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Fibronectin immobilized by covalent conjugation or physical adsorption shows different bioactivity on aminated-PET
Materials Science and Engineering C 27 (2007) 213 – 219 www.elsevier.com/locate/msec
Fibronectin immobilized by covalent conjugation or physical adsorption shows different bioactivity on aminated-PET Yue Zhang a , Chou Chai b , Xue Song Jiang b , Swee Hin Teoh c , Kam W. Leong b,d,⁎ a
Graduate Programme in Bioengineering, National University of Singapore, Singapore Tissue and Therapeutics Engineering Laboratory, Division of Johns Hopkins, Singapore c Deparment of Mechanical Engineering, National University of Singapore, Singapore Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, USA b
d
Received 8 July 2005; accepted 3 March 2006 Available online 22 June 2006
Abstract To manipulate the cellular response to synthetic surfaces, extracellular matrix (ECM) proteins such as fibronectin (FN) and collagen are often immobilized on the surface to promote interaction between these ligands and the cell receptors. In this study we compared the biological properties of FN-decorated polyethylene terephthalate (PET) produced by two widely used immobilization techniques: adsorption and conjugation. As revealed by the micro-bicinchoninic acid (micro-BCA) assay and AFM, the modified surface topography was dependent on the immobilization methods. Adsorption method preserved the compact conformation of FN, reaching saturation when a monolayer of FN was formed. Covalent conjugation induced FN unfolding and fibrillogenesis, forming multiple layers of FN. Biological characterization by adhesion of baby hamster kidney 21 (BHK21) cells and enzyme-linked immunosorbent assay (ELISA) for active Arg-Gly-Asp (RGD) domains suggested that the difference in conformation of FN led to different bioactivities. Adsorption maintained a more active RGD domain, thereby promoting cell adhesion, whereas conjugation induced fibrillogenesis and blocked the access of RGD, consequently suppressing cell adhesion as the surface density of FN increased. This study suggests that in addition to choosing the nature of the adhesion molecule, the mode of immobilization may also significantly influence the bioactivity of the surface. © 2006 Elsevier B.V. All rights reserved. Keywords: Surface modification; Conjugation; Adsorption; Fibronectin; Bioactivity
1. Introduction Surface modification can modify the interaction between the biomaterials surface and the biological environment and yet preserve the mechanical properties of the base biomaterials. Design of functionalized polymer surfaces for biomaterial applications often rely on immobilization of ECM proteins or peptides to control the integrin-mediated cell attachment. Among the ECM proteins, FN as a family of structurally related soluble and matrix glycoproteins have been extensively studied in numerous biological processes including cell adhesion, wound healing, and ⁎ Corresponding author. Department of Biomedical Engineering, Johns Hopkins School of Medicine720 Rutland Ave, Baltimore, MD 21205, USA. Tel.: +1 410 614 3741. E-mail address: [email protected] (K.W. Leong). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.03.013
organogenesis [1–4]. Following tissue injury, fibroblasts invade the fibrin clot in the wound space [5,6], where they are activated [7]. This activation to accelerate the wound healing process appears to initiate a switch of the fibroblast cell surface receptors from binding type I collagen to those that bind provisional matrix proteins such as fibrin and FN [8,9]. Cells producing a sufficient amount of FN are able to spread on material surface in a serum free medium, whereas cells that do not secrete adequate levels of FN cannot spread even with added FN in the medium [10]. This is one of the many examples showing the important role of FN in mediating the interaction between cells and biomaterials surface. The schematic of the FN molecule shows the repeated arrangement of the three module types (Fig. 1). The most abundant module is Type III, which contains the RGD domains. RGD has been identified as a key motif interacting with cells via the α5β1 integrin. The type III connecting segment (III CS) domain and
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Fig. 1. Structural scheme of FN (adapted from Magnusson [52]).
the adjacent heparin-binding domain are the other major cellbinding regions of FN, able to recognize the α4β1 and α4β7 integrins [11–13]. In the past 20 years different strategies have been used to immobilize FN onto various biomaterials surfaces. Adsorption is the most commonly adopted method. Although less stable than covalent immobilization, adsorption is convenient and may be less damaging to the conformation of the adsorbed molecules. The adsorption approach is nevertheless highly sensitive to the materials surface chemistry. Grinnell and Feld first showed that plasma FN would be adsorbed in higher content but with lower bioactivity on hydrophobic compared to hydrophilic surfaces [14], a finding subsequently confirmed in other studies [15,16]. Recently there has been increasing interest to immobilize FN to surfaces using covalent coupling chemistries via the amine, thiol, and aldehyde groups [17–20]. Covalent coupling or conjugation
presumably would be more efficient and stable against detachment. A critical issue of the covalent strategy is the conservation of the desired bioactivity of the immobilized FN. The degree of activity depends on the binding procedure and the substrate [20]. A combination of conjugation and adsorption can sometimes produce the optimal results. By conjugating FN to the terminals of Pluronic™ F108 adsorbed on polystyrene, Biran et al. concluded that the FN was more bioactive than the FN adsorbed directly onto polystyrene as revealed by the neurite adhesion and outgrowth assays [18]. Comparing the adsorption method with the covalent conjugation approach in the literature is difficult because of the different chemistries and surfaces used. In addition, the presence of serum and the use of trypsin in the cell adhesion study, which may alter the composition of the immobilized protein [21] or degrade the cell surface integrins, can confound the comparison. In this study,
Fig. 2. Reaction scheme of FN immobilization on aminated PET surface: (a) FN-conjugated pathway; (b) FN adsorption pathway.
