Biochemical characterization of the cell-biomaterial interface by quantitative proteomics.

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.

Nano-scale surface morphology, wettability and osteoblast adhesion on nitrogen plasma-implanted NiTi shape memory alloy.

Plasma immersion ion implantation (PIII) is an effective method to increase the corrosion resistance and inhibit nickel release from orthopedic NiTi shape memory alloy. Nitrogen was plasma-implanted into NiTi using different pulsing frequencies to investigate the effects on the nano-scale surface morphology, structure, wettability, as well as biocompatibility. X-ray photoelectron spectroscopy (XPS) results show that the implantation depth of nitrogen increases with higher pulsing frequencies. Atomic force microscopy (AFM) discloses that the nano-scale surface roughness increases and surface features are changed from islands to spiky cones with higher pulsing frequencies. This variation in the nano surface structures leads to different surface free energy (SFE) monitored by contact angle measurements. The adhesion, spreading, and proliferation of osteoblasts on the implanted NiTi surface are assessed by cell culture tests. Our results indicate that the nano-scale surface morphology that is altered by the implantation frequencies impacts the surface free energy and wettability of the NiTi surfaces, and in turn affects the osteoblast adhesion behavior.

Effects of Salivary Mg on Head and Neck Carcinoma via TRPM7.

Magnesium (Mg) has been known to play vital roles in regulating growth and various metabolic processes. In recent years, the association between Mg and tumorigenesis has raised more and more attention. However, the effects of Mg on the progression of head and neck carcinoma (HNC), as well as the mechanism behind it, remain undefined. In this study, the roles of Mg in tumorigenic activities were tested in CAL27 and FaDu cells as well as in a xenograft tumor model in nude mice. We demonstrated that a moderate increase in extracellular Mg contributed to the proliferation, migration, and invasion of 2 HNC cell lines, while the addition of Mg in drinking water promoted the growth of xenograft tumors in mice without altering their serum Mg levels. Moreover, TRPM7, a major Mg transporter, was shown to be essential for the tumorigenic activities of HNC and the Mg-induced promotive effects on HNC cells and was further shown to be associated with the activation of AKT/mTOR (mammalian target of rapamycin) signaling. In a preliminary clinical study, we determined the Mg ion concentrations in the stimulated saliva from 72 patients with nasopharynx carcinoma and 12 healthy individuals. Our data revealed that the salivary Mg levels of subjects with nasopharynx carcinoma were significantly higher than those of the healthy controls. This is correlated with our finding showing TRPM7 to be overexpressed in tumor tissues harvested from 9 patients with HNC. Therefore, we can conclude that salivary Mg level, within a certain range, could act as a risk factor for the progression of HNC, which involves the activation of AKT/mTOR signaling pathways through the TRPM7 channel. The control of salivary Mg level and the intervention of TRPM7 should not be ignored during the study of HNC.

Nickel release behavior, cytocompatibility, and superelasticity of oxidized porous single-phase NiTi.

Porous NiTi shape memory alloys are one of the promising biomaterials for surgical implants because of their unique shape memory effects and porous structure with open pores. However, the complex surface morphology and larger area of porous NiTi compared to dense NiTi make it more vulnerable from the viewpoint of release of nickel, which can cause deleterious effects in the human body. It is also more difficult to modify the exposed surfaces of a porous structure using conventional surface modification technologies. In this work, oxidation in conjunction with postreaction heat treatment was used to modify the surfaces of porous single-phase NiTi prepared by capsule-free hot isostatic pressing to mitigate Ni leaching and enhance the surface properties. Differential scanning calorimetry thermal analysis, uniaxial compression tests, inductively-coupled plasma mass spectrometry, and cell cultures reveal that porous NiTi alloys oxidized at 450 degrees C for 1 h have an austenite transition temperature below 37 degrees C, excellent superelasticity, lower nickel release, and no cytotoxicity.

Surface mechanical properties, corrosion resistance, and cytocompatibility of nitrogen plasma-implanted nickel-titanium alloys: a comparative study with commonly used medical grade materials.

