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.     

Modified Titanium Surface-Mediated Effects on Human Bone Marrow Stromal Cell Response

Surface modification of titanium implants is used to enhance osseointegration. The study objective was to evaluate five modified titanium surfaces in terms of cytocompatibility and pro-osteogenic/pro-angiogenic properties for human mesenchymal stromal cells: amorphous microporous silica (AMS), bone morphogenetic protein-2 immobilized on AMS (AMS + BMP), bio-active glass (BAG) and two titanium coatings with different porosity (T1; T2). Four surfaces served as controls: uncoated Ti (Ti), Ti functionalized with BMP-2 (Ti + BMP), Ti surface with a thickened titanium oxide layer (TiO₂) and a tissue culture polystyrene surface (TCPS). The proliferation of eGFP-fLuc (enhanced green fluorescence protein-firefly luciferase) transfected cells was tracked non-invasively by fluorescence microscopy and bio-luminescence imaging. The implant surface-mediated effects on cell differentiation potential was tracked by determination of osteogenic and angiogenic parameters [alkaline phosphatase (ALP); osteocalcin (OC); osteoprotegerin (OPG); vascular endothelial growth factor-A (VEGF-A)]. Unrestrained cell proliferation was observed on (un)functionalized Ti and AMS surfaces, whereas BAG and porous titanium coatings T1 and T2 did not support cell proliferation. An important pro-osteogenic and pro-angiogenic potential of the AMS + BMP surface was observed. In contrast, coating the Ti surface with BMP did not affect the osteogenic differentiation of the progenitor cells. A significantly slower BMP-2 release from AMS compared to Ti supports these findings. In the unfunctionalized state, Ti was found to be superior to AMS in terms of OPG and VEGF-A production. AMS is suggested to be a promising implant coating material for bioactive agents delivery.     

Corrosion and Wear Behavior of TiO2/TiN Duplex Coatings on Titanium by Plasma Electrolytic Oxidation and Gas Nitriding

In this study, corrosion and wear behavior of three kinds of coatings by two processes, namely, plasma electrolytic oxidation (PEO) coatings (Ti/TiO₂), gas nitriding coating (Ti/TiN), and the duplex coating (Ti/TiO₂-N) by combination of PEO and gas nitriding methods were systematically investigated. X-ray diffraction tests, field-emission scanning electron microscopy, and adhesion tests are employed for the coating characterization, along with the wear and electrochemical test for evaluating the corrosion and tribological properties. The morphology and structure of the coating consist of micro-cavities known as the pancake structure on the surface. The electrolytic plasma oxidation process produces a typical annealing behavior with a low friction coefficient based on the wear test. The coating consists of nitride and nitrate/oxides titanium for nitrided samples. The surface morphology of nitrided oxide titanium coating shows a slight change in the size of the crystals and the diameter of the cavities due to the influence of nitrogen in the titanium oxide coating. The tribological behavior of the coatings showed that the wear resistance of the duplex coating (Ti/TiO₂-N) and Ti/TiO₂ coatings is significantly higher compared to Ti/TiN coatings and uncoated Ti samples. The polarization resistance of the Ti/TiO₂-N and Ti/TiO₂ coatings was 632.2 and 1451.9 kΩ cm², respectively. These values are considerably greater than that of the uncoated Ti (135.9 kΩ cm²). Likewise, impedance showed that the Ti/TiO₂-N and Ti/TiO₂ coatings demonstrate higher charge transfer resistance than that of other samples due to better insulating behavior and denser structure.     

