Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment
D. E. MacDonald , , a, b, d, B. E. Rapuano d, N. Deo a, M. Stranick c, P. Somasundaran a and A. L. Boskey d
a Langmuir Center for Colloids and Interfaces, School of Engineering, Columbia University, 911 S.W. Mudd Building, Mail Code 4711, 500 West 120th Street, New York, NY 10027, USA
b V.A. Medical Center, Bronx, NY 10468, USA
c Colgate Palmolive Company, 909 River Road, Piscataway, NJ 08854, USA
d Hospital for Special Surgery affiliated with the Weill Medical College of Cornell University, New York, NY 10021, USA
Received 23 July 2003; accepted 29 September 2003. Available online 21 November 2003.
Abstract
The microstructure, chemical composition and wettability of thermally and chemically modified Ti-6Al-4 V alloy disks were characterized and correlated with the degree of radiolabeled fibronectin-alloy surface adsorption and subsequent adhesion of osteoblast-like cells. Heating either in pure oxygen or atmosphere (atm) resulted in an enrichment of Al and V within the surface oxide. Heating (oxygen/atm) and peroxide treatment both followed by butanol treatment resulted in a reduction in content of V, but not in Al. Heating (oxygen/atm) or peroxide treatment resulted in a thicker oxide layer and a more hydrophilic surface when compared with passivated controls. Post-treatment with butanol, however, resulted in less hydrophilic surfaces than heating or peroxide treatment alone. The greatest increases in the adsorption of radiolabeled fibronectin following treatment were observed with peroxide/butanol-treated samples followed by peroxide/butanol and heat/butanol, although binding was only increased by 20-40% compared to untreated controls. These experiments with radiolabeled fibronectin indicate that enhanced adorption of the glycoprotein was more highly correlated with changes in chemical composition, reflected in a reduction in V content and decrease in the V/Al ratio, than with changes in wettability. Despite promoting only a modest elevation in fibronectin adsorption, the treatment of disks with heat or heat/butanol induced a several-fold increase in the attachment of MG63 cells promoted by a nonadhesive concentration of fibronectin that was used to coat the pretreated disks compared to uncoated disks. Therefore, results obtained with these modifications of surface properties indicate that an increase in the absolute content of Al and/or V (heat), and/or in the Al/V ratio (with little change in hydrophilicity; heat+butanol) is correlated with an increase in the fibronectin-promoted adhesion of an osteoblast-like cell line. It would also appear that the thermal treatment-induced enhancement of cell adhesion in the presence of this integrin-binding protein is due to its increased biological activity, rather than a mass effect alone, that appear to be associated with changes in chemical composition of the metallic surface. Future studies will investigate the influence of the surface chemical composition of various implantable alloys on protein adsorption and receptor-mediated cell adhesion. In addition, by altering the properties of bound osteogenic protein enhancing exposure to cell integrin binding domains, it may be possible to develop implant surfaces which enhance the attachment, adhesion and developmental response of osteoblast precursors leading to accelerated osseointegration.
Author Keywords: Author Keywords: Titanium alloy; Surface treatment; Surface analysis; Wettability; Cell attachment; Protein adsorption; Fibronectin
Article Outline
1. Introduction
Commercially pure titanium (cpTi) and its titanium-aluminum-vanadium alloy Ti-6Al-4 V (Ti6Al4 V) have been used as implants due to their excellent biocompatibility and ability to allow bone-implant integration. Ti6Al4 V has been also used preferentially in orthopedic-prosthetic replacement due to its added mechanical strength, while cpTi has been employed for some dental implants [1]. Titanium forms a biocompatible surface oxide layer capable of interacting with surrounding biological fluids and cells when implanted in situ [2 and 3]. This layer, composed primarily of TiO2, is found superficially on both cpTi and Ti6Al4 V metals [2 and 4]. A multiplicity of implant surface forms exist, engineered with mechanical features that physically interlock the implant with bone. Various strategies have been utilized to improve bone integration of titanium-based implants. For example, radiofrequency glow discharge has been used to increase surface energy and to enhance cell binding [5 and 6]. Plasma spraying of calcium phosphate (hydroxyapatite) coatings onto the titanium surface is reported to enhance osteoconductivity [7, 8, 9 and 10], yet human studies have shown coating dissolution shortly after implantation from soluble nonapatitic phases [11 and 12]. Alteration in surface topography by physical placement of grooves and depressions along titanium surfaces has been shown to influence cell orientation through contact guidance [13 and 14]. Surface grit blasting and polishing methods enhance cell growth, improving implant fixation through increases in interlocking surface area [13, 14, 15, 16, 17 and 18] and alterations of oxide thickness [19 and 20]. However, the effects of chemical modifications [21, 22, 23, 24 and 25] of the surface oxide of Ti-alloys on their physical, chemical and biological properties have not been extensively studied. The objective of this investigation was to examine the effects of chemical and thermal modifications of Ti6Al4 V on surface chemical composition, wettability, topography and oxide thickness. Furthermore, the effects of these treatments on the adsorption of a cell-adhesive glycoprotein (fibronectin) and the attachment of osteoblast-like cells (MG63) to the treated surfaces were examined. The aim of this study was to characterize the particular implant surface properties that are most highly associated with enhanced biological activity of cell-adhesive proteins and attachment of osteogenic target cells.
2. Materials and methods
2.1. Materials
All chemicals were from Sigma-Aldrich (Milwaukee, WI) and were of spectroscopic grade. Fetal bovine serum (FBS) and cell culture media were from Gibco Laboratories (Grand Island, NY). Bovine serum albumin (BSA) (Fraction V; essentially fatty acid-free) and p-nitrophenol-N-acetyl-B-
-glucoseaminide and human plasma fibronectin were purchased from Sigma (St. Louis, MO). Tissue culture flasks (75 cm2), 96-well tissue culture plates, and 96-well nontissue culture treated plates were obtained from Laboratory Disposable products (North Haledon, NJ). MG63 osteoblast-like cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA).
Cylindrical implant disks were prepared at the Hospital for Special Surgery from titanium-aluminum-vanadium alloy (Ti6Al4 V) stock (President Titanium, Hanson, MA) to 6 mm diameter and 1.0 mm in thickness using a CNC SL-3 lathe (Mori Seiki, Nara, Japan). Each disk was milled to a 2° taper to enable the disks to form a tight seal when inserted into the well of the cell culture plates. The disks were wet-ground with 320-400 grit silicon carbide paper (smooth samples), deburred, and further polished with 85FNEXL and 7S Scotch Brite nonwoven nylon mesh wheels (3 M, Indianapolis, IN) and then with coarse and fine aluminum oxide compound (Matchless Metal Polish Company, Chicago, IL) on clean Grade NWY 86/87 buff wheels (Divine Brothers Co., Utica, NY). The disks were rinsed with distilled water at each step, precleaned ultrasonically in Chem-Crest 2000 cleaning solution (Crest Ultrasonic Corp., Trenton, NJ) at 66°C and spray washed twice in distilled water.
2.2. Disk preparation
2.2.1. Controls
Disks were washed successively in isopropanol, acetone, xylene, acetone, and 1
ammonium hydroxide, and rinsed with deionized water according to the ASTM-F86 protocol [26]. The disks were then passivated in 40% nitric acid and rinsed three times with deionized water. Disks were then dried and transferred into acid-washed scintillation vials in a HEPA filtered isolation hood (USA/Scientific, Ocala, FL) and stored closed in an auto-desiccator cabinet (Sanplatec Corp, Osaka, Japan). All disks were sterilized using a rapid dry heat oven (Alpha Medical, Hempsted, NY) for 5 min.
