Interactions between Exogenous FGF-2 and Sulfonic Groups: in Situ

May 31, 2013 - We have chosen SAMs as a simple mimic of HSPG because in this .... antibody (1:500 in 1% w/v BSA/PBS) for 1 h at room temperature. ...
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Interactions between Exogenous FGF‑2 and Sulfonic Groups: in Situ Characterization and Impact on the Morphology of Human AdiposeDerived Stem Cells Sara Amorim, Ricardo A. Pires,* Diana Soares da Costa, Rui L. Reis, and Iva Pashkuleva* 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal, and ICVS/3B’s PT Government Associate Laboratory, Guimarães/Braga, Portugal S Supporting Information *

ABSTRACT: FGF-2 is often used as a supplement to stem cells culture medium aiming at preserving their self-renewal capacity and plasticity through the passages. However, little is known on the influence of the underlying substrate in these interactions. In this study, we have used mixed self-assembled monolayers with different ratios of −SO3H and −OH tail groups to investigate the influence of substrate properties (e.g., charge) on the FGF-2 adsorption and activity. QCM-D data demonstrated that, in the presence of −OH groups, the quantity of the adsorbed FGF-2 is proportional to the percentage of surface −SO3H groups. The bioactivity of the adsorbed FGF-2 follows the same tendency as demonstrated by its interactions with anti-FGF-2. Surprisingly, the adlayer of FGF-2 formed on the surface containing only SO3H-tailed SAMs was similar to the surface with 25% of −SO3H groups, demonstrating that FGF-2 adsorption is not solely driven by electrostatic interactions. We related these results with changes in the morphology of adipose-derived stem cells (ASCs) cultured on the same surfaces.

1. INTRODUCTION Basic fibroblast growth factor, also known as bFGF, FGF-2, and FGF-b, belongs to the 22-member family of polypeptide fibroblast growth factors (FGFs). Among the five known isoforms of FGF-2, four (22, 22.5, 24, and 34 kDa) are localized in the nucleus and trigger an active intracrine signaling and the 18 kDa one is mostly cytosolic.1−3 FGF-2 spans diverse biological roles (e.g., regulation of cell proliferation, migration, and differentiation) that are accomplished via its binding to two classes of receptors: high-affinity transmembrane receptor tyrosine kinases (fibroblast growth factor receptors, FGFRs) and low-affinity receptors, heparin and heparan sulfate proteoglycans (HSPG).1,4,5 HSPG are negatively charged glycosaminoglycans found on cell surfaces and in the extracellular matrix where they interact with FGF-2: they stabilize and protect the growth factor from denaturation and enzymatic degradation,6,7 play a role in its storage and enhancement of the spatial diffusion radius of bioactive FGF2,8 and mediate its binding to FGFRs, acting as a local regulator of FGF-2 activity.9−11 It is well established that multivalent, mainly ionic interactions are crucial for these processes; clusters of positively charged basic amino acids on FGF-2 (pI = 9.6) form ion pairs with spatially defined negatively charged sulfonic and sulfate groups on the HSPG chain. 9,12−15 These interactions are weak and thus difficult to measure. The © XXXX American Chemical Society

diversity of HSPG, their difficult isolation and purification, together with the complex environment in which the interactions FGF-2−HSPG takes place make the process even more challenging for investigation. Recently, we have demonstrated that single component and mixed self-assembled monolayers (SAMs) with −SO3H and −OH tail groups can be successfully applied as functional mimics of sulfated glycosaminoglycans (GAGs) in studies with mesenchymal stem cells (MSCs).16 In HSPG, these two functionalities are the main ones that account for noncovalent, usually multivalent interactions such as H-bonding (−SO3H and −OH) and ionic interactions (−SO3H) associated with recognition processes. We have chosen SAMs as a simple mimic of HSPG because in this model there are not sterically hindered functional groups as in some synthetic analogues17,18 of HSPG, i.e. all functionalities are available on the surface for interactions with the different bioentities (e.g., proteins and cells) in the media. Besides, the surface composition of SAMs can be tailored relatively easily and they are compatible with a number of characterization techniques. Received: March 8, 2013 Revised: May 29, 2013

