Differentiation of human bone-derived cells grown on GRGDSP-peptide bound titanium surfaces

Various surface modifications have been applied to titanium alloy (Ti-6Al-4V) implants, in an attempt to enhance osseointegration; crucial for ideal prosthetic fixation. Despite the numerous studies demonstrating that peptide-modified surfaces influence in vitro cellular behavior, there is relatively little data reporting their effects on bone remodeling. The objective of this article was to examine the effects of chemically modifying Ti-6Al-4V surfaces with a common RGD sequence, a 15-residue peptide containing GRGDSP (glycine-arginine-glycine-aspartate-serine-proline), on the modulation of bone remodeling. The expression of proteins known to be associated with osseous matrix and bone resorption were studied during the growth of human bone-derived cells (HBDC) on these peptide-modified surfaces. HBDC grown for 7 days on RGD surfaces displayed significantly increased levels of osteocalcin, and pro-collagen Ialpha1 mRNAs, compared with the production by HBDC grown on the native Ti-6Al-4V. A pattern that was also reflected at the protein levels for osteocalcin, type I collagen, and bone sialoprotein. Moreover, HBDC grown for 7 and 14 days on RGD-modified Ti-6Al-4V expressed significantly higher level of osteoclast differentiation factors and lower levels of osteoprotegerin and IL-6 proteins compared with other surfaces tested. These results suggest that different chemical treatments of implant material (Ti-6Al-4V) surface result in differential bone responses, not only their ability to form bone but also to stimulate osteoclastic formation.     

A scanning electron microscopy study of human osteoblast morphology on five orthopedic metals

Despite the long-standing use of metals as orthopedic implants there still are unsolved problems with these materials and open questions about their behavior in a biological environment. Cell-culture studies provide a useful tool for investigations. In addition to the determination of biochemical or molecular biological parameters, the morphology of adhering cells reflects their interaction with the substrata. This article describes an investigation of the morphology of human osteoblasts on stainless steel, cobalt chromium alloy, commercially pure titanium, Ti-6Al-4V, and Ti-6Al-7Nb with surface designs similar to those used as clinical implants. A cell culture plastic surface was used as a control material. The materials were examined by scanning electron microscopy at different points of time. The cells spread, proliferated, and formed nodules on all test substrates in a time-dependent manner, without signs of a disturbing influence from any of the materials. On the smooth surfaces the cells showed a flattened fibroblast-like morphology and only slight differences could be detected. Therefore, the cellular morphology seems not to be markedly affected by the different chemical material compositions. In contrast, the titanium alloy with a rough, sandblasted surface induced a three-dimensional growth. This three-dimensional cellular network could be the basis for the known earlier differentiation of osteoblasts on rough surfaces in vitro and a better osseointegration in vivo.     

Effect of hydrogen peroxide on titanium surfaces: in situ imaging and step-polarization impedance spectroscopy of commercially pure titanium and titanium, 6-aluminum, 4-vanadium

To analyze titanium's response to representative surgical wound environments, a study was conducted on commercially pure titanium (CPTi) and titanium, 6-aluminum, 4-vanadium (Ti-6Al-4V) exposed to phosphate-buffered saline (PBS) with 30 mM of hydrogen peroxide (H(2)O(2)) added. The study was characterized by simultaneous electrochemical atomic force microscopy (EC AFM) and step-polarization impedance spectroscopy (SPIS). Surfaces were covered with protective oxide domes that indicated topography changes with potential and time of immersion. Less oxide dome coarsening was noted on surfaces treated with PBS containing H(2)O(2) than on surfaces exposed to pure PBS. Electrical data deduced from current transients collected while stepping voltage between 0 V and 1 V indicated that charge transfer in hydrogen peroxide solutions was an order of magnitude larger than it was in pure PBS. Oxide (early) resistances of CPTi samples were higher than were Ti-6Al-4V oxide resistances in both types of solutions, but CPTi oxide resistance was lower in the hydrogen peroxide solution compared to pure PBS. Capacitance data suggest that CPTi oxide films thicken in hydrogen peroxide solution more than they do in pure PBS. Differences in electrical properties between CPTi and Ti-6Al-4V surfaces suggest that CPTi, but not Ti-6Al-4V, has catalytic activity on H(2)O(2) and that the catalytic activity of CPTi oxide affects its ability to grow TiO(2). Differences in electrical properties are related to catalytic and oxidative mechanisms that take place directly on the titanium oxide surface and in wound environments. The study provides a foundation and theoretic basis for the porous oxide model on commercially pure titanium exposed to hydrogen peroxide.     

