Response of MC3T3-E1 osteoblast cells to the microenvironment produced on Co–Cr–Mo alloy using laser surface texturing

Osteoblast responses to Co–Cr–Mo alloy depend on not only the chemistry of alloy but also the physical properties of alloy surface, such as its microtopography and roughness. This study was undertaken to examine changes in cell adhesion, morphology, differentiation and gene expression during osteoblast interaction with different textured Co–Cr–Mo alloys. With laser surface texturing, three kinds of textured surfaces were fabricated. It showed that the microenvironment processed by laser surface texturing leads to an increase in surface roughness and DMEM contact angles of samples. Adhesion and differentiation tests demonstrated that osteoblast cells can discriminate surfaces with different roughness and surfaces with comparable roughness but different topographies such as triangle, circle and square textures. Morphological characteristics obtained by SEM imaging showed that osteoblast cells are elongated and are in polygonal shape on the textured surface. OPG/RANKL mRNA expression studies showed a significant and marked decrease in RANKL gene expression on the square-textured surface; by contrast, the ratio of OPG/RANKL showed a significant increase. These results indicate that Co–Cr–Mo surface textures affect osteoblast proliferation, morphology and gene expression; the microenvironment of implant should be considered in the future design.     

Adhesive B-doped DLC films on biomedical alloys used for bone fixation

The long-term failure of the total hip and knee prostheses is attributed to the production of wear particles at the articulating interface between the metals, ceramics and polymers used for surgical implants and bone-fixtures. Therefore, finding an adhesive and inert coating material that has low frictional coefficient should dramatically reduce the production of wear particles and hence, prolong the life time of the surgical implants. The novel properties of the non-toxic diamond-like carbon (DLC) coatings have proven to be excellent candidates for biomedical applications. However, they have poor adhesion strength to the alloys and biomaterials. The addition of a thin interfacial layer such as Si, Ti, TiN, Mo and Cu/Cr and/or adding additives such as Si, F, N, O, W, V, Co, Mo, Ti or their combinations to the DLC films has been found to increase the adhesion strength substantially. In our study, grade 316L stainless steel and grade 5 titanium alloy (Ti-6Al-4V) were used as biomaterial substrates. They were coated with DLC films containing boron additives at various levels using various Si interfacial layer thicknesses. The best film adhesion was achieved at 8% and 20% on DLC coated Ti-6Al-4V and grade 316L substrates, respectively. It has been demonstrated that doping the DLC with boron increases their adhesion strength to both substrates even without silicon interfacial layer and increases it substantially with optimum silicon layer thickness. The adhesion strength is also correlated with the hydrogen contents in the B-DLC films. It is found to reach its maximum value of 700 kg/cm² and 390 kg/cm² at 2/7 and 3/6 for CH₄/Ar partial pressures (in mTorr ratio) for Ti-6Al-4V and 316L substrates, respectively.     

Oberflächenmodifizierung von Titan zur Verbesserung der Grenzflächenverträglichkeit

Surface Modification of Titanium for Improvement of the Interfacial Biocompatibility We report the CVD‐polymerisation of amino‐functionalized [2,2]‐paracyclophane for polymer coating and functionalization of titanium surfaces. Additionally, the functionalization was carried out by silanization with 3‐aminopropyl‐triethoxysilane. The generated amino‐groups were used for covalent immobilization of bioactive substances to stimulate the adhesion and growth of osteoblasts. As bioactive substances the pentapeptide GRGDS and the growth factor BMP‐2 were chosen. The covalent bonding was achieved by activation with hexamethylene diisocyanate. Each modification step was characterized by X‐ray‐photoelectron‐spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The covalent bonding of the bioactive substances was proven by radiolabelling and surface‐MALDI‐ToF‐MS. In vitro‐biocompatibility tests with primary, human osteoblasts demonstrated the improved cell adhesion and spreading on the bioactive modified titanium surfaces.     

