Adsorption Force of Fibronectin on Various Surface Chemistries and

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Adsorption force of fibronectin on various surface chemistries and its vital role in osteoblast adhesion Manping Lin, Huaiyu Wang, Changshun Ruan, Juan Xing, Jinfeng Wang, Yan Li, Yuan-Liang Wang, and Yanfeng Luo Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501873g • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Adsorption force of fibronectin on various surface chemistries and its vital role in osteoblast adhesion †

‡

‡

†

†

Manping Lin ,§, Huaiyu Wang , Changshun Ruan , Juan Xing ,§, Jinfeng Wang ,§, Yan †

†

†

Li ,§, Yuanliang Wang ,§, Yanfeng Luo*, ,§

†Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing, 400030, China

§Research Center of Bioinspired Materials Science and Engineering, College of Bioengineering, Chongqing University, Chongqing 400030, China

‡Center for Human Tissue and Organs Degeneration, Institute Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.

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ABSTRACT: The amount, type and conformation of proteins adsorbed on an implanted biomaterial are believed to influence cell adhesion. Nevertheless, only a few research works have been dedicated to the contribution of protein adsorption force. To verify our hypothesis that the adsorption force of protein on biomaterial is another crucial mediator to cell adhesion, fibronectin (FN) adsorbed on self-assembled monolayers (SAMs) with terminal -OH, -CH3 and -NH2 was quantified for FN adsorption force (Fad) by utilizing a sphere/plane adsorption model and parallel plate flow chamber. As revealed, Fad on SAMs followed a chemistry-dependence of -NH2 > -CH3 >> -OH. It is further demonstrated that Fad together with FN conformation could regulate the late osteoblast adhesion and the consequent reorganization of the adsorbed FN and fibrillogenesis of the endogenous FN. Our study suggests that protein adsorption force plays a key role in cell adhesion and should be involved for better biomaterial design.

KEYWORDS: Protein adsorption force, Fibronectin, Self-assembled monolayers, Osteoblast adhesion


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1. INTRODUCTION When an implanted biomaterial gets involved into the physiological environment as a foreign matter, protein adsorption onto the surface is the initial step before cell adhesion,1,2 which plays a key role in the surface biocompatibility of a well-defined biomaterial.3,4 Nevertheless, most scientists in biomaterial field focus on the status of adsorbed proteins with their amount, type and conformation.3,5-8 Rare attentions have been paid to the adsorption force of proteins on biomaterial surfaces, regardless of its effect to cell behaviors.

It is well recognized that the status of adsorbed proteins can be regulated by surface properties of biomaterials such as surface chemistry,3 wettability9,10 and topography,11 which further mediate cell adhesion and subsequent cellular responses including cell morphology, proliferation, differentiation and secretion of extracellular matrix (ECM).3-4,12-14 This is defined as a bottom-up cell-material interaction originated from biomaterial to upper cells. There is another mode defined as top-down cell-material interaction, which is from cells to underlying biomaterial. In particular, when cells have adhered onto biomaterial, focal adhesions (FAs) and fibrillar adhesions (FBs) may form and lead to generation of cell traction force via contraction of actin cytoskeleton.15,16 This cell traction force can be transmitted to the adsorbed protein layer via FAs and FBs and then influence the underlying biomaterial via biomaterial-protein interface. The bottom-up and top-down biomaterial-cell interactions are in a mode of cooperation and competition, among which the proteins adsorbed onto biomaterial are crucial. Only when the cell traction force (top-down)


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can be resisted by the bottom-up cell-material interaction, FAs will form and grow up with increase of cell traction force. Otherwise, FAs will be degraded with reduction of cell traction force17-19 to achieve a dynamic equilibrium between these 2 kinds of biomaterial-cell interactions. Consequently, the biomaterial-protein interfacial force, as a key factor for transfer of cell traction, should have important role in cell adhesion. However, the contributions of this interfacial force, i.e. protein adsorption force were generally neglected in previous studies. Only a few research works have been devoted to the detection of protein adsorption force,20-22 though some works have noticed the importance of the interaction strength between protein and the underlying substrate. 11, 23

In this respect, establishing a method to quantify protein adsorption force, and

further exploring the contributions of protein adsorption force to cell adhesion, may gain a deeper insight into the aforementioned cell-material interactions for better biomaterial design.

To this aim, our study initially quantified the protein adsorption force on a biomaterial surface by employing fibronectin (FN) as a model protein and self-assembled monolayers (SAMs) with terminal -OH, -CH3 and -NH2 as a model surface. The adsorption force of FN on various SAMs was determined by utilizing a sphere/plane adsorption model and parallel plate flow chamber (PPFC). The effects of protein adsorption force on osteoblast adhesion (FAs and FBs formation) were further investigated by assaying the reorganization of the adsorbed FN and the fibrillogenesis of endogenous FN. This present work may provide a more comprehensive insight into biomaterial-protein-cell interactions involving protein adsorption force, which could


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help for designing more desirable surface chemistry of biomaterials to orchestrate protein adsorption and cell functions.

