Osteoblast behavior on TiO2 microgrooves prepared by soft-lithography and sol–gel methods

This study focused on the effects of microgrooved TiO₂ surfaces on osteoblast behavior. Microgrooved TiO₂ surfaces with different widths (12 μm and 40 μm) and flat surfaces were fabricated on glass substrates based on the combination of a sol–gel technique and soft-lithography. Osteoblasts (MC3T3-E1) were cultured on the as-prepared microgrooved and flat TiO₂ surfaces. Optical microscopy and scanning electron microscopy were used to analyze the adherent cell behavior by examining the cell morphology. Orientation angle analysis indicated that the cells tended to align along the microgrooves. This tendency was stronger on the microgrooves with smaller widths and became weak with increasing width. Alamar Blue assay indicated that the microgrooves restricted cell proliferation and the alkaline phosphatase assay revealed that the microgrooves limited the differentiation rate. This restriction increased with decreasing microgroove width. The surface energy of the TiO₂ surfaces was size-dependent and followed the order γ _12 μm < γ _40 μm < γ _{flat surfaces}. Osteoblast proliferation and differentiation on the surface with high surface energy exhibited high proliferation and differentiation rates. These results indicated that surface energy appeared to be a dominant factor for cell activity. Thus, surface energy would be a valuable index for the cell compatibility of a micropatterned surface.     

Osteoblast interaction with laser cladded HA and SiO2-HA coatings on Ti–6Al–4V

In order to improve the bioactivity and biocompatibility of titanium endosseous implants, the morphology and composition of the surfaces were modified. Polished Ti–6Al–4V substrates were coated by a laser cladding process with different precursors: 100 wt.% HA and 25 wt.% SiO₂-HA. X-ray diffraction of the laser processed samples showed the presence of CaTiO₃, Ca₃(PO₄)₂, and Ca₂SiO₄ phases within the coatings. From in vitro studies, it was observed that compared to the unmodified substrate all laser cladded samples presented improved cellular interactions and bioactivity. The samples processed with 25 wt.% SiO₂-HA precursor showed a significantly higher HA precipitation after immersion in simulated body fluid than 100 wt.% HA precursor and titanium substrates. The in vitro biocompatibility of the laser cladded coatings and titanium substrate was investigated by culturing of mouse MC3T3-E1 pre-osteoblast cell line and analyzing the cell viability, cell proliferation, and cell morphology. A significantly higher cell attachment and proliferation rate were observed for both laser cladded 100 wt.% HA and 25 wt.% SiO₂-HA samples. Compared to 100 wt.% HA sample, 25 wt.% SiO₂-HA samples presented a slightly improved cellular interaction due to the addition of SiO₂. The staining of the actin filaments showed that the laser cladded samples induced a normal cytoskeleton and well-developed focal adhesion contacts. Scanning electron microscopic image of the cell cultured samples revealed better cell attachment and spreading for 25 wt.% SiO₂-HA and 100 wt.% HA coatings than titanium substrate. These results suggest that the laser cladding process improves the bioactivity and biocompatibility of titanium. The observed biological improvements are mainly due to the coating induced changes in surface chemistry and surface morphology.     

Theoretical study on the electronic properties and stabilities of low-index surfaces of WC polymorphs

The low-index surfaces of WC polymorphs are calculated using the first-principles method based on density functional theory. It is found that there are large relaxations within the top three layers for all termination surfaces, and charge density falls greatly toward the vacuum. The outermost and second interlayer relaxations for C-terminated surfaces are much larger than those for W-terminated surfaces. The surface energies for all low-index surfaces are large which is due to the breaking of strong W–C bonds. For both WC polymorphs, the C-termination surfaces are thermodynamically more unstable than W-terminated surfaces over the whole range of carbon chemical potentials considered in this paper; the most stable surfaces correspond to the (0 0 1) surface with W termination for α-WC, and the (0 0 1) surface with WC termination for β-WC.     