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Previously, we observed stimulation of filopodia formation and higher mobility of MSCs when cultured on SAMs rich in −SO3H groups. In the present work, we investigate a possible contribution of FGF-2 in this process. FGF-2 is used often as a supplement to the adipose-derived stem cells (ASCs) culture medium aimed at preserving or promoting their self-renewal capacity,3,19 maintaining their plasticity through the passages,3,19 or modulating their differentiation (e.g., enhanced chondrogenesis).19−21 Besides the evidence of its benefits, additional investigations are required to elucidate the role(s) of FGF-2 in ASCs and to understand the molecular mechanisms behind this role.3,19,21

v BSA/PBS) for 1 h at room temperature. Labeled samples were washed with PBS and observed under an Imager Z1 fluorescence microscope (Zeiss). 2.5. Isolation and Expansion of Human Adipose Stem Cells (ASC). Human subcutaneous adipose tissue samples (age range of 20− 36 years) were obtained from lipoaspiration procedures under the scope of a cooperation agreement with Hospital da Prelada (Porto, Portugal). The adipose tissue was washed with PBS containing 10% antibiotic/antimycotic and then digested with a 0.1% collagenase from Clostridium histolyticum (Sigma-Aldrich) solution in PBS for 45 min at 37 °C under gentle stirring. The digested tissue was gently pressed through a strainer and centrifuged at 1000 g for 10 min. The cell pellet was resuspended and incubated in lysis buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA) 10 min before centrifugation at 800 g for 10 min. Cells were expanded in α-modified Eagle’s medium (Sigma-Aldrich) supplemented with 1% Antibiotic/Antimycotic (Gibco), 10% FBS (Gibco). Cells from the third and fourth passage were used in this study. 2.6. Cell Culture and Characterization. SAMs (n = 3 for each condition) were seeded with ASCs at a concentration of 3000 cells.cm−2 in either serum free medium or medium supplemented with FGF-2 (10 ng/mL) and incubated for 24 h at 37 °C under a humidified atmosphere of 5% CO2. Tissue culture polystyrene (TCPS) coverslips were processed as the other samples and used as controls. Afterward the samples were washed twice with PBS, fixed in 10% neutral buffered formalin for 30 min at 4 °C, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked with 3% BSA in PBS for 30 min at room temperature. To evaluate focal adhesion formation, a primary antibody against vinculin (clone h-VIN1, 1:400 in 1% w/v BSA/PBS, Sigma) was employed, followed by rabbit antimouse Alexafluor-488 (1:500 in 1% w/v BSA/PBS, Invitrogen). A phalloidin−TRITC conjugate was used (1:200 in PBS for 30 min, Sigma) to assess cytoskeleton organization. Nuclei were counterstained with 1 mg/mL 4,6-diamidina-2-phenylin (DAPI; Sigma) for 30 min. Samples were washed with PBS, mounted with Vectashield (Vector) on glass slides and observed under an Imager Z1 fluorescence microscope (Zeiss) and photographed using an Axio Cam MRm (Zeiss). Scanning electron microscopy (SEM) was employed to evaluate cell morphology. The samples used for immunostaining were washed twice in PBS, dehydrated in a graded series of ethanol, and finally, dried using hexamethyldisilazane. The samples were examined at an accelerating voltage of 15 kV in a Leica Cambridge S-360 scanning electron microscope. The size and number of the formed filopodia was measured from the SEM micrographs (ImageJ software 1.46r) of the cells. 2.7. Statistical Analysis. The normality of the data was evaluated using Shapiro−Wilk test (p < 0.05). When the data did not follow a normal distribution an initial Kruskal−Wallis test was executed followed by Mann−Whitney test. In all the cases, a significant variation was only considered for a p < 0.01.