2.2.2. Heat treatment
The alloy disks were heated to a temperature of 600°C for 1 h in a Lindberg Hevi-duty tube furnace (Watertown, Wisconsin) in either air or pure oxygen. Disks were sterilized and stored as described for controls.
2.2.3. Peroxide-treatment
The alloy disks were washed in a mixture (1:1:5 by volume) of 25% NH4OH(aq), 30% H2O2 (aq), and DI water at 80°C on a magnetic stirrer for 5 min [27]. Samples were then treated with 32% HCl (aq), 30% H2O2 (aq), and DI water (1:1:5 by volume) at 80°C on a magnetic stirrer for an additional 5 min. The disks were then washed twice with triple distilled water followed by ethanol, and dried in a dessicator, and sterilized.
2.2.4. Butanol-treatment
Heated and peroxide treated alloy disks were reacted with butanol to create a less wettable surface [28]. Samples were placed in a mixture of butanol and water (9:1 by volume) and ultrasonicated (L&R Quantrex S280, Kearny, NJ) at room temperature for 30 min [28]. The samples were then removed, rinsed in absolute ethanol, washed twice with triple distilled water, dried in a dessicator, and stored in stoppered, acid-washed Pyrex glass containers.
2.3. Surface analysis
2.3.1. Atomic force microscopy (AFM)
Titanium disks were carefully handled at the disk edges and loaded into the AFM (Digital Instruments, Santa Barbara, CA). The samples were analyzed at room temperature and humidity. The MultiMode AFM and NanoScope IIIa controller equipped with an Extender Electronic Module for phase-lag detection were used for the analysis as previously described [29]. Five representative disks from each treatment group were scanned with AFM in tapping mode using standard silicon tips with a nominal tip radius of 20 nm. Three random regions were imaged on each specimen. In view of the consideration that the average length of an osteoblast is 10
m [30], the scanning area selected was 10
m×10
m. The scan rate was 0.1 Hz at the largest size and 10 Hz or less for the smallest scan. Instrument adjustments were made to achieve optimal data resolution normal to the image plane under the given scan conditions, taking into account sample tilt and the height of surface features.
Quantitative measurements of surface area and the local root mean square (RMS) surface roughness and average roughness (Ra) were determined using a surface area of 10
m×10
m. The RMS roughness is defined as the height fluctuations in a given area. Ra is defined as the arithmetic average of the absolute height value of all points in the profile, or center line average height. Measurements of surface area, RMS and Ra roughness were made in three random areas per uncoated specimen and computed with a surface area and roughness analysis program (Digital Instruments, Santa Barbara, CA).
2.3.2. Electron spectroscopy for chemical analysis
Electron spectroscopy for chemical analyses (ESCA) was used for determination of surface composition, elemental oxidation state and oxide-layer thickness of both the treated and untreated titanium alloy samples using a Physical Electronics (PHI) model 5600 ESCA spectrometer (Eden Prairie, MN) as previously described [29]. The oxide layer thickness for the alloy disks was estimated using ESCA depth profiling. The disks were sputtered using a 50
A/cm2 Ar+ ion beam. Disks were rotated during sputtering to ensure uniform removal of the surface oxide layer. Using these conditions, the sputtering rate was estimated to be 0.2 nm/min. The atomic percentages of the elements present on the titanium surfaces were calculated using software and atomic sensitivity factors included with the instrument data system. The binding energies of the photoelectron peaks were referenced to the C1s line at 284.6 eV.
2.3.3. Wettability properties
The wettability of the control and modified disks was determined using contact angle analysis. Drops of distilled water were placed on the specimens and contact angles measured using a NRL-100-00-115-S contact angle goniometer (Rame-Hart, Mountain Lakes, NJ) automated with image analysis software. A total of 10 titanium alloy specimens from each group of control and treated samples were analyzed. An auto pipetter (Rame-Hart, Mountain Lakes, NJ) was employed with the goniometer to insure uniformity of the DI water droplet volume (2
l) for each specimen. Five timed measurements over a 15 s interval were made for each surface type and treatment, and all analyses were performed at room temperature. To reduce the effect of environmental moisture on the disks, especially the heated samples, all contact angle measurements were made within 1 h of treatment.
2.4. Titanium-aluminum-vanadium-protein interaction
2.4.1. Effects of treatments on binding of 125I-fibronectin to titanium alloy disks
To determine the effect of surface modification on extracellular glycoprotein adsorption, the model protein human plasma fibronectin (500
g) was radiolabeled with 125I by via the Chloramine T procedure to a specific radioactivity of 7.6
Ci/
g (PerkinElmer Life Sciences/New England Nuclear, Billerica, MA). Following open column purification of the radiolabeled product, the purity was checked by TLC and the iodinated protein was found to contain less than 5% unbound iodide. Titanium alloy disks for controls and each chemical and thermal treatment group were incubated in 96-well nontissue culture plates overnight at room temperature with 125I-fibronectin/1X PBS (phosphate-buffered saline) solution containing 0.1% bovine serum albumin (BSA). After incubation, the 125I-fibronectin medium was completely aspirated, disks were washed once with 1×PBS, aspirated, and 200
l of 0.1% BSA/1X PBS was added to each well and aspirated 1 h later. A total of 200
l of Wallac Optiphase Supermix scintillation cocktail was added to each well and the radioactivity (CPM) remaining bound to each disk was counted using the upper detector of a Wallac Model 1450 MicroBeta Trilux 96-well plate liquid scintillation counter (PerkinElmer Wallac, Gaithersburg, MD) for 1 min.
2.5. Cell attachment studies
MG63 (ATCC) cells were cultured in Modified Eagles Medium+nonessential amino acids (MEM+ NEAA) with 10% FBS (complete culture media) in tissue culture flasks. Cells were harvested from monolayers at 75-90% confluence by treatment with 0.25% trypsin in 0.53
sodium EDTA, centrifuged, resuspended in complete culture media and incubated for 2 h at 37°C to allow cells to recover from trypsinization. Recovered cells were centrifuged and resuspended in MEM+NEAA (serum-free media) and used for cell attachment experiments in 96-well nontissue culture plates containing titanium alloy disks.
2.5.1. Attachment of MG63 cells to titanium alloy disks
To determine the effect of surface treatment on basal or fibronectin-promoted cell attachment, titanium alloy disks were placed into 96-well nontissue culture plates (12 total disks per treatment group and control). MG63 cells were trypsinized and 150,000 cells applied to each disk in a final volume of 200
l of serum-free media. The samples were incubated for 2 h at 37°C, media aspirated, and further washed five times with serum-free media. The number of attached cells were quantified using a hexosaminidase assay [31]. In brief, 7.5 m
p-nitrophenol-N-acetyl-B-
-glucoseaminide in 0.1
citrate buffer (pH 5) was mixed with an equal volume of 0.5% Triton X-100 in water, and 60
l of the resultant solution added per cell culture well. The 96-well tissue culture plate was then incubated for 2 h at 37°C in 5% CO2. The color reaction was developed and enzyme activity blocked by adding 90
l per well of 50 m
glycine buffer (pH 10.4) containing 5 m
EDTA. Adsorbances were then measured at 405 nm in a Titerek Multiskan Plus spectrophotometer (LabSystems, Finland).
