Aggregation of HSA, IgG, and Fibrinogen on Methylated Silicon Surfaces

JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.

207, 228 –239 (1998)

CS985624

Aggregation of HSA, IgG, and Fibrinogen on Methylated Silicon Surfaces Juan Luis Ortega-Vinuesa,*,1 Pentti Tengvall,† and Ingemar Lundstro¨m† *Granada University, Department of Applied Physics, Biocolloid and Fluid Physics Group, 18071, Granada, Spain; and †Linko¨ping University, Department of Physics and Measurements Technology, Laboratory of Applied Physics, S-581 83 Linko¨ping, Sweden Received December 30, 1997; accepted May 1, 1998

Ellipsometry was used to quantify adsorption and tapping mode atomic force microscopy to study surface aggregation of human serum albumin (HSA), immunoglobulin G (IgG), and fibrinogen (Fib) adsorbed from aqueous solutions onto methylated silicon surfaces. After exposure to air the protein monolayers were spontaneously restructured, exposing disorganized areas with heterogeneity depending on the degree of surface methylation. The aggregation patterns also depended on some properties of the adsorbed protein (such as the number of contact points with the surface), but seemed to be almost independent of the adsorption time. The results indicate that aggregates were formed due to lateral reorganization on the adsorbed layer at the air-liquid interface during the drying process. The interpretation is that the heterogeneous structures result from a thermodynamically driven interaction between the hydrophobic surface and the similarly hydrophobic air. The main conclusion that can be extracted from this work is that fibrinogen (hydrophobic and large protein) interacts more irreversibly with the silicon surfaces than IgG, and much more so than HSA, which is less hydrophobic and smaller than fibrinogen. © 1998 Academic Press Key Words: protein adsorption; AFM imaging; aggregation structures.

INTRODUCTION

Atomic force microscopy (AFM) (1, 2) has recently become a method of choice to investigate “soft” biological samples, thereby eliminating the need for elaborate sample preparation techniques used in, for example, electronic microscopy. One major concern in the investigation of adsorbed protein layers is the extent to which the samples are affected by our preparation procedures and measuring devices. In contact mode AFM, the lateral force exerted by the tip often results in image artefacts by disruption of the adsorbed protein layer (3). This problem has been solved by the use of cantilevers oscillating at low amplitude (around 1 nm), sensitive to the weak attractive force between the tip and the surface (noncontact AFM) (4). The technique has been successfully used to image proteins with a high lateral resolution (5). 1

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0021-9797/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

Recently, a novel technique, called tapping mode AFM, was developed by Digital Instruments, and employs a silicon cantilever oscillating with a high amplitude (around 100 nm), thereby contacting (or tapping) the surface once every period. This mode largely avoids unfavorable tip–surface interactions sometimes encountered when using noncontact modes, and reduces the lateral forces exerted on the sample. Another critical concern is how the protein film is affected by removing it from its aqueous environment. It is suggested that protein adsorption is an irreversible process (6–8). The multiple contact formation leads to a strong interaction between the surface and the protein in the adsorbed state. However, desorption or displacement of adsorbed proteins can take place when changing the external conditions or adding other macromolecules. The extent of this kind of process depends on the degree of hydrophobicity of the solid surface as well as other many factors. The readiness of proteins to desorb is also related to the hydrophobicity of the sorbent. At hydrophilic surfaces, proteins seem to be less tightly bound than at the hydrophobic surfaces (6, 9, 10). Wa¨livaara et al. (11) found recently that IgG adsorbed onto hydrophilic silicon does not move after drying under a N2 stream, whereas formation of dendrite-like aggregates appears after air exposure on those sample where silicon was hydrophobic. The protein molecules undergo structural changes upon adsorption; the rearrangements, and thus the number of contact points with the surface, depend on both the nature of the protein (that is, if “soft” or “hard”) and the adsorption time. It is well known that desorption becomes difficult after long incubation times (12, 13). In this paper we studied the role of the adsorption time and the surface hydrophobicity during the immobilization of three different proteins (albumin, IgG, and fibrinogen) when the samples were dried in nitrogen. Our aim is to gain a better understanding of the air–liquid interface effects. MATERIALS AND METHODS