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(Maryland, USA). Anti-VCIP and RGD domains were purchased from Oncogene Science Inc. (Cambridge, USA). 2.2. FN conjugation and adsorption
Fig. 3. Density of FN immobilized on aminated PET surface as determined by micro-BCA (mean ± S.D.; n N 5).
we investigated the bioactivity of FN immobilized on PET, one of the most widely studied biomaterials surfaces [22,23], using both the conjugation and adsorption methods. In the first case, FN was covalent conjugated to an aminated PET surface via glutaraldehyde crosslinking, and then the surface was blocked by ethanolamine. In the second case, FN was physically adsorbed to the ethanolamine blocked animated-PET surface without FN conjugation. By minimizing the variation in surface characteristics we aim to more meaningfully compare the bioactivities of the adsorbed and conjugated FN. The chemical and physical properties of the immobilized surfaces were studied by micro-BCA, contact angle and AFM. The bioactivity of the immobilized surfaces was evaluated by the fibroblast adhesion assay (BHK21 cell line: FN dependent [24]) in serum-free medium and ELISA for the active RGD domain.
Fig. 2 shows the schematic of FN immobilization. In the conjugation pathway, clean PET surface aminated by ethylenediamine solution (ethylenediamine/ethanol= 1 :1) at 50 °C for 40 min was immersed in freshly prepared 2% glutaraldehyde solution with 0.05 M sodium cyanoborohydride, and then reacted for 12–20 h at room temperature. After washing, the glutaraldehyde-conjugated surface was transferred to FN solution in sodium carbonate buffer with 0.05 M sodium cyanoborohydride, and reacted for 24 h at room temperature. To block the unreacted aldehyde sites, 0.06 M ethanolamine was added and reacted for another 12 h at room temperature (20 °C to 25 °C). Finally the surface was washed with 5 M sodium chloride solution for 24 h, and then washed with the desired buffer (PBS). For FN adsorption, the same protocol described above to produce the glutaraldehyde-conjugated surface was used. After the surface was blocked by ethanolamine, FN was added into the system, reacted for 24 h, washed with 5 M sodium chloride for 24 h, and then washed with PBS. 2.3. Chemical and physical characterization of the surface 2.3.1. Quantification of immobilized FN The prepared surfaces were added to micro-BCA working solution (A/B/C/sodium carbonate buffer = 25 : 24 : 1 : 50), and incubated at 37 °C for 2 h. The total protein level was then measured by absorbance at 562 nm.
2. Materials and methods 2.1. Materials Biaxially oriented PET films about 100 um thickness were purchased from Goodfellow Inc. (Cambridge, UK). Lyophilized human plasma, sterile FN, and MTT were purchased from Roche Ltd. (Indiana, USA). Ethylenediamine, glutaraldehyde, ethanolamine, cyanoborohydride, RPMI medium, penicillin, and streptomycin were obtained from Sigma-Aldrich Co. (St. Louis, USA). Micro-BCA, BCA, and goat anti-rabbit IgG peroxidase were obtained from PIERCE Biotechnology Inc. (Rockford, USA). BHK21 cell line was obtained from Cambrex Walkersville Inc.
2.3.2. Surface contact angle measurement Clean PET surface and FN-immobilized PET surfaces were measured at room temperature and 60% relative humidity, using sessile drop method on a telescope goniometer. More than five measurements were carried out for each sample and the resulting values were averaged. 2.3.3. AFM measurement The surface topography of FN-immobilized PET was examined on a Nanoscope IIIa AFM (Digital Instruments Inc., USA) in air. Atomic force microscope images were obtained by scanning surface in the tapping mode. The driven frequency was
Fig. 4. Surface contact angle on clean, modified and FN-immobilized PET surface (mean ± S.D.; n N 5; ⁎p b 0.05).
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Fig. 5. AFM images of FN-immobilized PET surface. (A) FN-conjugated PET surface; scanning area: 4.8 mm * 4.8 mm, Ra: 119 nm. (B) FN-adsorbed PET surface; scanning area: 10 mm * 10 mm, Ra: 11.2 nm. (C1): Clean PET surface control. (C2): Modified PET surface control without FN adsorption or conjugation.
330 ± 50 kHz. The applied voltage was between 3.0 and 4.0 V, the drive amplitude at 300 mV, and the scanning rate was 1.0 Hz. An arithmetic mean of the surface roughness profile was determined. 2.4. ELISA FN-immobilized PET surfaces were first captured by primary antibody, rabbit anti-human VCIP/RGD domain (1 : 5000 dilution in BSA solution for 2 h at 37 °C). After washing, the surfaces were stained with secondary antibody, goat anti-rabbit IgG conjugated with peroxidase (1: 5000 dilutions in BSA solution for 2 h at 37 °C). After washing off the unbound secondary antibody, SlowTMB-solution was added to each well and incubated for 5–30 min at room temperature in darkness, which was oxidized by peroxidase, showing the absorbance value at wavelength of 450 nm. The ELISA results for active RGD domains were expressed as equivalent FN according to the linear FN adsorption isotherm on ethanolamine blocked PET surface as a function of total FN surface density (by micro-BCA).