In-vivo study of adhesion and bone growth around implanted laser groove/RGD-functionalized Ti-6Al-4V pins in rabbit femurs

Titanium surfaces were designed, produced, and evaluated for levels of osseointegration into the femurs of rabbits. A total of 36 Ti-6Al-4V pins (15 mm length, 1.64 mm diameter) were prepared into three experimental groups. These were designed to test the effects of osseointegration on laser grooved, RGD coated, and polished control surfaces, as well as combined effects. Circumferential laser grooves were introduced onto pin surfaces (40 μm spacing) using a UV laser (λ = 355 nm). The tripeptide sequence, Arginine-Glycine-Aspartic acid (RGD), was functionalized onto laser grooved surfaces. Of the prepared samples, surface morphology and chemistry were analyzed using scanning electron microscopy (SEM) and Immunoflourescence (IF) spectroscopy, respectively. The experimental pin surfaces were surgically implanted into rabbit femurs. The samples were then harvested and evaluated histologically. Sections of the sample were preserved in a methylmethacralate mold, sliced via a hard microtome, and polished systematically. In the case of the RGD coated and laser grooved surfaces, histological results showed accelerated bone growth into the implant, pull-out tests were also used to compare the adhesion between bone and the titanium pins with/without laser textures and/or RGD coatings.     

Insights into the structures,energies and electronic properties of nesquehonite surfaces by first-principles calculations

By using the first-principles calculations, the structure, energies and electronic properties of four commonly exposed surfaces for the nesquehonite crystal were investigated. The needle-like nesquehonite whisker is well developed with smooth side faces and irregular hexagonal end faces. Surface energy results indicate that the (1 0 1) surface is the most stable surface and corresponds to the side face. The density of dangling bond has a positive relationship with surface energy and the (1 0 1) surface has the least dangling bonds. In terms of relaxed surface energy, the order of relaxed surfaces is (1 0 1) < (2 0 0)-H < (3 0 1) < (2 0 0)-M < (0 0 4). During surface relaxation, the changes in the length of Mg-O bonds and hydrogen bonds contribute to generating a more stable surface with a lower surface energy. The PDOS (partial density of states) of these surfaces are mainly dominated by Mg and O atoms. A small peak value is found in the PDOS of (1 0 1) and (3 0 1) surfaces, which have less exposed Mg-O bonds. Electron transfer causes changes in the length of Mg-O bonds. A more active surface will obtain a larger value of transferred electrons during surface relaxation.     

Laser surface modification of 316 L stainless steel with bioactive hydroxyapatite

Laser-engineered net shaping (LENS?), a commercial additive manufacturing process, was used to modify the surfaces of 316 L stainless steel with bioactive hydroxyapatite (HAP). The modified surfaces were characterized in terms of their microstructure, hardness and apatite forming ability. The results showed that with increase in laser energy input from 32 J/mm² to 59 J/mm² the thickness of the modified surface increased from 222 ± 12 μm to 355 ± 6 μm, while the average surface hardness decreased marginally from 403 ± 18 HV_0.3 to 372 ± 8 HV_0.3. Microstructural studies showed that the modified surface consisted of austenite dendrites with HAP and some reaction products primarily occurring in the inter-dendritic regions. Finally, the surface-modified 316 L samples immersed in simulated body fluids showed significantly higher apatite precipitation compared to unmodified 316 L samples.     

Enhanced endothelialization guided by fibronectin functionalized plasma polymerized acrylic acid film

The plasma polymerized acrylic acid (PPAA) films with high carboxyl concentration were fabricated by low temperature plasma polymerization technology. Vacuum thermal treatment was used to modify the PPAA films. The stability of the PPAA film was evidenced by scanning electron microscopy (SEM). The result of toluidine blue-O (TBO) method showed that the –COOH concentration on the surface of the PPAA films decreased from 20.73 nM to17.90 nM after vacuum thermal treatment. Fibronectin (Fn) was successfully covalently immobilized onto the modified surface PPAA via a covalent bond reacted with carboxyl groups. Diffuse reflectance Fourier infrared transform spectroscopy (DRFTIR) and X-ray photoelectron spectroscopy (XPS) were utilized to characterize the layer-by-layer PPAA surface modification. From the evaluation of immunostaining for actin and Cell Counting Kit-8 (CCK-8), the surface of Fn-immobilized thermal treated PPAA films could be used to enhance the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs). Especially, the exposure of RGD domain existing in the immobilized Fn was ascertained by ELISA, which was one of main improvement factors of HUVEC's adhesion and proliferation. This study demonstrated a promising surface modification used for vascular devices.     