2.5.2. Effect of fibronectin concentration on MG63 cell attachment to titanium alloy surfaces
To determine the effect of coating concentration on MG63 cell attachment to Ti6Al4 V, control disks were incubated with increasing concentrations of fibronectin (0.1-1000 n
in PBS) in covered nontissue culture plates overnight at room temperature under a cell culture hood. The supernatant was then aspirated. The disks were washed with PBS, 200
l of 0.1% BSA/1 X PBS added to each well, and liquid aspirated 1 h later. MG63 cells were then added to each well (150,000 cells) and incubated for 2 h at 37°C. Samples were then washed five times with serum-free media and cell attachment numbers quantitated using a hexosaminidase assay [31]. Controls for these experimental plates were fibronectin free. A standard curve of hexosaminidase activity vs. cell number (r2 was ordinarily 0.95 and above) measured by a Coulter counter was run for every experiment. All of the measurements of cell number in the experimental ("unknown") wells were based on hexosaminidase activity and extrapolation from a standard curve (measured at cell numbers from 10,000 to 150,000 per well) that was prepared from the same culture of the same cell line used to prepare the unknown curve plate. Both unknown and standard curve plates also run at the same time with the same reagents.
2.5.3. Surface treatment effects on subadhesive fibronectin concentration on cell attachment
To examine potential effects of pretreatments on conformation/adhesive biological activity of coated phase fibronectin, a nonadhesive concentration of fibronectin was chosen. A nonadhesive concentration of fibronectin is defined as a concentration of the protein that does not promote cell attachment when coated on the unmodified metallic surface. For these experiments, both control and modified titanium alloy disks were placed into 96-well nontissue culture plates (12 total disks per treatment group and control) and incubated with fibronectin (0.1 n
in PBS) overnight at room temperature under a cell culture hood. Uncoated samples in PBS served as negative controls. The supernatant was aspirated, disks were washed once with 1×PBS, 200
l of 0.1% BSA/1 X PBS added to each well, incubated for 1 h, and aspirated. A total of 150,000 cells were applied to each disk in a final volume of 200
l of serum-free media, incubated for 2 h at 37°C, and cell adhesive quantitated as above [31].
2.6. Statistical analysis
All of the data (except for Table 2) were summarized and analyzed using a one-way analysis of variance (ANOVA) for all surfaces. The alpha level was set at the 0.05 level. For radiolabeled fibronectin adsorption experiments and cell culture experiments, data are presented as mean±SEM (N=total number of disks). Statistical comparisons were performed using a paired Students t-test and ANOVA with the alpha level set at 0.05. For radiolabeled fibronectin or cell culture experiments, individual data is expressed as % control (control defined at 100%).
3. Results
3.1. Surface analysis
AFM images demonstrated differences in surface topography due to various treatments (Fig. 1). The control surfaces were smooth and relatively free of scratches at micron levels (Fig. 1a). All the treated surfaces exhibited a roughened oxide topography with sharply defined crests and pits ( Fig. 1b-d). Surface topography of these thickened oxide surfaces did not change when thermally or peroxide treated samples were further exposed to butanol (data not shown). The roughness values (RMS, Ra) as determined by AFM were higher than the other specimens for the heated alloy, irrespective of whether the heating was accomplished in an atmospheric or oxygen environment (P<0.001) (Table 1). Peroxide treatment resulted in lower RMS and Ra values than the heated samples, yet markedly greater than controls. Therefore, heat treatment increased both roughness and surface area above control levels to a substantially greater value than any of the other treatments (Table 1 and Table 2).
Fig. 1. Atomic force microscopy (AFM) of Ti-6Al-4V samples in Tapping ModeTM of (a) control, (b) peroxide treated, (c) heated (atmosphere), and (d) heated (oxygen). AFM images for the combination of treatments with butanol were the same as (b), (c), and (d).
Table 1. Surface area measurements for titanium-aluminum-vanadium samples chemically and thermally modifieda
One-way ANOVA of the data demonstrates that differences among treatments are significantly greater than expected by chance; P<0.0006.
One-way ANOVA of the data demonstrates that differences among treatments are significantly greater than expected by chance; P<0.0001.
3.2. Oxide thickness and chemical analysis
The oxide thickness as measured by ESCA for unheated disk surfaces was approximately 5 nm, whereas the H2O2 treated disks were on the order of 10 nm. The oxide layer on the H2O2+butanol treated disks was 6.5 nm which is less than that for H2O2 treatment alone. Heated samples in either oxygen or air, including postbutanol treatment, produced thick oxide layers that were unsuitable for depth profiling.
Titanium, aluminum, vanadium, oxygen, carbon and nitrogen were detected on all samples by ESCA (Table 3). Due to the low concentration in the surface layer, vanadium can only be detected by ESCA using monochromated excitation radiation [22]. Low levels of Si were observed only with the control specimens, possibly due to minor silica or silicone contamination. Carbon was detected on all treated and untreated disk surfaces. For control and H2O2 treated disks, the C surface organic contamination was primarily in the form of — CHx and C-O bonded species. Nitrogen, also from organic surface contamination, was detected on all surfaces, regardless of treatment. The levels of the metals varied in accordance with the amounts of surface organics. Higher surface organic levels translate into less metals being detected.
Table 3. Surface composition as determined by ESCA
Atomic percent values are reproducible to±10% rsd or better.
For the control disks, Ti was the metal detected at the highest concentration, followed by Al with only minor amounts of V (Table 3). The Al/Ti and V/Ti ratios were less than the theoretical values for the bulk alloy, indicating that the surface is enriched in Ti relative to the bulk alloy. The V/Al ratio is also less than the theoretical value for the alloy, indicating that V is also deficient with respect to Al in the surface layer. The Ti2p3/2, Al2p, and V2p3/2 binding energies for the control disks averaged 458±0.3, 74.3±0.2, and 515.4±0.3 and reflect the presence of Ti in the +4 state, Al in the +3 state, and V in the +3. This is consistent with the assignments for TiO2 [32], Al2O3 [33, 34 and 35], and V2O3 [36] in the surface oxide layer. Low intensity metallic Ti and Al peaks were also observed, reflecting the relatively thin nature of the native oxide on the alloy surface.
For the heated samples, the Al and V levels were significantly higher than those determined for the control disks (Table 3). The Al/Ti and V/Ti ratios for the heated disks were also higher than the unheated disks and higher than the theoretical ratios for the alloy itself. This indicates that, in comparison with the native oxide on the alloy surfaces, heat treatment causes an enrichment of Al and V oxides on the surface. In fact, heat treatment produces a surface oxide with a higher concentration of Al than Ti. Heat treatment in the presence of air resulted in a greater enrichment of Al and V in the surface oxide than oxygen heat treatment alone. The V/Al ratios for the heated disks were also greater than those for the controls. For both heat treatments, the Ti2p3/2 and Al2p binding energies for the heated disks averaged 458.9±0.1 and 74.8±0.1 eV indicating the presence of TiO2 and Al2O3 in the oxide layer. Two V peaks were observed having V2p3/2 binding energies of 516.7±0.1 and 517.8±0.1 eV indicating the presence of V as both VO2 and V2O5 (VOx). The V2O5 is twice as prevalent as VO2 within the surface oxide layer. In the heated samples, V decreased after immersion in butanol. The atomic ratio data reflect these changes, as decreases in the Al/Ti, V/Ti, and V/Al ratios were observed following butanol treatment. The most significant reductions occurred in the V/Ti and V/Al ratios suggesting that surface vanadium oxides for the heated disks do not adhere strongly to the disk surfaces. The reduction in Al/Ti was less, indicating that most of the surface Al2O3 does adhere well to the disk surfaces. The percentages of C-O following butanol treatment of the heated disks increased, 16-20% and 17-24% for heated disks in the presence of oxygen or atmospheric conditions suggesting that butanol adsorbs on the surfaces of the heated disks.