2. MATERIALS AND METHODS 2.1. Materials. Unless otherwise stated, chemicals were bought from Sigma Aldrich and used without further purification. Recombinant Human FGF-2 was purchased from Peprotech (AF-100-18B). FGF-2 Antibody (anti-FGF-2), clone bFM-2, 17.5 kDa was purchased from Milipore (05-118). The secondary antibody, Alexa Fluor 488 Rabbit Anti-Mouse IgG, was obtained from Invitrogen (A21206). 2.2. Formation and Characterization of Self-Assembling Monolayers. The substrates used in this study were gold-coated ATcut quartz crystals (QSX301, Q-Sense, Sweden) and glass slides uniformly coated with gold (∼20 nm) by the e-beam technique. Titanium (3−5 nm film) was used as a primer improving the adhesion between the gold and the glass. The substrates were prepared as previously reported by da Costa et al.16 Briefly, the cleaned substrates (5:1:1 solution of H2O:NH3:H2O2 at 80 °C for 10 min) were immersed into 20 μM ethanol solution of HS(CH2)11OH (sample designated as SO3H_0) or HS(CH2)11SO3H (sample SO3H_100) for at least 48 h to ensure well formed monolayers. Mixed SAMs were formed by coadsorption from binary solutions prepared by mixing pure solutions of HS(CH2)11OH and HS(CH2)11SO3H at ratios of 1:3 and 3:1 in order to obtain 25% and 75% of −SO3H groups on the surface. These samples are referred as SO3H_25 and SO3H_75, respectively. The obtained substrates were washed several times with ethanol, dried under N2 and characterized or used in the further studies. Contact angle measurements (1 μL water, OCA15+, DataPhysics Instruments), ellipsometry (SpecEL 2000-VIS, Mikropack), X-ray photoelectron spectroscopy (K-Alpha instrument, Thermo Scientific), time-of-flight secondary ion mass spectrometry (ToF-SIMS IV instrument, ION-TOF GmbH, Germany) and ζpotential measurements (SurPASS instrument, Anton Paar) were used to confirm the SAM formation (Supporting Information). 2.3. Quartz Crystal Microbalance (QCM-D) Measurements and Modeling. The crystals coated with SAMs were washed several times with ethanol, dried under N2, and immediately placed in the QCM-D flow chamber (E4 instrument, Q-Sense). A stable baseline was acquired in 5 mM solution of Trizma hydrochloride in PBS buffer (referred as buffer in the text below) at 25 °C. This buffer was also used to prepare all of the solutions. The FGF-2 solution (4 mL of 1 or 2 μg/mL) was added to the chamber under closed circuit at a flow rate of 50 μL/min until surface saturation. The sensor was rinsed with buffer to remove loosely bound material and 3% BSA solution was introduced (50 μL/min) in the flow chamber for 30 min. After rinsing, a primary antibody anti-FGF-2 (10 or 20 μg/mL) was added to the deposited FGF-2 until saturation. The sensor was finally rinsed with buffer to remove loosely bound antibody. QCM-D measurements were performed at several harmonics (n = 3, 5, 7, 9, 11 and 13). Δf/n and ΔD were fitted for the ninth overtone using the Q-Tools software (v 3.0.6.213) and Voigt element-based model. The thickness and mass of the wet films was calculated by fitting in an iterative fashion and by assuming a fluid density of 1010 kg/m3, a fluid viscosity of 0.001 kg/ms, and a layer density of 1125 kg/ m3. 2.4. Protein Immunolabeling. The crystals coated with antiFGF-2 were removed from the microbalance and incubated with a rabbit antimouse Alexa Fluor 488 secondary antibody (1:500 in 1% w/

3. RESULTS AND DISCUSSION 3.1. SAMs Formation and Characterization. Surfaces containing more than one functional group can be generated by coadsorption of two (or more) thiols from the solution. Previously, it has been observed that the ratio of the components in solution can be different than the obtained monolayers; differences in relative solubility and chain length are main parameters that can generate phase-segregation and/ or preferential adsorption of a certain component.22−24 The tail groups of the thiols used in this study (−OH and −SO3H) are both polar, capable of hydrogen bonding and therefore, they do have similar solubility. Additionally, we have used thiols with the same length (C11) aiming elimination of the abovementioned undesired effects. Finally, low total concentration (20 μM) was chosen to ensure the similarity between the B