Modulation of poly (d,l-lactic acid) with amniotic fluid for improvement of the cell affinity

The objective of this study was to investigate the effects of naturally occurring amniotic fluid modified poly(d,l-lactic acid) (PDLLA) film on the culture of rat calvaria osteoblast. The characteristics of surfaces (both modified and control) were examined by contact angle measurement and electron spectroscopy for chemical analysis (XPS). Cell adhesion and proliferation were used to assess the cell behavior on modified films and control one. MTT assay was used to determine cell viability and alkaline phosphatase (ALP) activity was taken to evaluate differentiated cell function. Compared with the untreated films, cell adhesion of osteoblast was significantly higher (P < 0.05) than that found on control, and osteoblast proliferation was also greater than control one (P < 0.01) at the time interval of 4 and 7 days. Moreover, the alkaline phosphatase (ALP) activity exhibited statistic difference (P < 0.05) and cell viability demonstrated significant difference (P < 0.01) between amniotic fluid modified PDLLA films and control one. These results suggested that amniotic fluid was a suitable material when used to modify PDLLA in order to improve its biocompatibility.     

Fabrication of NiCr alloy parts by selective laser melting: Columnar microstructure and anisotropic mechanical behavior

NiCr alloy, because of its wide applications in electrical elements and dental field was widely studied in the past. In this work, NiCr cubes and tensile specimens were fabricated by using a new processing technique-selective laser melting (SLM). Microstructural and mechanical behavior characterization of SLM-processed NiCr components was performed. An unusual columnar microstructural architecture composed of 〈1 0 0〉 texture (corresponding to (2 0 0) plane) oriented the building direction was observed. Moreover, it was found that the columnar grain growth across the melt pools occurred during the SLM process and the growth trend became stronger with the decrease of the laser scanning speed. Associated with the microstructural characteristic, an anisotropic mechanical behavior at different reference planes (i.e., at the horizontal and vertical surfaces) was demonstrated for the samples fabricated using different processing parameters. The results showed that with increasing the laser scanning speed, the microhardness at the horizontal surface decreased, while at the vertical surface it increased; an increase of the yield strength (YS) and the ultimate tensile strength (UTS) was observed.     

Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm², FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm², 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO₂ on each sample; after Nd:YAG treatment a reduced state of Ti (Ti^3 +) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly.     

Effect of chemical composition on corneal cellular response to photopolymerized materials comprising 2-hydroxyethyl methacrylate and acrylic acid

Characterization of corneal cellular response to hydrogel materials is an important issue in ophthalmic applications. In this study, we aimed to investigate the relationship between the feed composition of 2-hydroxyethyl methacrylate (HEMA)/acrylic acid (AAc) and material compatibility towards corneal stromal and endothelial cells. The monomer solutions of HEMA and AAc were mixed at varying volume ratios of 92:0, 87:5, 82:10, 77:15, and 72:20, and were subjected to UV irradiation. Results of electrokinetic measurements showed that an increase in absolute zeta potential of photopolymerized membranes is observed with increasing the volume ratios of AAc/HEMA. Following 4 days of incubation with various hydrogels, the primary rabbit corneal stromal and endothelial cell cultures were examined for viability, proliferation, and pro-inflammatory gene expression. The samples prepared from the solution mixture containing 0–10 vol.% AAc displayed good cytocompatibility. However, with increasing volume ratio of AAc and HEMA from 15:77 to 20:72, the decreased viability, inhibited proliferation, and stimulated inflammation were noted in both cell types, probably due to the stronger charge–charge interactions. On the other hand, the ionic pump function of corneal endothelial cells exposed to photopolymerized membranes was examined by analyzing the Na⁺,K⁺-ATPase alpha 1 subunit (ATP1A1) expression level. The presence of material samples having higher anionic charge density (i.e., zeta potential of ? 38 to ? 56 mV) may lead to abnormal transmembrane transport. It is concluded that the chemical composition of HEMA/AAc has an important influence on the corneal stromal and endothelial cell responses to polymeric biomaterials.     

Influence of sterilization methods on cell behavior and functionality of osteoblasts cultured on TiO2 nanotubes