2. EXPERIMENTAL SECTION Preparation and characterization of SAMs on Au-sputtered silica wafers (Au-SAMs). Au-thiol method was utilized to produce SAMs with terminal -CH3, -NH2 and -OH according to the method previously reported by Keselowsky et al.3 Firstly, bottom Cr (10 nm) and upper Au (15 nm) thin films were deposited on silica wafers via sputter coating at a vacuum of around 2 × 10-5 mbar. The obtained Au-sputtered silica wafers were cleaned with Piranha solution (98% H2SO4 : 30% H2O2 = 7:3 v/v) at room temperature for 1 hour, rinsed with 99% ethanol and deionized H2O, and dried by a nitrogen stream. Afterward, the wafers were immersed in

1-dodecanethiols

(HS-(CH2)11-CH3,

11-amino-undecanethiol

(HS-(CH2)11-NH2,

Acros

Organics,

Sigma-Aldrich,

USA)

USA), and

11-mercapto-1-undecanol (HS-(CH2)11-OH, Sigma-Aldrich, USA), respectively, at room temperature overnight. The wafers were subsequently rinsed by ethanol and deionized H2O, and dried by a nitrogen stream, endowing the SAMs with -CH3, -NH2 and -OH terminals. All the obtained SAMs were equilibrated in PBS buffer prior to other experiments. All the protein adsorption and cell adhesion experiments were performed on Au-SAMs unless otherwise stated.

The static water contact angles were measured by the sessile drop method with a


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Model 200 video-based optical system (Future Scientific Ltd. Co., Taiwan, China) at room temperature and ambient humidity.

Covalent conjugation of FN to microspheres. According to the method reported by Vallieres et al.,24 FN was covalently bound to NH2-functionalized microspheres (diameter 9 µm, Aladdin, China) by using sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB) (Pierce, Thermo Scientific, USA) as a coupling agent. In particular, the NH2-functionalized microspheres were collected by centrifugation for 10 min at 5000 rpm and rinsed three times with PBS. Subsequently, Sulfo-SMPB (1 mM) in conjugation buffer (1 mM EDTA in PBS, pH 7.2) was added to react for 2 hours at room temperature. The obtained Sulfo-SMPB conjugated microspheres were further incubated in 200 µg/mL FN solution at 4 oC overnight, and the density of conjugated FN on microspheres was quantified by using a microBCA Protein Assay Kit (Thermo Scientific, USA) according to the manufacturer’s instructions. The thickness of the conjugated FN layer was measured by atomic force microscopy (AFM, Icon Dimension with ScanAsyst, Bruke, USA) in a Scanasyst mode at room temperature.

XPS

analysis

of

FN-conjugated

microspheres.

XPS

spectra

of

NH2-functionalized microspheres, Sulfo-SMPB conjugated microspheres and FN-conjugated microspheres were collected by X-ray Photoelectron Spectroscopy (XPS), utilizing a XSAM 800 photoelectron spectroscope (Kratos, UK) with an Al Kα X-ray source (hν =1486.6 eV) under ultrahigh vacuum conditions (2×10-7 Pa). The


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binding energy scale was set with C-C/C-H bonds at 284.8 eV.

Data analysis was

carried out with a commercial software package (XPS PEAK, Version 4.1).

FN adsorption force on various SAMs. Since detection of FN adsorption force requires transparent SAMs, SAMs on glass slides (Glass-SAMs) were prepared according to the previously described method.25,26 In brief, the cleaned slides were treated with Piranha solution at 80 oC for 1 hour, the obtained slides were labeled as -OH slides. The -OH slides were dipped into 5% solution of chloro(dimethyl) octadecylsilane (Sigma-Aldrich, USA) in hexane for 1 hour to obtain -CH3 slides, whereas the -OH slides were dipped into 1% solution of (3-aminopropyl) triethoxysilane (Alfa Aesar, USA) in acetone for 1 hour to obtain -NH2 slides. Static water contact angles and XPS spectra of various Glass-SAMs were both characterized to ensure that they are equivalent to the corresponding Au-SAMs.

FN adsorption force on various SAMs was detected by using a sphere/plane adhesion model. The experimental set-up has been described previously by Lorthois et al.27 Particularly, a glass slide with SAMs was mounted into a parallel flow chamber (PPFC), serving as the bottom of PPFC. The bubbles in PPFC were removed by filling the PPFC with PBS, and then FN-conjugated microspheres were slowly injected into PPFC by a syringe. After 37 oC incubation for 2 hours, flow shear stress in PPFC was increased step by step. At the end of each step, microspheres remaining on the SAMs were photographed by a phase contrast optical microscopy for counting. FN adsorption force was calculated according to the detachment of FN-conjugated


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microspheres.

Determination of the conformation for the adsorbed FN. Adsorption of human plasma FN (R&D Systems, USA) on SAMs was performed by immersing SAMs into 20 µg/mL FN solution for 2 hours. The amount of adsorbed FN was quantified by using a microBCA Protein Assay Kit (Thermo Scientific, USA).