21 cells were pretreated with serum-free medium for 2 h, and then detached by incubation with non-enzymatic cell dissociation solution, in order to avoid the serum effect and minimize the cell surface receptors from degradation. For the cell viability assay, cells (104 per 96-well TCPS well) were cultured for 24 h, and then measured by MTT assay. For the cell adhesion assay, cells (2 × 105 per 24-well TCPS well) were incubated for 2 h. The unattached cells were washed by warmed PBS several times. The adherent cells were lysed by 4% SDS and centrifuged. The supernatant was subjected to BCA assay. Based on the assumption that the cells had equal amount of proteins, the total protein content quantified by BCA was correlated with cell numbers. As the FN surface density was very low, it would not affect the cell number determination. 2.6. Statistical analysis Data were presented as the mean ± S.D. The statistical significance of the data was determined by Student's t test. The significant level was set at 0.05.
2.5. In vitro cell culture experiment
3. Results
The BHK21 cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (10 mg/ml). The cell studies were carried below passage 20. Before the cell assay on the surface, the BHK
3.1. FN immobilization on substrates
Fig. 6. ELISA measurement of active RGD domains on immobilized FN compared with Micro-BCA result (FN input concentration of 30 ng/ml; mean ± S.D.; n N 4).
Fig. 3 shows the surface density of FN as a function of the input concentration in solution. The conjugation method produced a higher FN content onto the surface than the adsorption method. With adsorption, the content of FN adsorbed reached a saturation
Fig. 7. Cell adhesion on FN-immobilized PET surface (mean ± S.D.; n N 4).
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level around 400 ng/cm2 once the FN input concentration exceeded 4 μg/ml. The saturation level is consistent with conventional adsorption isotherms, representing approximately the amount of FN necessary to produce a monolayer coating based on the dimension of the molecules [25]. In contrast, the conjugation method seemed to be able to form multiple layers of FN despite extensive washing. At an FN input concentration of 30 μg/ml, 918 ± 134 ng/cm2 of FN could be stably immobilized onto the surface.
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(Fig. 5). On the FN-adsorbed PET surface, the protein appeared evenly distributed, with a mean roughness of Ra = 11.2 nm and a maximal roughness of 217 nm. In contrast, the protein on the FNconjugated PET surface appeared to be more closely packed in layers, with a mean roughness of Ra = 119 nm and a maximal roughness of 522 nm. The modification process alone did not affect the surface morphology much. 3.4. ELISA test for RGD domain
3.2. Contact angle measurement The cleaned and unmodified PET surface showed a static contact angle of 74.1° ± 2.6° (Fig. 4). Amination of the PET surface with ethylenediamine, which introduced the polar amino groups on the surface, lowered the contact angle to 62.9° ± 3.1°. The conjugation of FN on the aminated surface decreased the angle further to 56.7° ± 2.0°. The ethanolamine-blocked surface had similar contact angle with the aminated surface (61.5° ± 2.9°), and the FN adsorption onto this surface did not change the contact angle further (61.8° ± 2.1°). While the mechanism is not clear, the difference in the contact angles between the FN-conjugated and the FN-adsorbed surfaces suggests dissimilarity in the conformation of FN presented on the surface. 3.3. Surface morphological analysis by AFM Different morphology was observed when FN was conjugated or adsorbed to the PET surfaces in the AFM topographic images
Although the FN surface density was higher in the FN-conjugated surface according to the micro-BCA result, the ELISA test for RGD domains showed a lower FN bioactivity than the FN-adsorbed surface (Fig. 6). There is little difference between the micro-BCA and ELISA measurements for the surface density of adsorbed FN, 433.1 ± 45.8 ng/cm2 vs. 410.8 ±13.7 ng/ cm2, respectively, at the FN input concentration of 30 μg/ml. In contrast, the corresponding values for the conjugation method are 917.6 ± 133.8 ng/cm2 and 220.3 ± 10.1 ng/cm2, indicating a loss of 76% of the active RGD domains. 3.5. In vitro cell culture The cell viability over 24 h on the two FN-immobilized PET surfaces did not show any significant difference according to the MTT assay. However, the adhesion of BHK21 cells at the 2h time point was quite different (Fig. 7). The FN-adsorbed PET surface supported cell adhesion (from 53.6 ± 10.2% to 70.3 ±
Fig. 8. Hypothetical scheme of fibrillogenesis induced by covalent conjugation of FN on PET. (A) Compact conformation of FN in solution is stabilized by III12–14/ III2–3 and III12–14/I domains. (B) FN unfolds when the amino groups of the FN are immobilized to the surface. (C) The unfolded FN exposes new layer of negatively charged “surface” for extra FN adsorption. (D) FN adsorbs and partially unfolds on the FN layer through electronic association, further exposes new “surface” for FN immobilization.