Based on ESCA analysis, the surfaces of the H2O2 treated disks were comprised primarily of titanium in the form of Ti4+ as TiO2. A low intensity metallic Ti peak was also observed in the ESCA spectrum of these samples, indicating a thickness of the oxide film is on the order of 5 nm. Aluminum was present primarily as Al3+, Al2O3, with a small amount of metallic Al detected as well. Vanadium was present in a range of oxidation states, from metallic V to V+5, with the highest concentration of vanadium present in the lower oxidation states. The Al/Ti atomic ratios for the H2O2 treated disks were lower than that of the control disks, indicating that the H2O2 treatment depletes the surface of aluminum (Table 3). The V/Ti ratio for the H2O2 and control disks were the same. The V/Al ratios for the H2O2 disks were higher than for the control disks, further indicating a slight depletion of aluminum at the H2O2 treated surface. The metallic composition of the H2O2 treated disk did not change significantly as a result of immersion in butanol. Similar to the heated disks, the percentage of C-O, however, did increase from 16% to 24% after butanol treatment and the increase in C-O strongly suggests interaction of butanol to the disk surface (Table 3).
3.3. Wettability properties
Contact angle measurements for the control titanium alloy disks showed an average contact angle of 43.0±5.3 (Table 4). Heat treatment (either in air or oxygen atmosphere) resulted in a more hydrophilic surface when compared with controls (P<0.001) but with no significant difference between the heat treatment in air or oxygen environment (P>0.05). Similarly, peroxide treatment rendered the specimens more hydrophilic than controls (P<0.001). Posttreatment with butanol, however, resulted in less hydrophilic surfaces than heating or peroxide treatment alone ( P<0.01).
Table 4. Wettability measurements for titanium-aluminum-vanadium samples chemically and thermally modifieda
One-way ANOVA of the data demonstrates that differences among treatments are significantly greater than expected by chance; P<0.0001.
3.4. Protein-surface interaction
When 125I-Fibronectin was incubated with titanium alloy disks at a solution phase concentration of 0.1, 0.2, and 1.0 n
, respectively and subsequently washed, 0.63±0.14% (SEM; N=18), 0.67±0.15% (SEM; N=6), and 0.3±0.01% (SEM; N=6) remained bound to the titanium alloy surface. At a solution phase volume of 200
l per disk, control disks (not chemically or thermally pretreated) incubated with solution phase concentrations of 0.1, 0.2, and 1.0 n
fibronectin (MW=225,000) were coated with 28, 60 and 135 pg per disk, respectively. Pretreatment with heat (atm)/butanol, peroxide, and peroxide/butanol showed a statistically significant increase in the binding of 125I-Fibronectin by Student's t-tests but not by ANOVA (Fig. 2).
Fig. 2. Effects of chemical and thermal treatments on binding of 125I-fibronectin (FN) to titanium alloy disks. Titanium alloy disks were either untreated (C) or pretreated with heat+oxygen (H+O2), heat under normal atmosphere (H), heat+oxygen+butanol (H+O2+B), heat under normal atmosphere+butanol (H+B), peroxide alone (P), or peroxide+butanol (P+B), and the disks were coated overnight with 125I-fibronectin (0.1 n
). Control disks bound radioactivity of 304±67 DPM per disk (mean±SEM; N=17); background radioactivity was 9.5±2.2 DPM. Data are from at least three independent replicate experiments. *P<0.03, **P<0.003; fibronectin bound to pretreated disk vs. uncoated control disk based on Student's t-test. One-way ANOVA of the data demonstrates that differences among treatments are not significantly greater than expected by chance; P<0.13.
3.5. Cell binding
Cell binding to different surfaces was dependent on the concentration of fibronectin (Fig. 3). Fibronectin did not promote a statistically significant increase in the attachment of MG63 cells at solution phase concentrations of 0.1 and 1 n
. Concentrations of 25 n
or higher caused substantial increases in the attachment of cells to the coated titanium alloy surface, although statistical significance for these effects was only reached at concentrations of 500 n
or higher measured with control disks (N=5; P<0.01) that were not precoated with fibronectin. At a concentration of 1000 n
, the number of attached cells measured 633±152% of that measured with control disks (N=5; P<0.01) that were not precoated with fibronectin (Fig. 3).
Fig. 3. Effects of fibronectin on MG63 cell attachment to titanium alloy disks. Disks were precoated overnight with increasing solution concentrations of fibronectin (0.1-1000 n
) and cell attachment quantified as described in methods. The basal level of cell attachment of cells to control (uncoated, fibronectin-free) disks was 19,144±4658 cells per disk (mean±SEM; N=4 independent cultures). Data are from at least three independent cultures. One-way ANOVA of the data demonstrates that differences among groups (control and different concentrations of fibronectin) are significantly greater than expected by chance; P<0.0001. *Significantly greater than control (P<0.01) and fibronectin concentrations from 0.1 to 25 n
(P<0.05) based on Tukey-Kramer analysis. **Significantly greater than control (P<0.001), fibronectin concentrations from 0.1 to 25 n
(P<0.01) and 50 n
fibronectin (P<0.05) based on Tukey-Kramer analysis.
None of the pretreatments induced a statistically significant increase in basal attachment except for heat/butanol (Fig. 3). Although a concentration of 0.1 n
fibronectin alone did not by itself promote cell attachment to untreated disks, several of the pretreatments increased the attachment of MG63 cells to (0.1 n
) fibronectin-coated disks above that observed after pretreatment alone (with no subsequent fibronectin coating). Thermal pretreatments augmented cell attachment in the presence of a nonadhesive concentration of fibronectin to a greater degree than nonthermal treatments. Following pretreatment with heat under normal atmosphere, heat under oxygen with butanol posttreatment or heat under normal atmosphere with butanol posttreatment, the attachment of cells to fibronectin-coated disks was increased to 478±38% (N=3; P<0.05), 572±252% (N=3; P<0.01) or 549±179% (N=3; P<0.01), respectively, that of untreated control disks. Since none of these three treatments alone nor fibronectin alone significantly increased cell adhesion (Fig. 4), the thermal treatments appeared to enhance fibronectin-promoted adherence. This was the case even though the pretreatment with heat under normal atmosphere or heat (with oxygen or under normal atmosphere) with butanol posttreatment had no statistically significant effect (based on ANOVA) on 125I-Fibronectin (0.1 n
) binding to the titanium alloy disks (Fig. 2).
