TiO2/HA and Titanate/HA Double-Layer Coatings on Ti6Al4V Surface and Their Influence on In Vitro Cell Growth and Osteogenic Potential.

Hydroxyapatite (HA) layers are appropriate biomaterials for use in the modification of the surface of implants produced inter alia from a Ti6Al4V alloy. The issue that must be solved is to provide implants with appropriate biointegration properties, enabling the permanent link between them and bone tissues, which is not so easy with the HA layer. Our proposition is the use of the intermediate layer ((IL) = TiO2, and titanate layers) to successfully link the HA coating to a metal substrate (Ti6Al4V). The morphology, structure, and chemical composition of Ti6Al4V/IL/HA systems were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectrometry (EDS). We evaluated the apatite-forming ability on the surface of the layer in simulated body fluid. We investigated the effects of the obtained systems on the viability and growth of human MG-63 osteoblast-like cells, mouse L929 fibroblasts, and adipose-derived human mesenchymal stem cells (ADSCs) in vitro, as well as on their osteogenic properties. Based on the obtained results, we can conclude that both investigated systems reflect the physiological environment of bone tissue and create a biocompatible surface supporting cell growth. However, the nanoporous TiO2 intermediate layer with osteogenesis-supportive activity seems most promising for the practical application of Ti6Al4V/TiO2/HA as a system of bone tissue regeneration.

Evaluation of the Cathodic Electrodeposition Effectiveness of the Hydroxyapatite Layer Used in Surface Modification of Ti6Al4V-Based Biomaterials.

The important issue associated with the design and the fabrication of the titanium and titanium alloy implants is the increase of their biointegration with bone tissue. In the presented paper, the research results concerning the conditions used in the cathodic deposition of hydroxyapatite on the surface Ti6Al4V substrates primarily modified by the production of TiO2 nanoporous coatings, TiO2 nanofibers, and titanate coatings, are discussed. Despite excellent biocompatibility with natural bone tissue of materials based on hydroxyapatite (HA), their poor adhesion to the substrate caused the limited use in the implants' construction. In our works, we have focused on the comparison of the structure, physicochemical, and mechanical properties of coating systems produced at different conditions. For this purpose, scanning electron microscopy images, chemical composition, X-ray diffraction patterns, infrared spectroscopy, wettability, and mechanical properties are analyzed. Our investigations proved that the intermediate titanium oxide coatings presence significantly increases the adhesion between the hydroxyapatite layer and the Ti6Al4V substrate, thus solving the temporary delamination problems of the HA layer.

Assessment of Titanate Nanolayers in Terms of Their Physicochemical and Biological Properties.

The surface modification of titanium substrates and its alloys in order to improve their osseointegration properties is one of widely studied issues related to the design and production of modern orthopedic and dental implants. In this paper, we discuss the results concerning Ti6Al4V substrate surface modification by (a) alkaline treatment with a 7 M NaOH solution, and (b) production of a porous coating (anodic oxidation with the use of potential U = 5 V) and then treating its surface in the abovementioned alkaline solution. We compared the apatite-forming ability of unmodified and surface-modified titanium alloy in simulated body fluid (SBF) for 1-4 weeks. Analysis of the X-ray diffraction patterns of synthesized coatings allowed their structure characterization before and after immersing in SBF. The obtained nanolayers were studied using Raman spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), and scanning electron microscopy (SEM) images. Elemental analysis was carried out using X-ray energy dispersion spectroscopy (SEM EDX). Wettability and biointegration activity (on the basis of the degree of integration of MG-63 osteoblast-like cells, L929 fibroblasts, and adipose-derived mesenchymal stem cells cultured in vitro on the sample surface) were also evaluated. The obtained results proved that the surfaces of Ti6Al4V and Ti6Al4V covered by TiO2 nanoporous coatings, which were modified by titanate layers, promote apatite formation in the environment of body fluids and possess optimal biointegration properties for fibroblasts and osteoblasts.

The Photocatalytic Activity of Titania Coatings Produced by Electrochemical and Chemical Oxidation of Ti6Al4V Substrate, Estimated According to ISO 10678:2010The Photocatalytic Activity of Titania Coatings Produced by Electrochemical and Chemical Oxidation of Ti6Al4V Substrate, Estimated According to ISO 10678:2010.

The last twenty years have been a period of intense investigations of materials based on titanium dioxide, which have unique properties and functionalities, and which can be used in various areas of medicine. As a part of this issue, the results of our works for the assessment of the photocatalytic activity of titanium dioxide nanocoatings of different nanoarchitecture (nanoporous, nanotubular, nanosponge-like and nanofibrous examples), which were earlier checked in terms of their biocompatibility and usability for the modification of medical devices' surfaces, are presented. The studied materials were produced on the surface of Ti6Al4V substrates using electrochemical and chemical oxidation methods. The activity of produced titania materials was studied on the base of the methylene blue (MB) degradation effect, in accordance to ISO 10678:2010. In our works, we have focused on the analysis of the correlation between the photocatalytic activity of nanoarchitecturally different TiO2 coatings, their morphology and structure. The obtained results prove that all studied coatings, both amorphous and amorphous containing crystalline domains, revealed photocatalytic activity in the photoinduced degradation of the organic pollution standard. This activity may be an additional advantage of medical device coatings, being adequate for use in sterilization processes applying UVA light.