The effect of Pt and Pd alloying additions on the corrosion behavior of titanium in fluoride-containing environments

In this study, we examined the corrosion behaviors of pure titanium, the alloys Ti-6Al-4V and Ti-6Al-7Nb, and the new experimental alloys Ti-Pt and Ti-Pd using anodic polarization and corrosion potential measurements in an environment containing fluoride. Before and after immersion in the test solutions, we made observations using a scanning electron microscope. The test solutions included an artificial saliva containing 0.2% NaF (corresponding to 905 ppm F) and an artificial saliva with a low concentration of oxygen. Although the surfaces of the Ti-Pt and Ti-Pd alloys were not affected by an acidic environment containing fluoride, the surfaces of the pure titanium, the Ti-6Al-4V alloy, and the Ti-6Al-7Nb alloy were markedly roughened by corrosion. The surfaces of the pure titanium, the Ti-6Al-4V alloy, and the Ti-6Al-7Nb alloy were microscopically damaged by corrosion when they were immersed in the solution containing a low concentration of dissolved oxygen, even with a fluoride concentration included in the commercial dentifrices. In this situation, however, the surfaces of the new Ti-Pt and Ti-Pd alloys were not affected. These alloys are expected to be of use in dental work as new titanium alloys with high corrosion resistances.     

The influence of surface microroughness and hydrophilicity of titanium on the up-regulation of TGFβ/BMP signalling in osteoblasts

The topography of titanium implants has been identified as an important factor affecting the osseointegration of surgically placed dental implants. Further modification to produce a hydrophilic microrough titanium implant surface has been shown to increase osseointegration compared with microrough topology alone. This study aimed to determine possible molecular mechanisms to explain this clinical observation by examining differences in the whole genome mRNA expression profile of primary human osteoblasts in response to sand-blasted acid-etched (SLA) and hydrophilic SLA (modSLA) titanium surfaces. A decrease in osteoblast proliferation associated with the titanium surfaces (modSLA > SLA > control) correlated with an increase in expression of the osteogenic differentiation markers BSPII and osteocalcin. Pathway analysis demonstrated that a number of genes associated with the TGFβ?BMP signalling cascade (BMP2, BMP6, SP1, CREBBP, RBL2, TBS3, ACVR1 and ZFYVE16) were significantly differentially up-regulated with culture on the modSLA surface. BMP2 was shown to have the largest fold change increase in expression which was subsequently confirmed at the protein level by ELISA. Several other genes associated with the functionally important mechanisms relevant to bone healing, such as Wnt signalling (CTNNA1, FBX4, FZD6), angiogenesis (KDR), osteoclastogenesis (HSF2, MCL1) and proteolysis (HEXB, TPP1), were also differentially regulated. These results suggest that chemical (hydrophilic) modification of the SLA surface may result in more successful osseointegration through BMP signalling.     

Wear properties of Ti and Ti-6Al-7Nb castings for dental prostheses

Titanium has been increasingly applied to dental prostheses because of its biocompatibility. However, application remains limited, due to the low strength and poor wear resistance of unalloyed titanium. The purpose of this study is to evaluate the wear resistance of high-strength Ti-6Al-7Nb alloy castings for dental application. Test specimens were cast from commercially pure titanium (CP-Ti grades 2 and 3) and Ti-6Al-7Nb alloy ingots, and subjected to a wear test simulating the occlusal loading pattern. Wear resistance was evaluated by the weight loss during the test. Ti-6Al-7Nb alloy was found to exhibit lower weight loss than CP-Ti. Moreover, scanning electron microscopic (SEM) observation after the test revealed that the worn surface of Ti-6Al-7Nb alloy is much smoother than that of CP-Ti. These results indicate that Ti-6Al-7Nb alloy castings can be used to produce dental prostheses of improved wear resistance and mechanical strength.     

