Comparison of Different Approaches to Surface Functionalization of Biodegradable Polycaprolactone Scaffolds.

Due to their good mechanical stability compared to gelatin, collagen or polyethylene glycol nanofibers and slow degradation rate, biodegradable poly-ε-caprolactone (PCL) nanofibers are promising material as scaffolds for bone and soft-tissue engineering. Here, PCL nanofibers were prepared by the electrospinning method and then subjected to surface functionalization aimed at improving their biocompatibility and bioactivity. For surface modification, two approaches were used: (i) COOH-containing polymer was deposited on the PCL surface using atmospheric pressure plasma copolymerization of CO2 and C2H4, and (ii) PCL nanofibers were coated with multifunctional bioactive nanostructured TiCaPCON film by magnetron sputtering of TiC-CaO-Ti3POx target. To evaluate bone regeneration ability in vitro, the surface-modified PCL nanofibers were immersed in simulated body fluid (SBF, 1×) for 21 days. The results obtained indicate different osteoblastic and epithelial cell response depending on the modification method. The TiCaPCON-coated PCL nanofibers exhibited enhanced adhesion and proliferation of MC3T3-E1 cells, promoted the formation of Ca-based mineralized layer in SBF and, therefore, can be considered as promising material for bone tissue regeneration. The PCL-COOH nanofibers demonstrated improved adhesion and proliferation of IAR-2 cells, which shows their high potential for skin reparation and wound dressing.

Structure and antibacterial properties of Ag-doped micropattern surfaces produced by photolithography method.

Photolithography methods offer ample opportunities for creating biological surface patterns over large areas. Herein, samples with patterned surface having the same Ag total coverage area and content, but different surface topography made of periodically spaced Ag/Si pillars with a diameter of 10 and 50 µm and a height of 3, 1, and 0.2 µm were produced by photolithography technique and studied to uncover the dependences of bactericide ion release on surface topography and antibacterial effect on Ag+ ion concentration. Reactive ion etching of Si wafers in areas unprotected by Ag capping layer was accompanied by a number of competing processes: (i) formation of Ag particles on the tops of pillars due to temperature-activated diffusion and coalescence, (ii) sputtering of Ag from the pillar to surface and redeposition into the etching cavities, resulting in the formation of small Ag nanoparticles located in areas between pillars, (iii) precipitation of AgSix phase as a result of chemical interaction of sputtered Si ions with Ag ions and atoms in surrounding plasma. Samples with the largest pillar heights which had also Ag particles formed between pillars demonstrated the fastest Ag+ ion release and, correspondingly, a noticeable antibacterial effect toward antibiotic-resistant hospital Escherichia coli K-261 strains already after 3 h. All samples showed 100% antibacterial effect after 24 h. Thus our results open up new possibilities for the production of scalable micropattern surfaces with controlled bactericide ion release and pronounced antibacterial characteristics for future applications in the orthopedic field.

Ultrasharp h-BN Nanocones and the Origin of Their High Mechanical Stiffness and Large Dipole Moment.

We report on experimental synthesis and theoretical studies of ultrasharp BN-nanocones. Using scanning and transmission electron microscopy, the cone-like morphology of synthesized products was confirmed. Theoretical analysis of the dipole moment nature in h-BN nanocones reveals that the moment has contributions from the polarity of B-N bonds and electronic flexoelectric effect associated with a curved h-BN lattice. The latter phenomenon is predicted on the basis of the extension of the theory of flexoelectric effects in the h-BN lattice through establishing universality of the linear dependence of flexoelectric dipole moments on local curvature in various nano- h-BN networks (nanotubes and fullerenes). Our study of the atomic structure response and its polarization under deformation of nanocones with different apex angles shows the advantageous properties of cones with the smallest angles.

Antibacterial Performance of TiCaPCON Films Incorporated with Ag, Pt, and Zn: Bactericidal Ions Versus Surface Microgalvanic Interactions.