Silicon Surfaces Silicon wafers (Okmetic) of 0.5 mm thickness were cut into 1 3 1 cm pieces and cleaned using the following protocol: The 228

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TABLE 1 Surface Methylation Procedure, Water Contact Angles, and Hysteresis Calculated from the Difference between the Advancing and Receding Contact Angles ml of DDS in 40 ml of TCE

DDS concentration (%)

Advancing c. angle (ua)

Receding c. angle (ur)

Hysteresis (ua-ur)

0 3 10 32 45 100 400

0 0.008 0.025 0.08 0.12 0.25 1.00

7° 6 1° 34° 6 1° 78° 6 1° 89° 6 1° 92° 6 1° 93° 6 1° 94° 6 1°

6° 6 1° 27° 6 1° 65° 6 1° 78° 6 1° 84° 6 1° 86° 6 1° 87° 6 1°

'1° 7° 6 2° 13° 6 2° 11° 6 2° 8° 6 2° 7° 6 2° 7° 6 2°

surfaces were immersed for 5 to 10 min. at 80°C in a basic peroxide solution (H2O : H2O2 (30% v/v) : NH4OH (25% v/v), ratio 5:1:1 (v/v)). After being rinsed in deionized water, the samples were again immersed for 5 min. in an acidic peroxide solution at 80°C, (H2O : H2O2 : HCl, ratio 6:1:1), where H2O2 and HCl final concentrations were 1.5 M. Surfaces were finally rinsed in deionized water and dried in N2 gas just before use. This procedure leads to hydrophilic surfaces. Methylated silicon was obtained in the following way; After the last washing

FIG. 2. Advancing water contact angle (E) and thickness (F) of silicon oxide layers for different degrees of methylation. (The arrows match each curve for its own Y-axis).

step in the procedure above, silicon pieces were extensively rinsed twice in ethanol and once in trichloroethylene (Merck). The samples were then incubated at room temperature for 10 min. in nonstirred solutions of dimethyldichlorosilane (DDS, Cl2(CH3)2Si; Fluka) at 0.008%, 0.025%, 0.08%, 0.12%, 0.25%, and 1.0% concentrations in trichloroethylene (TCE). After this, the samples were rinsed in ethanol, TCE, and ethanol, in that order. Finally, prior to the protein incubations, the surfaces were rinsed in water and dried under a gentle N2 stream. In order to quantify the hydrophobic/hydrophilic character of the silicon surfaces, advancing and receding contact angles were measured. These were monitored in laboratory atmosphere with a Rame´-Hart NRL model 100 goniometer. Water was used to probe the surfaces by the sessile drop technique. Reported data were obtained by averaging the values given by three different and independent experiments for each sample. The values for the silicon samples are shown in Table 1. Protein Solutions Human serum albumin (HSA; Sigma, code F3879) and fibrinogen (Fib; Sigma, code A9511) were purchased as lyophilized powders obtained from pooled human plasma. A polyclonal immuno-g globulin G (IgG; Kabi) was from a 165 mg/ml stock solution. All of them were individually diluted in Hank’s balanced buffer. The protein concentrations were 20 mg/ml throughout the experimental series, and solutions were freshly prepared before use. Protein Adsorption

FIG. 1. Schematic picture where roughness and thickness of methylated Si-surfaces can be appreciated. This figure helps to understand the hysteresis values of Table I and the ellipsometry data in Fig. 2.

Three different sets of experiments were performed. a) Silicon pieces treated in the 0.12% DDS solution (ua 5

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TABLE 2 Adsorbed Protein Film Thickness, Protein Surface Concentration, and Water Contact Angles for the Three Different Protein Monolayers Obtained under the Experimental Conditions that Lead to a Maximum Surface Coverage (see Text)

Protein

Thickness (Å)

Gads (mg/m2)

Advancing c. angle (ua)

Receding c. angle (ur)

Hysteresis (ua-ur)