Fig. 4. Effect of treatments on basal and fibronectin-promoted MG63 cell attachment to titanium alloy disks. Chemical and thermal pretreatments were either applied to disks or not applied prior to overnight incubation with or without 0.1 n
fibronectin. Disks were then incubated with MG63 cells and the number of attached cells measured as described in methods. Basal level of cell attachment of cells to control (untreated, fibronectin-free) disks was 22,671±5431 cells per disk (mean±SEM; N=6 independent cultures). Data are from at least three independent cultures. One-way ANOVA of the data obtained in the absence of fibronectin demonstrates that differences among treatments are not significantly greater than expected by chance; P<0.0896. One-way ANOVA of the data obtained in the presence of fibronectin demonstrates that differences among treatments are significantly greater than expected by chance; P<0.0009. *Significantly greater than control samples based on Tukey-Kramer analysis; P<0.05. **Significantly greater than control samples based on Tukey-Kramer analysis; P<0.01.
4. Discussion
In this study, thermal and chemical modification of titanium disks was shown to result in an alteration of surface topography, oxide chemistry, wettability, as well as protein- and cell-binding affinities. Changes in % atomic composition (particularly that of Al) that appear to be associated with enhanced protein-induced attachment of MG63 cells to the implant surface were identified. Such findings may further the understanding of the particular surface properties that are most closely related to osteoblast cell attachment and function. For this reason it is suggested that either the surface modifications investigated here or knowledge gained from their study may be used to help improve the biocompatibility and osteoinductive properties of titanium based implants.
The characterization of titanium alloy surface composition and properties prior to treatment provided an important baseline from which to evaluate the effects of chemical and thermal treatments on surface characteristics, protein adsorption and cell attachment. The influence of chemical composition and oxide content on cell attachment to implant surfaces has not been extensively investigated. Biocompatibility of titanium implants is associated with the oxide on its surface [2] suggesting that alteration in oxide chemistry can influence the biological properties of the implant materials. The thickness of the TiO2 surface film of 5 nm, as observed in this study, is in the previously reported range [1, 34 and 37]. Reports using angle resolved XPS showed that TiO2 is located throughout the oxide [4] with minor suboxides of Ti2O3 and TiO observed near the metal/oxide interface [1, 4 and 38]. Our ESCA analysis found aluminum to be present throughout the control oxide confirming literature reports [1, 4 and 33]. We observed that titanium was 76% greater in atomic percent than aluminum while the level of vanadium was lower than the bulk concentration [1, 33 and 35]. In view of these considerations, it would be expected that the complex surface compositional environment of Ti6Al4 V disks would exhibit interesting biological properties particularly following thermal or chemical modification.
Heating the titanium disks was found to alter a number of surface properties. Heat treatment increased surface roughness and significant differences were seen in the surface topography among the heated samples. Even though treatments employing heat increased the surface roughness of our Ti6Al4 V disks, the resulting Ra values (298-392 nm) were equivalent to those reported by others for control "polished" Ti6Al4 V surfaces [22, 39 and 40]. In view of this marginal effect on roughness it is not surprising that even though MG63 cells have been found to attach less well to rough surfaces [17] (including surfaces made 7-10 times rougher than our surfaces by sandblasting [41]), we observed that heat treatment had no inhibitory effect on basal MG63 cell attachment. This suggests that the stimulatory effect of heat followed by butanol on basal MG63 cell attachment is most likely to be attributable to changes in surface properties other than roughness alone. Furthermore, since the heat-induced change in roughness is of such small magnitude compared to the accompanying changes in atomic composition (see below), increased roughness is also unlikely to explain our observed effects of heat treatment on peptide-induced cell attachment.
The surface property of Ti6Al4 V disks that was most closely associated with receptor-mediated cell attachment was the percent composition of Al relative to that of Ti in the oxide layer. Heating samples in air or oxygen increased surface Al2O3 content and fibronectin-promoted cell attachment on these surfaces to a much greater degree than treatments that did not include high temperature. Heating Ti6Al4 V between 650°C and 850°C encourages the diffusion of Ti and Al from the substrate into the oxide forming a multilayer of Al2O3 and TiO2 [42]. The formation of Al2O3 and VOx enriched layers would explain the 42-55% decrease in the at% of Ti observed and the increased Al/Ti and V/Ti ratios. Heating in atmosphere encouraged the formation of a higher at% Al2O3 layer compared to heating in oxygen alone with no significant difference between VOx with either treatment. The findings that the oxidation of Ti6Al4 V is faster in air than oxygen at 900°C and that the oxidation in pure oxygen hinders the formation of an Al2O3 barrier layer oxide [43] would help explain the lower at% of Al in our specimens heated in pure oxygen compared with those heated in air. Importantly, heat with oxygen did not significantly increase protein-induced cell-adhesion, whereas heat (atm) with or without butanol did. These findings demonstrate that fibronectin adhesive activity appears to increase with increasing percent composition of aluminum. Other studies have similarly reported heat treatment increases the Al content of the surface layer and concurrently cell attachment [1, 44, 45 and 46]. Our results suggest that this Al-enriched surface layer may influence protein conformation potentially exposing cellular binding domains and thus facilitating cell attachment.
The findings of this study suggest that changes in overall surface wettability induced by butanol treatment [28] may not be as critical for protein adsorption and biological activity as surface chemistry. Local surface charge can be influenced by the acid-base balance of metal hydroxo-complexes. Hydroxide ions (OH−) attach to metal cations. The titanium alloy oxide surface in contact with water is believed to be highly hydroxylated. The hydroxo-complexes of multivalent cations (i.e., TiIV or AlIII) are amphoteric:
Ti-OH+H2O |
Ti-OH+H2O |
Treatments, such as high temperature, that decrease the content of [M-OH2]+ groups through thermal oxidation and/or increase the relative surface content of metals that are acidic at physiological pH, leading to the predominance of [M-O]− over [M-OH2]+ groups, would be expected to increase negative surface charge and the concentration of specific negatively charged groups that may interact with proteins. This may in turn promote conformational changes that lead to increased protein adsorption and/or biological activity. The heating of alumina surfaces has been shown to enhance the acidity of the surface hydroxyl groups, especially at high calcination temperatures [47]. Therefore, the temperature-induced change in the surface concentration of aluminum oxides and/or acid-base balance of aluminum hydroxo-complexes (depending on the pKa) may be particularly important for the surface behavior of adsorbed proteins at physiological pH, thereby helping to explain the observed correlation between aluminum content and protein-induced cell attachment.
Oxide surface topography also was less important for determining protein and cell attachment than surface chemistry. Surface oxidation with hydrogen peroxide [48, 49, 50 and 51] resulted in a topography of numerous elevations and a thickened oxide layer. Oxidation with hydrogen peroxide leads to increased oxide content through the formation of peroxide complexes that convert into Ti(IV)O2−, Ti(IV)O22−, and Ti(III)O2 [48, 52 and 53]. The RMS and Ra values were lower than those in heated samples suggesting less surface roughness for peroxide treated surfaces. In accordance with other reports, peroxide treatment of Ti6Al4 V resulted in a highly hydrophilic surface [54]. Furthermore, hydrophilicity can be enhanced by the formation of surface hydroxyl groups, which was encouraged by peroxide treatments in our study and has previously been shown to favor cell attachment [55]. Notably, peroxide treatment did not augment protein-induced cell attachment. This finding indicates that none of the peroxide-induced changes in surface properties, including increased overall surface wettability, increased oxide thickness and hydroxyl group formation, were correlated with increased cell attachment.