The conformation of FN adsorbed on SAMs, represented by the cell binding domains in FN, was determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. After being rinsed in PBS and blocked in 1% BSA at room temperature for 1 hour, the FN-adsorbed SAMs were incubated with HFN7.1 monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA) at 4 o

C overnight. For ELISA assay, the obtained SAMs were incubated with

HRP-conjugated goat anti-mouse IgG antibody (Cwbiotech, China) at room temperature

for

1

hour,

and

subsequently

incubated

with

3,

3’,

5,

5’-Tetramethylbenzidine (Beyotime, China) at room temperature for 20 minutes in darkness. The reaction supernatants were transferred to 96-well plates to measure the absorbance at 370 nm by using Multifunctional Microplate Reader (Bio-Rad Model 680,

USA).

For

immunofluorescence assay,

SAMs

were

incubated

with

FITC-conjugated goat anti-mouse IgG antibody (ZSGB-Bio, China) at room temperature for 1 hour. After being rinsed, the SAMs were mounted in glycerol and determined by Confocal Laser Scanning Microscopy (CLSM, TCS SP5, Leica, Germany). The conformation of adsorbed FN on various SAMs was also


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characterized by AFM (Icon Dimension with ScanAsyst, Bruke, USA) in a peakforce tapping mode at room temperature. The images of the adsorbed FN were recorded with a ScanAsyst Air tip (Bruke, USA).

Osteoblast culture. Primary osteoblasts were isolated from newborn SD rat calvaria,28 and expanded in DMEM (Gibico, USA) supplemented with 10% fetal calf serum (FCS, Sijiqing, China), penicillin (100 U/mL), streptomycin(100 µg/mL) and 0.05% L-glutamine. After reaching confluence, cells were sub-cultured and identified by von Kossa staining method according to previously reported procedure.29 All the cells were cultured in a humidified atmosphere of 5% carbon dioxide at 37 oC. For all the following experiments, osteoblasts at passage 3 were employed to seed on the SAMs at a density of 2 × 105 cells/cm2.

Organization and expression of tensin and vinculin. The organization of tensin and vinculin was assayed by immunofluorescence staining. FN-adsorbed SAMs were triply rinsed with PBS, and then seeded with osteoblasts in serum free medium for 2 hours and 12 hours, respectively. The obtained osteoblasts were fixed in 4% (w/v) paraformaldehyde for 30 minutes and permeabilized in 0.25% (v/v) Triton X-100 for 10 minutes. The samples were incubated with 5% BSA for 1 hour at room temperature to block the non-specific binding sites, followed by the respective additions of primary antibody against tensin (Sigma-Aldrich, USA) or vinculin (abcam, UK) for overnight incubation at 4 oC. Afterwards, FITC-conjugated secondary antibody (ZSGB-Bio, China) was added for 1 hour incubation at room


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temperature. Finally, the cells were incubated with Rhodamine-Phalloidin (Molecular Probes, Invitrogen, USA) overnight at 4 oC for F-actin staining, and incubated with H33258 (Sigma-Aldrich, USA) for 10 minutes at room temperature for nuclei staining. After being rinsed, the SAMs were mounted in glycerol and determined by CLSM (TCS SP5, Leica, Germany).

Western blot was also employed to quantify the expression of tensin and vinculin. After osteoblasts were seeded onto FN-adsorbed SAMs for 2 and 12 hours, total protein extraction was performed by lysing the cells with RIPA Lysis Buffer (Beyotime, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, China). Equal amounts of cell lysates were separated in 5%-8% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The transferred membranes were blocked with a block buffer (Cwbiotech, China) and then incubated separately with primary antibody against tensin (Sigma-Aldrich, USA), vinculin (abcam, UK) and β-actin (Cwbiotech, China). The employed secondary antibodies were all HRP conjugated. After the incubation of the antibodies was completed, the obtained membranes were visualized with eECL Kit (Cwbiotech, China). The gray values were measured by GS-800 imaging densitometer (Bio-Rad, USA) and quantified by Quantity One (Bio-Rad, USA) software.

Osteoblast-driven reorganization of the adsorbed FN on SAMs. The capability of osteoblasts to reorganize the adsorbed FN was assayed by


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immunofluorescence staining. After being seeded on FN-adsorbed SAMs in serum free medium for 12 hours, osteoblasts were fixed. The immunofluorescence staining of the absorbed FN, F-actin cytoskeleton and nuclei were subsequently performed.

Fibrillogenesis of endogenous FN on various FN-adsorbed SAMs. Immunofluorescence staining was also utilized to determine the endogenous FN fibrillogenesis by employing an anti-FN antibody ab6328 (abcam, UK) specific to cellular FN. After being cultured in serum free medium for 12 hours and 24 hours, osteoblasts on FN-adsorbed SAMs were fixed and stained for endogenous FN, F-actin cytoskeleton and nuclei.

Statistical analysis. Experiments were performed in triplicate at least unless otherwise stated. Quantitative values were expressed as mean ± SD.

Single factor

analysis of variance (ANOVA) technique with OriginPro (version 8.1) was utilized to assess the level of statistical significance.