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9.4%) as a function of the adsorbed FN surface density (from 0 to 433.1 ± 45.8 ng/cm2). The FN-conjugated surface on the other hand suppressed cell adhesion, from 53.6 ± 10.2% to 12.2 ± 6.0%, as the FN density, as determined by micro-BCA, increased from 0 to 917.6 ± 133.8 ng/cm2. 4. Discussion 4.1. FN conformation and its bioactivity Many studies have documented the dependence of the biological properties of FN on its conformation. Certain activities of FN are latent in the intact molecule and become fully expressed in its proteolytic fragments [26–29]. For example, 29 and 40 kDa heparin-binding proteolytic fragments of FN are potent inhibitors of endothelial growth in vitro, while native FN is not [30]. Generally it is believed that the FN molecule is secreted by cells and folded into globular domains specialized for particular functions, such as binding to integrins, collagen, heparin sulfate, and hyaluronic acid; therefore, the change of their conformation would affect their bioactivity [31–34]. 4.2. FN adsorption Langmuir isotherm is often used for the rudimentary description of the protein adsorption phenomena: the near-linear dependence of adsorbed molecules on the bulk concentration at low concentration regimens and the saturation effect at higher concentrations [35–38]. Our results are consistent with this trend. The saturation level represents the formation of monolayer of FN on the surface. The monolayer coverage theoretically depends on the shape and size of the protein molecule, and has been reported to range between 200 and 500 ng/cm2 [39–42]. In this experiment, we reported a saturation surface density around 400 ng/cm2. The AFM images showed that the adsorbed FN maintained its compact conformation, was uniformly distributed on the surface, and exhibited less structural alteration compared with the conjugated FN. 4.3. FN conjugation and fibrillogenesis In solution, the compact conformation of FN is maintained by only a small number of electrostatic associations between the distant segments of the molecule: III12–14/III2–3, and III12–14/ amino-terminal Heparin I domains [43]. However, the electrostatic associations can be destroyed in a regulated stepwise process, fibrillogenesis, in which the soluble FN is converted into insoluble fibrillar network. At initiation, integrins at the cell surface recognize the cell-binding domains of FN, usually the α5β1 binding to the RGD domains or the α4β1 binding to the CS domains. This is followed by the unfolding of FN, which exposes the FN binding sites and promotes intermolecular interactions with other FN molecules. Finally the integrin clustering and binding of additional FN leads to fibril formation [44,45]. Based on the FN-conjugation result and the morphology observed in AFM, we hypothesize a cell-free fibrillogenesis model for the covalent conjugation process (Fig. 8). At first the amino
groups of the protein are randomly immobilized to the surface, which induces FN to unfold (Fig. 8A). As the positively charged amino groups are conjugated to the substrate, the negatively charged domains of the protein are driven away from the surface, forming a negative charge concentrated “surface” (Fig. 8B). This negatively charged “surface” then facilitates immobilization of FN through electrostatic interaction, and induces the FN to unfold, forming new adsorption “surface” repeatedly. In this manner the FN can be immobilized in multiple layers and initiate the fibrillogenesis process. This proposed model suggests that the orientation of the FN molecules on the aminated surface and subsequent unfolding of the molecule may be the key to the induction of fibrillogenesis. At high surface charge densities, the ζ-potential would repel the negative domains and attract the positive domains, leaving open and accessible the domains implicated in the fibrillogenesis process. By this model we can explain how the covalent conjugation method can stably immobilize multiple layers of FN, and how fibril-like structures can form on the aminated-PET surface. Pernodet et al. has also reported similar cell-free fibrillogenesis on sulfonated polystyrene surface after 130 h of incubation with 100 μg/ml FN [46]. It is increasingly apparent that the FN matrix can have profound effects on cell functions. Data from structural modeling of repeat III10 indicate that the unfolding of the FN and the following fibrillogenesis process might control the accessibility of the RGD sequence, and enable cells to retract from the FN matrices in order to migrate and proliferate [47]. Studies on adherence of endothelial cells also report that after fibrillogenesis the FN matrix prevents the adhesion of extra cells [48]. Consistent with these data, our experiments suggest that the conjugated FN induced fibrillogenesis, suppressed the accessibility of RGD domains, and subsequently suppressed cell adhesion. 5. Summary This study examined the regulation of FN-mediated cell– material interaction at the level of FN immobilization methods. The adsorption method achieved a monolayer coverage, preserved higher bioactivity in maintaining more RGD domains, and supported cell adhesion. In contrast, the conjugation method achieved a higher content of surface immobilized, but led to lower bioactivity as evidenced by fewer RGD domains and suppressed cell adhesion. This is postulated as a consequence of fibrillogenesis that lowers the bioactivity of FN. The combination of these two methods may offer some degree of control to vary the surface bioactivity of the immobilized FN. While it may be easy to generate a highly adhesive substrate by direct adsorption of cell adhesion molecules like FN, it may be advantageous to produce a less adhesive and stably decorated surface for certain applications. For instance, a difficulty encountered in the transplantation paradigm is the inability of cells, once attached to an overly adhesive bridging substrate, to leave the substrate for the comparatively less adhesive surrounding tissue [49–51]. In this regard, promoting cell migration away from the device surface probably will require that the surface become less adhesive in comparison with the surrounding tissue, which often is not recognized in the development of bioactive surfaces. Findings in this study may aid the