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Table 1. Surface Characteristics of the Obtained SAMs χ0SO3H surface SO3H_0 SO3H_25 SO3H_75 SO3H_100 a

surface thickness (nm) 1.4 1.7 1.9 2.2

(0.2) (0.2) (0.1) (0.2)

a

theoretical

SAM (XPS)

0 25 75 100

0 26 70 100

water contact angleb 20.9 18.0 31.8 41.1

(3.7) (2.5) (7.7) (6.3)

isoelectric point (pI) 3.8 (0.0) 3.1 (0.3) H3O+ ≫ K+ ≈ Cl−) in the surface D

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Table 2. Frequency and Dissipation Shifts and the Respective Voigt Mass for the FGF-2 Adsorption on Different SAM Surfaces (9th Overtone) ΔDFGF‑2 (10−6)

|Δf FGF‑2| (Hz) sample SO3H_0 SO3H_25 SO3H_75 SO3H_100

FGF-2 (1 μg/mL) 16.7 19.5 27.9 20.4

(0.1) (0.1) (0.1) (0.1)

FGF-2 (2 μg/mL) 9.3 (0.1) 12.2 (0.2) 23.2 (0.1) 12.2 (0.2)

FGF-2 (1 μg/mL) 5.94 3.76 3.74 4.59

(0.03) (0.04) (0.05) (0.05)

charge formation of nondissociating organic surfaces.23,29,30 The absence of a plateau range in the zeta potential vs pH plot for SO3H_0 confirms that there are no dissociating groups on this surface.29 Addition of a −SO3H tailed thiol to the SAM at low concentration (χ0SO3H = 0.25, SO3H_25) results in shifting the isoelectric point (pI) to 3, but the shape of the curve remains the same; that is, no plateau is observed. These results indicate that acidic surface sites are present on the SO3H_25 sample but they do not dominate at the interface. The plots for SO3H_75 and SO3H_100 are different as compared to those for SO3H_0 and SO3H_25; they do contain a plateau confirming the presence of charged surface units. The measured lower ζ potentials are in good agreement with previously reported values for sulfonic terminated layers obtained by electroosmotic mobility measurements.28 However, it must be noted the coincidence of the plots for SO3H_75 and SO3H_100 at acidic pH. Several reasons, all based on the electrostatic interactions, have been previously suggested to explain similar behavior.23,27 Because we are working with surface confined groups, the generated local potentials via ionizations can counteract further dissociation of neighboring −SO3H groups, and most probably this is what we are observing in the case of SO3H_100. Additionally, the counterion distribution in the double layer is different: elevated surface charge densities are associated with higher degrees of charge compensation within the stagnant part of the double layer (Figure 1A) and therefore decrease the value of the ζ potential. 3.2. FGF-2 Interactions with the Formed SAMs. FGF-2 is a globular protein31,32 with hydrodynamic radius of 1.45 nm33 that contains a number of basic residues and has an isoelectric point of pI = 9.62. The heparin binding site of FGF-2 is a highly positive charged environment31,32 as can be seen from the electrostatic map of the growth factor (Figure 2). It is therefore expected that the augmentation of χ0SO3H will result in an increasing adsorption of FGF-2 if the process is driven solely by electrostatic forces. We followed the adsorption in real-time by QCM-D (Figure 3). This technique is based on a quartz crystal disk (i.e., the QCM-D sensor) that oscillates at its resonance frequency when an alternating potential is applied. The resonance frequency ( f) changes upon mass adsorption on the surface. The driving potential is continuously switched on and off, and in this way the damping of the oscillatory motion, the energy dissipation (D), is also measured. We observed similar (but not the same) kinetics for all studied surfaces (Figure 3A,C): a rapid protein adsorption upon addition of the protein solution at t = 0 (fast frequency decrease) that is slowing as surface coverage increases (slower frequency decrease). Accordingly, the fast phase of the adsorption process is shorter when higher concentration of FGF-2 is used (Figure 2, panel A vs panel C, Supporting Information, Figure S3). As expected for globular proteins,34,35