It is very important to prevent bacterial colonization at the early postoperative stages. There are four major strategies and their corresponding types of antibacterial surfaces specifically designed to fight infection: bactericide release, anti-adhesion, pH-sensitive, and contact-killing. Herein, we aimed at determining the antibacterial efficiency of different types of bactericidal ions and revealing the possible contribution of surface microgalvanic effects arising from a potential difference on heterogeneous surfaces. We considered five types of TiCaPCON films, with Ag, Zn, Pt, Ag + Zn, and Pt + Zn nanoparticles (NPs) on their surface. The Ag-modified film demonstrated a pronounced antibacterial effect at a very low Ag ion concentration of 0.11 ppb in physiological solution that was achieved already after 3 h of immersion in Escherichia coli ( E. coli) bacterial culture. The Zn-containing sample also showed a noticeable antibacterial effect against E. coli and Staphylococcus aureus ( S. aureus) strains, wherein the concentration of Zn ions was 2 orders of magnitude higher (15 ppb) compared with the Ag ions. The presence of Ag NPs accelerated the leaching of Zn ion out of the TiCaPCON-Ag-Zn film, but no synergistic effect of the simultaneous presence of the two bactericidal components was observed. After the incubation of the samples with Ag, Zn, and Ag + Zn NPs in E. coli and S. aureus suspensions for 24 and 8 h, respectively, all bacterial cells were completely inactivated. The Pt-containing film showed a very low Pt ion release, and therefore the contribution of this type of ions to the total bactericidal effect could be neglected. The results of the electrochemical studies and Kelvin probe force microscopy indicated that microgalvanic couples were formed between the Pt NPs and the TiCaPCON film, but no noticeable antibacterial effect against either E. coli or S. aureus strains was observed. All ion-modified samples provided good osteoblastic cell attachment, spreading, and proliferation and therefore were concluded to be nontoxic for cells. In addition, the TiCaPCON films with Ag, Pt, and Zn NPs on their surface demonstrated good osteoconductive characteristics.

Synergistic and long-lasting antibacterial effect of antibiotic-loaded TiCaPCON-Ag films against pathogenic bacteria and fungi.

Implant-related bacterial infections remain a serious problem that is not solved yet. Herein we combined several antibacterial agents to achieve synergistic effects and broader protection of widely used metallic implants. Titanium samples with microcontainers for drug, produced by selective laser sintering, were coated with Ag-doped biocompatible and bioactive TiCaPCON film and loaded with an antibiotic (gentamicin or a mixture of gentamicin and amphotericin B). Bactericide release tests demonstrated that the release rate of one bactericide agent (Ag+ ions or gentamicin) depended on the presence of the other antibacterial component. The antibacterial activity of the biocide-doped samples was evaluated against clinically isolated Escherichia coli O78 (E. coli), Staphylococcus aureus (S. aureus) bacteria, and Neurospora crassa wt-987 (N. crassa) spores. It was found that samples loaded with low gentamicin concentration (0.2 and 0.02 mg/cm2), i.e. 10 and 100 times less than the standard gentamicin concentration (2 mg/cm2), demonstrated a superb antibacterial activity against E. coli bacteria. We showed that a crosslinking reaction between gentamicin and TiCaPCON film proceeded either through the formation of amide bonds or via the electrostatic interaction between amine groups of gentamicin and COOH groups of TiCaPCON and led to the formation of relatively stable drug/film conjugates that prevented a rapid dissolution of gentamicin and ensured its long-term (for 72 h) antibacterial protection. Leaching of silver ions provided an effective antibacterial protection after the depletion of the drug reservoirs. The obtained results clearly show a synergistic antibacterial action of Ag+ ions and gentamicin against S. aureus bacteria. In addition, in the presence of Ag+ ions, the antifungal activity of samples loaded with a mixture of gentamicin and amphotericin B against N. crassa fungus was observed to increase. Thus, it is demonstrated that silver can be successfully coupled with different types of antibiotics to provide innovative hybrid metal-ceramic bioconstructions that are able to deliver precise doses of bactericide agents within a certain period of time and are equally effective against Gram-negative E. coli bacteria, Gram-positive S. aureus, and N. crassa fungus.