Stainless steel and titanium alloys are the most common metallic orthopedic materials. Recently, nickel-titanium (NiTi) shape memory alloys have attracted much attention due to their shape memory effect and super-elasticity. However, this alloy consists of equal amounts of nickel and titanium, and nickel is a well known sensitizer to cause allergy or other deleterious effects in living tissues. Nickel ion leaching is correspondingly worse if the surface corrosion resistance deteriorates. We have therefore modified the NiTi surface by nitrogen plasma immersion ion implantation (PIII). The surface chemistry and corrosion resistance of the implanted samples were studied and compared with those of the untreated NiTi alloys, stainless steel, and Ti-6Al-4V alloy serving as controls. Immersion tests were carried out to investigate the extent of nickel leaching under simulated human body conditions and cytocompatibility tests were conducted using enhanced green fluorescent protein mice osteoblasts. The X-ray photoelectron spectroscopy results reveal that a thin titanium nitride (TiN) layer with higher hardness is formed on the surface after nitrogen PIII. The corrosion resistance of the implanted sample is also superior to that of the untreated NiTi and stainless steel and comparable to that of titanium alloy. The release of nickel ions is significantly reduced compared with the untreated NiTi. The sample with surface TiN exhibits the highest amount of cell proliferation whereas stainless steel fares the worst. Compared with coatings, the plasma-implanted structure does not delaminate as easily and nitrogen PIII is a viable way to improve the properties of NiTi orthopedic implants.

Surface characteristics, biocompatibility, and mechanical properties of nickel-titanium plasma-implanted with nitrogen at different implantation voltages.

NiTi shape memory alloy is one of the promising orthopedic materials due to the unique shape memory effect and superelasticity. However, the large amount of Ni in the alloy may cause allergic reactions and toxic effects thereby limiting its applications. In this work, the surface of NiTi alloy was modified by nitrogen plasma immersion ion implantation (N-PIII) at various voltages. The materials were characterized by X-ray photoelectron spectroscopy (XPS). The topography and roughness before and after N-PIII were measured by atomic force microscope. The effects of the modified surfaces on nickel release and cytotoxicity were assessed by immersion tests and cell cultures. The XPS results reveal that near-surface Ni concentration is significantly reduced by PIII and the surface TiN layer suppresses nickel release and favors osteoblast proliferation, especially for samples implanted at higher voltages. The surfaces produced at higher voltages of 30 and 40 kV show better adhesion ability to osteoblasts compared to the unimplanted and 20 kV PIII samples. The effects of heating during PIII on the phase transformation behavior and cyclic deformation response of the materials were investigated by differential scanning calorimetry and three-point bending tests. Our results show that N-PIII conducted using the proper conditions improves the biocompatibility and mechanical properties of the NiTi alloy significantly.

Surface structure and properties of biomedical NiTi shape memory alloy after Fenton's oxidation.

Fenton's oxidation is traditionally used to remove inorganic and organic pollutants from water in waster water treatment. It is an advanced oxidation process in which H2O2 is catalytically decomposed by ferrous irons into hydroxyl radicals (*OH) which have a higher oxidation potential (2.8V) than H2O2. In the work reported here, we for the first time use Fenton's oxidation to modify the surface of biomedical NiTi shape memory alloy (SMA). The influences of Fenton's oxidation on the surface microstructure, blood compatibility, leaching of harmful Ni ions and corrosion resistance in simulated body fluids is assessed using scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, inductively coupled plasma mass spectrometry, electrochemical tests, hemolysis analysis and the blood platelet adhesion test. The mechanical stability of the surface titania film produced by Fenton's oxidation as well as their effects on the shape memory behavior of the SMA are studied by bending tests. Our results show that Fenton's oxidation produces a novel nanostructured titania gel film with a graded structure on the NiTi substrate without an intermediate Ni-rich layer that is typical of high-temperature oxidation. Moreover, there is a clear Ni-free zone near the top surface of the titania film. The surface structural changes introduced by Fenton's oxidation improve the electrochemical corrosion resistance and mitigate Ni release. The latter effects are comparable to those observed after oxygen plasma immersion ion implantation reported previously and better than those of high-temperature oxidation. Aging in boiling water improves the crystallinity of the titania film and further reduces Ni leaching. Blood platelet adhesion is remarkably reduced after Fenton's oxidation, suggesting that the treated SMA has improved thrombo resistance. Enhancement of blood compatibility is believed to stem from the improved hemolysis resistance, the surface wettability and the intrinsic electrical characteristics of the titania film. The titania film produced by Fenton's oxidation has good mechanical stability and does not adversely impact the shape memory behavior of NiTi. Our work suggests that Fenton's oxidation is a promising low-temperature, low-cost surface modification method for improving the surface properties of biomedical NiTi SMA.