Modell zur Analyse des knöchernen Anwachsverhaltens auf bioaktiv beschichteten Implantatoberflächen

Model to analyse the bone on‐growth on bioactive coated implant surfaces Especially on the field of bone regeneration, transient and permanent implants are an important method of therapy in the Orthopaedic Surgery. In this context, bioactive surfaces on metallic implants provide an improved contact to the surrounding bone. The goal of our study was to establish an in‐vitro test system to evaluate the on‐growth of bone‐derived cells on different surface coatings. Therefore, we invented a special kind of clamps made of commercially‐pure (c‐p) titanium and blasted with hydroxyapatite particles followed by electrochemically coating with calcium phosphate (BONIT®‐HA, BONIT®). Definite pieces of human cancellous bone were attached to these clamps, inserted onto tissue culture plates and cultivated in DMEM for ten days. Finally, the contact area between human cancellous bone and the implant surface was analyzed and the spreading of osteoblast‐like cells evaluated by scanning electron microscopy (SEM). A well‐spread morphology of bone cells was observed on the implant surfaces coated with calcium phosphate (CaP). In comparison the clamps without CaP coatings showed only a marginal growth of bone cells on the clamp surface. The presented newly in‐vitro test setup using titanium clamps coated with bioactive layers attached to human cancellous bone represents a well‐functioning model for qualitative evaluation of bone on‐growth.     

Biomimetic Titanium Alloy with Sparsely Distributed Nanotubes Could Enhance Osteoblast Functions

Designing the bone implant surface by mimicking the structure of natural tissue is an intriguing means to achieve better osseointegration. In this study, a biomimetic surface with sparsely distributed 80 nm diameter nanotubes (SNT) with a spacing of about 20–80 nm from each other, contrary to the closely distributed nanotubes on pure titanium, is fabricated by anodization of near β titanium alloy Ti‐5Zr‐3Sn‐5Mo‐15Nb (TLM). The structure is more similar to the cross‐section of collagen fibrils than commonly reported nanotubes on pure titanium. The SNT‐textured Ti‐alloy also shows good wettability and low elastic modulus more compatible with bone tissues. The surface bioactivity is evaluated in vitro by primary osteoblast cultures. Compared with the polished non‐textured TLM, the SNT texture exhibits satisfactory bioactivity with protein adsorption, initial cell adhesion, cell differentiation as revealed by ALP activity and osteogenesis‐related gene expression. Although cell proliferation is slightly suppressed, ECM deposition is enhanced. The enhanced cell functions are probably related to the biomimetic structure, biochemical characteristics, and mechanical properties of SNT‐textured Ti‐alloy as well as the potential fluid exchange effect among the nanotubes. The SNT texture which constitutes a bio‐interface with fluid supply from the implant surface can be tailored to enhance biological properties such as cell immigration and differentiation.     

Cytocompatibility evaluation of Ni-free stainless steel manufactured by nitrogen adsorption treatment

Cytocompatibility of nickel-free austenitic stainless steel manufactured by nitrogen adsorption treatment, Fe–Cr–Mo–N, was evaluated and compared with a conventional austenitic stainless steel, 316L, and nickel-free ferrite stainless steel, Fe–Cr–Mo, before nitrogen adsorption treatment. Two types of cytotoxicity tests were performed; static and dynamic conditions. In static conditions, human normal diploid fibroblast HEL299 was cultured on the disks of the three materials and cell growth was examined. In dynamic conditions, the disks were rotated in extracting medium with zirconia balls at 37 °C for 14 days, and the collected extracting medium was added into HEL299 culture to examine its inhibitive effect on cell growth. Quantification of metallic elements in collected extracting medium was also performed. As a result, Fe–Cr–Mo–N had higher cell growth than 316L in static and dynamic conditions. Nickel was detected in the extracting media of 316L and Fe–Cr–Mo, whereas no nickel was detected in that of Fe–Cr–Mo–N. These facts indicate that Fe–Cr–Mo–N has higher cytocompatibility than 316L and that the nitrogen adsorption treatment contributes to the higher corrosion resistance of Fe–Cr–Mo–N in the presence of wear.     