Mechanical properties of decellularized extracellular matrix coated with TiCaPCON film.

For the first time the surface of decellularized extracellular matrix (DECM) was modified via deposition of a multicomponent bioactive nanostructured film for improvement of the DECM's mechanical properties. TiCaPCON films were deposited onto the surface of intact and decellularized ulna, radius, and humerus bones by magnetron sputtering of TiC0.5 + 10%Ca3(PO4)2 and Ti targets in a gaseous mixture of Ar + N2. The film structure was studied using x-ray diffraction, scanning and transmission electron microscopy, and Raman spectroscopy. The films were characterized in terms of their wettability, as well as adhesion strength to the intact bone and DECM substrates. The mechanical properties of TiCaPCON-coated samples were investigated by compression testing. In addition, humerus bones were evaluated during three-point bending tests. The results indicate that the tightly adhered films, uniformly covering the DECM surfaces, possessed hydrophilic characteristics. A maximum improvement in mechanical properties (250%) was observed for coated humerus samples. In case of decellularized radius bones, the compressive strength also increased by 150% after coating. The positive role of TiCaPCON films was less noticeable for ulna bones because of large data scattering. These results clearly indicate that the films acted as a rigid frame that increased the material compressive strength. Compared with intact bones, fracture in the TiCaPCON-coated DECM samples was characterized by rarer and larger cracks generated under higher critical loads. As a result, the samples were crushed into several large pieces and numerous tiny fragments. Although the film deposition increased the bone stiffness, the bending tests revealed that the flexural strength of the coated samples became 20%-25% lower than the strength of the film-free samples.

Approaches for Controlled Ag+ Ion Release: Influence of Surface Topography, Roughness, and Bactericide Content.

Silver is the most famous bactericidal element known from ancient times. Its antibacterial and antifungal effects are typically associated with the Ag ionization and concentration of Ag+ ions in a bacterial culture. Herein we thoroughly studied the influence of surface topography and roughness on the rate of Ag+ ion release. We considered two types of biocompatible and bioactive TiCaPCON-Ag films with 1 and 2 at. % of Ag and nine types of Ti surfaces with an average roughness varying in the range from 5.4 × 10-2 to 12.6 µm and different topographic features obtained through polishing, sandblasting, laser treatment, and pulsed electrospark deposition. It is demonstrated that the Ag+ ion release rates do not depend on the Ag content in the films as the main parameter, and it is other factors, such as the state of Ag agglomeration, surface topography and roughness, as well as kinetics of surface oxidation, that play a critical role. The obtained results clearly show a synergistic effect of the Ag content in the film and surface topography and roughness on Ag+ ion release. By changing the surface topographical features at a constant content of bactericidal element, we showed that the Ag+ ion release can be either accelerated by 2.5 times or almost completely suppressed. Despite low Ag+ ion concentration in physiological solution (<40 ppb), samples with specially fabricated surface reliefs (flakes or holes) showed a pronounced antibacterial effect already after 3 h of immersion in E. coli bacterial culture. Thus, our results open up new possibilities for the production of cost-effective, scalable, and biologically safe implants with pronounced antibacterial characteristics for future applications in the orthopedic field.

In vitro bioactivity study of TiCaPCO(N) and Ag-doped TiCaPCO(N) films in simulated body fluid.