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design of such functional surfaces with controlled bioactivity in the future. Acknowledgment This project is supported by a grant from Agency for Science, Technology and Research (A⁎STAR) of Singapore and the Division of Johns Hopkins in Singapore. We also want to specially thank Mr Lu Thong Beng (Electron Microscopy Unit, National University of Singapore) and Mr Chua Kian Ngiap (Graduate Program of Bio Engineering, National University of Singapore) for their valuable support in experiments. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]
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Fibronectin immobilized by covalent conjugation or physical adsorption shows different bioactivity on aminated-PET Yue Zhang a , Chou Chai b , Xue Song Jiang b , Swee Hin Teoh c , Kam W. Leong b,d,⁎ a
Graduate Programme in Bioengineering, National University of Singapore, Singapore Tissue and Therapeutics Engineering Laboratory, Division of Johns Hopkins, Singapore c Deparment of Mechanical Engineering, National University of Singapore, Singapore Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, USA b
d
Received 8 July 2005; accepted 3 March 2006 Available online 22 June 2006
Abstract To manipulate the cellular response to synthetic surfaces, extracellular matrix (ECM) proteins such as fibronectin (FN) and collagen are often immobilized on the surface to promote interaction between these ligands and the cell receptors. In this study we compared the biological properties of FN-decorated polyethylene terephthalate (PET) produced by two widely used immobilization techniques: adsorption and conjugation. As revealed by the micro-bicinchoninic acid (micro-BCA) assay and AFM, the modified surface topography was dependent on the immobilization methods. Adsorption method preserved the compact conformation of FN, reaching saturation when a monolayer of FN was formed. Covalent conjugation induced FN unfolding and fibrillogenesis, forming multiple layers of FN. Biological characterization by adhesion of baby hamster kidney 21 (BHK21) cells and enzyme-linked immunosorbent assay (ELISA) for active Arg-Gly-Asp (RGD) domains suggested that the difference in conformation of FN led to different bioactivities. Adsorption maintained a more active RGD domain, thereby promoting cell adhesion, whereas conjugation induced fibrillogenesis and blocked the access of RGD, consequently suppressing cell adhesion as the surface density of FN increased. This study suggests that in addition to choosing the nature of the adhesion molecule, the mode of immobilization may also significantly influence the bioactivity of the surface. © 2006 Elsevier B.V. All rights reserved. Keywords: Surface modification; Conjugation; Adsorption; Fibronectin; Bioactivity
1. Introduction Surface modification can modify the interaction between the biomaterials surface and the biological environment and yet preserve the mechanical properties of the base biomaterials. Design of functionalized polymer surfaces for biomaterial applications often rely on immobilization of ECM proteins or peptides to control the integrin-mediated cell attachment. Among the ECM proteins, FN as a family of structurally related soluble and matrix glycoproteins have been extensively studied in numerous biological processes including cell adhesion, wound healing, and ⁎ Corresponding author. Department of Biomedical Engineering, Johns Hopkins School of Medicine720 Rutland Ave, Baltimore, MD 21205, USA. Tel.: +1 410 614 3741. E-mail address: [email protected] (K.W. Leong). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.03.013
organogenesis [1–4]. Following tissue injury, fibroblasts invade the fibrin clot in the wound space [5,6], where they are activated [7]. This activation to accelerate the wound healing process appears to initiate a switch of the fibroblast cell surface receptors from binding type I collagen to those that bind provisional matrix proteins such as fibrin and FN [8,9]. Cells producing a sufficient amount of FN are able to spread on material surface in a serum free medium, whereas cells that do not secrete adequate levels of FN cannot spread even with added FN in the medium [10]. This is one of the many examples showing the important role of FN in mediating the interaction between cells and biomaterials surface. The schematic of the FN molecule shows the repeated arrangement of the three module types (Fig. 1). The most abundant module is Type III, which contains the RGD domains. RGD has been identified as a key motif interacting with cells via the α5β1 integrin. The type III connecting segment (III CS) domain and
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Fig. 1. Structural scheme of FN (adapted from Magnusson [52]).
the adjacent heparin-binding domain are the other major cellbinding regions of FN, able to recognize the α4β1 and α4β7 integrins [11–13]. In the past 20 years different strategies have been used to immobilize FN onto various biomaterials surfaces. Adsorption is the most commonly adopted method. Although less stable than covalent immobilization, adsorption is convenient and may be less damaging to the conformation of the adsorbed molecules. The adsorption approach is nevertheless highly sensitive to the materials surface chemistry. Grinnell and Feld first showed that plasma FN would be adsorbed in higher content but with lower bioactivity on hydrophobic compared to hydrophilic surfaces [14], a finding subsequently confirmed in other studies [15,16]. Recently there has been increasing interest to immobilize FN to surfaces using covalent coupling chemistries via the amine, thiol, and aldehyde groups [17–20]. Covalent coupling or conjugation
presumably would be more efficient and stable against detachment. A critical issue of the covalent strategy is the conservation of the desired bioactivity of the immobilized FN. The degree of activity depends on the binding procedure and the substrate [20]. A combination of conjugation and adsorption can sometimes produce the optimal results. By conjugating FN to the terminals of Pluronic™ F108 adsorbed on polystyrene, Biran et al. concluded that the FN was more bioactive than the FN adsorbed directly onto polystyrene as revealed by the neurite adhesion and outgrowth assays [18]. Comparing the adsorption method with the covalent conjugation approach in the literature is difficult because of the different chemistries and surfaces used. In addition, the presence of serum and the use of trypsin in the cell adhesion study, which may alter the composition of the immobilized protein [21] or degrade the cell surface integrins, can confound the comparison. In this study,
Fig. 2. Reaction scheme of FN immobilization on aminated PET surface: (a) FN-conjugated pathway; (b) FN adsorption pathway.