The microstructure and mechanical properties of prolonged and lower temperature aged Fe–Ni–Mn–Mo–Ti–Cr maraging steel

In this study, the microstructure and mechanical properties of Fe–Ni–Mn–Mo–Ti–Cr maraging steel at low temperature and prolonged aging condition were investigated. Optical and scanning electron microscopy examinations, tensile and hardness tests were conducted to study the microstructure, aging behavior and mechanical properties of the cold‐rolled steel. The results showed that aging of cold rolled Fe–Ni–Mn–Mo–Ti–Cr maraging steel resulted in the formation of Mo rich and Ti rich Lave phase precipitates. Existence of many dislocation cores due to cold rolling and subsequently, low temperature aging caused to formation of uniform distribution of very fine precipitates. The presence of these precipitates increased the yield and ultimate tensile strengths but couldn't improve the uniform tensile ductility. This alloy showed ultra‐high fracture stress of about 1950 MPa with a negligible tensile elongation (about 2 %) at the peak aged condition. The fractographic studies indicated this alloy shows semi‐brittle fracture in the subsequent aging treatment.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

The generation of nanocolloidal wear from stainless steel and titanium alloy and its toxic effects in a monocyte cell culture model

This study addresses the aspect of submicron‐sized / nanocolloidal wear comparing stainless steel (316L) and titanium alloy (TiAl6V4) for their toxic and inflammatory potentials. Wear was generated in a tribometer using the disc‐on‐pin‐method with pin and disc submerged in a sterile cell culture medium setting. The wear medium was separated according to Stokes' law into a fraction smaller 200 nm (nanocolloids) and a fraction greater 200 nm (particles). Vitality and inflammatory potential was measured in a cell culture model employing murine macrophages (J774). Cells were incubated with increasing concentrations (12.5, 25 and 50vol%) of either wear medium (particles and nanocolloids) from 316L and TiAl6V4. Vitality was measured by MTT assay and inflammatory reactions were quantified by TNF‐α ELISA. Nanocolloids from stainless steel and titanium induced strong, dose dependant toxic effects in the MTT assay while particles did not affect vitality in a dose dependant manner. The inflammatory response remained unaltered in all four groups. We conclude that interactions between soluble metallic wear and proteins forming nanocolloidal wear should be considered when conduction experiments addressing the aspect of biocompatibility in metallic implant materials.     

A comparative study of the impurity segregation from commercially pure Ti, Ti6Al4V and Ti3Al8V6Cr4Zr4Mo

The surface composition of commercially pure Ti, Ti6Al4V and Ti3Al8V6Cr4Zr4Mo during annealing at different constant temperatures was experimentally investigated. Auger electron spectroscopy was used to monitor the APPHs of the specified elements present on the surfaces. The surfaces of Ti and its alloys were contaminated by oxygen and carbon, and the contamination is attributed to the continual uptake of the background gases, even in the UHV chamber. It was found that mainly C and S segregated at 400 °C, and Cl at higher temperatures (500–630 °C) for commercially pure Ti. However, S was the main segregating species for all three samples. The segregation of Al was measured for the Ti6Al4V and Ti3Al8V6Cr4Zr4Mo samples at higher temperatures. The linear least-square fit method was employed to determine the contribution of pure Ti and TiC from the measured APPH's. The AES fitting confirmed the formation of TiC on the surface at temperatures 400–500 °C.     