HSA IgG Fibrinogen

23.5 6 0.3 33.2 6 1.1 52.2 6 0.4

3.2 6 0.1 4.5 6 0.2 7.1 6 0.1

56° 6 1° 71° 6 2° 82° 6 1°

10° 6 1° 13° 6 1° 10° 6 1°

46° 6 2° 58° 6 3° 72° 6 2°

92°) were incubated in the protein solutions for varying times from 10 s to 30 min. b) Silicon samples with varying hydrophobicities were incubated in the protein solutions for 5 min. c) Silicon samples with varying hydrophobicies were incubated in the protein solutions for 1 h. During adsorption, samples were gently and continuously shaken. Finally, the surfaces were rinsed in Hank’s buffer and deionized water, and dried under a N2 stream. Ellipsometry The dried samples were analyzed with an Auto-Ell III null ellipsometer (Rudolph Research). The thickness of the deposited protein layer (d) was determined from two angles (C and D) that are used in the McCracking evaluation algorithm (14). The protein layer thickness was determined at 5 points for duplicate surfaces, and then an average value was calculated. The surface mass concentration (G) was calculated according to Stenberg and Nygren (15), that is, by multiplying the d value with the density of a dry protein layer (1.37 g/cm3). Atomic Force Microscopy

angle (u) by the sessile drop technique (see Table 1). It is observed that contact angle values increase for increasing DDS added to TCE, indicating that methylation reaction successfully took place. The differences between the advancing and receding contact angles, that is, the hysteresis, give a qualitative idea about the heterogeneity and roughness of the surface (16, 17). As can be seen, the silicon surfaces were quite smooth (i.e., homogeneous), with higher u-hysteresis values in those cases where the degree of methylation was intermediate. This result can be visually explained in the scheme shown in Fig. 1. Nonmethylated and totally methylated Si-surfaces present a more homogeneous and plane interface than the intermediate cases, where roughness, and thus u-hysteresis, is higher. The thickness of methylation was measured by ellipsometry. The union of Si(CH3)2 groups enlarges by some Ångstroms the average silicon oxide thickness (see Fig. 1). Thickness data are shown in Fig. 2, where the agreement between both analytical methods, contact angle and ellipsometry, is good. As protein adsorption depends not only on surface hydrophobicity but also on the nature of the macromolecules, the contact angle of monolayers of each kind of protein (HSA, IgG, and Fib) deposited on hydrophilic Si-surfaces was mea-

AFM measurements were made using a commercial Nanoscope III system (Digital Instruments). The instrument was operated in tapping mode using silicon cantilevers (Nanoprobe) oscillating with a typical amplitude of 100 nm, at a resonance frequency of 300 –350 kHz. The tip diameter reported by the manufacturer was around 10 nm. Image sizes were equal to 5 3 5 mm2 with a resolution of 512 3 512 measurement points (pixels). Measurements were made on two individually prepared surfaces in each case; a total of 70 pictures was taken. Only representative images are shown. RESULTS AND DISCUSSION

Methylation–Hydrophobicity of the Silicon Surfaces The degree of methylation, that is, the surface hydrophobicity, affects the protein adsorption patterns. The extent of surface methylation has been quantified by two independent methods. The most sensitive consists of measuring the water contact

FIG. 3. Adsorption isotherms in Hank’s buffer for HSA (F), IgG (h), and fibrinogen (Œ) to Si-surfaces methylated in a 0.12% DDS solution.

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FIG. 4. Adsorption of HSA (F), IgG (h), and fibrinogen (Œ) as function of degree of methylation (hydrophobicity) for incubation times (a) 5 min. and (b) 1 h.