Construction of poly (vinyl alcohol)/poly (lactide-glycolide acid)/vancomycin nanoparticles on titanium for enhancing the surface self-antibacterial activity and cytocompatibility.

Comparing with traditional drug dosage form, controlled release systems offer more effective and favorable route to deliver drugs in optimum dose to specific sites with long term release duration. In this work, an effective drug delivery system composed of poly (vinyl alcohol) (PVA)/poly (lactide-glycolide acid) (PLGA) nanoparticles (NPs) with encapsulated vancomycin (Van), is constructed on the surface of biomedical titanium. The PVA/PLGA/Van NPs synthesized via double emulsion route are grafted onto the surface of titanium plates modified by alkaline-heat treatment and subsequent aminopropyltriethoxysilane (APTES) deposition. In vitro tests disclose that NPs can release a small amount of drugs continuously due to the slow swelling or hydrolysis of polymer chain segments as the immersion time increases. As the pH value reduces, the ester bonds rupture with releasing more drugs, which is why this drug delivery system exhibits the highest antibacterial efficiency at the lowest pH value of 4.5 in this work. Cell culture results reveal that this smart surface system on titanium facilitates the cell attachment and proliferation on implants. Hence, this pH controlled drug delivery system can be successfully applied as a bio-platform for improving both the osteoblasts adhesion and antibacterial activity of metallic implants.

Construction of N-halamine labeled silica/zinc oxide hybrid nanoparticles for enhancing antibacterial ability of Ti implants.

The traditional antibiotic treatment for bacterial infections often induces antibiotic resistance in bacteria. In this work, we developed hybrid nanoparticles (NPs) with a self-antibacterial ability on Ti implants using monodispersed polystyrene-acrylic acid (PSA) nanoparticles as colloidal templates followed by the electrostatic adsorption of zinc oxide (ZnO) and the subsequent deposition of silica (SiO2) membrane on the outside. These synthesized PSA-ZnO-SiO2 NPs were pretreated by 5,5-dimethylhydantoin (DMH) before chlorination in a diluted NaClO solution. These nanoparticles (PSA-ZnO-SiO2-DMH) were subsequently labeled by N-halamines and then immobilized on the surface of titanium plates through hydrogen bonding. Field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) were utilized to characterize the modified surface. Antibacterial tests disclosed that the PSA-ZnO-SiO2-DMH-Cl NPs modified surface exhibited excellent antibacterial activity against both Pseudomonas aeruginosa (P.au), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In vitro cell culture results revealed that PSA-ZnO-SiO2-DMH-Cl had no obvious cytotoxicity for an MC3T3-E1 preosteoblast. This novel surface system provides a promising self-antibacterial bioplatform for metallic implants without using antibiotics.

Construction of poly(lactic-co-glycolic acid)/ZnO nanorods/Ag nanoparticles hybrid coating on Ti implants for enhanced antibacterial activity and biocompatibility.