Characterization of Hydroxyapatite Film Obtained by Er:YAG Pulsed Laser Deposition on Sandblasted Titanium: An In Vitro Study

The surface of titanium (Ti) dental implants must be modified to improve their applicability, owing to the biological inertness of Ti. This study aims to use sandblasting as a pretreatment method and prepare a hydroxyapatite (HA) coating on Ti to improve its biocompatibility and induce bone bonding and osteogenesis. In this paper, sandblasted Ti discs were coated with α-tricalcium phosphate (α-TCP) via Er:YAG pulsed laser deposition (Er:YAG-PLD). An HA coating was then obtained via the hydrothermal treatment of the discs at 90 °C for 10 h. The surface characteristics of the samples were evaluated by SEM, SPM, XPS, XRD, FTIR, and tensile tests. Rat bone marrow mesenchymal stem cells were seeded on the HA-coated discs to determine cellular responses in vitro. The surface characterization results indicated the successful transformation of the HA coating with a nanorod-like morphology, and its surface roughness increased. In vitro experiments revealed increased cell attachment on the HA-coated discs, as did the cell morphology of fluorescence staining and SEM analysis; in contrast, there was no increase in cell proliferation. This study confirms that Er:YAG-PLD could be used as an implant surface-modification technique to prepare HA coatings with a nanorod-like morphology on Ti discs.     

Hydroxyapatite Formation on Coated Titanium Implants Submerged in Simulated Body Fluid

Titanium implants are commonly used in the field of dentistry for prosthetics such as crowns, bridges, and dentures. For successful therapy, an implant must bind to the surrounding bone in a process known as osseointegration. The objective for this ongoing study is to determine the potential of different implant surface coatings in providing the formation of hydroxyapatite (HA). The coatings include titanium nitride (TiN), silicon dioxide (SiO₂), and quaternized titanium nitride (QTiN). The controls were a sodium hydroxide treated group, which functioned as a positive control, and an uncoated titanium group. Each coated disc was submerged in simulated body fluid (SBF), replenished every 48 h, over a period of 28 days. Each coating successfully developed a layer of HA, which was calculated through mass comparisons and observed using scanning electron microscopy (SEM) and energy dispersive analysis x-rays (EDX). Among these coatings, the quaternized titanium nitride coating seemed to have a better yield of HA. Further studies to expand the data concerning this experiment are underway.     

Novel Coatings to Minimize Corrosion of Titanium in Oral Biofilm

The aim of this work is to investigate the effects produced by polymicrobial biofilm (Porphyromonas gingivalis, Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius) on the corrosion behavior of titanium dental implants. Pure titanium disks were polished and coated with titanium nitride (TiN) and silicon carbide (SiC) along with their quarternized versions. Next, the disks were cultivated in culture medium (BHI) with P. gingivalis, S. mutans, S. sanguinis, and S. salivarius and incubated anaerobically at 37 °C for 30 days. Titanium corrosion was evaluated through surface observation using Scanning Electron Microscope (SEM) and Atomic Force Microscopy (AFM). Furthermore, the Ti release in the medium was evaluated by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). SEM images showed that coated Ti disks exhibited lower corrosion compared to non-coated disks, except for the quartenized TiN. This was confirmed by AFM, where the roughness was higher in non-coated Ti disks. ICP showed that Ti levels were low in all coating disks. These results indicate that these SiC and TiN-based coatings could be a useful tool to reduce surface corrosion on titanium implant surfaces.     

Synthesis of Biocompatible Hydroxyapatite Using Chitosan Oligosaccharide as a Template

In this study, a novel biocompatible hydroxyapatite (HA) was synthesized by using chitosan oligosaccharide (COS) as a template. These HA samples were studied by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The biocompatibility of HA samples was evaluated via cell viability, cell morphology and alkaline phosphatase staining of MG-63 cell lines. The results show that HA synthesized in the presence of COS was favorable to proliferation and osteogenic differentiation of MG-63 cells. These hydroxyapatites are potentially attractive biomaterials for bone tissue engineering applications.     