Titanium substrata composition influences osteoblastic phenotype: In vitro study.

In spite of observed differences at the interface between boon and either commercially pure titanium [Ti(cpi)] or titanium alloy (Ti-6Al-4V), the mechanism of such a response is ill understood. This prompted further investigation of the influence of similar metals on human bone-derived cells (HBDCs). This study investigated the influence of Ti(cpi) and its alloy on osteoblastic proteins formed by HBDCs grown for 5, 7, 10, and 14 days on these metals and compared them to cells grown on tissue culture polystyrene plates. Messenger RNA and translated proteins that form an array of osteogenic parameters were determined: alkaline phosphatase (ALP), thrombospondin, osteopontin, osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I) and bone sialoprotein (BSP). At the four predetermined time points, cells grown on either Ti(cpi) or Ti-6Al-4V generally expressed similar mRNA levels, while levels of their respective proteins differed. Cells on Ti(cpi) had peak levels for most proteins at day 7, whereas those on Ti-6Al-4V peaked at either day 5 and/or day 7. At day 5 cells grown on Ti-6Al-4V had higher levels of ALP, Col I, ON/SPARC, OC, and BSP than those in Ti(cpi); this difference was not maintained at later time points in culture. The differential regulation of proteins occurring between cells from the same patient grown on titanium and its alloy implies that HBDCs respond to small differences in the surface chemistry and/or microcrystallinity.     

Titanium alloy mini-implants for orthodontic anchorage: immediate loading and metal ion release

Removable osseointegrated titanium mini-implants were successfully used as anchorage devices in orthodontics. The early load is necessary to simplify the mini-implant methodology, but can lead to failure during osseointegration. The Ti-6Al-4V alloy was used instead of commercially pure Ti due to its superior strength. However, the corrosion resistance is low, allowing for metal ion release. The purpose of this work was to analyze the immediately loaded mini-implant fixation and to gauge the vanadium ion release during the healing process. Titanium alloy mini-implants were inserted in the tibiae of rabbits. After 1, 4 and 12 weeks, they were submitted to removal torque testing. There was no increase in the removal torque value between 1 and 4 weeks of healing, regardless of the load. Nevertheless, after 12 weeks, a significant improvement was observed in both groups, with the highest removal torque value for the unloaded group. The kidney, liver and lung were also extracted and analyzed by atomic absorption spectrometry. In comparison with the control values, the content of vanadium increased slightly after 1week, significantly increased after 4 weeks and decreased slightly after 12 weeks, without reaching the 1 week values. A stress analysis was carried out which enables both the prediction of the torque at which commercially pure (CP) Ti and Ti-6Al-4V deform plastically and the shear strength of the interface. This analysis reveals that the removal torques for CP Ti dangerously approach the yield stress. The results of this rabbit model study indicate that titanium alloy mini-implants can be loaded immediately with no compromise in their stability. The detected concentration of vanadium did not reach toxic levels in the animal model.     

Wear resistance of experimental Ti-Cu alloys

After using cast titanium prostheses in clinical dental practice, severe wear of titanium teeth has been observed. This in vitro study evaluated the wear behavior of teeth made with several cast titanium alloys containing copper (CP Ti+3.0 wt% Cu; CP Ti+5.0 wt% Cu; Ti-6Al-4V +1.0 wt% Cu; Ti-6Al-4V+4.0 wt% Cu) and compared the results with those for commercially pure (CP) titanium, Ti-6Al-4V, and gold alloy. Wear testing was performed by repeatedly grinding upper and lower teeth under flowing water in an experimental testing apparatus. Wear resistance was assessed as volume loss (mm(3)) at 5kgf (grinding force) after 50,000 strokes. Greater wear was found for the six types of titanium than for the gold alloy. The wear resistance of the experimental CP Ti+Cu and Ti-6Al-4V+Cu alloys was better than that of CP titanium and Ti-6Al-4V, respectively. Although the gold alloy had the best wear property, the 4% Cu in Ti-6Al-4V alloy exhibited the best results among the titanium metals. Alloying with copper, which introduced the alpha Ti/Ti(2)Cu eutectoid, seemed to improve the wear resistance.     