Structure and mechanical properties of Ti–5Cr based alloy with Mo addition

The effects of molybdenum (Mo) on the structure and mechanical properties of a Ti–5Cr-based alloy were studied with an emphasis on improving its strength/modulus ratio. Commercially pure titanium (c.p. Ti) was used as a control. As-cast Ti–5Cr and a series of Ti–5Cr–xMo (x = 1, 3, 5, 7, 9 and 11 wt.%) alloys were prepared by using a commercial arc-melting vacuum-pressure casting system, and investigated with X-ray diffraction (XRD) for phase analysis. Three-point bending tests were performed to evaluate the mechanical properties of all specimens and their fractured surfaces were observed by using scanning electron microscopy (SEM). The experimental results indicated that Ti–5Cr–7Mo, Ti–5Cr–9Mo and Ti–5Cr–11Mo alloys exhibited ductile properties, and the β-phase Ti–5Cr–9Mo alloy exhibited the lowest bending modulus. However, the Ti–5Cr–3Mo and Ti–5Cr–5Mo alloys had much higher bending moduli due to the formation of the ω phase during quenching. It is noteworthy that the Ti–5Cr–9Mo alloy exhibited the highest bending strength/modulus ratios at 26.0, which is significantly higher than those of c.p. Ti (8.5) and Ti–5Cr (13.3). Furthermore, the elastically recoverable angle of the Ti–5Cr–9Mo alloy (30°) was greater than that of c.p. Ti (2.7°). The reasonably high strength (or high strength/modulus ratio) β-phase Ti–5Cr–9Mo alloy exhibited a low modulus, ductile property, and excellent elastic recovery capability, which qualifies it as a novel implant materials.     

Biological Multi‐layer Systems as Implant Surface Modification

In living organisms the natural contact areas between cells are the cell membranes. These membranes separate the individual cells or build reaction compartments in an aqueous environment. Beside their structural role they also have specific functions that are due to the great variety of their components which are lipids (about 40 %) and proteins (about 60 %). In terms of implant development the next neighbors of the cells are artificial materials which do not belong to the natural cellular environment. Therefore, a biocompatible implant surface is needed which is achieved by either the correct choice of the material and surface roughness or a functionalization of the surface. To date little is known of the role lipids could play in this context. However, from literature we know that phospholipids can cause calcification and that modified phosphorylcholine polymers ('MPC polymers') are used to decrease cell adhesion and to improve blood compatibility. In the last few years it became obvious that the lipid contribution in the membrane is not only important as support for proteins but that the lipid membrane itself can also be a target for drug design and its structure can influence the function of the proteins. We therefore focused our interest on this class of amphiphilic molecules. In this work we present initial observations on the modification of metallic implant surfaces of Ti‐6Al‐7Nb (in mass percent) by phospholipid multilayers, using contact angle measurements and surface sensitive characterization techniques such as scanning electron microscopy (SEM) and scanning force microscopy (SFM). Preliminary data concerning cell adhesion experiments are also presented.     

Characterization of neutrophil adhesion to different titanium surfaces

Although titanium (Ti) is known to elicit a foreign body response when implanted into humans, Ti implant healing resembles normal wound healing in terms of inflammatory cell recruitment and inflammation persistence. Rough implant surfaces may present better conditions for protein adsorption and for the adhesion of platelets and inflammatory cells such as neutrophils. Implanted biomedical devices initially interact with coagulating blood; however, direct contact between the oxide layer of the implant and neutrophils has not been completely described. The aim of the present study is to compare the behaviours of neutrophils in direct contact with different Ti surfaces. Isolated human neutrophils were placed into contact with Ti discs, which had been rendered as ‘smooth’ or ‘rough’, following different surface treatments. Scanning electron microscopy and flow cytometry were used to measure cell adhesion to the surfaces and exposure of membrane proteins such as CD62L and CD11b. Topographic roughness was demonstrated as higher for SLA treated surfaces, measured by atomic force microscopy and elemental analysis was performed by energy dispersive X-ray, showing a similar composition for both surfaces. The adhesion of neutrophils to the ‘rough’ Ti surface was initially stronger than adhesion to the ‘smooth’ surface. The cell morphology and adhesion marker results revealed clear signs of neutrophil activation by either surface, with different neutrophil morphological characteristics being observed between the two surface types. Understanding the cellular mechanisms regulating cell–implant interactions should help researchers to improve the surface topography of biomedical implant devices.     

