with a focal length of 10 cm was used for the experiments with the actual experimentation done at a defocused distance of 0.5 mm. Bare metal (control) and surface modified samples were characterized using optical microscopy and scanning electron microscopy (SEM). Optical and SEM results clearly show a radically different surface morphology for laser patterned samples when compared to control samples. Since laser parameters were kept constant, response varied for each material type. At 300X magnification, SEM results clearly show a ~100 µm width of the raster patterned zone of varying geometry. Ti seems to have a more uniform surface pattern when compared to Co-Cr alloy suggesting a need to further optimize Co-Cr alloy laser parameters. In summary, our results demonstrate the effect of laser treatment in creating micro-nano structured surface topographies on Ti and Co-Cr alloy which can be subsequently modified to address current orthopedic clinical needs.

Metallic biomedical devices with nanometer-sized grains (NGs) provide surfaces that are different from their coarse-grained (CG) (tens of micrometer) counterparts in terms of increased fraction of grain boundaries (NG > 50%; CG