Poly(lactic-co-glycolic acid)/Ag/ZnO nanorods coating were successfully prepared on the surface of Ti metallic implants using a hydrothermal method and subsequent spin-coating of mixtures of poly(lactic-co-glycolic acid) and silver nanoparticles. The poly(lactic-co-glycolic acid)/Ag/ZnO nanorods coating exhibited excellent antibacterial efficacy of over 96% against both Staphylococcus aureus and Escherichia coli when the initial content of Ag nanoparticles was over 3wt%. In addition, the release of both silver and zinc could last for over a hundred days due to the enwrapping of poly(lactic-co-glycolic acid). Proliferation of mouse calvarial cells exhibited minimal cytotoxicity on the poly(lactic-co-glycolic acid)/Ag/ZnO coating with an initial content of Ag nanoparticles of 1wt% and 3wt%, while it inhibited cell proliferation once this value was increased to 6wt%. The results revealed that this poly(lactic-co-glycolic acid)/Ag/ZnO composite could provide a long-lasting antibacterial approach and good cytocompatibility, thus exhibiting considerable potential for biomedical application in orthopedic and dental implants with excellent self-antibacterial activity and good biocompatibility.

The controlled drug release by pH-sensitive molecularly imprinted nanospheres for enhanced antibacterial activity.

In this study, we prepared pH-sensitive hybrid nanospheres through the implementation of a facile molecularly imprinted polymer (MIP) technique combined with a UV-initiated precipitation polymerization method using vancomycin (VA) for the templates. During the course of this investigation, both 2-hydroxyethyl methacrylate (HEMA) and 2-(diethylamino) ethyl methacrylate (DEAEMA) were utilized as the functional monomers, while ethylene glycol dimethacrylate (EGDMA) was used as a cross-linker. The obtained MIP nanospheres exhibited well-controlled particle size, with a drug loading capacity of about 17%, much higher than that of the non-imprinted polymer (NIP) nanospheres (5%). In addition, the VA loading quantity was closely correlated with the dosage of the cross-linking agent, and the MIP nanospheres exhibited a slower and more controlled VA release rate than the NIP nanospheres. Moreover, these MIP nanospheres were sensitive to pH values, and consequently showed an increasing release rate of VA as the pH level was decreased. The VA-loaded MIP nanospheres showed the higher antibacterial ratio of over 92% against Staphylococcus aureus (S. aureus) while the NIP nanospheres were friendly to S. aureus. These MIP nanospheres can be promising for targeting drug delivery system to achieve specific therapies such as preventing bacterial infections and killing cancer cells without damaging health cells and tissues.

Metal Ion Coordination Polymer-Capped pH-Triggered Drug Release System on Titania Nanotubes for Enhancing Self-antibacterial Capability of Ti Implants.

The current work reports a novel hybrid system with a highly efficient, bioresponsive, and controlled release of antibacterial activity via the metal ion coordination polymer on titania nanotubes (TNTs). These hybrid systems exhibited a self-defense behavior that is triggered by the change of the ambient environment acidity due to bacterial infection with Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli). The antibacterial agents, including antibiotics and nanosilver particles, can be loaded into TNTs and then sealed with coordination polymers (CPs) through the attachment of metallic ions such as Zn2+ or Ag+. The zinc and silver ions work as intermediate coordination bonds, and they are sensitive to the change in H+. Because of the strong bonding of CPs, the amount of released antimicrobial agents is maintained at a nonsignificant level when pH is maintained at 7.4. However, the coordination bond of the capped CPs was triggered to open and release antibacterial agents from TNTs once the environment becomes acidic. The release rate gradually increased as the pH value further decreased. Subsequently, the antibacterial efficiency of the hybrid system is accelerated as the local microenvironment becomes more acidic during bacterial infection. In addition, the metal ions that are used for intermediate bond bridging are also favorable for specific biological functions. For example, Zn2+ can promote the proliferation of osteoblastic cells, while Ag+ can further enhance the antibacterial capability. In conclusion, this smart surface coating system not only demonstrates excellent self-antibacterial properties and biocompatibility but also formulates a controllable delivery system for the long-lasting treatment of biomaterial-related bacterial infections.

Surface characteristics, mechanical properties, and cytocompatibility of oxygen plasma-implanted porous nickel titanium shape memory alloy.