In Vitro Studies on Nanoporous, Nanotubular and Nanosponge-Like Titania Coatings, with the Use of Adipose-Derived Stem Cells.

In vitro biological research on a group of amorphous titania coatings of different nanoarchitectures (nanoporous, nanotubular, and nanosponge-like) produced on the surface of Ti6Al4V alloy samples have been carried out, aimed at assessing their ability to interact with adipose-derived mesenchymal stem cells (ADSCs) and affect their activity. The attention has been drawn to the influence of surface coating architecture and its physicochemical properties on the ADSCs proliferation. Moreover, in vitro co-cultures: (1) fibroblasts cell line L929/ADSCs and (2) osteoblasts cell line MG-63/ADSCs on nanoporous, nanotubular and nanosponge-like TiO2 coatings have been studied. This allowed for evaluating the impact of the surface properties, especially roughness and wettability, on the creation of the beneficial microenvironment for co-cultures and/or enhancing differentiation potential of stem cells. Obtained results showed that the nanoporous surface is favorable for ADSCs, has great biointegrative properties, and supports the growth of co-cultures with MG-63 osteoblasts and L929 fibroblasts. Additionally, the number of osteoblasts seeded and cultured with ADSCs on TNT5 surface raised after 72-h culture almost twice when compared with the unmodified scaffold and by 30% when compared with MG-63 cells growing alone. The alkaline phosphatase activity of MG-63 osteoblasts co-cultured with ADSCs increased, that indirectly confirmed our assumptions that TNT-modified scaffolds create the osteogenic niche and enhance osteogenic potential of ADSCs.

Comprehensive Evaluation of the Biological Properties of Surface-Modified Titanium Alloy Implants.

An increasing interest in the fabrication of implants made of titanium and its alloys results from their capacity to be integrated into the bone system. This integration is facilitated by different modifications of the implant surface. Here, we assessed the bioactivity of amorphous titania nanoporous and nanotubular coatings (TNTs), produced by electrochemical oxidation of Ti6Al4V orthopedic implants' surface. The chemical composition and microstructure of TNT layers was analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). To increase their antimicrobial activity, TNT coatings were enriched with silver nanoparticles (AgNPs) with the chemical vapor deposition (CVD) method and tested against various bacterial and fungal strains for their ability to form a biofilm. The biointegrity and anti-inflammatory properties of these layers were assessed with the use of fibroblast, osteoblast, and macrophage cell lines. To assess and exclude potential genotoxicity issues of the fabricated systems, a mutation reversal test was performed (Ames Assay MPF, OECD TG 471), showing that none of the TNT coatings released mutagenic substances in long-term incubation experiments. The thorough analysis performed in this study indicates that the TNT5 and TNT5/AgNPs coatings (TNT5-the layer obtained upon applying a 5 V potential) present the most suitable physicochemical and biological properties for their potential use in the fabrication of implants for orthopedics. For this reason, their mechanical properties were measured to obtain full system characteristics.

Titania Nanofiber Scaffolds with Enhanced Biointegration Activity-Preliminary In Vitro Studies.

The increasing need for novel bone replacement materials has been driving numerous studies on modifying their surface to stimulate osteogenic cells expansion and to accelerate bone tissue regeneration. The goal of the presented study was to optimize the production of titania-based bioactive materials with high porosity and defined nanostructure, which supports the cell viability and growth. We have chosen to our experiments TiO2 nanofibers, produced by chemical oxidation of Ti6Al4V alloy. Fibrous nanocoatings were characterized structurally (X-ray diffraction (XRD)) and morphologically (scanning electron microscopy (SEM)). The wettability of the coatings and their mechanical properties were also evaluated. We have investigated the direct influence of the modified titanium alloy surfaces on the survival and proliferation of mesenchymal stem cells derived from adipose tissue (ADSCs). In parallel, proliferation of bone tissue cells-human osteoblasts MG-63 and connective tissue cells - mouse fibroblasts L929, as well as cell viability in co-cultures (osteoblasts/ADSCs and fibroblasts/ADSCs has been studied. The results of our experiments proved that among all tested nanofibrous coatings, the amorphous titania-based ones were the most optimal scaffolds for the integration and proliferation of ADSCs, fibroblasts, and osteoblasts. Thus, we postulated these scaffolds to have the osteopromotional potential. However, from the co-culture experiments it can be concluded that ADSCs have the ability to functionalize the initially unfavorable surface, and make it suitable for more specialized and demanding cells.

"To Be Microbiocidal and Not to Be Cytotoxic at the Same Time "-Silver Nanoparticles and Their Main Role on the Surface of Titanium Alloy Implants.