sured. This is a rough estimation about the hydrophobicity of the external part of the protein molecules. The monolayers were obtained at room temperature after four hours of incubation of Si pieces immersed in the protein solution (40 mg/ml; ionic strength 5 0.002). The medium pH was adjusted to be equal to the isoelectric point of each protein (4.7 for HSA, 7.0 for IgG, 6.0 for Fib), where the adsorbed amount is the maximum (18). The protein film thickness, adsorbed amount (Gads), and u values are shown in Table 2. The hysteresis, given as the difference between the advancing and receding contact angle, is high for all of the proteins, as could be expected. This is caused by both the inherent heterogeneity and roughness of the macromolecule surfaces, and by the fact that protein molecules are not perfectly packed on the surface, thus causing an additional interfacial roughness. Therefore, this heterogeneity is responsible for this important u-hysteresis. Nevertheless, from the advancing contact angle values (Table 2) we see that albumin surfaces are less hydrophobic than IgG, which in turn is less hydrophobic than fibrinogen. Adsorption Isotherms Initially, adsorption isotherms were carried out on Si-surfaces methylated in a 0.12% DDS solution (ua 5 92°). The results are shown in Fig. 3. Both HSA and IgG adsorbed rapidly, while the fibrinogen adsorption was slower. These experiments provide an indication about the protein–sorbent affinity (19, 20). The reason why fibrinogen accommodates more slowly than HSA and IgG during the adsorption process becomes difficult to explain. The adsorption pH (Hank’s buffer) was around 7, and both the Fib molecules and the Si-surface were negatively charged. The electrostatic repulsion

is a barrier against adsorption, although the high ionic strength of the medium (around 150 mM) considerably screens such a repulsion effect. At pH 7, however, albumin is even more negatively charged than fibrinogen, as it is a protein with high charge density, a property that has been used to stabilize colloidal systems (21). Thus, the electrostatic repulsion should also affect the HSA deposition, but the adsorption kinetic for HSA indicates high affinity. Therefore, the electrostatic repulsion alone would not explain the slow adsorption of fibrinogen. Another factor to be considered is the diffusion coefficient (DHSA 5 6.7 1027 cm2/s, and DFib 5 2.0 1027 cm2/s), which would control the approach velocity to the surface. Bigger molecules usually possess lower mobility to reach the interface and therefore, adsorption would last longer. Our experiments were, however, performed under continuous agitation, so “D” would not explain the above results at all. Thus the slow deposition of fibrinogen must be caused by some type of interaction barrier originating in the structural nature of fibrinogen, in addition to the above-noted effects of electrostatic repulsion and low D. The above results could be easily explained if fibrinogen was a fibrous kind of protein with helical structure, and HSA and IgG were both random coil-like structures with more internal flexibility. The differences in the adsorption kinetics in Fig. 3 might then be explained by the slower rate of accommodation of fibrinogen than those of HSA and IgG, giving rise to a thermodynamically higher adsorption barrier for fibrinogen. However, as Andrade and Hlady have stated (22) fibrinogen has quite a labile conformation and very high interfacial activity and spreading pressure, which would lead to an adsorption of high affinity.

FIG. 5. Fibrinogen adsorption in Fig. 3 shown by AFM imaging: (a) an empty Si-surface, (b) t 5 10 s, Gads 5 0.55 mg/m2, (c) t 5 30 s, Gads 5 0.80 mg/m2, (d) t 5 1 min., Gads 5 1.00 mg/m2, (e) t 5 3 min., Gads 5 1.60 mg/m2, (f) t 5 10 min., Gads 5 3.45 mg/m2, and (g) t 5 30 min., Gads 5 3.60 mg/m2. The white stains that appear in some pictures (5a, 5c, and 5g) correspond with contamination or protein aggregation. Scan side 5 mm.

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FIG. 6. AFM images of HSA adsorbed to silicon surfaces methylated in a 0.12% DDS solution (ua 5 92°): (a) incubation time 5 3 min., Gads 5 1.90 mg/m2, (b) incubation time 5 30 min., Gads 5 1.90 mg/m2. Scan side 5 mm.

Another set of experiments, where protein deposition was studied as a function of surface hydrophobicity for a fixed incubation time, was performed. The results are shown in Fig. 4a (t 5 5 min.) and Fig. 4b (t 5 1 h). The largest adsorbed amounts were always obtained on the most hydrophobic surfaces. Independent of this, the adsorption patterns were different for different proteins. For example, IgG adsorption occurred extremely rapidly (Gads after 5 min. incubation ' Gads after 1 h incubation), indicating that IgG molecules quickly saturate the surfaces. On the other hand, adsorption of Fib is again quite slow for the whole methylation range. The deposition is, as expected, slower for hydrophilic Si-surfaces (see Figs. 4a and 4b), where electrostatic repulsion is more relevant due to the presence of a high number of negative charges in the non- (and/or low-)methylated silicon. AFM One of the main goals of this work was to study the protein layer reorganization caused by the drying process with AFM imaging. Wa¨livaara et al. (11) demonstrated that formation of protein aggregates after air exposure highly depended on the degree of hydrophobicity of the Si-surface. They interpreted that the hydrophobic surface–air interactions thermodynami-