Formation of Micro- and Nanostructures on the Nanotitanium Surface by Chemical Etching and Deposition of Titania Films by Atomic Layer Deposition (ALD)

In this study, an integrated approach was used for the preparation of a nanotitanium-based bioactive material. The integrated approach included three methods: severe plastic deformation (SPD), chemical etching and atomic layer deposition (ALD). For the first time, it was experimentally shown that the nature of the etching medium (acidic or basic Piranha solutions) and the etching time have a significant qualitative impact on the nanotitanium surface structure both at the nano- and microscale. The etched samples were coated with crystalline biocompatible TiO₂ films with a thickness of 20 nm by Atomic Layer Deposition (ALD). Comparative study of the adhesive and spreading properties of human osteoblasts MG-63 has demonstrated that presence of nano- and microscale structures and crystalline titanium oxide on the surface of nanotitanium improve bioactive properties of the material.     

Surface Modifications of Titanium Aluminium Vanadium Improve Biocompatibility and Osteogenic Differentiation Potential

Osteogenic cells are strongly influenced in their behaviour by the surface properties of orthopaedic implant materials. Mesenchymal stem and progenitor cells (MSPCs) migrate to the bone–implant interface, adhere to the material surface, proliferate and subsequently differentiate into osteoblasts, which are responsible for the formation of the bone matrix. Five surface topographies on titanium aluminium vanadium (TiAl₆V₄) were engineered to investigate biocompatibility and adhesion potential of human osteoblasts and the changes in osteogenic differentiation of MSPCs. Elemental analysis of TiAl₆V₄ discs coated with titanium nitride (TiN), silver (Ag), roughened surface, and pure titanium (cpTi) surface was analysed using energy-dispersive X-ray spectroscopy and scanning electron microscopy. In vitro cell viability, cytotoxicity, adhesion behaviour, and osteogenic differentiation potential were measured via CellTiter-Glo, CytoTox, ELISA, Luminex^® technology, and RT-PCR respectively. The Ag coating reduced the growth of osteoblasts, whereas the viability of MSPCs increased significantly. The roughened and the cpTi surface improved the viability of all cell types. The additive coatings of the TiAl₆V₄ alloy improved the adhesion of osteoblasts and MSPCs. With regard to the osteogenic differentiation potential, an enhanced effect has been demonstrated, especially in the case of roughened and cpTi coatings.     

成骨样细胞MG-63中的甲状腺激素受体Ⅰ——细胞核可溶性甲状腺激素受体与激素(T3)结合的动力学

建立人类成骨样细胞系MG-63中甲状腺激素受体的结合实验的方法,测定受体动力学参数.体外培养MG-63细胞,制备细胞核提取液,对其细胞核提取物中的甲状腺激素受体与激素(主要是T3)结合的动力学进行了Scatchard分析.MG-63细胞核内存在高亲和力(Ka为7.68×109L/mol)和有限结合容量的甲状腺激素受体,最大结合容量为(111.25+10.73)fmol/mg蛋白.建立了一个简易的自培养细胞制备纯净细胞核的方法,为进一步研究成骨样细胞及骨组织中的甲状腺激素受体奠定基础.

Abstract：

To establish a method for radioligand binding assay of thyroid hormone receptors(TR)in human osteoblast-like osteosarcoma cell line MG-63 and to estimate the kinetic parameters of putative receptors. The MG-63cell was cultured in Ham's F12, the soluble TR was prepared from the intact nuclear extracts. The binding properties between TR and T3 were performed by using the traditional Scatchard analysis. The apparent Ka of TR in MG-63 is 7.68× 109 L/mol, and MBC(111. 25+ 10.77)fmol/mg protein. The study indicated that MG-63 cells possessed high affinity and limited-capacity of TR in its nuclear extracts. This may serve as the starting and basic work about TR in bone cell.     