Voigt mass (ng/cm2)

FGF-2 (2 μg/mL) 3.47 2.37 1.57 1.59

(0.03) (0.04) (0.05) (0.05)

FGF-2 (1 μg/mL) 261.7 334.7 458.0 351.3

(4.3) (2.7) (4.9) (4.2)

FGF-2 (2 μg/mL) 171.2 255.5 476.6 276.4

(7.9) (9.3) (3.9) (13.1)

the derivatives of the frequency changes (Supporting Information, Figure S3) demonstrate a simple adsorption profile that does not include any conformational changes and/ or reorganization upon adsorption. These results agree with previous reports demonstrating no conformational changes in FGF-2 upon binding to sulfate groups.5,36 The protein was adsorbed irreversible to all studied surfaces as demonstrated by exchanging the protein solution with a buffer: no changes in the frequency or dissipation were detected upon washing the adsorbed FGF-2 layers with a buffer. The respective frequency and dissipation shifts at this point as well as the calculated Voigt masses are presented in Table 2. We have observed the expected trend for all −OH containing surfaces (SO3H_0, SO3H_25, and SO3H_75): an augmentation in the χ0SO3H resulted in rise of the adsorbed FGF-2. Further increasing of χ0SO3H from 0.75 to 1.00 does not change dramatically the surface ζ-potential as demonstrated above. Thus, we were expecting similar quantities of FGF-2 to adsorb on these two substrates. However, our results indicate smaller f shifts for SO3H_100 in comparison with SO3H_75 (Table 2, Figure 3A,C). Because Δf accounts for both protein and water deposition on the QCM crystal, this result may indicate less adsorbed protein on SO3H_100 or more hydrated protein layer (more mass) on SO3H_75. In fact, the SO3H_100 substrate is the only one without −OH groups. Moreover, the most dissipative layers (Table 2, ΔD) were measured for the pure −OH surfaces (and ΔD decreases with the increase of χ0SO3H) and can be related with their high hydration state. To relate the dissipation caused by a unit frequency (mass) change, we have plotted ΔD vs Δf (Figure 3B,D).37−39 As expected, the curves obtained for SO3H_0 surfaces are distinguished by very large dissipation shifts. Two slopes are visible in the ΔD/Δf plots for the rest of the studied surfaces, indicating bilayer formation: a relatively rigid first layer (small dissipation shifts) and a loosely bound second one. Higher FGF-2 concentration (Figure 3, panel B vs panel D) causes formation of less dissipative layers with betterpronounced and larger first slope especially in the case of SO3H_75 SAMs. The formation of the first layer is electrostatically driven: the positively charged amino acids from FGF-2 interact with the negatively charged −SO3H groups available on the surface and, as a result, a strongly attached adlayer is formed, although, it can result in a partial denaturation of the adsorbed protein.39,40 This interpretation of the results is supported by two more observations: (i) the first phase (slope) is missing in the ΔD/Δf plots for SO3H_0 substrates (no negatively charged groups on the surface) and (ii) the rate of the first phase is a concentration dependent, it increases at higher FGF-2 concentration. The second layer is composed by a loosely bound protein, i.e. not fixed by strong electrostatic interactions. These more flexible and weakly bound protein molecules allow for larger amount of trapped water that does also contribute to the higher dissipation shifts. E

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Figure 4. Bioactivity of FGF-2 adsorbed on surfaces with different χ0SO3H: interactions with anti-FGF-2 measured by QCM-D (A and C) and immunostaining with rabbit antimouse Alexa Fluor 488 secondary antibody (B and D).