cally drive a restructuring of the adsorbed protein during the drying process. Similar aggregate formation has been observed by other authors (5, 23, 24). Cullen and Lowe (25) have demonstrated with AFM used in an aqueous environment that IgG adsorption resulted in a homogeneous distribution of adsorbed molecules. Therefore, disruption of the adsorbed protein layer in dried samples most likely is caused only by the air–surface interaction. However, there are still some experimental parameters related to the above phenomena that could be interesting to study, e.g., the nature of protein, surface hydrophobicity, and adsorption time. All affect the strength of the surface–molecule interactions and thus the degree of immobilization of the protein layer. Therefore, formation of aggregates could be avoided if the molecule–sorbent interaction were strong enough to make the immobilization irreversible. Our adsorption experiments provided many samples to be analyzed by AFM in order to study the importance of such parameters. First, the impact of adsorption time was studied. It is well known that the desorption probability of adsorbed proteins decreases with adsorption time, due to conformational changes in the protein molecules that strengthen the protein–surface interactions (26, 27). Nevertheless, dendrite-like aggregates

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FIG. 7. AFM images of IgG adsorbed to silicon surfaces methylated in a 0.12% DDS solution (ua 5 92°): (a) incubation time 5 3 min., Gads 5 3.35 mg/m2, (b) incubation time 5 30 min., Gads 5 3.70 mg/m2. Scan side 5 mm.

and small “droplets” were found in every sample corresponding to the data shown in Fig. 3. AFM images of the fibrinogen following the adsorption isotherm are shown in Figs. a–5g. In this case, the differences between the adsorbed protein features depend mainly on the adsorbed amount, and not on the incubation time. As can be seen, the results obtained by AFM and ellipsometry are mutually supportive. For low coverages the protein aggregates are dendrite-like (Figs. 5b and 5c), while for high-protein loads, “droplet” structures appear (Figs. 5f and 5g). The adsorbed amounts (Gads) are the same only in the latter samples, where adsorption time differs. For that reason, it is better to study the “time” parameter for HSA and IgG samples located in the “plateaus” shown in Fig. 3, as in these cases there are a number of samples where Gads coincides for varying incubation times. The AFM images for HSA are shown in Figs. 6a and 6b, and for IgG in Figs. 7a and 7b. As can be seen, adsorption time does not influence the aggregation patterns. However, the drying process always disrupted the protein layer for all times (up to 1 h) when silicon was highly methylated. Therefore, it is possible to conclude that long adsorption times do not yield protein–surface interactions strong enough to prevent the disrupting effect caused by the hydrophobic surface– hydrophobic air interactions.

Second, AFM imaging was used to observe the influence of both the degree of methylation and the protein nature for the formation of “droplet” structures. Figures 8 –10 correspond to some data shown in Fig. 4b (where adsorption time was equal to 1 h). Images of adsorbed HSA are shown in Figs. 8a– 8c; IgG in Figs. 9a–9c; and fibrinogen in Figs. 10a–10c. The “droplet” formation took place for all proteins on surfaces that were methylated in the 0.08% (ua 5 89°), 0.12% (ua 5 92°), 0.25% (ua 5 93°), and 1.0% DDS (ua 5 94°) solutions (figures not shown). The droplet boundaries were more irregular and heterogeneous for HSA than for IgG and Fib. With regard to the results shown in Figs. 8 –10, the formation of droplets occurs for HSA even on the nonmethylated (highly hydrophilic) Si-surface (Fig. 8a). This feature was not found for IgG (Fig. 9a) or fibrinogen (Fig. 10a). When the surfaces were slightly methylated, that is, 0.008% DDS (ua 5 34°), protein restructuring caused by the drying is extremely relevant for albumin (Fig. 8b), not too high although important for IgG (Fig. 9b), and negligible for fibrinogen (Fig. 10b). Only when the hydrophobicity of the surface increases to 0.025% DDS (ua 5 78°), do “droplet” aggregates begin to appear for fibrinogen (Fig. 10c). The above patterns can be explained on the basis of the

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FIG. 8. AFM images of HSA adsorbed to (a) nonmethylated Si-surface (Gads 5 0.90 mg/m2), (b) Si-surface methylated in a 0.008% DDS solution (ua 5 34°) (Gads 5 1.60 mg/m2), and (c) Si-surface methylated in a 0.025% DDS solution (ua 5 78°) (Gads 5 2.00 mg/m2). Adsorption time 5 1 h. Scan side 5 mm.