亚砷酸和顺铂对骨肉瘤MG-63细胞生长的抑制作用比较

目的 观察亚砷酸和顺铂对骨肉瘤MG-63细胞生长的抑制作用.方法 采用细胞培养的方法,体外培养人骨肉瘤株MG-63细胞.应用倒置相差显微镜、透射电镜、四氮唑盐(MTT)比色、流式细胞仪检测等方法,观察亚砷酸和顺铂[各加入100μl(1 g/L)]对骨肉瘤MG-63细胞生长的抑制作用和对细胞凋亡、细胞周期的影响.结果 相差显微镜下,对照组MG-63细胞贴壁生长良好,细胞核呈圆形,细胞呈上皮样细胞排列;亚砷酸组MG-63细胞排列无规则,有细胞死亡;顺铂组MG-63细胞多变小,变圆,胞质的空泡化明显.电镜下,对照组MG-63细胞表面可见球形质膜突起,线粒体嵴粗大,双层核膜结构清晰;亚砷酸组MG-63细胞表面的球形突起消失,线粒体嵴变细,细胞核缩小,凋亡细胞核膜清晰;顺铂组MG-63细胞表面球形突起脱离,细胞核膜增厚,核内染色质形成细纱状.亚砷酸和顺铂均能抑制MG-63细胞生长,抑制率(%)组间比较差异有统计学意义(F=78.859,P<0.05);在2、4、8、16、32、64、128 h,亚砷酸(56.31±0.03、70.00±0.06、79.84±0.03、87.31±0.13、84.70±0.09、90.68±0.06、91.18±0.05)和顺铂(7.55±0.15、15.70±0.17、30.72±0.07、49.80±0.05、45.11±0.13、61.62±0.08、93.80±0.12)对细胞生长的抑制率与对照组(2.03±0.07、2.78±0.08、3.11±0.01、5.67±0.04、12.23±0.04、18.65±0.04、24.45±0.04)比较明显升高(P均<0.05);但与顺铂比较,亚砷酸起效快,在2～32 h抑制率明显高于同时间顺铂(P均<0.05).亚砷酸和顺铂均可导致MG-63细胞凋亡,凋亡率组间比较差异有统计学意义(F=13.317,P<0.05);亚砷酸在24、36、48 h(20.5±3.78、45.76±9.90、25.16±15.41),顺铂在24、36、48 h(12.55±1.51、18.85±3.40、12.37±5.43),MG-63细胞凋亡率(%)明显高于同时间对照组(6.57±1.48、8.03±2.08、6.54±1.30,P<0.05或<0.01).亚砷酸和顺铂均对MG-63细胞周期G1、S、G2/M期有抑制作用,抑制率组间比较差异有统计学意义(F值分别为54.579、43.429、21.795,P<0.05或<0.01);对G1期抑制率(%)亚砷酸(78.26±5.24)和顺铂(80.48±2.81)与对照组(57.49±6.65)比较明显升高(P<0.05或<0.01).结论 亚砷酸和顺铂都可抑制骨肉瘤MG-63细胞生长,诱导细胞凋亡;亚砷酸作用快于顺铂,亚砷酸和顺铂对MG-63细胞的抑制作用发生在细胞周期中的G1期.     

Refractory Metal Coated Alumina Foams as Support Material for Stem Cell and Fibroblasts Cultivation

Ceramics are widely used as implant materials; however, they are brittle and may emit particles when used in these applications. To overcome this disadvantage, alumina foams, which represent a 3D cellular structure comparable to that of human trabecular bone structures, were sputter coated with platinum, tantalum or titanium and modified with fibronectin or collagen type I, components of the extracellular matrix (ECM). To proof the cell material interaction, the unmodified and modified materials were cultured with (a) mesenchymal stem cells being a perfect indicator for biocompatibility and releasing important cytokines of the stem cell niche and (b) with fibroblasts characterized as mediators of inflammation and therefore an important cellular component of the foreign body reaction and inflammation after implantation. To optimize and compare the influence of metal surfaces on cellular behavior, planar glass substrates have been used. Identified biocompatible metal surface of platinum, titanium and tantalum were sputtered on ceramic foams modified with the above-mentioned ECM components to investigate cellular behavior in a 3D environment. The cellular alumina support was characterized with respect to its cellular/porous structure and niche accessibility and coating thickness of the refractory metals; the average cell size was 2.3 mm, the average size of the cell windows was 1.8 mm, and the total foam porosity was 91.4%. The Pt, Ti and Ta coatings were completely dense covering the entire alumina foam surface. The metals titanium and tantalum were colonized very well by the stem cells without a coating of ECM components, whereas the fibroblasts preferred components of the ECM on the alumina foam surface.     