3. RESULTS Characterization of SAMs. Various SAMs with terminal -OH, -NH2 and -CH3 were prepared on Au-sputtered silica wafers and glass slides. The static contact angles determined on various SAMs (Table 1) were 24.5 ± 1.0° (-OH), 49.0 ± 1.0° (-NH2) and 107.1 ± 1.8° (-CH3) for Au-SAMs, and were 16.0 ± 0.4° (-OH), 47.0 ± 2.3° (-NH2) and 105.7 ± 0.7° (-CH3) for Glass-SAMs. According to the Berg limit of 65° for defining hydrophilicity and hydrophobicity,30 only CH3-SAMs were hydrophobic,


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which is consistent with the reported data.14,31,32 Further XPS characterizations of the Au-SAMs (Figure S1) and Glass-SAMs (Figure S2) both indicated that the desired SAMs have been successfully prepared.

Table 1. Static contact angles of various SAMs with terminal -OH, -NH2 and -CH3 on Au-sputtered silica wafers and glass slides. SAMs -OH -NH2 -CH3

Au (degree) 24 ± 1 49 ± 1 107 ± 2

Glass (degree) 16 ± 1 47 ± 2 106 ± 1

Preparation and characterization of FN-conjugated microspheres. The -SH in FN molecules were conjugated to NH2-functionalized microspheres by utilizing Sulfo-SMPB as a coupling agent,24 and the FN-conjugated microspheres were achieved. A single FN molecule contains only 2-3 free -SH groups yet tens of free – NH2.33 Therefore, employing -SH instead of -NH2 in FN is supposed to minimize the influence of chemical conjugation to natural FN conformation. Figure 1 illustrates the preparation process of FN-conjugated microspheres (Figure 1a), together with the XPS

characterizations

in

each

step

(Figure

1b

and

c).

Compared

to

NH2-functionalized microspheres, Sulfo-SMPB modified microspheres demonstrated an increase in nitrogen and oxygen contents (Figure 1b). Following FN conjugation, the nitrogen percentage was further increased from 1.96% on Sulfo-SMPB modified microspheres to 9.35% on FN-conjugated microspheres (Figure 1b). Moreover, the comparison and analysis of high-resolution C1s spectra in Figure 1c revealed that, a


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288.8 eV new peak on Sulfo-SMPB microspheres was attributed to the imide groups of SMPB, and a 287.5 eV new peak on FN-conjugated microspheres was attributed to the amide groups on FN. XPS characterizations indicated the successful preparation of FN-conjugated microspheres. The FN density and thickness on FN-conjugated microspheres were also determined by microBCA Protein Assay Kit and AFM, respectively, therein the density was 1.64 ± 0.38 µg/cm2 and the thickness was 2.67 ± 0.35 nm.

Figure 1. Schematic diagram for preparation of FN-conjugated microspheres from amine latex particles (a) and the XPS spectra (b) and high resolution C1s spectra (c) of NH2-, Sulfo-SMPB-, and FN-conjugated microspheres.


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Evaluation of FN adsorption force on various SAMs. To evaluate FN adsorption force on various SAMs, the FN-conjugated microspheres were injected onto SAMs for adherence, following which flow shear stress was applied for microspheres detachment. Microspheres remaining on the SAM were photographed for counting, as pictures revealed in Figure S3. Since the flow field in the vicinity of a microsphere can significantly influence the microsphere detachment,34 only the microspheres with a distance from their neighbor microspheres greater than 5 times of microsphere radius were counted. As indicated in Figure S3a, the microsphere agglomerates labeled by rectangle and the sliding microspheres labeled by arrow were excluded. Consequently, percentage of the remained microspheres on SAMs was plotted as a function of the shear stress (Figure 2), within which the introduced shear stress capable of removing 50% of the initially adhered microspheres is denoted by τ50% as a critical shear stress.

Figure 2. Percentage of the remained FN-conjugated microspheres versus flow shear stress.


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According to the sphere/plane adhesion model analysis reported previously,27,35 the adsorption force of FN on SAMs (Fad) could be calculated by the following equation:

r r Fad = τ 50% r 2 1024 + 964 = τ 50% r 2 1024 + 964 l l0 + Ra

where, r is the radius of microspheres, Ra is the roughness of SAMs, l0 is the thickness of the conjugated FN layer on microspheres (2.67 ± 0.35 nm), and l is the thickness of the conjugated FN meshwork (l = l0 + Ra). l0 and Ra values were determined by AFM in a Scanasyst mode and Tapping mode, respectively. The calculated Fad values, including Fad per microsphere and Fad per FN monomer are summarized in Table 2. It is clear from Table 2 that Fad on various SAMs follows an order of -NH2 > -CH3 >> -OH. Particularly, Fad on OH-SAMs was one order of magnitude lower than Fad on NH2-SAMs and CH3-SAMs.