Joint replacement components made of hot-forged and surface-treated Ti-6Al-7Nb alloy.

We have developed a titanium-aluminium alloy with the inert alloying element niobium. The optimal composition was found to be Ti-6Al-7Nb (Protasul-100). This custom-made alloy designed for implants shows the same alpha/beta structure as Ti-6Al-4V and exhibits equally good mechanical properties. The corrosion resistance of Ti-6Al-7Nb in sodium chloride solution is equivalent to that of pure titanium and Ti-6Al-4V. This is due to a very dense and stable passive layer. Highly stressed anchorage stems of different hip prosthesis designs have been made from hot-forged Ti-6Al-7Nb. The polished surfaces of hip, knee and wrist joints made of Ti-6Al-7Nb and articulating against polyethylene are surface-treated by means of a very hard and 3-5 microns thick titanium nitride coating (Tribosul-TiN) or by oxygen diffusion hardening (Tribosul-ODH) to a depth of 30 microns.     

The effect of plasma chemical oxidation of titanium alloy on bone-implant contact in rats

Many different technologies have been used to enhance osseointegration in orthopaedic and dental implant surgery. Hydroxyapatite coatings, pure or in combination with growth factors or bisphosphonates, showed improved osseointegration of titanium alloy implants. We choose a different approach to enhance osseointegration: plasma chemical oxidation was used to modify the surface of titanium alloy implants. This technique converts the nm-thin natural occurring titanium oxide layer on an implant to a 4?μm thick ceramic coating (TiOB surface). Bioactive TiOB surfaces have a macroporous structure and were loaded with calcium and phosphorus, while bioinert TiOB surfaces are smooth. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bone response to TiOB surfaces in?vivo. 64 rats were randomly assigned to four groups of implants: (1) titanium alloy (control), (2) titanium alloy, type III anodization, (3) bioinert TiOB surface and (4) bioactive TiOB surface. Mechanical fixation, peri-implant-bone area and bone contact were evaluated by pull-out tests and histology at three and eight weeks. Shear strength and bone contact at eight weeks were significantly increased in the bioactive TiOB group compared to all other groups. The results of plasma chemical oxidation in a rat model showed that the bioactive TiOB surface has a positive effect on implant anchorage by enhancing the bone-implant contact in normal bone.     

Surface modification by alkali and heat treatments in titanium alloys

Pure titanium and titanium alloys are normally used for orthopedic and dental prostheses. Nevertheless, their chemical, biological, and mechanical properties still can be improved by the development of new preparation technologies. This has been the limiting factor for these metals to show low affinity to living bone. The purpose of this study is to improve the bone-bonding ability between titanium alloys and living bone through a chemically activated process and a thermally activated one. Two kinds of titanium alloys, a newly designed Ti-In-Nb-Ta alloy and a commercially available Ti-6Al-4V ELI alloy, were used in this study. In this study, surface modification of the titanium alloys by alkali and heat treatments (AHT), alkali treated in 5.0M NaOH solution, and heat treated in vacuum furnace at 600 degrees C, is reported. After AHT, the effects of the AHT on the bone integration property were evaluated in vitro. Surface morphologies of AHT were observed by optical microscopy (OM) and scanning electron microscopy (SEM). Chemical compositional surface changes were investigated by X-ray diffractometry (XRD), energy dispersive spectroscopy (EDS), and auger electron spectroscopy (AES). Titanium alloys with surface modification by AHT showed improved bioactive behavior, and the Ti-In-Nb-Ta alloy had better bioactivity than the Ti-6Al-4V ELI alloy in vitro.     