ΔD/Δf plots confirmed less dissipative adlayers on SO3H_75 when compared to SO3H_100 SAMs. This result indicates that FGF-2 adsorption is not solely driven by electrostatic forces and that −OH groups do also contribute to this process most probably by their involvement in Hbonding with the growth factor. Similar results have been reported by Ornitz et al.,36 who compared FGF-2 binding to several synthetic heparan-derived di- and trisaccharides with different sulfation degree. Sucrose octasulfate, the fully sulfated mimic used in that study, was not the most bioactive. The crystal structure of the complexes between the growth factor and the synthetic analogs reveal formation of different Hbonding that determine the affinity and activity of FGF-2. The direct analysis of the adsorbed mass calculated by the Voigt model also indicates maximum adsorption on the SO3H_75 surface. The obtained values are comparable for both tested FGF-2 concentrations indicating a surface saturation. Assuming that the hydrodynamic radius of FGF-2 is 1.45 nm33 and considering its globular structure, we have estimated a surface coverage of 6.6 nm2 per FGF-2 molecule. This value corresponds to 1.515 × 1013 molecules/cm2 (2.515 × 10−11 mol/cm2) or 432.6 ng/cm2 (molecular weight of FGF2 is 17.2 kDa) for densely packed protein monolayer. The theoretically calculated mass is close although lower than the

Voigt mass obtained for SO3H_75 (Table 2) and indicates a 6 and 10% of hydration for the layers using FGF-2 concentrations of 1 and 2 μg/mL, respectively. These hydration values are low in comparison with the reported ones, typically ranging between 25 and 75% of the protein mass.41 Moreover, ΔD/ Δf plots indicate formation of two layers with different dissipations and thus, it is difficult to estimate the exact hydration of the adsorbed FGF-2. The bioactivity of the adsorbed FGF-2 was further evaluated using anti-FGF-2 (Figure 4). The obtained FGF-2 adlayers were first treated with BSA solution to eliminate possible nonspecific interactions. Upon washing to remove loosely bound BSA, part of the deposited FGF-2 (1 μg/mL) layers was also washed out (frequency ≥ 0, Figure 4A). As a result, we detected similar, unexpectedly low values for the adsorbed anti-FGF-2 on all tested substrates. As described above less dissipative layers were obtained from more concentrated FGF-2 solutions (Figure 3D) and therefore, we expected those to be more stable upon washing. Indeed, in this case (Figure 4C) we did not detect any loss of growth factor and measured minimal nonspecific adsorption (very small quantities of BSA on all tested SAMs after washing). The shifts in frequency and dissipation were different for the tested surfaces demonstrating formation of larger and less dissipative F

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Figure 5. Representative fluorescence micrographs (immunostaining of vinculin (green), actin (red), and nuclei (blue)) of ASCs cultured for 24 h on surfaces with different −SO3H content (SO3H_0, SO3H_25, SO3H_75, and SO3H_100) and in the presence or absence of exogenous FGF-2 in the culture medium. Insets represent SEM images of cells cultured onto the same SAM surfaces (in all the cases scale = 20 μm).

adlayer on SO3H_75. Surfaces lacking in −SO3H groups (SO3H_0) demonstrated an opposite behavior: few and very dissipative anti-FGF-2 deposition was detected in this case. The immunostaining with secondary antibody confirms these results (Figure 4D): most intensive fluorescence was visible for SO3H_75 and SO3H_100 while no fluorescence was detected for SO3H_0. 3.3. Effect of Exogenous FGF-2 on Cell Morphology. From a cell therapy perspective, adipose-derived stem cells (ASCs) present several advantages: they do have plasticity similar to bone marrow stem cells but the isolation of ASCs is simpler and the access to subcutaneous adipose tissue is easier. However, they do share the common for all mesenchymal stem cells problem: losing self-renewal capacity (ability to proliferate while maintaining an undifferentiated phenotype) through the passages. Among different soluble supplements, FGF-2 has been shown to have remarkable effect on preserving the self-

renewal capacity and morphology of ASCs.3,19,20 Thus, we have investigated the influence of this growth factor in combination with different surface chemistries created by mixed SAMs on the morphology of ASCs. Previously, we have shown that in the presence of proteins in the culture medium, e.g. serum, the effect of surface chemistry on the cellular behavior can be hidden or overcome by the effect of the proteins adsorbed on the surface.16 Therefore, we performed two sets of experiments: (i) in the first one the ASCs were cultured alone, in the absence of any proteins, and therefore, the surface chemistry is the only factor that can influence cellular behavior; (ii) in the second one we added to the culture medium only FGF-2, i.e., changes in the cellular state will be due to the interactions between FGF-2 and the underlying SAMs. ASCs adhere to all studied substrates and the addition of FGF-2 does not induce any significant difference in the number G