Good surface properties and biocompatibility are crucial to porous NiTi shape memory alloys (SMA) used in medical implants, as possible nickel release from porous NiTi may cause deleterious effects in the human body. In this work, oxygen plasma immersion ion implantation (O-PIII) was used to reduce the amount of nickel leached from porous NiTi alloys with a porosity of 42% prepared by capsule-free hot isostatic pressing. The mechanical properties, surface properties, and biocompatibility were studied by compression tests, X-ray photoelectron spectroscopy (XPS), and cell culturing. The O-PIII porous NiTi SMAs have good mechanical properties and excellent superelasticity, and the amount of nickel leached from the O-PIII porous NiTi is much less than that from the untreated samples. XPS results indicate that a nickel-depleted surface layer predominantly composed of TiO(2) is produced by O-PIII and acts as a barrier against out-diffusion of nickel. The cell culturing tests reveal that both the O-PIII and untreated porous NiTi alloys have good biocompatibility.

Dopamine Modified Organic-Inorganic Hybrid Coating for Antimicrobial and Osteogenesis.

A hybrid coating composed of hydroxyapatite (HA), Ag nanoparticles (NPs), and chitosan (CS) was successfully prepared on a Ti substrate by a layer-by-layer assembly process. A polydopamine-assisted (PDA-assisted) coating showed a good bond with HA. Ag NPs were uniformly distributed into the hybrid coating through a solution method and ultraviolet light reduction. A CS nanofilm was deposited via spin-coating to control the release of Ag+ from the hybrid coating. The results disclosed that the 3-layer CS coating could efficiently control the release of Ag+ from the hybrid coating via the Fickian diffusion mechanism and that the PDA/HA/Ag/CS-1 coating exhibited antibacterial ratios of 63.0% and 51.8% against E. coli and S. aureus, respectively. Furthermore, the normal structure of E. coli was obviously destroyed by two types of Ag doped coatings. The cell viability assay showed that CS effectively reduced the cytotoxicity of the hybrid coating after a 7 day incubation. The hybrid coating presented high ALP activities at days 3 and 14. The results reveal that hybrid coatings can endow Ti implants with good antibacterial capability as well as cell viability and osteogenic activity.

Design of magnesium alloys with controllable degradation for biomedical implants: From bulk to surface.

The combination of high strength, light weight, and natural biodegradability renders magnesium (Mg)-based alloys promising in orthopedic implants and cardiovascular stents. Being metallic materials, Mg and Mg alloys made for scaffolds provide the necessary mechanical support for tissue healing and cell growth in the early stage, while natural degradation and reabsorption by surrounding tissues in the later stage make an unnecessarily follow-up removal surgery. However, uncontrolled degradation may collapse the scaffolds resulting in premature implant failure, and there has been much research in controlling the degradation rates of Mg alloys. This paper reviews recent progress in the design of novel Mg alloys, surface modification and corrosion mechanisms under different conditions, and describes the effects of the structure, composition, and surface conditions on the degradation behavior in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Owing to their unique mechanical properties, biodegradability, biocompatibility, Mg based biomaterials are becoming the most promising substitutes for tissue regeneration for impaired bone, vascular and other tissues because these scaffolds can provide not only ideal space for the growth and differentiation of seeded cells but also enough strength before the formation of normal tissues. The most important is that these scaffolds can be fully degraded after tissue regeneration, which can satisfy the increasing demand for better biomedical devices and functional tissue engineering biomaterials in the world. However, the rapid degradation rate of these scaffolds restricts the wide application in clinic. This paper reviews recent progress on how to control the degrdation rate based on the relevant corrosion mechanisms through the design of porous structure, phase structure, grains, and amorphous structure as well as surface modification, which will be beneficial to the better understanding and functional design of Mg-based scaffolds for wide clinical applications in tissue reconstruction in near futures.

The modulation of stem cell behaviors by functionalized nanoceramic coatings on Ti-based implants.