Table 2. The detected τ50% and calculated Fad values on various SAMs. SAMs -OH -NH2 -CH3

τ50% (Pa) 0.24 4.29 2.40

r (µm) 4.3 ± 0.3 4.3 ± 0.3 4.3 ± 0.3

Ra (nm) 0.48 0.54 0.57

l* Fad (nm) (nN/microsphere) 3.15 ± 0.35 5.0 ± 0.8 3.21 ± 0.35 87.6 ± 13.7 3.24 ± 0.35 48.8 ± 7.6

Fad (pN/FN) 1.5 ± 0.4 25.6 ± 7.5 14.1 ± 4.1

* l = l0 + Ra , where l0 is 2.67 ± 0.35 nm.

Conformation of FN adsorbed on various SAMs. It is well documented that the conformation of adsorbed protein could directly influence cell adhesion and cell traction force.3,7,19,36 In addition to assaying FN density (Figure 3a), the conformation


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of FN molecules was characterized by HFN7.1 monoclonal antibody (Figure 3b and c) to detect the cell-binding domains of FN. The intensity of HFN7.1 on various SAMs, which indicates the accessibility of cell binding domains by cells,3 is shown in Figure 3b. In particular, the highest HFN7.1 intensity was detected on OH-SAMs (0.72 ± 0.06 a.u./cm2), whereas the HFN7.1 intensities on CH3-SAMs (0.52 ± 0.03 a.u./cm2) and NH2-SAMs (0.55 ± 0.04 a.u./cm2) demonstrated no significant difference. Moreover, the immunofluorescent results (Figure 3c) is consistent with the ELISA assays (Figure 3b), implying that the accessibility of cell-binding domains follows an order of -OH > -NH2 ≈ -CH3.

Figure 3. FN adsorption on various SAMs (FN concentrations: 20 µg/mL). a) Surface density of adsorbed FN; b) HFN7.1 intensity measured by ELISA; c) Immunofluorescence images of cell-binding domains. * denoted p＜0.05, ** denoted p＜0.01.

The conformation of adsorbed FN was further examined by visualizing the FN


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organization via AFM37 in a peakforce tapping mode (Figure 4). AFM characterization reveals 3 kinds of FN organization on SAMs, including imbedded globular-like aggregates (solid arrows), protruding irregular aggregates (dotted arrows), and network-like structure (dashed arrows). In detail, the protruding irregular aggregates, which may be responsible for the best accessibility of cell binding domains, were only detected on OH-SAMs. On the other hand, dense imbedded globular-like aggregates were detected on CH3-SAMs, whereas network-like structure was observed on NH2-SAMs. Similar FN organization on OH surface was reported by Llopis-Hernandez et al.14 However, their observed dense network-like organization on CH3 surface is divergent with our observation. This divergence is possibly due to the different FN adsorption duration (30 minutes in Llopis-Hernandez’s work14 yet 2 hours in this work). Bough et al.38 performed FN adsorption on hydrophobic fluorosilane-treated glass for 1 hour and observed similar compact and global FN organization by using the fluorescence resonance energy transfer spectroscopy.

Figure 4. Organization of adsorbed FN on various SAMs visualized by AFM (500×500 nm).


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Osteoblast traction force on various FN-adsorbed SAMs. Formation of FAs and FBs is the prerequisite for generation of cell traction force. Once cells become specifically adhered, the magnitude of cell traction force depends on the number and size of FAs and can be remarkably enhanced by FBs.39-41 Since FAs are vinculin rich and FBs are tensin rich,19 vinculin and tensin are often used to represent FAs and FBs, respectively, and further to indicate cell traction force.15,36,42,43 In this study, the expressions and organizations of vinculin and tensin were detected to indicate cell traction force. To examine the dynamic contribution of the absorbed FN, both 2 hours (Figure 5) and 12 hours (Figure 6) of osteoblasts cultured in serum free medium were determined for cell traction force.

As indicated in Figure 5a, when osteoblasts were cultured in serum free medium for 2 hours, the vinculin and tensin expression on various FN-adsorbed SAMs followed an order of -OH > -NH2 ≈ -CH3. Similar order was observed from the imaged organization (Figure 5b) and the quantified total area of FAs and FBs (Figure 5c), which demonstrated that the traction force of osteoblasts on various SAMs follows an order of -OH > -NH2 ≈ -CH3 after 2 hours of cells seeding. This chemistry-dependence of cell traction force takes the same trend as HFN7.1 intensity (Figure 3), indicating that HFN7.1 intensity, i.e. the accessibility of cell binding domains, might be the main mediator for early cell traction force.


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Figure 5. Expression and organization of vinculin and tensin after osteoblasts were seeded in serum free medium for 2 hours on various FN-adsorbed SAMs. a) Western


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blotting

images

and

quantitative

analysis

of

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tensin

and

vinculin.

b)

Immunofluorescence staining images of tensin (green) and vinculin (green), together with F-actin (red) and nuclei (blue). C) Total area of FAs and FBs on various SAMs, quantified by immunofluorescence staining image analysis. The measurements were performed at least 20 cells per group. Scale bar indicates 10 µm. * denoted p＜0.05, ** denoted p＜0.01.