In vitro evaluation of human osteoblast adhesion to a thermally oxidized γ-TiAl intermetallic alloy of composition Ti–48Al–2Cr–2Nb (at.%)

Ti–48Al–2Cr–2Nb (at.%) (γ-TiAl), a gamma titanium aluminide alloy originally designed for aerospace applications, appears to have excellent potential as implant material. Thermal treatment of γ-TiAl renders this alloy extremely corrosion resistant in vitro, which could improve its biocompatibility. In this study, the surface oxides produced by thermal oxidation (at 500°C, and at 800°C for 1 h in air) on γ-TiAl were characterized by X-ray photoelectron spectroscopy (XPS). hFOB 1.19 cell adhesion on thermally oxidized γ-TiAl was examined in vitro by a hexosaminidase assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) after 1, 7 and 14 days. Ti–6Al–4V surfaces were used for comparison. Hexosaminidase assay data and CLSM analysis of focal contacts and cytoskeleton organization showed no differences in cell attachment on autoclaved and both heat-treated γ-TiAl surfaces at the different time points. SEM images showed well organized multi-layers of differentiated cells adhered on thermally oxidized γ-TiAl surfaces at day 14. Unexpectedly, thermally oxidized Ti–6Al–4V surfaces oxidized at 800°C exhibited cytotoxic effects on hFOB 1.19 cells. Our results indicate that thermal oxidation of γ-TiAl seems to be a promising method to generate highly corrosion resistant and biocompatible surfaces for implant applications.     

铝含量对（Ti,Al）N镀层结构及耐磨性的影响

运用多弧离子镀在Ｗ６Ｍｏ５Ｃ４Ｖ２高速钢表面沉积了（Ｔｉ,Ａｌ）Ｎ镀层,并经ＳＥＭ,ＸＲＤ和ＥＤＳ等方法分析表明,多靶联合工作获得的（Ｔｉ,Ａｌ）Ｎ镀层,其成分可连续变化,随弧流增大,铝含量增多;随负偏压增大,铝含是降低。弧流与偏压二者对镀层的成分均有影响,其中弧流较为显著。     

Laser polishing of aluminum by remelting with high energy pulses

Laser polishing is a contact‐free, quick and automated method to smooth surfaces. The method has been applied to different forging and casting aluminum alloys. The surfaces of the samples were belt‐grinded with a grain size of mesh 240. The samples are protected from ambient air in a gas shield chamber. The used laser system is an Nd : YAG Laser with maximum pulse energy of 65 J. The initial and the laser polished surfaces have been analyzed by microscopy, roughness spectroscopy, white light interferometry and cross‐section polishes. The surfaces of the laser polished forging alloys are covered by multiple lateral and horizontal cracks. Unlike the forging alloys, the casting alloys could be processed well by laser polishing. The initial surface roughness of Ra₂₄₀ = 1.37 µm was reduced up to Ra_LP ≈ 0.47 µm. This represents a roughness reduction of 66%. The roughness spectroscopy of the laser polished surface shows for structural wavelengths from 2.5 µm to 500 µm a Ra‐value close to 0.1 µm and from 500 µm to 800 µm higher values. The remelted area extends up to100 µm into the material.     

Development of a β-type Ti–12Mo–5Ta alloy for biomedical applications: cytocompatibility and metallurgical aspects