The bone tissue compatibility of a new Ti-Nb-Sn alloy with a low Young's modulus

A Ti-Nb-Sn alloy was developed as a new β-type titanium alloy which had a low Young's modulus and high strength. The Young's modulus of the Ti-Nb-Sn alloy was reduced to about 45 GPa by cold rolling, much closer to human cortical bone (10-30 GPa) than that of Ti-6Al-4V alloy (110 GPa) and other β-type titanium alloys developed for biomedical applications. The tensile strength of the Ti-Nb-Sn alloy was increased to a level greater than that of Ti-6Al-4V alloy by heat treatment after severe cold rolling. In this study the cytotoxicity of Ti-25Nb-11Sn alloy was evaluated in direct contact cell culture tests using metal disks and the bone tissue compatibility - examined using metal rods inserted into the medullary canal of rabbit femurs. The remarkable findings were that: (1) there were no significant differences in the relative growth ratio and relative absorbance ratio between cells grown with the Ti-Nb-Sn alloy, Ti-6Al-4V alloy and CP-Ti in direct contact cell culture tests; (2) there were no significant differences in the load at failure between the Ti-Nb-Sn alloy and Ti-6Al-4V alloy in pull-out metal rods tests; (3) there were no significant differences in new bone formation around metal rods between the Ti-Nb-Sn alloy and Ti-6Al-4V alloy in histological evaluations. The new Ti-Nb-Sn alloy with an elasticity closer to that of human bone is thus considered to be bioinert while also having a high degree of bone compatibility similar to that of Ti-6Al-4V alloy.     

Varying Ti-6Al-4V surface roughness induces different early morphologic and molecular responses in MG63 osteoblast-like cells

Osteoblast response to Ti implants depends not only on the chemistry of the implant but also on the physical properties of the implant surface, such as microtopography and roughness. This study was undertaken to examine early changes in cell morphology and gene expression during the early phase of osteoblast interaction with titanium alloy (Ti-6Al-4V) surfaces of two different roughnesses. MG63 osteoblast-like cells were cultured for 2, 6, 24, and 72 h on smooth (Ra=0.18+/-0.03 microm) and rough (Ra=2.95+/-0.23 microm) Ti-6Al-4V surfaces. Changes in cell proliferation were assessed by measuring cell number after 72 h in culture. Morphological characteristics were observed by scanning electron microscopy after 2, 6, and 24 h of culture. Changes in gene expression for extracellular signal-regulated kinase 2 (Erk2), type I collagen (alpha2[I] collagen), phospholipase C-gamma2 (Plc-gamma2), and beta-actin were measured by RT-PCR after 6 and 24 h in culture. Cell number was significantly higher on the smooth surface. In scanning electron micrographs, cells on smooth Ti-6Al-4V were spherical and raised up from the surface after 2 h in culture. In contrast, cells on the rough surface adopted an irregular, elongated shape that spanned across pits in the surface. At 24 h, cells on the smooth surface had flattened, become elongate, and covered the surface. In contrast, cells on the rough surface appeared more differentiated in shape and the margins of the cells were irregular, with many processes extending out, following the contour of the surface. Of the genes examined, only Erk2 and beta-actin showed a change in expression with surface roughness. Both genes were upregulated (p<0.05) on the rough surface at 6 h. These results indicate that Ti-6Al-4V surface roughness affects osteoblast proliferation, morphology, and gene expression, and that these effects can be measured after periods as short as 2-6 h.     

Effects of topographical surface modifications of electron beam melted Ti-6Al-4V titanium on human fetal osteoblasts

The aim of the study was to assess the suitability of different Ti-6Al-4V surfaces produced by the electron beam melting (EBM) process as matrices for attachment, proliferation, and differentiation of human fetal osteoblasts (hFOB 1.19). Human osteoblasts were cultured in vitro on smooth and rough-textured Ti-6Al-4V alloy disks. By means of cell number and vitality and SEM micrographs cell attachment and proliferation were observed. The differentiation rate was examined by using quantitative real-time PCR analysis for the gene expression of alkaline phosphatase (ALP), type I collagen (Coll-I), bone sialoprotein (BSP) and osteocalcin (OC). After 3 days of incubation there was a significant higher vitality (p < 0.02) and proliferation (p < 0.02) of hFOB cells on smooth surfaces (R(a) = 0.077 microm) and compact surfaces with adherent partly molten titanium particles on the surface (R(a) /= 56.9 microm) reduced proliferation of hFOB cells. Surface characteristics of titanium can easily be changed by EBM in order to further improve proliferation.     