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(Maryland, USA). Anti-VCIP and RGD domains were purchased from Oncogene Science Inc. (Cambridge, USA). 2.2. FN conjugation and adsorption
Fig. 3. Density of FN immobilized on aminated PET surface as determined by micro-BCA (mean ± S.D.; n N 5).
we investigated the bioactivity of FN immobilized on PET, one of the most widely studied biomaterials surfaces [22,23], using both the conjugation and adsorption methods. In the first case, FN was covalent conjugated to an aminated PET surface via glutaraldehyde crosslinking, and then the surface was blocked by ethanolamine. In the second case, FN was physically adsorbed to the ethanolamine blocked animated-PET surface without FN conjugation. By minimizing the variation in surface characteristics we aim to more meaningfully compare the bioactivities of the adsorbed and conjugated FN. The chemical and physical properties of the immobilized surfaces were studied by micro-BCA, contact angle and AFM. The bioactivity of the immobilized surfaces was evaluated by the fibroblast adhesion assay (BHK21 cell line: FN dependent [24]) in serum-free medium and ELISA for the active RGD domain.
Fig. 2 shows the schematic of FN immobilization. In the conjugation pathway, clean PET surface aminated by ethylenediamine solution (ethylenediamine/ethanol= 1 :1) at 50 °C for 40 min was immersed in freshly prepared 2% glutaraldehyde solution with 0.05 M sodium cyanoborohydride, and then reacted for 12–20 h at room temperature. After washing, the glutaraldehyde-conjugated surface was transferred to FN solution in sodium carbonate buffer with 0.05 M sodium cyanoborohydride, and reacted for 24 h at room temperature. To block the unreacted aldehyde sites, 0.06 M ethanolamine was added and reacted for another 12 h at room temperature (20 °C to 25 °C). Finally the surface was washed with 5 M sodium chloride solution for 24 h, and then washed with the desired buffer (PBS). For FN adsorption, the same protocol described above to produce the glutaraldehyde-conjugated surface was used. After the surface was blocked by ethanolamine, FN was added into the system, reacted for 24 h, washed with 5 M sodium chloride for 24 h, and then washed with PBS. 2.3. Chemical and physical characterization of the surface 2.3.1. Quantification of immobilized FN The prepared surfaces were added to micro-BCA working solution (A/B/C/sodium carbonate buffer = 25 : 24 : 1 : 50), and incubated at 37 °C for 2 h. The total protein level was then measured by absorbance at 562 nm.
2. Materials and methods 2.1. Materials Biaxially oriented PET films about 100 um thickness were purchased from Goodfellow Inc. (Cambridge, UK). Lyophilized human plasma, sterile FN, and MTT were purchased from Roche Ltd. (Indiana, USA). Ethylenediamine, glutaraldehyde, ethanolamine, cyanoborohydride, RPMI medium, penicillin, and streptomycin were obtained from Sigma-Aldrich Co. (St. Louis, USA). Micro-BCA, BCA, and goat anti-rabbit IgG peroxidase were obtained from PIERCE Biotechnology Inc. (Rockford, USA). BHK21 cell line was obtained from Cambrex Walkersville Inc.
2.3.2. Surface contact angle measurement Clean PET surface and FN-immobilized PET surfaces were measured at room temperature and 60% relative humidity, using sessile drop method on a telescope goniometer. More than five measurements were carried out for each sample and the resulting values were averaged. 2.3.3. AFM measurement The surface topography of FN-immobilized PET was examined on a Nanoscope IIIa AFM (Digital Instruments Inc., USA) in air. Atomic force microscope images were obtained by scanning surface in the tapping mode. The driven frequency was
Fig. 4. Surface contact angle on clean, modified and FN-immobilized PET surface (mean ± S.D.; n N 5; ⁎p b 0.05).
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Fig. 5. AFM images of FN-immobilized PET surface. (A) FN-conjugated PET surface; scanning area: 4.8 mm * 4.8 mm, Ra: 119 nm. (B) FN-adsorbed PET surface; scanning area: 10 mm * 10 mm, Ra: 11.2 nm. (C1): Clean PET surface control. (C2): Modified PET surface control without FN adsorption or conjugation.
330 ± 50 kHz. The applied voltage was between 3.0 and 4.0 V, the drive amplitude at 300 mV, and the scanning rate was 1.0 Hz. An arithmetic mean of the surface roughness profile was determined. 2.4. ELISA FN-immobilized PET surfaces were first captured by primary antibody, rabbit anti-human VCIP/RGD domain (1 : 5000 dilution in BSA solution for 2 h at 37 °C). After washing, the surfaces were stained with secondary antibody, goat anti-rabbit IgG conjugated with peroxidase (1: 5000 dilutions in BSA solution for 2 h at 37 °C). After washing off the unbound secondary antibody, SlowTMB-solution was added to each well and incubated for 5–30 min at room temperature in darkness, which was oxidized by peroxidase, showing the absorbance value at wavelength of 450 nm. The ELISA results for active RGD domains were expressed as equivalent FN according to the linear FN adsorption isotherm on ethanolamine blocked PET surface as a function of total FN surface density (by micro-BCA).