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FIG. 9. AFM images of IgG adsorbed to (a) nonmethylated Si-surface (Gads 5 2.80 mg/m2), (b) Si-surface methylated in a 0.008% DDS solution (ua 5 34°) (Gads 5 2.40 mg/m2), and (c) Si-surface methylated in a 0.025% DDS solution (ua 5 78°) (Gads 5 3.60 mg/m2). Adsorption time 5 1 h. Scan side 5 mm.

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FIG. 10. AFM images of fibrinogen adsorbed on (a) nonmethylated Si-surface (Gads 5 3.30 mg/m2), (b) Si-surface methylated in a 0.008% DDS solution (ua 5 34°) (Gads 5 4.50 mg/m2), and (c) Si-surface methylated in a 0.025% DDS solution (ua 5 78°) (Gads 5 4.60 mg/m2). Adsorption time 5 1 h. Scan side 5 mm.

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strength of the surface–protein interaction. This depends, on one hand, on the number of contacts between a single macromolecule and its sorbent, since it is well known that a multiplecontact formation leads to irreversible adsorption (7). Fibrinogen is a large protein (MW 5 340000 Daltons), so the number of molecular segments in contact with the surface could be higher than in IgG (MW 5 150000 Daltons) and HSA (MW 5 66000 Daltons). On the other hand, the hydrophobic interaction is one of the most important contributions that controls the protein adsorption processes (6, 8, 28). The release of water from the hydrophobic components of two approaching surfaces results in a decrease of the Gibbs energy of the system due to the entropy gain of the water molecules. Therefore, this energy diminution must be higher for the most “hydrophobic” protein, that is, fibrinogen (see Table 2), and lower for the less “hydrophobic” one, HSA (see Table 2). Therefore, both factors contribute to the immobilization of the adsorbed molecules; this immobilization is thus more irreversible and stronger for the fibrinogen and more labile and weaker for albumin, as shown by AFM analysis. When silicon is hydrophobic enough, the hydrophobic air–surface interaction competes with the protein–surface interaction. Dendrite-like aggregates and droplet formation only occur when the magnitude of the former exceeds the magnitude of the latter. In that case, the initially homogeneous protein layer is somewhat disrupted, leading to a net Gibbs energy diminution of the system during the drying process. That is, the hydrophobic surface– hydrophobic gas interaction is optimized, and the contact area between the hydrophilic areas of the protein and the hydrophobic air is minimized. CONCLUSIONS

In this paper, the competitive effect between air–surface and protein–surface interactions was studied. Droplet-like structures were formed for intermediate and highly methylated Si-surfaces for all three proteins. The adsorption time is not an important parameter and seems not to strengthen the protein– surface interactions in order to prevent protein reorganizations. The extent and strength of the protein–surface interaction, however, does depend on the nature of the protein: its size (multiple contact formation), rigidity, and superficial hydrophobicity. This last property was estimated semiquantitatively by contact angle measurements on protein monolayers deposited on hydrophilic Si-surfaces. The main conclusion that can be extracted from this work is that fibrinogen (hydrophobic and large protein) interacts more irreversibly with the silicon sur-

faces than IgG, and much more so than HSA, which is less hydrophobic and smaller than fibrinogen. ACKNOWLEDGMENTS This study was supported by the Swedish Biomaterials Consortium, funded by the Swedish National Board for Industrial and Technical Development (NUTEK) and the Swedish Natural Science Research Council (NFR). J.L.O.V. would like to thank to the Spanish Ministry of Education and Culture for supporting his stay at the University of Linko¨ping (Sweden).

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