Effect of Multi-Phosphonate Coating of Titanium Surfaces on Osteogenic Potential

The aim of this study was to evaluate the impact of a novel multi-phosphonate (MP) coating strategy of dental implant surfaces on the expression of osteogenesis-related factors in vitro. MG-63 human osteoblast-like cells, bone marrow mesenchymal stem cells (BM-MSCs), and human periodontal ligament stem cells (hPDLSCs) were cultured separately on titanium disks with and without MP coating. Cell attachment was visualized by focal adhesion and actin cytoskeleton staining. The proliferation and gene expression of the markers related to osteogenesis and bone turnover were measured after 48 and 120 h of cell culture. Actin cytoskeleton assembly and focal adhesion were similar between test surfaces within each cell type but differed from those on tissue culture plastic (TCP). The proliferation of MG-63 cells and PDLSCs was comparable on all surfaces, while BM-MSCs showed an increase on tissue culture plastic (TCP) versus titanium. The gene expression of osteoprotegerin and receptor activator of nuclear factor-kappa B ligand was higher in MG-63 cells grown on MP-coated surfaces. At the same time, osteocalcin was decreased compared to the other surfaces. Collagen type I gene expression after 120 h was significantly lower in hPDLSCs cultivated on MP-coated surfaces. Within the limitations of this study, MP coating on titanium surfaces might have a slight beneficial effect on bone turnover in vitro.     

Stress Distribution Pattern in Zygomatic Implants Supporting Different Superstructure Materials

The aim of this study was to assess and compare the stress–strain pattern of zygomatic dental implants supporting different superstructures using 3D finite element analysis (FEA). A model of a tridimensional edentulous maxilla with four dental implants was designed using the computer-aided design (CAD) software. Two standard and two zygomatic implants were positioned to support the U-shaped bar superstructure. In the computer-aided engineering (CAE) software, different materials have been simulated for the superstructure: cobalt–chrome (CoCr) alloy, titanium alloy (Ti), zirconia (Zr), carbon-fiber polymers (CF) and polyetheretherketone (PEEK). An axial load of 500 N was applied in the posterior regions near the zygomatic implants. Considering the mechanical response of the bone tissue, all superstructure materials resulted in homogeneous strain and thus could reconstruct the edentulous maxilla. However, with the aim to reduce the stress in the zygomatic implants and prosthetic screws, stiffer materials, such Zr, CoCr and Ti, appeared to be a preferable option.     

Ion Release and Surface Changes of Nickel–Titanium Archwires Induced by Changes in the pH Value of the Saliva—Significance for Human Health Risk Assessment

The aim of this study was to explore whether changes in the salivary pH influence mechanical properties, surface roughness, and ion release from NiTi archwires with various surface coatings, and discuss the clinical significance of the findings. The uncoated, rhodium-coated, and nitrified NiTi wires were immersed into artificial saliva of different pH values (4.8, 5.1, 5.5, and 6.6). Released nickel and titanium ions were measured with inductively coupled plasma-optical emission spectroscopy at the end of 28 days. Atomic force microscopy was used to measure the arithmetic average surface roughness R_a, the root-mean-square roughness R_q, and the maximum height of the asperities R_Z. The nanoindentation hardness (H_IT) and Young’s modulus (E_IT) measurements were performed. The change in the pH of artificial saliva is inversely proportional to the release of titanium from both coated and uncoated wires, and the release of nickel from uncoated wires. The surface roughness parameters of both coated and uncoated wires are unaffected by the change in the pH of artificial saliva. The change in the pH of saliva has minor influence on the hardness and Young’s modulus of elasticity of both coated and uncoated wires. The concentration of released metal ions measured was below the recommended upper limit for daily intake; nevertheless, hypersensitivity effects cannot be excluded, even at lower concentrations and at low pH.     