21 cells were pretreated with serum-free medium for 2 h, and then detached by incubation with non-enzymatic cell dissociation solution, in order to avoid the serum effect and minimize the cell surface receptors from degradation. For the cell viability assay, cells (104 per 96-well TCPS well) were cultured for 24 h, and then measured by MTT assay. For the cell adhesion assay, cells (2 × 105 per 24-well TCPS well) were incubated for 2 h. The unattached cells were washed by warmed PBS several times. The adherent cells were lysed by 4% SDS and centrifuged. The supernatant was subjected to BCA assay. Based on the assumption that the cells had equal amount of proteins, the total protein content quantified by BCA was correlated with cell numbers. As the FN surface density was very low, it would not affect the cell number determination. 2.6. Statistical analysis Data were presented as the mean ± S.D. The statistical significance of the data was determined by Student's t test. The significant level was set at 0.05.
2.5. In vitro cell culture experiment
3. Results
The BHK21 cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (10 mg/ml). The cell studies were carried below passage 20. Before the cell assay on the surface, the BHK
3.1. FN immobilization on substrates
Fig. 6. ELISA measurement of active RGD domains on immobilized FN compared with Micro-BCA result (FN input concentration of 30 ng/ml; mean ± S.D.; n N 4).
Fig. 3 shows the surface density of FN as a function of the input concentration in solution. The conjugation method produced a higher FN content onto the surface than the adsorption method. With adsorption, the content of FN adsorbed reached a saturation
Fig. 7. Cell adhesion on FN-immobilized PET surface (mean ± S.D.; n N 4).
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level around 400 ng/cm2 once the FN input concentration exceeded 4 μg/ml. The saturation level is consistent with conventional adsorption isotherms, representing approximately the amount of FN necessary to produce a monolayer coating based on the dimension of the molecules [25]. In contrast, the conjugation method seemed to be able to form multiple layers of FN despite extensive washing. At an FN input concentration of 30 μg/ml, 918 ± 134 ng/cm2 of FN could be stably immobilized onto the surface.
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(Fig. 5). On the FN-adsorbed PET surface, the protein appeared evenly distributed, with a mean roughness of Ra = 11.2 nm and a maximal roughness of 217 nm. In contrast, the protein on the FNconjugated PET surface appeared to be more closely packed in layers, with a mean roughness of Ra = 119 nm and a maximal roughness of 522 nm. The modification process alone did not affect the surface morphology much. 3.4. ELISA test for RGD domain
3.2. Contact angle measurement The cleaned and unmodified PET surface showed a static contact angle of 74.1° ± 2.6° (Fig. 4). Amination of the PET surface with ethylenediamine, which introduced the polar amino groups on the surface, lowered the contact angle to 62.9° ± 3.1°. The conjugation of FN on the aminated surface decreased the angle further to 56.7° ± 2.0°. The ethanolamine-blocked surface had similar contact angle with the aminated surface (61.5° ± 2.9°), and the FN adsorption onto this surface did not change the contact angle further (61.8° ± 2.1°). While the mechanism is not clear, the difference in the contact angles between the FN-conjugated and the FN-adsorbed surfaces suggests dissimilarity in the conformation of FN presented on the surface. 3.3. Surface morphological analysis by AFM Different morphology was observed when FN was conjugated or adsorbed to the PET surfaces in the AFM topographic images
Although the FN surface density was higher in the FN-conjugated surface according to the micro-BCA result, the ELISA test for RGD domains showed a lower FN bioactivity than the FN-adsorbed surface (Fig. 6). There is little difference between the micro-BCA and ELISA measurements for the surface density of adsorbed FN, 433.1 ± 45.8 ng/cm2 vs. 410.8 ±13.7 ng/ cm2, respectively, at the FN input concentration of 30 μg/ml. In contrast, the corresponding values for the conjugation method are 917.6 ± 133.8 ng/cm2 and 220.3 ± 10.1 ng/cm2, indicating a loss of 76% of the active RGD domains. 3.5. In vitro cell culture The cell viability over 24 h on the two FN-immobilized PET surfaces did not show any significant difference according to the MTT assay. However, the adhesion of BHK21 cells at the 2h time point was quite different (Fig. 7). The FN-adsorbed PET surface supported cell adhesion (from 53.6 ± 10.2% to 70.3 ±
Fig. 8. Hypothetical scheme of fibrillogenesis induced by covalent conjugation of FN on PET. (A) Compact conformation of FN in solution is stabilized by III12–14/ III2–3 and III12–14/I domains. (B) FN unfolds when the amino groups of the FN are immobilized to the surface. (C) The unfolded FN exposes new layer of negatively charged “surface” for extra FN adsorption. (D) FN adsorbs and partially unfolds on the FN layer through electronic association, further exposes new “surface” for FN immobilization.