Nanoceramic coating on the surface of Ti-based metallic implants is a clinical potential option in orthopedic surgery. Stem cells have been found to have osteogenic capabilities. It is necessary to study the influences of functionalized nanoceramic coatings on the differentiation and proliferation of stem cells in vitro or in vivo. In this paper, we summarized the recent advance on the modulation of stem cells behaviors through controlling the properties of nanoceramic coatings, including surface chemistry, surface roughness and microporosity. In addition, mechanotransduction pathways have also been discussed to reveal the interaction mechanisms between the stem cells and ceramic coatings on Ti-based metals. In the final part, the osteoinduction and osteoconduction of ceramic coating have been also presented when it was used as carrier of BMPs in new bone formation.

Antibacterial Activity of Silver Doped Titanate Nanowires on Ti Implants.

A nanostructured film composed of one-dimensional titanate nanowires (TNWs) was employed as a carrier of Ag nanoparticles and chitosan (CS) to improve the surface antibacterial activity and biocompatibility of titanium implants. A TNWs film was produced on a Ti substrate by an alkali hydrothermal reaction and subsequently doped by Ag nanoparticles through an ultraviolet light chemical reduction. The CS nanofilm was deposited on the Ag nanoparticles through a spin-assisted layer by layer assembly method. The results disclosed that Ag nanoparticles were successfully carried by TNWs and homogeneously distributed on the entire surface. Moreover, a CS nanofilm was also successfully deposited on the Ag nanoparticles. Antibacterial tests showed that the samples modified with a higher initial concentration of AgNO3 solution exhibited better antibacterial activity, and that a CS nanofilm could further improve the antibacterial activity of the TNWs. Cell viability and ALP tests revealed that the release of Ag(+) was detrimental for the growth, proliferation, and differentiation of MC3T3, and that CS could lower the negative effects of Ag gradually as the incubation time increased.

Carbon plasma immersion ion implantation of nickel-titanium shape memory alloys.

Nickel-titanium (NiTi) shape memory alloys possess super-elasticity in addition to the well-known shape memory effect and are potentially suitable for orthopedic implants. However, a critical concern is the release of harmful Ni ions from the implants into the living tissues. We propose to enhance the corrosion resistance and other surface and biological properties of NiTi using carbon plasma immersion ion implantation and deposition (PIII&D). Our corrosion and simulated body fluid tests indicate that either an ion-mixed amorphous carbon coating fabricated by PIII&D or direct carbon PIII can drastically improve the corrosion resistance and block the out-diffusion of Ni from the materials. Our tribological tests show that the treated surfaces are mechanically more superior and cytotoxicity tests reveal that both sets of plasma-treated samples favor adhesion and proliferation of osteoblasts.

Corrosion resistance, surface mechanical properties, and cytocompatibility of plasma immersion ion implantation-treated nickel-titanium shape memory alloys.

Nickel-titanium shape memory alloys are promising materials in orthopedic applications because of their unique properties. However, for prolonged use in a human body, deterioration of the corrosion resistance of the materials becomes a critical issue because of the increasing possibility of deleterious ions released from the substrate to living tissues. We have investigated the use of nitrogen, acetylene, and oxygen plasma immersion ion implantation (PIII) to improve the corrosion resistance and mechanical properties of the materials. Our results reveal that the corrosion resistance and mechanical properties such as hardness and elastic modulus are significantly enhanced after surface treatment. The release of nickel is drastically reduced as compared with the untreated control. In addition, our in vitro tests show that the plasma-treated surfaces are well tolerated by osteoblasts. Among the three types of samples, the best biological effects are observed on the nitrogen PIII samples.

Investigation of nickel suppression and cytocompatibility of surface-treated nickel-titanium shape memory alloys by using plasma immersion ion implantation.

Nickel-titanium (NiTi) shape memory alloys are increasingly being used in orthopedic applications. However, there is a concern that Ni is harmful to the human body. We have recently investigated the use of nitrogen, or oxygen plasma immersion ion implantation to mitigate this deleterious effect. Our results reveal that the near-surface Ni concentration in all the treated samples is significantly suppressed. In addition, our in vitro tests show that the plasma-treated surfaces are cytologically compatible allowing the attachment and proliferation of osteoblasts. Among the two types of samples, the best biological effects are found on the samples with nitrogen implantation.