Modification of the oxide surface topography and surface chemistry changes the affinity of these surfaces for cells and proteins [55 and 56]. Protein adsorption is the initial event occurring when a biomaterial surface is in contact with a biological environment. Fibronectin, a glycoprotein made by numerous cell types including odontoblasts and osteoblasts [57 and 58], plays an important initial role in the interaction between implant surfaces and the surrounding biological milieu. Fibronectin binds rapidly to exposed titanium dioxide surfaces in vitro [59 and 60] and is detected as early as 1 week post Ti-implant implantation [61]. The presence of low levels of fibronectin, a protein involved in cell-substrate interaction [23 and 62], enhances cell binding [23, 63, 64 and 65]. It is interesting to note that, at a nonadhesive concentration of fibronectin (0.1 n
), treatment with heat (atm) or heat/butanol (atmosphere or oxygen) led to a dramatic statistically significant increase in cell attachment (compared to the corresponding treatments without fibronectin) that greatly exceeded the marginal effects of these treatments on fibronectin adsorption. These changes in activity are better explained by altered biological activity, possibly resulting from protein conformational changes, in the adsorbed fibronectin molecule, than by mass effects alone. A number of environmental and surface variables (e.g., ionic strength, pH, temperature, surface charge) influence the fibronectin molecular shape and alter the protein conformation upon binding to the metallic surface [29, 62, 63, 66, 67, 68, 69, 70, 71 and 72]. Relatively small sequences within the protein chain (e.g., RGD) are responsible for protein-cell binding that emphasizes the importance of correct protein strand orientation [63, 64 and 73]. Thus, altered fibronectin conformation may unmask potential cell binding domains.
In summary, we have examined various chemical/thermal modifications of Ti6Al4 V surface properties, including roughness, wettability, oxide chemistry, and atomic composition, in relation to protein adsorption and receptor-induced MG63 cell attachment. We have shown that (a) receptor-mediated cell attachment is more highly correlated with atomic percent composition, specifically Al content, than other properties, and (b) that chemical modifications which alter Al content can regulate the biological activity of a cell-adhesive protein independently of protein adsorption. Future studies will investigate the influence of changes in the metallic composition of various implantable alloys on protein adsorption and receptor-mediated cell attachment.
Acknowledgements
Generous support was received from VA Merit Grant No. 2894-005 during the preparation of this article and for the studies described herein. Special thanks goes to George Boutis, Christina Silva, and Komal Patel for their assistance with the cell culture experiments.
References
1. Ask M, Lausmaa J, Kasemo B. Preparation and surface spectroscopic characterization of oxide films on Ti6Al4V. Appl Surf Sci 1988-89;35:283-301.
2. B. Kasemo and J. Lausmaa, Surface science aspects of inorganic biomaterials. CRC Crit Rev Biocompat 2 (1986), pp. 335-380. Abstract-EMBASE
3. B.D. Ratner, D.G. Castner, T.A. Horbett, T.J. Lenk, K.B. Lewis and R.J. Rapoza, Biomolecules and surfaces. J Vac Sci Technol A 8 (1990), pp. 2306-2317. Full Text via CrossRef
4. I. Milo
ev, M. Metiko
-Hukovic and H-.H. Stehblow, Passive film on orthopaedic TiAlV alloy formed in physiological solution investigated by X-ray photoelectron spectroscopy. Biomaterials 21 (2000), pp. 2103-2113. SummaryPlus | Full Text + Links | PDF (410 K)
5. C.M. Michaels, J.C. Keller and C.M. Stanford, In vitro periodontal ligament fibroblast attachment of plasma-cleaned titanium surfaces. J Oral Implantol 17 (1991), pp. 132-139. Abstract-MEDLINE
6. D. Kawahara, J.L. Ong, G.N. Raikar, L.C. Lucas, J.E. Lemons and M. Nakamura, Surface characterization of radio-frequency glow discharge and autoclaved titanium surfaces. Int J Oral Maxillofac Implants 11 (1996), pp. 435-442. Abstract-MEDLINE
7. K. Sřballe, E.S. Hansen, H. Brockstedt-Rasmussen and C. Bünger, Hydroxyapatite coating converts fibrous tissue to bone around loaded implants. J Bone Joint Surg Am 75-B (1993), pp. 270-278. Abstract-EMBASE
8. R.G.T. Greesink and N.H.M. Hoefnagels, Six-year results of hydroxyapatite-coated total hip replacement. J Bone Joint Surg Am 77-B (1995), pp. 534-547.
9. W.J.A. Dhert, C.P.A.T. Klein, J.A. Jansen et al., A histological and histomorphometric investigation of fluorapatite, magnesium whitlockite and hydroxylapatite plasma-sprayed coatings in goats. J Biomed Mater Res 27 (1993), pp. 127-138. Abstract-MEDLINE | Abstract-Compendex | Abstract-EMBASE
10. W.J.A. Dhert, P. Thomsen, A.K. Blomgren, M. Esposito, L.E. Ericson and A.J. Verbout, Integration of press-fit implants in cortical bone: a study on interface kinetics. J Biomed Mater Res 41 (1998), pp. 574-583. Abstract-MEDLINE | Abstract-Compendex | Abstract-EMBASE | Full Text via CrossRef
11. D.E. MacDonald, F. Betts, S.B. Doty and A.L. Boskey, A methodological study for the analysis of apatite-coated dental implants retrieved from humans. Ann Periodontol 5 (2000), pp. 175-184. Abstract-MEDLINE
12. D.E. MacDonald, F. Betts, M. Stranick, S. Doty and A.L. Boskey, Physiochemical study of plasma-sprayed hydroxyapatite-coated implants in humans. J Biomed Mater Res 54 (2001), pp. 480-490. Abstract-MEDLINE | Abstract-EMBASE | Full Text via CrossRef
13. D.M. Brunette, J. Ratkay and B. Chehroudi, Behaviour of osteoblasts on micromachined surfaces. In: J.E. Davies, Editor, The bone-biomaterial interface, University of Toronto Press, Toronto (1991), pp. 49-61.
14. B. Boyan, T. Hummert, K. Kieswetter, D. Schraub, D. Dean and Z. Schwartz, Effect of titanium surface characteristics on chondrocytes and osteoblasts in vitro. Cells Mater 5 (1995), pp. 323-335.