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9.4%) as a function of the adsorbed FN surface density (from 0 to 433.1 ± 45.8 ng/cm2). The FN-conjugated surface on the other hand suppressed cell adhesion, from 53.6 ± 10.2% to 12.2 ± 6.0%, as the FN density, as determined by micro-BCA, increased from 0 to 917.6 ± 133.8 ng/cm2. 4. Discussion 4.1. FN conformation and its bioactivity Many studies have documented the dependence of the biological properties of FN on its conformation. Certain activities of FN are latent in the intact molecule and become fully expressed in its proteolytic fragments [26–29]. For example, 29 and 40 kDa heparin-binding proteolytic fragments of FN are potent inhibitors of endothelial growth in vitro, while native FN is not [30]. Generally it is believed that the FN molecule is secreted by cells and folded into globular domains specialized for particular functions, such as binding to integrins, collagen, heparin sulfate, and hyaluronic acid; therefore, the change of their conformation would affect their bioactivity [31–34]. 4.2. FN adsorption Langmuir isotherm is often used for the rudimentary description of the protein adsorption phenomena: the near-linear dependence of adsorbed molecules on the bulk concentration at low concentration regimens and the saturation effect at higher concentrations [35–38]. Our results are consistent with this trend. The saturation level represents the formation of monolayer of FN on the surface. The monolayer coverage theoretically depends on the shape and size of the protein molecule, and has been reported to range between 200 and 500 ng/cm2 [39–42]. In this experiment, we reported a saturation surface density around 400 ng/cm2. The AFM images showed that the adsorbed FN maintained its compact conformation, was uniformly distributed on the surface, and exhibited less structural alteration compared with the conjugated FN. 4.3. FN conjugation and fibrillogenesis In solution, the compact conformation of FN is maintained by only a small number of electrostatic associations between the distant segments of the molecule: III12–14/III2–3, and III12–14/ amino-terminal Heparin I domains [43]. However, the electrostatic associations can be destroyed in a regulated stepwise process, fibrillogenesis, in which the soluble FN is converted into insoluble fibrillar network. At initiation, integrins at the cell surface recognize the cell-binding domains of FN, usually the α5β1 binding to the RGD domains or the α4β1 binding to the CS domains. This is followed by the unfolding of FN, which exposes the FN binding sites and promotes intermolecular interactions with other FN molecules. Finally the integrin clustering and binding of additional FN leads to fibril formation [44,45]. Based on the FN-conjugation result and the morphology observed in AFM, we hypothesize a cell-free fibrillogenesis model for the covalent conjugation process (Fig. 8). At first the amino
groups of the protein are randomly immobilized to the surface, which induces FN to unfold (Fig. 8A). As the positively charged amino groups are conjugated to the substrate, the negatively charged domains of the protein are driven away from the surface, forming a negative charge concentrated “surface” (Fig. 8B). This negatively charged “surface” then facilitates immobilization of FN through electrostatic interaction, and induces the FN to unfold, forming new adsorption “surface” repeatedly. In this manner the FN can be immobilized in multiple layers and initiate the fibrillogenesis process. This proposed model suggests that the orientation of the FN molecules on the aminated surface and subsequent unfolding of the molecule may be the key to the induction of fibrillogenesis. At high surface charge densities, the ζ-potential would repel the negative domains and attract the positive domains, leaving open and accessible the domains implicated in the fibrillogenesis process. By this model we can explain how the covalent conjugation method can stably immobilize multiple layers of FN, and how fibril-like structures can form on the aminated-PET surface. Pernodet et al. has also reported similar cell-free fibrillogenesis on sulfonated polystyrene surface after 130 h of incubation with 100 μg/ml FN [46]. It is increasingly apparent that the FN matrix can have profound effects on cell functions. Data from structural modeling of repeat III10 indicate that the unfolding of the FN and the following fibrillogenesis process might control the accessibility of the RGD sequence, and enable cells to retract from the FN matrices in order to migrate and proliferate [47]. Studies on adherence of endothelial cells also report that after fibrillogenesis the FN matrix prevents the adhesion of extra cells [48]. Consistent with these data, our experiments suggest that the conjugated FN induced fibrillogenesis, suppressed the accessibility of RGD domains, and subsequently suppressed cell adhesion. 5. Summary This study examined the regulation of FN-mediated cell– material interaction at the level of FN immobilization methods. The adsorption method achieved a monolayer coverage, preserved higher bioactivity in maintaining more RGD domains, and supported cell adhesion. In contrast, the conjugation method achieved a higher content of surface immobilized, but led to lower bioactivity as evidenced by fewer RGD domains and suppressed cell adhesion. This is postulated as a consequence of fibrillogenesis that lowers the bioactivity of FN. The combination of these two methods may offer some degree of control to vary the surface bioactivity of the immobilized FN. While it may be easy to generate a highly adhesive substrate by direct adsorption of cell adhesion molecules like FN, it may be advantageous to produce a less adhesive and stably decorated surface for certain applications. For instance, a difficulty encountered in the transplantation paradigm is the inability of cells, once attached to an overly adhesive bridging substrate, to leave the substrate for the comparatively less adhesive surrounding tissue [49–51]. In this regard, promoting cell migration away from the device surface probably will require that the surface become less adhesive in comparison with the surrounding tissue, which often is not recognized in the development of bioactive surfaces. Findings in this study may aid the
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design of such functional surfaces with controlled bioactivity in the future. Acknowledgment This project is supported by a grant from Agency for Science, Technology and Research (A⁎STAR) of Singapore and the Division of Johns Hopkins in Singapore. We also want to specially thank Mr Lu Thong Beng (Electron Microscopy Unit, National University of Singapore) and Mr Chua Kian Ngiap (Graduate Program of Bio Engineering, National University of Singapore) for their valuable support in experiments. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]
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