Bioactivity of multicomponent TiCaPCO(N) and Ag-doped TiCaPCO(N) films was evaluated in vitro using simulated body fluid (SBF) and compared with that of bioactive glass Biogran. The first group of films was fabricated by magnetron sputtering of composite TiС0.5 -Ti3 POx -CaO target produced via the self-propagating high-temperature synthesis (SHS) method (TiCaPCON films), after which their surface was implanted with Ag+ ions to obtain Ag-doped TiCaPCON films. The second group of films was fabricated by pulsed electrospark deposition (PED) using SHS-produced composite TiС0.5 -Ti3 POx -CaO and TiС0.5 -Ti3 POx -CaO-Ag electrodes. After immersion in SBF, the structure and chemistry of surface were well characterized using a combination of various microanalytical techniques, such as scanning electron microscopy, X-ray diffractometry (both in conventional and grazing incidence mode), Fourier transform infrared spectroscopy, Raman spectroscopy, and glow discharge optical emission spectroscopy. The results showed that the surfaces of the TiCaPCO(N) and Ag-doped TiCaPCO(N) films were bioactive in vitro and induced the formation of an apatite layer during exposure in SBF. In the case of the magnetron-sputtered films, the apatite layer was formed over 14 days, while 28 days were needed to form CaP phase on the surface of PED-modified samples. Various factors (film structure, surface roughness, surface functional groups, surface charge, and composition, supersaturation, and near-surface local supersaturation of SBF) affecting the kinetics of bone-like apatite formation on a bioactive surface are discussed. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 193-203, 2017.

Mechanical properties and current-carrying capacity of Al reinforced with graphene/BN nanoribbons: a computational study.

Record high values of Young's modulus and tensile strength of graphene and BN nanoribbons as well as their chemically active edges make them promising candidates for serving as fillers in metal-based composite materials. Herein, using ab initio and analytical potential calculations we carry out a systematic study of the mechanical properties of nanocomposites constructed by reinforcing an Al matrix with BN and graphene nanoribbons. We consider a simple case of uniform distribution of nanoribbons in an Al matrix under the assumption that such configuration will lead to the maximum enhancement of mechanical characteristics. We estimate the bonding energy and the interfacial critical shear stress at the ribbon/metal interface as functions of ribbon width and show that the introduction of nanoribbons into the metal leads to a substantial increase in the mechanical characteristics of the composite material, as strong covalent bonding between the ribbon edges and Al matrix provides efficient load transfer from the metal to the ribbons. Using the obtained data, we apply the rule of mixtures in order to analytically assess the relationship between the composite strength and concentration of nanoribbons. Finally, we study carbon chains, which can be referred to as the ultimately narrow ribbons, and find that they are not the best fillers due to their weak interaction with the Al matrix. Simulations of the electronic transport properties of the composites with graphene nanoribbons and carbyne chains embedded into Al show that the inclusion of the C phase gives rise to deterioration in the current carrying capacity of the material, but the drop is relatively small, so that the composite material can still transmit current well, if required.

Characteristics and in vitro response of thin hydroxyapatite-titania films produced by plasma electrolytic oxidation of Ti alloys in electrolytes with particle additions.

The enhancement of the biological properties of Ti by surface doping with hydroxyapatite (HA) is of great significance, especially for orthodontic applications. This study addressed the effects of HA particle size in the electrolyte suspension on the characteristics and biological properties of thin titania-based coatings produced on Ti-6Al-4V alloy by plasma electrolytic oxidation (PEO). Detailed morphological investigation of the coatings formed by a single-stage PEO process with two-step control of the electrical parameters was performed using the Minkowski functionals approach. The surface chemistry was studied by glow discharge optical emission spectroscopy and Fourier transform infrared spectroscopy, whereas mechanical properties were evaluated using scratch tests. The biological assessment included in vitro evaluation of the coating bioactivity in simulated body fluid (SBF) as well as studies of spreading, proliferation and osteoblastic differentiation of MC3T3-E1 cells. The results demonstrated that both HA micro- and nanoparticles were successfully incorporated in the coatings but had different effects on their surface morphology and elemental distributions. The micro-particles formed an irregular surface morphology featuring interpenetrated networks of fine pores and coating material, whereas the nanoparticles penetrated deeper into the coating matrix which retained major morphological features of the porous TiO2 coating. All coatings suffered cohesive failure in scratch tests, but no adhesive failure was observed; moreover doping with HA increased the coating scratch resistance. In vitro tests in SBF revealed enhanced bioactivity of both HA-doped PEO coatings; furthermore, the cell proliferation/morphometric tests showed their good biocompatibility. Fluorescence microscopy revealed a well-organised actin cytoskeleton and focal adhesions in MC3T3-E1 cells cultivated on these substrates. The cell alkaline phosphatase activity in the presence of ascorbic acid and ß-glycerophosphate was significantly increased, especially in HA nanoparticle-doped coatings.