15. R.M. Pilliar, Overview of surface variability of metallic endosseous dental implants: textured and porous surface-structured designs. Implant Dent 7 (1998), pp. 305-314. Abstract-MEDLINE
16. A. Wennerberg, C. Hallgren, C. Johansson and S. Danelli, A histomorphometric evaluation of screw-shaped implants each prepared with two surface roughnesses. Clin Oral Implant Res 9 (1998), pp. 11-19. Abstract-MEDLINE | Full Text via CrossRef
17. J.Y. Martin, Z. Schwartz, T.W. Hummert et al., Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). J Biomed Mater Res 29 (1995), pp. 389-401. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex
18. D. Buser, R.K. Schenk, S. Steinemann, J.P. Fiorelini, C.H. Fox and H. Stich, Influence of surface characteristics on bone integration on titanium implants. J Biomed Mater Res 25 (1991), pp. 889-902. Abstract-EMBASE | Abstract-MEDLINE
19. C. Larsson, P. Thomsen, J. Lausmaa, M. Rodahl, B. Kasemo and L.E. Ericson, Bone response to surface modified titanium implants: studies on electropolished implants with different oxide thicknesses and morphology. Biomaterials 15 (1994), pp. 1062-1074. Abstract
20. Y-.T. Sul, C.B. Johansson, S. Petronis et al., Characteristics of the surface oxides on turned and electrochemically oxidized pure titanium implants up to dielectric breakdown: the oxide thickness, micropore configurations, surface roughness, crystal structure, and chemical composition. Biomaterials 23 (2002), pp. 491-501. SummaryPlus | Full Text + Links | PDF (361 K)
21. D.K. Pettit, T.A. Horbett and A.S. Hoffman, Influence of the substrate binding characteristics of fibronectin on corneal epithelial cell outgrowth. J Biomed Mater Res 26 (1992), pp. 1259-1275. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex
22. C. Sittig, M. Textor, N.D. Spencer, M. Wieland and P-.H. Vallotton, Surface characterization of implant materials c.p. Ti, Ti-6Al-7Nb and Ti-6Al-4 V with different pretreatments. J Mater Sci: Mater Med 10 (1999), pp. 35-46. Abstract-Compendex | Abstract-EMBASE | Full Text via CrossRef
23. G. Altankov, V. Thom, T. Groth, K. Jankova, G. Jonsson and M. Ulbricht, Modulating the biocompatibility of polymer surfaces with poly(ethylene glycol): effect of fibronectin. J Biomed Mater Res 52 (2000), pp. 219-230. Abstract-EMBASE | Abstract-MEDLINE | Abstract-Compendex | Full Text via CrossRef
24. H-.M. Kim, F. Miyaji, T. Kokubo, S. Nishiguchi and T. Nakamura, Graded surface structure of bioactive titanium prepared by chemical treatment. J Biomed Mater Res 45 (1999), pp. 100-107. Abstract | Abstract + References | PDF (170 K) | Full Text via CrossRef
25. H.B. Wen, Q. Liu, J.R. de Wijn, K. de Groot and F.Z. Cui, Preparation of bioactive microporous titanium surface by a new two-step chemical treatment. J Mater Sci: Mater Med 9 (1998), pp. 121-128. Abstract-Compendex | Abstract-EMBASE | Full Text via CrossRef
26. F-86 AS. Standard practice for surface preparation and marking of metallical surgical implants. Annual book of ASTM standards, vol. 13.01. USA: American Society for Testing and Materials; 1996. p. 6-8.
27. M. Malmsten, Ellipsometry studies of the effects of surface hydrophobicity on protein adsorption. Colloids Surf B 3 (1995), pp. 297-308. SummaryPlus | Full Text + Links | PDF (922 K)
28. H. Nygren, Initial reactions of whole blood with hydrophilic and hydrophobic titanium surfaces. Colloids Surf B 6 (1996), pp. 329-333. SummaryPlus | Full Text + Links | PDF (360 K)
29. D.E. MacDonald, B. Markovic, M. Allen, P. Somasundaran and A.L. Boskey, Surface analysis of human plasma fibronectin adsorbed to commercially pure titanium materials. J Biomed Mater Res 41 (1998), pp. 120-130. Abstract-MEDLINE | Abstract-Compendex | Abstract-EMBASE | Full Text via CrossRef
30. Boyan B, Dean DD, Lohmann CH, Cochran DL, Sylvia VL, Schwartz DK. The titanium-bone cell interface in vitro: the role of the surface in promoting osteointegration. In: Brunette DM, Tengvall P, Textor M, Thomsen P, editors. Titanium in medicine: material science surface science engineering biological responses and medical applications. Berlin: Springer; 2001. p. 561-85.
31. U. Landegren, Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Immunol Methods 67 (1984), pp. 379-388. Abstract
32. I. Milo
ev, H-.H. Strehblow, B. Navin
ek and M. Metiko
-Hukovic, Electrochemical and thermal oxidation of tin coatings studied by xps. Surf Interface Anal 23 (1995), pp. 529-539. Abstract-Compendex | Abstract-INSPEC
33. R.N.S. Sodhi, A. Weninger, J.E. Davies and K. Sreenivas, X-ray photoelectron spectroscopic comparison of sputtered Ti, Ti6Al4 V, and passivated bulk metals for use in cell culture techniques. J Vac Sci Technol, A 9 (1991), pp. 1329-1333. Abstract-INSPEC | Full Text via CrossRef
34. M.T. Pham, I. Zyganow, W. Matz et al., Corrosion behavior and microstructure of titanium implanted with a and b stabilizing elements. Thin Solid Films 310 (1997), pp. 251-259. Abstract-Compendex | Abstract-INSPEC
35. Y. Okazaki, T. Tateishi and Y. Ito, Corrosion resistance of implant alloys in pseudo physiological solution and role of alloying elements in passive films. Mater Trans JIM 38 (1997), pp. 78-84. Abstract-Compendex | Abstract-INSPEC
36. C.N.R. Rao, D.D. Sarma, S. Vasudevan and M.S. Hegde, Study of transition metal oxides by photoelectron spectroscopy. Proc R Soc London A 67 (1979), pp. 239-252. Abstract-INSPEC
37. T. Albrektsson, P.I. Brĺnemark, H.A. Hansson et al., The interface zone of inorganic implants in vivo — titanium implants in bone. Ann Biomed Eng 11 (1983), pp. 1-27. Abstract-Compendex | Abstract-Compendex | Abstract-EMBASE
38. P.A. Lee, K.F. Stork, B.F. Maschhoff, K.W. Nebesny and N.R. Armstrong, Oxide formation on Fe and Ti thin films and on Fe thin films modified with ultrathin layers of Ti. Surf Sci Interf Anal 17 (1991), pp. 48-56. Abstract-Compendex
39. J. Lincks, B.D. Boyan, C.R. Blanchard et al., Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition. Biomaterials 19 (1998), pp. 2219-2232. Abstract | PDF (752 K)
40. D.D. Deligianni, N. Katsala, S. Ladas, D. Sotiropoulou, J. Amedee and Y.F. Missirlis, Effect of surface roughness of the titanium alloy Ti-6Al-4 V on human bone marrow cell response and on protein adsorption. Biomaterials 22 (2001), pp. 1241-1251. SummaryPlus | Full Text + Links | PDF (427 K)
41. K. Anselme, M. Bigerelle, B. Noel et al., Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses. J Biomed Mater Res 49 (2000), pp. 155-166. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex | Full Text via CrossRef
42. H.L. Du, P.K. Datta, D.B. Lewis and J.S. Burnell-Gray, Air oxidation behavior of Ti-6Al-4 V alloy between 650 and 850°C. Corros Sci 36 (1994), pp. 631-642. Abstract
43. S. Becker, A. Rahmel, M. Schorr and M. Schütze, Mechanism of isothermal oxidation of the intermetallic TiAl and of TiAl alloy. Oxidat Metals 38 (1992), pp. 425-464. Abstract-Compendex | Abstract-INSPEC
44. M. Browne and P.J, .R. Gregson, Characterization of titanium alloy implant surfaces with improved dissolution resistance. J Mater Sci: Mater Med 7 (1996), pp. 323-329. Abstract-EMBASE | Abstract-Compendex
45. H. Zreigat and C.R. Howlett, Titanium substrata composition influences osteoblastic phenotype: in vitro study. J Biomed Mater Res 47 (1999), pp. 360-366.