Ti-based biocompatible alloys are especially used for replacing failed hard tissue. Some of the most actively investigated materials for medical implants are the beta-Ti alloys, as they have a low elastic modulus (to inhibit bone resorption). They are alloyed with elements such as Nb, Ta, Zr, Mo, and Fe. We have prepared a new beta-Ti alloy that combines Ti with the non-toxic elements Ta and Mo using a vacuum arc-melting furnace and then annealed at 950 degrees C for one hour. The alloy was finally quenched in water at room temperature. The Ti-12Mo-5Ta alloy was characterised by X-ray diffraction, optical microscopy, SEM and EDS and found to have a body-centred-cubic structure (beta-type). It had a lower Young's modulus (about 74 GPa) than the classical alpha/beta Ti-6Al-4V alloy (120 GPa), while its Vickers hardness remained very high (about 303 HV). This makes it a good compromise for a use as a bone substitute. The cytocompatibility of samples of Ti-12Mo-5Ta and Ti-6Al-4V titanium alloys with various surface roughnesses was assessed in vitro using organotypic cultures of bone tissue and quantitative analyses of cell migration, proliferation and adhesion. Mechanically polished surfaces were prepared to produce unorientated residual polished grooves and cells grew to a particularly high density on the smoother Ti-12Mo-5Ta surface tested.     

Rezyklieren von metallischen Spänen mittels Electro‐Discharge Sintering

Composite material Ferro‐Titanit^® is produced powder‐metallurgical by Deutsche Edelstahlwerke GmbH (DEW) and is commonly used for wear and corrosion resistant component parts. Materials properties can be attributed to the microstructure which consists of a corrosion resistant metallic matrix and a huge amount of approx. 50 vol.% of hard Ti‐monocarbides. Although Ferro‐Titanit^® possesses a high amount of hard particles, the material can be machined by turning and drilling in solution annealed condition. Due to the alloying content (Mo, Cr, TiC) of Ferro‐Titanit^®, there is a high motivation to recover those elements by a recycling process of the chips, thus expensive and limited resources can be saved. On idea of a recycling process can be found in the redensification of those chips by electro discharge sintering (EDS). In this work, chips of the material Ferro‐Titanit^® were densified by EDS technique and the resulting microstructure was investigated by optical and scanning electron microscopy. Furthermore, microstructure and hardness of the EDS densified specimens was discussed with regard to the microstructure of conventionally sintered Ferro‐Titanit^®‐samples in laboratory conditions.     

Fabrication of thin film TiO2 nanotube arrays on Co–28Cr–6Mo alloy by anodization

Titanium oxide (TiO₂) nanotube arrays were prepared by anodization of Ti/Au/Ti trilayer thin film DC sputtered onto forged and cast Co–28Cr–6Mo alloy substrate at 400 °C. Two different types of deposited film structures (Ti/Au/Ti trilayer and Ti monolayer), and two deposition temperatures (room temperature and 400 °C) were compared in this work. The concentrations of ammonium fluoride (NH₄F) and H₂O in glycerol electrolyte were varied to study their effect on the formation of TiO₂ nanotube arrays on a forged and cast Co–28Cr–6Mo alloy. The results show that Ti/Au/Ti trilayer thin film and elevated temperature sputtered films are favorable for the formation of well-ordered nanotube arrays. The optimized electrolyte concentration for the growth of TiO₂ nanotube arrays on forged and cast Co–28Cr–6Mo alloy was obtained. This work contains meaningful results for the application of a TiO₂ nanotube coating to a CoCr alloy implant for potential next-generation orthopedic implant surface coatings with improved osseointegrative capabilities.     

Elektronenstrahlbehandlung von PVD‐Hartstoffschichten

Electron beam treatment of PVD – hard coatings Coatings of the type CrN_x, (Ti, Cr)N, (Ti, Al)N, Ti(C, N) and Ti(B,N) were deposited on the quenched and tempered steel C45 to investigate the effect of electron beam treatment on the structure and the properties of hard coated steels. A controlled energy input by electron beams was used to investigate the thermal behaviour of hard coatings with fixing the transformation levels by self‐quenching. Simultaneously a different case hardening of the substrate was caused providing a different effect of supporting the hard layer. There are big differences in the thermal stability of the investigated coatings. The surface hardness, adhesion and wear resistance of the composit hard coating/steel was improved in dependence on the energy input. The use of electron beam technologies enables the generation of support layers which locally increase the working behaviour of hard coated steel.