Toward bioactive yet antibacterial surfaces.

The fabrication of antibacterial yet biocompatible and bioactive surfaces is a challenge that biological and biomedical community has faced for many years, while no "dream material" has been developed so far. The primary goal of this study was to establish an optimal range of Ag concentration and its state of agglomeration in bioactive nanocomposite TiCaPCON films which would provide a strong bactericidal effect without compromising the material biocompatibility and bioactivity. To obtain samples with different Ag content and redistribution, two different methods were employed: (i) TiCaPCON films deposition by magnetron sputtering of composite TiС0.5-Ca3(РО4)2 target followed by Ag(+) ion implantation and (ii) Ag-doped TiCaPCON films obtained by co-sputtering of composite TiС0.5-Ca3(РО4)2 and Ag targets. In order to reveal the antibacterial role of Ag nanoparticles and Ag(+) ions, both separate and in synergy, part of the samples from the first and second groups was subjected to additional ion etching to remove an Ag rich surface layer heavily populated with Ag nanoparticles. All resultant films were characterized with respect to surface morphology, chemical composition, surface roughness, wettability, and Ag(+) ion release. The antibacterial and antifungal effects of the Ag-doped TiCaPCON films were evaluated against clinically isolated Escherichia coli O78 (E. coli) and Neurospora crassa wt-987 spores. The influence of the surface chemistry on spreading, proliferation, and early stages of MC3T3-E1 osteoblastic cell differentiation was also studied. Our data demonstrated that under optimal conditions in terms of Ag content and agglomeration, the Ag-doped TiCaPCON films are highly efficient against E. coli bacteria and, at the same time, provide good adhesion, spreading, proliferation and differentiation of osteoblastic cells which reflect high level of biocompatibility and bioactivity of the films. The influence of Ag(+) ions and nanoparticles on the MC3T3-E1 osteoblastic cells and E. coli bacteria is also discussed.

Fabrication method, structure, mechanical, and biological properties of decellularized extracellular matrix for replacement of wide bone tissue defects.

The present paper was focused on the development of a new method of decellularized extracellular matrix (DECM) fabrication via a chemical treatment of a native bone tissue. Particular attention was paid to the influence of chemical treatment on the mechanical properties of native bones, sterility, and biological performance in vivo using the syngeneic heterotopic and orthotopic implantation models. The obtained data indicated that after a chemical decellularization treatment in 4% aqueous sodium chlorite, no noticeable signs of the erosion of compact cortical bone surface or destruction of trabeculae of spongy bone in spinal channel were observed. The histological studies showed that the chemical treatment resulted in the decellularization of both bone and cartilage tissues. The DECM samples demonstrated no signs of chemical and biological degradation in vivo. Thorough structural characterization revealed that after decellularization, the mineral frame retained its integrity with the organic phase; however clotting and destruction of organic molecules and fibers were observed. FTIR studies revealed several structural changes associated with the destruction of organic molecules, although all organic components typical of intact bone were preserved. The decellularization-induced structural changes in the collagen constituent resulted changed the deformation under compression mechanism: from the major fracture by crack propagation throughout the sample to the predominantly brittle fracture. Although the mechanical properties of radius bones subjected to decellularization were observed to degrade, the mechanical properties of ulna bones in compression and humerus bones in bending remained unchanged. The compressive strength of both the intact and decellularized ulna bones was 125-130 MPa and the flexural strength of humerus bones was 156 and 145 MPa for the intact and decellularized samples, respectively. These results open new avenues for the use of DECM samples as the replacement of wide bone tissue defects.

Multifunctional Ti-(Ca,Zr)-(C,N,O,P) films for load-bearing implants.