46. K. Anselme, P. Linez, M. Bigerelle et al., The relative influence of the topography and chemistry of TiAl6V4 surfaces on osteoblastic cell behavior. Biomaterials 21 (2000), pp. 1567-1577. SummaryPlus | Full Text + Links | PDF (1813 K)
47. F. Deng, G. Wang, Y. Du, C. Ye, Y. Kong and X. Li, 1 h mas adn 1h[23na] double resonance nmr studies on the modification of surface hydroxyl groups of gamma-alumina by sodium. Solid State Nucl Magn Reson 7 (1997), pp. 281-290. Abstract-MEDLINE | Abstract-Compendex
48. B. Wälivaara, I. Lundström and P. Tengvall, An in vitro study of H2O2—treated titanium surfaces in contact with blood plasma and a simulated body fluid. Clin Mater 12 (1993), pp. 141-148. Abstract-MEDLINE | Abstract-Compendex | Abstract-EMBASE
49. B. Wälivaara, B.O. Aronsson, M. Rodahl, J. Lausmaa and P. Tengvall, Titanium with different oxides: in vitro studies of protein adsorption and contact activation. Biomaterials 15 (1994), pp. 827-834. Abstract
50. J. Pan, D. Thierry and C. Leygraf, Hydrogen peroxide toward enhanced oxide growth on titanium in PBS solution: blue coloration and clinical relevance. J Biomed Mater Res 30 (1996), pp. 393-402. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex | Full Text via CrossRef
51. J. Pan, H. Liao, C. Leygraf, D. Thierry and J. Li, Variation of oxide films on titanium induced by osteoblast-like cell culture and the influence of an H2O2 pretreatment. J Biomed Mater Res40 (1998), pp. 244-256. Abstract-MEDLINE | Abstract-EMBASE | Full Text via CrossRef
52. J. Mühlebach, K. Müller and G. Schwarzenbach, The peroso complexes of titanium. Inorg Chem 9 (1970), pp. 2381-2390.
53. P. Tengvall and I. Lundström, Physico-chemical considerations of titanium as a biomaterial. Clin Mater 9 (1992), pp. 115-134. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex
54. D.E. MacDonald, N. Deo, B. Markovic, M. Stranick and P. Somasundaran, Adsorption and dissolution behavior of human plasma fibronectin on thermally and chemically modified titanium dioxide particles. Biomaterials 23 (2002), pp. 1269-1279. SummaryPlus | Full Text + Links | PDF (205 K)
55. B. Kasemo, Biological surface science. Surf Sci 500 (2002), pp. 656-677. SummaryPlus | Full Text + Links | PDF (1296 K)
56. D.M. Brunette, Principles of all behavior on titanium surfaces and their application to implanted devices. In: D.M. Brunette, P. Tengvall, M. Textor and P. Thomsen, Editors, Titanium in medicine, Springer, Berlin (2001), pp. 485-512.
57. A.G. Brownell, C.C. Bessem and H.C. Slavkin, Possible function of mesenchyme cell-derived fibronectin during formation of basal lamina. Proc Natl Acad Sci USA 78 (1981), pp. 3711-3715. Abstract-EMBASE | Abstract-MEDLINE
58. W. Dessau, J. Sasse, R. Timple, F. Jilek and K. von der Mark, Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. J Cell Biol 79 (1978), pp. 342-355. Abstract-EMBASE | Abstract-MEDLINE | Full Text via CrossRef
59. B. Ivarsson and I. Lundström, Physical characterization of protein adsorption on metal and metaloxide surfaces. CRC Crit Rev Biocompat 2 (1986), pp. 1-96.
60. D.E. MacDonald, B. Markovic, A.L. Boskey and P. Somasundaran, Physico-chemical properties of human plasma fibronectin binding to well characterized titanium dioxide. Colloids Surf B 11 (1998), pp. 131-139. SummaryPlus | Full Text + Links | PDF (199 K)
61. A. Rosengren, B.R. Hohansson, N. Danielsen, P. Thomsen and L.E. Ericson, Immunohistochemical studies on the distribution of albumin, fibrinogen, fibronectin, IgG and collagen around PTFE and titanium implants. Biomaterials 17 (1996), pp. 1779-1786. SummaryPlus | Full Text + Links | PDF (1485 K)
62. G. Altankov and T. Groth, Reorganization of substratum-bound fibronectin on hydrophilic and hydrophobic materials is related to biocompatibility. J Mater Sci: Mater Med 5 (1994), pp. 732-737. Abstract-Compendex | Abstract-EMBASE
63. F. Grinnell and M.K. Feld, Adsorption characteristics of plasma fibronectin in relationship to biological activity. J Biomed Mater Res 15 (1981), pp. 363-381. Abstract-EMBASE | Abstract-Compendex | Abstract-MEDLINE
64. F. Grinnell and M.K. Feld, Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during cell adhesion in serum-containing medium. J Biol Chem 257 (1982), pp. 4888-4893. Abstract-MEDLINE
65. D.J. Juliano, S.S. Saavedra and G.A. Truskey, Effect of the conformation and orientation of adsorbed fibronectin on endothelial cell spreading and the strength of adhesion. J Biomed Mater Res 27 (1993), pp. 1103-1113.
66. B. Sjöberg, M. Eriksson, E. Österlund, S. Pap and K. Österlund, Solution structure of human plasma fibronectin as a function of NaCl concentration determined by small-angle neutron diffraction. Eur J Biophys 17 (1989), pp. 5-11. Abstract-MEDLINE | Abstract-INSPEC | Abstract-EMBASE
67. M.J. Benecky, C.G. Kolvenbach, R.W. Wine, J.P. DiOrio and M.W. Mosesson, Human plasma fibronectin structure probed by steady-state fluorescence polarization: evidence for a rigid oblate structure. Biochemistry 29 (1990), pp. 3082-3091. Abstract-EMBASE | Abstract-MEDLINE
68. M.Y. Khan, M.S. Medow and S.A. Newman, Unfolding transitions of fibronectin and its domains, stabilization and structural alteration of the n-terminal domain by heparin. Biochem J 270 (1990), pp. 33-38. Abstract-MEDLINE | Abstract-EMBASE
69. H.P. Erickson and N.A. Carrell, Fibronectin in extended and compact conformations. J Biol Chem 258 (1983), pp. 14539-14544. Abstract-EMBASE | Abstract-MEDLINE
70. G.K. Iwamoto, L.C. Winterton, R.S. Stoker, R.A. van Wagenen, J.D. Andrade and D.F. Mosher, Fibronectin adsorption detected by interfacial fluorescence. J Colloid Interface Sci 106 (1985), pp. 459-464. Abstract-INSPEC | Abstract-Compendex
71. R. Emch, X. Clivaz, C. Taylor-Denes, P. Vaudaux and P. Descouts, Scanning tunneling microscopy for studying the biomaterial-biological tissue interface. J Vac Sci Technol A8 (1990), pp. 655-658. Abstract-INSPEC | Full Text via CrossRef
72. R. Emch, F. Zenhausern, M. Jobin, M. Taborelli and P. Descouts, Morphological difference between fibronectin sprayed on mica and on pmma. Ultramicroscopy 42-44 (1992), pp. 1155-1160. Abstract
73. B. Bergkvist, J. Carlsson, T. Karlsson and S. Oscarsson, Threshold analysis, a new method to determine the orientation of macromolecules onto surfaces by use of TM-AFM. J Colloid Interface Sci 206 (1998), pp. 475-481.
Corresponding author. Langmuir Center for Colloids and Interfaces, School of Engineering Columbia University, 911 S.W. Mudd Building, Mail Code 4711, 500 West 120th Street, New York, NY 10027, USA. Tel.:+1-1212-854-2926; 1-718-584-9000; fax: +1-212-854-8362
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