We investigated the adhesion, proliferation and osteogenic functionality of osteoblasts cultured on titanium dioxide (TiO₂) nanotubes in response to different sterilization methods (dry autoclaving vs. wet autoclaving). We prepared various sizes (30–100 nm diameter) of TiO₂ nanotubes on titanium substrates by anodization, sterilized nanotubes by different conditions, and seeded osteoblast cells onto the nanotube surfaces with two different cell seeding densities (10,000 vs. 50,000 cells/well in 12-culture well). The result of this study indicates that the adhesion, proliferation and alkaline phosphatase activity of osteoblasts cultured on only the larger 70 and 100 nm TiO₂ nanotube arrays were dramatically changed by the different sterilization conditions at a low cell seeding density. However, with a higher cell seeding density (50,000 cells/well in 12-cell culture well), the results revealed no significant difference among altered nanotube geometry, 30–100 nm diameters, nor sterilization methods. Next, it was revealed that the nanofeatures of proteins adhered on nanotubular TiO₂ morphology are altered by the sterilization method. It was determined that this protein adhesion effect, in combination with the cell density of osteoblasts seeded onto such TiO₂ nanotube surfaces, has profound effects on cell behavior. This study clearly shows that these are some of the important in vitro culture factors that need to be taken into consideration, as well as TiO₂ nanotube diameters which play an important role in the improvement of cell behavior and functionality.     

In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen

A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280 m²g^? 1. The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2 weeks with a cumulative release ~ 53%, 75% and 50%, respectively, over the first 7 days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time.     

Improvement in the morphology of micro-arc oxidised titanium surfaces: A new process to increase osteoblast response

This study describes a method for combining sandblast-acid etching and micro-arc oxidation to optimise titanium implant surfaces, and examines the effects of these surfaces on osteoblast response. Titanium discs were grouped as: micro-arc oxidised (MAO), sandblast-acid etched and micro-arc oxidised (MAO-SA), micro-arc oxidised and heated (MAO-HT), and untreated smooth surface. The combination of sandblast-acid etching and micro-arc oxidation in the MAO-SA group created an average surface roughness of 2.02 ± 0.15 μm compared to the untreated machined surface of 0.31 ± 0.06 μm. Scanning electron microscopy observations of the surface structures showed that the irregularly ordered valleys created by sandblast-acid etching remained after micro-arc oxidation and that micropores had also formed. These microstructures provided a better place for osteoblasts to spread compared with the other surfaces. In addition, our results indicated that adherent osteoblasts expressed greater alkaline phosphatase (ALP) activity and osteocalcin (OC) production on MAO-SA surfaces compared with MAO, MAO-HT, and smooth surfaces. The overall results clearly indicate that combining sandblast-acid etching and micro-arc oxidation techniques improves the titanium surface morphology and increases the roughness, which provides an optimal surface for cell differentiation and osseointegration.     

Dislocation density-based modeling of subsurface grain refinement with laser-induced shock compression

Laser shock peening (LSP) is an innovative surface treatment technique applied to improve the mechanical properties and surface microstructures of metallic components. This paper is concerned with prediction of the microstructural evolution of metallic components subjected to single or multiple LSP impacts. A numerical framework is developed to model the evolution of dislocation density and dislocation cell size using a dislocation density-based material model. It is shown that the developed model captures the essential features of the material mechanical behaviors and predicts that the total dislocation density reaches the order of 10¹⁴ m^?2 and a minimum dislocation cell size is below 250 nm for LSP of monocrystalline coppers using the laser energy density on the order of 500 GW/cm². It is further shown that the model is cable of predicting the material strengthening mechanism in terms of residual stress and microhardness of the LY2 aluminum alloy due to grain refinement in a LSP process with less laser energy densities on the order of several GW/cm².     

Investigation of in vitro bone cell adhesion and proliferation on Ti using direct current stimulation

Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 μA, was used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–material interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 μA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 μA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell–material interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model.     

Controlling immunoglobulin G orientation on a protein-A terminated bilayer system

In this study, a bilayer system composed of N-[3-trimethoxysilyl propyl]-ethylene diamine (TEDA) and protein-A on silicon wafer was prepared by a simple two-step procedure. Self-assembly deposition of TEDA at optimal conditions resulted in the formation of homogeneous self-assembled monolayers (SAMs) ~ 2.3 nm thick with the surface roughness ~ 0.38 nm. The height value of protein-A overlayer was found to be ~ 3.5 nm, which is within experimental error of the diameter of a single protein-A (3 nm). Immunoglobulin G (IgG) molecules were then immobilized on the bilayer system by protein-A – IgG specific interactions. Using this very simple approach, the IgG layer was formed almost of a monomolecular layer for longer adsorption time (~ 100 min), and it was packed densely for adsorption time longer than 100 min, which resulted in the increase of the amount of IgG immobilized. The use of a bilayer system composed of TEDA and protein A on silicon wafer opens the door for a fundamental understanding of how protein A affects IgG orientation on the surface and also indicates a useful guide to designing surfaces for applications such as immunosensors and biochips.     