Physicochemical and Biological Properties of Graphene-Oxide-Coated Metallic Materials

In this article, we present graphene oxide (produced by a modified Hummers’ method) coatings obtained using two different methods: electrophoretic deposition on 316L stainless steel and chemical modification of the surface of gold applied to the steel. The coating properties were characterized by microscopic and spectrometric techniques. The contact angle was also determined, ranging from 50° to 70°. Our results indicated that GO coatings on steel and gold were not toxic towards L929 cells in a direct cell adhesion test—on all tested materials, it was possible to observe the growth of L929 cells during 48 h of culture. The lack of toxic effect on cells was also confirmed in two viability tests, XTT and MTT. For most of the tested materials, the cell viability was above 70%. They showed that the stability of the coating is the crucial factor for such GO coatings, and prove that GO in the form of coating is non-toxic; however, it can show toxicity if detached from the surface. The obtained materials also did not show any hemolytic properties, as the percentage of hemolysis was on the level of the negative control, which is very promising in the light of future potential applications.     

The Effect of TiN and DLC Anti-Wear Coatings on the Tribofilm Formation and Frictional Heat Phenomena in Coated Metals vs. WC-Co

The aim of this work was to study the effect of anti-wear coatings on the selected frictional phenomena, i.a., frictional heating and tribofilm formation, of model tribological pairs. For this purpose, three popular metallic substrate materials were selected: AISI 316L and AISI 440B stainless steels, as well as Ti6Al4V two-phase titanium alloy. The substrates were tested in the dry sliding conditions in three states: uncoated, as well as titanium nitride (TiN) or diamond-like-carbon (DLC) coated. According to the results provided, under applied frictional conditions TiN coating, even if it is worn off the sample surface, contributes to excessive frictional heating of a tribological pair by altering the tribofilm formation. The analysis also showed that in some tribological pairs, rapid temperature alteration of a counter sample can be used to approximate the sliding distance after which the TiN coating becomes worn off. On the contrary, in all pairs tested, the DLC film became locally damaged, but it sustained its antifriction properties, contributing to low coefficients of friction (COFs) and the lowest frictional temperatures observed.     

Effects of 17beta-estradiol on the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in human osteoblastic MG-63 cell cultures

Estrogens are important regulators of bone cell function. Osteoblast-derived membrane type 1 matrix metalloproteinses (MT1-MMP) have recently been implied to play an important role in the process of bone resorption by proteolytically activating latent matrix metalloproteinase-2 (proMMP-2) at the cell surface and degrading tumor necrosis factor-alpha (TNF-alpha). In the present study, we observed the effects of 17beta-estradiol (E2) on MT1-MMP production and subsequent activation of latent matrix proMMP-2, and also proMMP-2 secretion in cultures of human osteoblastic MG-63 cells. Western immunoblot analysis showed that treatment with increasing doses of E2 in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein. Confocal immunohistochemistry analysis also confirmed that E2 induced MT1-MMP synthesis in MG-63 cells. We found unexpectedly that although MT1-MMP synthesis was up-regulated by E2 in cultures of MG-63 cells, activation of proMMP-2 was unchanged, which can be attributed partly to the undetectable tissue inhibitor of metalloproteinase-2 (TIMP-2) protein in MG-63 cells by Western immunoblotting. ProMMP-2 production was also not influenced by E2. In conclusion, E2 induces MT1-MMP protein expression in MG-63 cells while it is not followed by proMMP-2 activation, E2 may suppress bone resorption by accentuated degradation of TNF-alpha which mediated through increasingly MT1 -MMP production in osteoblastic cells.