On the other hand, when the osteoblasts culture in serum free medium is up to 12 hours, a sharp increase of vinculin and tensin expressions was detected on NH2-SAMs (Figure 6a). As a result, the vinculin and tensin expressions on NH2-SAMs were significantly

higher

than

those

on

OH-SAMs

and

CH3-SAMs.

The

immunofluorescence staining of vinculin and tensin demonstrated the remarkable FAs and FBs growth on NH2-SAMs (Figure 6b). More than 25% of FAs on NH2-SAMs were from 5µm2 to 15 µm2, whereas nearly all the FAs on CH3-SAMs were below 4 µm2 (Figure S4). According to the FAs and FBs area results shown in Figure 6c, the traction force of osteoblasts after 12 hours of cell incubation on various SAMs follows a chemistry-dependence as -NH2 > -OH > -CH3. This is quite different from the results of HFN7.1 intensity (-OH > -NH2 ≈ -CH3), implying that the late cell traction force is not only dependent on FN cell binding domains. In fact, FN adsorption force may also contribute to the late cell traction force.


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Figure 6. Expression and organization of vinculin and tensin after osteoblasts were seeded in serum free medium for 12 hours on various FN-adsorbed SAMs. a) Western


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blotting

images

and

quantitative

analysis

of

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tensin

and

vinculin.

b)

Immunofluorescence staining images of tensin (green) and vinculin (green), together with F-actin (red) and nuclei (blue). C) Total area of FAs and FBs on various SAMs, quantified by immunofluorescence staining image analysis. The measurements were performed at least 20 cells per group. Scale bar indicates 10 µm. * denoted p＜0.05, ** denoted p＜0.01.

Reorganization of the adsorbed FN on various SAMs. It is documented that FN reorganization is the outcome of cell traction force44 in a top-down mode. According to our hypothesis, FN adsorption force could modulate FN reorganization, which in turn regulates cell adhesion and cell morphology in a bottom-up mode. Therefore, to verify the role of FN adsorption force in late cell adhesion, the reorganization of the adsorbed FN (Figure 7-FN) and the osteoblast morphology represented by F-actin (Figure 7-F-actin) were photographed after 12 hours of cells incubation in serum free medium. The observed dark area surrounding and underlying osteoblasts represented the contribution of cell traction force16,39 from the initial cell adhesion to the end of 12h seeding, while the F-actin cytoskeleton indicated the instantaneous cell morphology at the end of 12h seeding.

As shown in Figure 7-FN, the dark area on OH-SAMs was sparse with FN. In contrast, almost intact FN molecules were remained on CH3-SAMs. Regarding NH2-SAMs, the underlying FN molecules could even orient along the upper osteoblasts, which is a strong evidence for FN reorganization. What is more


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interesting, the cell tracing dark area on OH-SAMs was significantly greater than the upper osteoblast cytoskeleton, implying that osteoblasts on OH-SAMs would experience good spreading during early cell adhesion and then contract at the late stage of cell adhesion. Meanwhile, no cell contraction was observed on CH3-SAMs and NH2-SAMs.

Figure 7. Reorganization of adsorbed FN on various SAMs after osteoblasts were seeded in serum free medium for 12 hours. Immunofluorescence staining images of F-actin (red), FN (green) and nuclei (blue). Scale bar indicates 10 µm.


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Figure 8. Fibrillogenesis of endogenous FN on various SAMs after osteoblasts were


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seeded in serum free medium for 12 hours and 24 hours. Immunofluorescence staining images of endogenous FN (green), F-actin (red) and nuclei (blue). The image areas within the rectangles were magnified. Scale bar indicates 10 µm in the magnified images and 20 µm in the others.

Fibrillogenesis of endogenous FN on various FN-adsorbed SAMs. It is reported that endogenous FN can be secreted by adherent cells and subsequently assembled into insoluble multimeric fibrils on biomaterial surface.45,46 FN fibrillogenesis is dependent on FN reorganization and mediated by cell traction force,36 hence it may be regulated by FN adsorption force as well according to our hypothesis.

Figure 8 reveals the fibrillogenesis of endogenous FN on various FN-adsorbed SAMs. After 12 hours of osteoblasts incubation in serum free medium, NH2-SAMs was abundant with FN matrix and obvious fibrils were observed. On the other hand, the endogenous FN secreted on CH3-SAMs and OH-SAMs was sparse with fibrils. When the cell culture time is up to 24 hours, the increase of FN fibrils was conspicuous on NH2-SAMs, but was negligible on CH3-SAMs and OH-SAMs. In one word, the fibrillogenesis of endogenous FN on various FN-adsorbed SAMs followed an order of NH2 >> CH3 ≈ OH.