The chemical vapor deposition (CVD) method has been used to produce dispersed silver nanoparticles (AgNPs) on the surface of titanium alloy (Ti6Al4V) and nanotubular modified titanium alloys (Ti6Al4V/TNT5), leading to the formation of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs systems with different contents of metallic silver particles. Their surface morphology and silver particles arrangement were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS), and atomic force microscopy (AFM). The wettability and surface free energy of these materials were investigated on the basis of contact angle measurements. The degree of silver ion release from the surface of the studied systems immersed in phosphate buffered saline solution (PBS) was estimated using inductively coupled plasma ionization mass spectrometry (ICP-MS). The biocompatibility of the analyzed materials was estimated based on the fibroblasts and osteoblasts adhesion and proliferation, while their microbiocidal properties were determined against Gram-positive and Gram-negative bacteria, and yeasts. The results of our works proved the high antimicrobial activity and biocompatibility of all the studied systems. Among them, Ti6Al4V/TNT5/0.6AgNPs contained the lowest amount of AgNPs, but still revealed optimal biointegration properties and high biocidal properties. This is the biomaterial that possesses the desired biological properties, in which the potential toxicity is minimized by minimizing the number of silver nanoparticles.

The Morphology, Structure, Mechanical Properties and Biocompatibility of Nanotubular Titania Coatings before and after Autoclaving Process.

The autoclaving process is one of the sterilization procedures of implantable devices. Therefore, it is important to assess the impact of hot steam at high pressure on the morphology, structure, and properties of implants modified by nanocomposite coatings. In our works, we focused on studies on amorphous titania nanotubes produced by titanium alloy (Ti6Al4V) electrochemical oxidation in the potential range 5⁻60 V. Half of the samples were drying in argon stream at room temperature, and the second ones were drying additionally with the use of immersion in acetone and drying at 396 K. Samples were subjected to autoclaving and after sterilization they were structurally and morphologically characterized using Raman spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) and scanning electron microscopy (SEM). They were characterized in terms of wettability, mechanical properties, and biocompatibility. Obtained results proved that the autoclaving of amorphous titania nanotube coatings produced at lower potentials (5⁻15 V) does not affect their morphology and structure regardless of the drying method before autoclaving. Nanotubular coatings produced using higher potentials (20⁻60 V) require removal of adsorbed water particles from their surface. Otherwise, autoclaving leads to the destruction of the architecture of nanotubular coatings, which is associated with the changing of their mechanical and biointegration properties.

Titania Nanotubes/Hydroxyapatite Nanocomposites Produced with the Use of the Atomic Layer Deposition Technique: Estimation of Bioactivity and Nanomechanical Properties.

Titanium dioxide nanotubes/hydroxyapatite nanocomposites were produced on a titanium alloy (Ti6Al4V/TNT/HA) and studied as a biocompatible coating for an implant surface modification. As a novel approach for this type of nanocomposite fabrication, the atomic layer deposition (ALD) method with an extremely low number of cycles was used to enrich titania nanotubes (TNT) with a very thin hydroxyapatite coating. X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used for determination of the structure and the surface morphology of the fabricated nanocoatings. The biointegration activity of the layers was estimated based on fibroblasts' proliferation on the TNT/HA surface. The antibacterial activity was determined by analyzing the ability of the layers to inhibit bacterial colonization and biofilm formation. Mechanical properties of the Ti6Al4V/TNT/HA samples were estimated by measuring the hardness, Young's module, and susceptibility to scratching. The results revealed that the nanoporous titanium alloy coatings enriched with a very thin hydroxyapatite layer may be a promising way to achieve the desired balance between biofunctional and biomechanical properties of modern implants.

Studies on Silver Ions Releasing Processes and Mechanical Properties of Surface-Modified Titanium Alloy Implants.

Dispersed silver nanoparticles (AgNPs) on the surface of titanium alloy (Ti6Al4V) and titanium alloy modified by titania nanotube layer (Ti6Al4V/TNT) substrates were produced by the chemical vapor deposition method (CVD) using a novel precursor of the formula [Ag5(O2CC2F5)5(H2O)3]. The structure and volatile properties of this compound were determined using single crystal X-ray diffractometry, variable temperature IR spectrophotometry (VT IR), and electron inducted mass spectrometry (EI MS). The morphology and the structure of the produced Ti6Al4V/AgNPs and Ti6Al4V/TNT/AgNPs composites were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Moreover, measurements of hardness, Young's modulus, adhesion, wettability, and surface free energy have been carried out. The ability to release silver ions from the surface of produced nanocomposite materials immersed in phosphate-buffered saline (PBS) solution has been estimated using inductively coupled plasma mass spectrometry (ICP-MS). The results of our studies proved the usefulness of the CVD method to enrich of the Ti6Al4V/TNT system with silver nanoparticles. Among the studied surface-modified titanium alloy implants, the better nano-mechanical properties were noticed for the Ti6Al4V/TNT/AgNPs composite in comparison to systems non-enriched by AgNPs. The location of silver nanoparticles inside of titania nanotubes caused their lowest release rate, which may indicate suitable properties on the above-mentioned type of the composite for the construction of implants with a long term antimicrobial activity.