Analysis on migration and activation of live macrophages on transparent flat and nanostructured titanium

The immunotoxicity of implanted nanostructured titanium is a paramount issue for vascular, dental and orthopedic applications. However, it has been unclear whether implanted surface nanostructures can inhibit or aggrevate inflammatory responses. Herein, macrophage activation, as evidence of migration, on transparent flat and nanostructured titanium correlated with pro-inflammatory protein synthesis and cytokine release. Through the real-time monitoring of initial cytoskeleton variations, this study identified that macrophage movement was restricted on nanostructured titanium compared to flat titanium surfaces. Furthermore, nanostructured titanium elicited secretion of fewer pro-inflammatory enzyme molecules and cytokines, as well as reduced nitric oxide production. All results collectively indicated that initial macrophage activation can be mitigated by nanoscale surface topography alone, without modification of surface chemistry or stiffness.     

Mesenchymal stem cell adhesion and spreading on microwave plasma-nitrided titanium alloy

Improved methods to increase surface hardness of metallic biomedical implants are being developed in an effort to minimize the formation of wear debris particles that cause local pain and inflammation. However, for many implant surface treatments, there is a risk of film delamination due to the mismatch of mechanical properties between the hard surface and the softer underlying metal. In this article, we describe the surface modification of titanium alloy (Ti-6Al-4V), using microwave plasma chemical vapor deposition to induce titanium nitride formation by nitrogen diffusion. The result is a gradual transition from a titanium nitride surface to the bulk titanium alloy, without a sharp interface that could otherwise lead to delamination. We demonstrate that vitronectin adsorption, as well as the adhesion and spreading of human mesenchymal stem cells to plasma-nitrided titanium is equivalent to that of Ti-6Al-4V, while hardness is improved 3- to 4-fold. These in vitro results suggest that the plasma nitriding technique has the potential to reduce wear, and the resulting debris particle release, of biomedical implants without compromising osseointegration; thus, minimizing the possibility of implant loosening over time.     

TIM-1 regulates macrophage cytokine production and B7 family member expression

T-cell immunoglobulin mucin-1 (TIM-1) is associated with the regulation of T helper type 2 (Th2) immune responses and has been associated with asthma susceptibility. Previous studies have shown that administration of TIM-1 results in T cell hyperproliferation and increased Th2 cytokine secretion. TIM-1 has also been shown to bind to macrophages, but the effects of TIM-1 administration on macrophage activity have not been assessed. In this study we demonstrate that TIM-1 binds to the mouse macrophage cell line RAW 264.7. Stimulation of the RAW264.7 cells with TIM-1 increases nitric oxide production. A dramatic increase in the pro-inflammatory cytokines TNF-alpha and IL-6 is seen upon TIM-1 stimulation of RAW 264.7 cells. Additionally, there is a moderate increase in the immuno-modulatory cytokine IL-10 when RAW 264.7 cells are stimulated with TIM-1. TIM-1 stimulation also alters the expression of some members of the B7 family of co-stimulatory/co-inhibitory proteins. TIM-1 stimulation leads to increased B7-1, B7-H1, and PD-L2 expression, while inhibiting B7-H2 expression. These studies suggest that TIM-1 can regulate macrophage activation and alter the co-stimulatory properties of macrophages and thus may contribute to allergic inflammatory diseases such as asthma.     

The machinability of cast titanium and Ti-6Al-4V

This study investigated the machinability (ease of metal removal) of commercially pure (CP) titanium and Ti-6Al-4V alloy. Both CP Ti and Ti-6Al-4V were cast into magnesia molds. Two types of specimens (with alpha-case and without alpha-case) were made for CP Ti and Ti-6Al-4V. Machinability (n = 5) was evaluated as volume loss (mm3) by cutting/grinding the 3.0 mm surface using fissure burs and silicon carbide (SiC) under two machining conditions: (1) two machining forces (100 or 300 gf) at two rotational speeds (15000 or 30000 rpm) for 1 min, and (2) constant machining force of 100 gf and rotational speed of 15000 rpm for 1, 2, 5, 10, and 30 min. As controls, conventionally cast Co-Cr and Type IV gold alloys were evaluated in the same manner as the titanium. When fissure burs were used, there was a significant difference in the machinability between CP titanium with alpha-case and without alpha-case. On the other hand, there was no appreciable difference in the amount of metal removed for each tested metal when using the SiC points.