Films of Ti-Ca-P-C-O-(N), Ti-Ca-C-O-(N) and Ti-Zr-C-O-(N) were deposited by DC magnetron sputtering or ion implantation-assisted magnetron sputtering of composite targets TiC0.5 + 10%Ca10(PO4)6(OH)2, TiC0.5 + 20%(CaO + TiO2) and TiC0.5 + 10%ZrO2 in an Ar atmosphere or reactively in a gaseous mixture of Ar + 14%N2. The microstructure, elemental and phase composition of films were studied by means of X-ray diffraction, transmission electron microscopy, scanning force microscopy, X-ray photoelectron spectroscopy and energy-dispersive X-ray spectroscopy. The films were characterized in terms of their hardness, Young's modulus, elastic recovery, adhesion strength, and friction and wear both in air and under physiological solution. Particular attention was paid to the analysis of deformation and fracture for various film/substrate systems during scratch testing. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of adhesion, spreading, and proliferation of MC3T3-E1 osteoblasts and IAR-2 epithelial cells, morphometric analysis, actin cytoskeleton, focal contacts staining, alkaline phosphatase activity and von Kossa staining of osteoblastic culture. Cell culture experiments demonstrated an increase of osteoblastic proliferation on Ca- and P-incorporated films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of adherent cells on their surfaces. The results obtained show that multicomponent nanostructured Ti-(Ca, Zr)-(C, N, O, P) films possess a combination of high hardness, wear resistance and adhesion strength, reduced Young's modulus, low friction coefficient and high biocompatibility.

Design, characterization and testing of Ti-based multicomponent coatings for load-bearing medical applications.

A comparative investigation of multicomponent thin films based on the systems Ti-Ca-C-O-(N), Ti-Zr-C-O-(N), Ti-Si-Zr-O-(N) and Ti-Nb-C-(N) is presented. TiC(0.5) + 10%CaO, TiC0.5 + 20%CaO, TiC0.5 + 10%ZrO2, TiC0.5 + 20%ZrO2, Ti5Si3 + 10%ZrO2, TiC0.5 + 10%Nb2C and TiC0.5 + 30%Nb2C composite targets were manufactured by means of self-propagating high-temperature synthesis, followed by DC magnetron sputtering in an atmosphere of argon or in a gaseous mixture of argon and nitrogen. The films were characterized in terms of their structure, chemical composition, surface topography, hardness, elastic modulus, elastic recovery, surface charge, friction coefficient, and wear rate. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of the proliferation of Rat-1 fibroblasts and IAR-2 epithelial cells on the tested films, morphometric analysis and actin cytoskeleton staining of the cells cultivated on the films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of cells on the surfaces. The films deposited under optimal conditions showed high hardness in the range of 30-37 GPa, significant reduced Young's modulus, low friction coefficient down to 0.1-0.2 and low wear rate in comparison with conventional magnetron-sputtered TiC and TiN films. The surface of all films was negatively charged with an outstanding shift between the Ar and Ar + N2 Zeta potential curves that reaches 5 mV at the highest pH values. We did not detect statistically significant differences in the attachment, spreading and cell shape of cultured IAR-2 and Rat-1 cells on the Ti-Ca-C-O-(N), Ti-Zr-C-O-(N) (TiC0.5 + 10%ZrO2 target), Ti-Si-Zr-O-(N) films and the uncoated substrata. The adhesion and proliferation of cultured cells in vitro was perfect at all investigated films. Assessment of the population of cells covering on the Teflon plates coated with the Ti-Ca-C-O-(N) and Ti-Zr-C-O-(N) films after 16 weeks of subcutaneous implantation revealed the high biocompatibility level of tested films and absence of inflammatory reactions in mice. Contrary, the epitheliocytes and fibroblasts cultivated on the Ti-Zr-C-O-(N) (TiC0.5 + 20%ZrO2 target) and Ti-Nb-C-(N) films had disturbing actin cytoskeleton.