4. DISCUSSION It is well recognized that the surface properties of biomaterials could determine


25

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their bio-functions by regulating cell adhesion,4,31,47,48 and therein protein adsorption plays a key role.1,49 Extensive studies have proved that the surface chemistry, wettability and topography of biomaterials influence the type, amount and conformation of adsorbed proteins. However, few attentions have been paid to the adsorption force of proteins on a biomaterial surface, though the interaction strength of proteins with material surface have been often proposed to explain the cell response to material surface.11,23 In our research, it is demonstrated that the protein adsorption force on a biomaterial surface relies on the surface chemistry, and cell adhesion and the consequent matrix reorganization could be regulated by protein adsorption force together with protein conformation. Our findings provide another perspective to understand cell-biomaterial interactions.

The combination of PPFC and sphere/plane adhesion model has been previously reported to detect the interaction force between functionalized microspheres (-NH2 and -OH) and BSA-conjugated stainless steel.35 The obtain results (6.2 ± 1.9 nN for -NH2 and 2.7 ± 1.5 nN for -OH) are reliable since they are in the same order of magnitude to the data from AFM measurements.22 In this study, similar method was employed to detect FN adsorption force on SAMs with various chemistries. The obtained adsorption force demonstrated an obvious chemistry-dependence of -NH2 > -CH3 >> -OH (Figure 2). This result seems divergent with the general view that stronger protein adsorption force should originate from more hydrophobic surfaces (-CH3 is much more hydrophobic than -OH and -NH2, as depicted in Table 1). But in actual fact, protein adsorption force is also modulated by the type of adsorbed protein


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and the surface charge of biomaterials. A typical example of this is, Wang et al. reported that the adsorption force of BSA on hydrophilic OH-SAMs is 2.54 ± 0.14 nN, significantly higher than that on hydrophobic CH3-SAMs (0.87 ± 0.39 nN).21 In our case, FN is negatively charged and NH2-SAMs are positively charged in pH 7.4 medium. The electrostatic interaction between FN and NH2-SAMs is positive to FN adsorption force, leading to the higher FN adsorption force on NH2-SAMs than on CH3-SAMs. In addition, FN organization on various SAMs (Figure 4) may partially explain the chemistry-dependence of FN adsorption force. The network-like FN organization on NH2-SAMs is quite different from the imbedded globular-like FN organization on CH3-SAMs, implying that the FN interacting sites on NH2-SAMs are more than those on CH3-SAMs.

The density and conformation of the adsorbed proteins are widely employed to characterize the adsorbed proteins.3,6-8 The conformation of FN molecules can be generally represented by the accessibility of cell binding domains which is of great importance for initial cell adhesion.3 Our results (Figure 3b and c) reveal that the accessibility of cell binding domains follows an order of -OH > -NH2 ≈ -CH3, which is consistent with the previous report.3 In addition, organization of the adsorbed FN determined by AFM (Figure 4) is the visual reflection of FN conformation on various SAMs. To the same conclusion, the protruding irregular FN aggregates on OH-SAMs demonstrate the best accessibility of cell binding domains.

It is well documented that cell-material interactions consist of a bottom-up mode


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(from-material-to-cell) and a top-down mode (from-cell-to-material), of which the relationship is interdependent and dynamic.[3, 23, 31] In theory, the initiation of early cell adhesion is in a bottom-up mode and should rely on the accessibility of cell binding domains. In this respect, the FAs and FBs formation after 2 hours of cells seeding was detected for cell adhesion. Selection of 2 hours seeding is because that the time-point of 2 hours after cell seeding is generally regarded as the stage of initial cell adhesion.50 Consistent with our deduction, the chemistry-dependence of initial FAs and FBs formation (-OH > -NH2 ≈ -CH3, Figure 5) is the same as that of the accessibility of cell binding domains (-OH > -NH2 ≈ -CH3, Figure 3).

Once the adhered cells generate cell traction force, it will be transferred in a top-down

mode,

and

dynamically

regulated

by

the

underlying

proteins,

protein-biomaterial interface and biomaterial. To observe the dynamic process, the cell traction force of osteoblasts after 12 hours seeding (characterized by FAs and FBs) was further detected. The obtained cell traction force demonstrated a different chemistry-dependence (-NH2 > -OH > -CH3, Figure 6) compared to that at early cell adhesion (-OH > -NH2 ≈ -CH3, Figure 5). Contribution of the FN adsorption force together with the organization of the adsorbed FN can explain this divergence.

In particular, the FN adsorption force on various SAMs is in an order of -NH2 > -CH3 >> -OH (Table 2). For OH-SAMs, the initial cell traction force (after 2 hours of cells seeding) is the strongest (Figure 5) but the FN adsorption force is the weakest. The weak FN adsorption force cannot resist the strong cell traction force, leading to


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the degradation of FAs and reduction of cell traction force in the late stage of cell adhesion (Figure 6).17-19 This viewpoint is further supported by the other result (Figure 7-merge) that cells on OH-SAMs obviously contracted after 12 hours of cells seeding. In contrast, the highest FN adsorption force on NH2-SAMs is strong enough to withstand the relatively low initial cell traction force. As a result, FAs grow up on NH2-SAMs and give a boost to the late cell traction force (Figure 6). Despite of the relatively high FN adsorption force and low initial cell traction force on CH3-SAMs, the imbedded globular-like FN organization on CH3-SAMs is much more rigid37 than the network-like FN organization on NH2-SAMs (Figure 4), which prevents FN on CH3-SAMs from reorganizing to expose other cell binding domains (Figure 7-FN). Correspondingly, formation of FAs and FBs is hampered and the cell traction force on CH3-SAMs remains low even after 12 hours of seeding. All in all, both the FN adsorption force and the organization of adsorbed FN play key roles in the late cell adhesion.

