BT 643: BIOINTERFACE ENGINEERING

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1. Refer to the appended research article “Effect of Functional Groups of Self-Assembled

Monolayers on Protein Adsorption and Initial Cell Adhesion” (ACS Biomater. Sci. Eng.
2018, 4, 9, 3224-3233) and answer the followings:
a) On which surface, the rate of cell adhesion was the maximum and why?
b) On which surface, the % of adhered cells was the maximum and why?
c) Explain the significance of cell shape index towards cell adhesion?
d) Which surface was found to be the best and why?
e) Why do the images in the figure 6 exhibit fluorescence in different colors in different
regions of the cell? [6]

2. In a cell adhesion experiment on a surface, the following data were obtained:

Time (min) % Surface Coverage

0 0
15 1
30 2
45 4
60 7
120 12
360 27

Determine the rate constant value (h-1) for the cell adhesion? [4]

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 Article

Cite This: ACS Biomater. Sci. Eng. 2018, 4, 3224−3233 pubs.acs.org/journal/abseba

Effect of Functional Groups of Self-Assembled Monolayers on

Protein Adsorption and Initial Cell Adhesion
Abshar Hasan,*,† Sudip K. Pattanayek,*,‡ and Lalit M. Pandey*,†
†
Bio-Interface & Environmental Engineering Lab, Department of Biosciences and Bioengineering, Indian Institute of Technology
Guwahati, Assam 781039, India
‡
Macromolecules and Interfaces Laboratory, Department of Chemical Engineering, Indian Institute of Technology Delhi, Hauz
Khas, New Delhi 110 016, India
*
S Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via INDIAN INST OF TECH GUWAHATI on May 9, 2020 at 12:44:24 (UTC).

ABSTRACT: Surface modification plays a vital role in regulating

protein adsorption and subsequently cell adhesion. In the present work,
we prepared nanoscaled modified surfaces using silanization and
characterized them using Fourier-transform infrared spectroscopy
(FTIR), water contact angle (WCA), and atomic force microscopy
(AFM). Five different (amine, octyl, mixed, hybrid, and COOH)
surfaces were prepared based on their functionality and varying
wettability and their effect on protein adsorption and initial cell
adhesion was investigated. AFM analysis revealed nanoscale roughness
on all modified surfaces. Fetal bovine serum (FBS) was used for protein
adsorption experiment and effect of FBS was analyzed on initial cell
adhesion kinetics (up to 6 h) under three different experimental
conditions: (a) with FBS in media, (b) with preadsorbed FBS on
surfaces, and (c) incomplete media, i.e., without FBS. Various cell features such as cell morphology/circularity, cell area and
nuclei size were also studied for the above stated conditions at different time intervals. The cell adhesion rate as well as cell
spread area were highest in the case of surfaces with preadsorbed FBS. We observed higher surface coverage rate by adhering
cells on hybrid (rate, 0.073 h−1) and amine (0.072 h−1) surfaces followed by COOH (0.062 h−1) and other surfaces under
preadsorbed FBS condition. Surface treated with cells in incomplete media exhibited least adhesion rate, poor cell spreading and
improper morphology. Furthermore, we found that initial cell adhesion rate and Δadhered cells (%) linearly increased with the
change in α-helix content of adsorbed FBS on surfaces. Among all the modified surfaces and under all three experimental
conditions, hybrid surface exhibited excellent properties for supporting cell adhesion and growth and hence can be potentially
used as surface modifiers in biomedical applications to design biocompatible surfaces.
KEYWORDS: silanization, protein adsorption, cell adhesion, kinetics, wettability

1. INTRODUCTION modified biomimetic surfaces to imitate the extracellular matrix

Interdisciplinary research in the field of tissue engineering
amalgamates basic biological sciences, material science,
engineering and medical technology to overcome the damage
or failure of tissue/organ due to injuries, diseases or trauma
and for many other applications.1,2 Bone implant, heart stents
and artificial tooth are the most commonly used biomaterial
implants nowadays.3 Protein adsorption and cell adhesion are
the primary events that take place on biomaterial surfaces as
soon as they come in contact with the body fluids. Nonspecific
protein adsorption from the protein rich plasma may cause
deterioration of biomaterial due to platelet adhesion and
activation resulting in thrombosis.4 The implant’s surface
interactions with biological system play an important role in
deciding its fate as these interactions describe the response of
proteins, cells, tissue, and organs.5 In this direction, biomimetic
surfaces are designed by various surface modification
techniques to minimize the foreign body response (FBR)
and maximize the purpose of implant.6−8 The tendency of
(ECM) provides platform to support cell adhesion and
proliferation. Surface modifications using various physical,
chemical, and biological processes had been reported to
circumvent such unwanted processes.8−10 Surfaces of ceramics,
polymers, and metals have been modified via physical (for
example, plasma treatment) and chemical methods (for
example, functionalization via self-assembled monolayers
[SAMs], immobilized ligands, etc.) to carefully modulate
protein adsorption, cell adhesion, and differentiation.11−23
In a recent study, it was found that surface functional groups
tune protein adsorption, which in turn regulate cell adhesion
and spreading.24 Dynamic behavior of adsorbed cell-adhesive
protein (e.g., vitronectin) due to adhering cells on the surface
regulate cell adhesion at the cell−material interface.25 Garcia et
Received: July 13, 2018
Accepted: August 13, 2018
Published: August 13, 2018