The corrosion resistance of Nitinol alloy in simulated physiological solutions: Part 1: The effect of surface preparation

The corrosion behaviour of Nitinol alloy containing nearly equi-atomic composition of nickel and titanium and its constituent metals (nickel and titanium) was investigated in simulated Hanks physiological solution (pH value 7.5) and pH modified simulated Hanks physiological solution (pH values 4.5 and 6.5) and by electrochemical method of anodic potentiodynamic polarization at 37 °C. In this chloride-rich medium the corrosion stability of Nitinol is limited by the susceptibility to localized corrosion and is in that sense more similar to nickel than to titanium. The corrosion stability of Nitinol is strongly dependent on the surface preparation—grinding, polishing or chemical etching. Whereas a ground surface is not resistant to localized corrosion, polished and chemically etched surfaces are resistant to this type of corrosion attack. The reasons for this behaviour were investigated through metallurgical, topographical and chemical properties of the surface as a function of surface preparation. For that purpose, scanning electron microscopy combined with chemical analysis, confocal microscopy and X-ray photoelectron spectroscopy were used. The surface roughness decreased in the following order: chemically etched > ground > polished surface. Besides differences in topography, distinct differences in the chemical composition of the outermost surface are observed. Ground, rough surfaces comprised mainly titanium oxides and small amounts of nickel metal. Chemically etched and, especially, polished surfaces are composed of a mixture of titanium, nickel and titanium oxides, as studied by angle resolved X-ray photoelectron spectroscopy. These results emphasize the importance of detailed investigation of the metal surface since small differences in surface preparation may induce large differences in corrosion stability of material when exposed to corrosive environments.     

In vitro bioactivity evaluation of nano- and micro-crystalline anodic TiO2: HA formation,cellular affinity and organ culture

Anodization is an easily viable technique useful for producing TiO₂ coatings on titanium substrates. Nano-crystalline anodic TiO₂ structure was produced on titanium at 20 V using 1 M Na₂SO₄ and 0.5% NaF and consolidated by a further heat-treatment. Micro-crystalline anodic TiO₂ was produced on titanium by applying a galvanostatic current density of 70 A/m² in water medium. To assess the usefulness of these nano- and micro-oxides for bone implant stability, physical properties and bone in vitro bioactivity including HA formation, cellular affinity and mouse-tissue morphogenesis, were evaluated. Bioactivity of the different anodic surfaces was evaluated by treating them in a simulated body fluid (SBF) to form hydroxyapatite (HA) and the rates of HA formation were compared. Deposits of HA could be seen on the nano-oxide surface within 7 days, whereas HA was detected only after 14 days on the micro-oxide surface. In vitro cell culture tests done using mouse osteoblasts indicated that the nano-oxides showed statistically significant cell activity than the micro-oxides and the machined titanium. Branching morphogenesis test done for 72 h on these surfaces showed more branching on the micro- and nano-oxides as compared with titanium surface.     

Thromboresistant and endothelialization effects of dopamine-mediated heparin coating on a stent material surface

Heparinization of surfaces has proven a successful strategy to prevent thrombus formation. Inspired by the composition of adhesive proteins in mussels, the authors used dopamine to immobilize heparin on a stent surface. This study aimed to assess the thromboresistant and endothelialization effects of dopamine-mediated heparin (HPM) coating on a stent material surface. The HPM was synthesized by bonding dopamine and heparin chemically. Cobalt–chromium (Co–Cr) alloy disks were first placed in the HPM solution and applied to surface stability then underwent thromboresistant tests and human umbilical vein endothelial cells (HUVEC) cytotoxicity assays. The results showed not only thromboresistant activity and a stable state of heparin on the surfaces after investigation with toluidine blue and thrombin activation assay but also proliferation of HUVEC in vitro. Studies on animals showed that the HPM-coated stent has no obvious inflammation response and increasing of restenosis rate compared to the bare metal stent (BMS) indicating good biocompatibility as well as safety in its in vivo application. Moreover, improving the endothelial cell (EC) proliferation resulted in a higher strut-covering rate (i.e., endothelialization) with shuttle-shaped EC in the HPM-coated stent group compared to that of the BMS group. These results suggest that this facile coating approach could significantly promote endothelialization and offer greater safety than the BMS for its much improved thromboresistant property. Moreover, it may offer a platform for conjugating secondary drugs such as anti-proliferative drugs.