As is well known, the vital step for the fibrillogenesis of endogenous FN is the cell-driven exposure of cryptic sites mainly localized within FN type Ⅲ domains.51 Cell traction force can stretch FN molecules via FAs or FBs, whereby the FN type Ⅲ domains are unfolded to expose the cryptic sites.16,52-54 Accordingly, FN stretching and reorganization driven by cell traction force is a prerequisite for FN fibrillogenesis. According to our hypothesis, the FN fibrillogenesis is also affected by FN adsorption force.


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According to Table 2, the FN adsorption force on OH-SAMs is 1.47 ± 0.43 pN/FN whereas the traction force derived by a single FN-integrin is tens of pN.55,56 At the early stage of cell adhesion, the FN adsorption force on OH-SAMs is too small to resist the highest cell traction force (Figure 5), leading to FN desorption with negligible fibrillogenesis of endogenous FN (Figure 8). On the other hand, the FN adsorption force on NH2-SAMs (25.56 ± 7.47 pN/FN) is strong enough, and FN stretching and reorganization can benefit from the network-like FN organization on NH2-SAMs (Figure 4). Consequently, the synergistic effects of cell traction force, FN adsorption force and FN organization on NH2-SAMs gave rise to the rearrangement of adsorbed FN molecules along cell orientation (Figure 7-FN). Reasonably, obvious fibrillogenesis of endogenous FN was observed (Figure 8). Similar to NH2-SAMs, the strong FN adsorption force on CH3-SAMs (14.10 ± 4.12 pN/FN) can resist the relatively low initial cell traction force. However, the imbedded globular-like FN organization on CH3-SAMs is too rigid37 for stretching and reorganizing. Accordingly, the hampered FN reorganization also leads to negligible FN fibrillogenesis on CH3-SAMs (Figure 8).

5. CONCLUSION In this study, FN adsorption force on SAMs with various surface chemistries (-OH, -NH2 and -CH3) was quantified, and FN conformation was characterized by both the accessibility of FN cell-binding domains and FN organization. It is


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demonstrated that the initial osteoblast adhesion (2 hours after cells seeding) is mainly governed by the accessibility of FN cell-binding domains. Nevertheless, the late cell adhesion (12 hours after cells seeding) is influenced by FN adsorption force and FN organization. The vital role of FN adsorption force in cell adhesion was further verified by the reorganization of the adsorbed FN and the fibrillogenesis of endogenous FN. These findings shed new light on the intricate role of protein adsorption in cell-material interactions, and may help for better biomaterial design.

ASSOCIATED CONTENT Supporting Information XPS characterizations of the Au-SAMs (Figure S1) and Glass-SAMs (Figure S2). Representative images of the detachment process of FN conjugated microspheres under laminar shear stress (Figure S3). Size distribution of FBs and FAs on various SAMs (Figure S4). Calculation of the fibronectin adsorption force per FN monomer in Table 2. These materials are available at free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Y.F. Luo); Fax: +86 23 65102507.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This work was supported by Grants from the National Natural Science Foundation of China (No. 31370946) and the Fundamental Research Funds for the Central Universities (No. CDJZR11235501).

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fibrinogen-coated surfaces. Biomaterials 2001, 22, 827-832. (51) Pankov, R.; Yamada, K.M. Fibronectin at a glance. J. Cell. Sci. 2002, 115(2), 3861-3863. (52) Vadillo-Rodriguez, V.; Bruque, J.M.; Gallardo-Moreno, A.M.; Gonzalez-Martin, M.L. Surface-dependent mechanical stability of adsorbed human plasma fibronectin on Ti6Al4V: Domain unfolding and stepwise unraveling of single compact molecules. Langmuir 2013, 29, 8554-8560. (53) Erickson, H.P. Reversible unfolding of fibronectin type Ⅲ and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin. Proc. Natl. Acad. Sci. 1994, 91, 10114-10118. (54) Lemmon, C.A.; Ohashi, T.; Erickson, H.P. Probing the folded state of fibronectin type Ⅲ domains in stretched fibrils by measuring buried cysteine accessibility. J. Biol. Chem. 2011, 286(30), 26375-26382. (55) Kong, F.; Garcia, A.J.; Mould, A.P.; Humphries, M.J.; Zhu, C. Demonstration of catch bonds between an integrin and its ligand. J. Cell. Biol. 2009, 185(7), 1275-1284. (56) Thoumine, O.; Kocian, P.; Kottelat, A.; Meister, J.J. Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis. Eur. Biophys. J. 2000, 29, 398-408.


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Biomacromolecules

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Adsorption Force of Fibronectin on Various Surface Chemistries and

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