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Figure 6. Influence of exogenous FGF-2 on average filopodia length (A) and filopodia number (B). hASC were cultured alone or with FGF-2 on −OH (SO3H_0) and −SO3H (SO3H_100) functionalized surfaces in a serum free media for 24 h. Significant differences (Kruskal−Wallis test followed by Mann−Withney test, p < 0.01) are marked with **.

of adherent cells. However, we noticed morphological changes in the case of −SO3H rich SAMs (SO3H_100 and especially SO3H_75): ASCs cultured in the presence of FGF-2 demonstrated more pronounced spindle-like morphology (Figure 5 and Figure S4, Supporting Information). This shape has been previously associated with ASCs fast-cycling state and their ability to proliferate at the single-cell level when cultured for long-term (higher passages).3 There are two possible scenarios for the observed different shape: FGF-2 addition can either select a subpopulation of ASCs with the referred shape that adheres better to the substrates or it changes the molecular machinery of a subset (or entire) population of ASCs enhancing their ability to respond to external clues. Recently, it was reported that SOX-9, collagen II and aggrecan are all significantly overexpressed in ASCs cultured in the presence of FGF-2,20 and therefore, the proposed second scenario is most probable. Another difference that is visible for cells cultured on sulfonic-rich surfaces and particularly on the pure sulfonic SAMs is the formation of filopodia (Figure 5). We have previously reported on this effect, observed in the absence of FGF-2 and showed that it is associated with higher cellular motility.16 Here, we demonstrate that this effect is induced by the sulfonic groups on the surface but it is reinforced in the presence of FGF-2: both filopodia number and length increase upon adding of FGF-2 in the culture medium and this effect was most significant for the SO3H_100 substrates (Figure 6). This result is also confirmation of our speculation for the possible scenario of FGF-2 effect, namely that it affects the cellular machinery making ASCs more responsive.

concentration but the kinetics of the process is concentrationdependent. Surfaces containing only −SO3H groups demonstrated unexpected behavior: a lower amount of FGF-2 was detected for these substrates as compared to the ones with −SO3H surface fraction of 0.75. We speculate that additionally to the electrostatic interactions, H-bonding between the −OH groups and the growth factor are contributing to the adsorption process. The lower dissipation of the adlayer formed on SO3H_75 confirms this speculation. The bioactivity of FGF-2 was tested by its interactions with anti-FGF-2 and decreases in the following order: −SO3H(0.75) > −SO3H(1.00) ≫ −SO3H(0.25) > −SO3H(0.00). Finally, we investigated the impact of these interactions on ASCs morphology. We found that, in addition to spindle-like cellular morphology, the exogenous FGF-2 induces significantly more and longer filopodia in ASCs cultured on −SO3H rich surfaces.

4. CONCLUSIONS We have demonstrated that self-assembled monolayers containing sulfonic groups are versatile and reliable tools applicable in model studies as HSPG mimics. Using these SAMs, we were able to investigate the interaction of FGF-2 with −SO3H groups in situ: in the presence of −OH groups the adsorbed mass increases with the rise of the −SO3H surface fraction. This mass increase is not a function of FGF-2

ACKNOWLEDGMENTS This work was performed in the framework of the EU 7th Framework Programme (FP7/2007-2013) under Grant Agreement No. NMP4-SL-2009-229292 (Find&Bind). The authors are grateful to M. Carmen Marquez-Posadas and Santos Merino from Fundacion Tekniker for providing gold coated glass slides and to Thomas Groth from University of Halle for his assistance on the ζ-potential measurements. R.A.P.

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ASSOCIATED CONTENT

S Supporting Information *

Details on the surface characterization of the SAM layers and lower magnification images of the cell studies. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +351 253 510 907. Fax: +351 253 510 909. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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acknowledges the Portuguese Foundation for Science and Technology (FCT) for his postdoc grant (BPD/39333/2007).

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