© 2018 American Chemical Society 3224 DOI: 10.1021/acsbiomaterials.8b00795

ACS Biomater. Sci. Eng. 2018, 4, 3224−3233
ACS Biomaterials Science & Engineering
al.26 reported the enhanced expression of integrins receptors
on hydrophilic surface indicating better cell adhesion on them
as compared to hydrophobic surfaces. Arima and Iwata further
elaborated that adhesion of HUVECs cells was best responded
by CH3/OH surface (θ = 40°), whereas HeLa cells adhered
best on CH3/OH and CH3/COOH surfaces (θ = 50°).27
Their results agrees well with the literature, which states that
cell adhesion and spreading takes place optimally on surfaces
with moderate wettability in the range of 50−80°. On the
contrary, the attachment and proliferation of osteoblastic cells
were found to increase with an increase in surface wettability,
which was correlated to fibronectin adsorption.28,29 FTIR and
XPS studies revealed that interactions between the internal
hydrophobic domains of protein and surfaces lead to the
change in the secondary structure of proteins and may expose
the cell adhesive sites resulting in enhanced cell adhesion and
spreading.13,30,31 Moreover, Inoue et al.32 emphasized that
surface ζ potentials of polymeric brush substrates play major
role in regulating protein adsorption and cell adhesion rather
than surface wettability. Therefore, it highlights the need to
investigate this very important yet complicated phenomenon
on model surfaces with wide range of wettability.
SAMs of silanes and thiols form highly organized covalently
attached organic molecules with nanothick coatings which in
turn provide tunable surface properties.33,34 Desirable surface
properties can be easily generated by choosing various
functionalized molecules from the huge available library.
Although SAMs with different functionalities such as −NH2,
−COOH, -Cl, −OH, −CH3, and their mixed combinations
have been explored for protein adsorption and cell
adhesion31,35−37 but their effect on initial cell adhesion kinetics
and spreading is less explored. This inspired use to undertake
this challenge to explore the underlying aspects of initial cell
adhesion on surfaces exhibiting different surface functionalities
(wettability) in the presence and absence of serum proteins.
In the present work, we prepared surfaces with different
wettability by varying the surface functionalities using various
mono, mixed and hybrid SAMs.13,38 The adsorption of serum
proteins was examined on these modified surfaces in terms of
adsorbed amounts using bicinchoninic acid (BCA) assay and
change in secondary structures using FTIR analysis. The
kinetics of initial L929 mouse fibroblast cell adhesion was
investigated in the presence of modified surfaces (a) without
FBS proteins in media, (b) with FBS in media, and (c) with
FBS preadsorbed on surfaces. With the change in the protein
behavior at differently functionalized surfaces, the behavior of
the adhering cells also changes. Along with kinetics studies
under all three experimental conditions stated above, we also
determined the effect of surface modifications on cell adhesion
and spreading, their morphology and nuclei size.
2. MATERIAL AND METHODS
2.1. Materials. Aminopropyl triethoxysilane (APTES, Cat. No.
440140), triethoxy(octyl)silane (TEOS, Cat. No. 440213), phalloi-
din- fluorescein isothiocyanate (FITC) labeled (Cat. No. P5282),
propidium iodide (PI, Cat. No. P4170), anhydrous toluene, dibutyltin
dilaurate, and p-tolyl isocyanate were purchased from Sigma-Aldrich,
India. Fetal bovine serum, vinculin primary antibody (Cat no.
700062) and secondary antibody-Alexa Fluor 350 (Cat. No. A11046)
were purchased from ThermoFisher Scientific, India. Methanol,
toluene, circular glass coverslip (14 mm diameter), sodium chloride
(NaCl), potassium chloride (KCl), monobasic potassium phosphate
(KH2PO4), dibasic sodium phosphate (Na2HPO4), sulfuric acid
(H2SO4), hydrogen peroxide (H2O2), and diiodomethane (DI) were
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procured from Himedia, India. Double-distilled water (Mili-Q, 18
MΩ) was used throughout the work.
2.2. Surface Silanization/Modification. Five different surfaces
with varying wettability and functionality were prepared using
aforementioned silane groups based on our previous reports.13,31,38−40
Briefly, glass coverslips were washed with piranha (H2SO4:H2O2 = 7:3
v/v), ammonia (water:H2O2:NH3 = 5:1:1 v/v), and HCl (water:-
H2O2:HCl = 3:1:1 v/v) solutions and dried overnight at 50 °C prior
to surface modification. For monotype amine and octyl SAMs
surfaces, cleaned substrates were dipped in 1% (v/v) solution of
APTES and TEOS in anhydrous toluene and incubated for 24 h
under inert conditions at room temperature. Mixed (amine-octyl)
SAM was prepared by mixing APTES and TEOS in 1:1 ratio (1% v/v)
under similar experimental conditions. Carboxylic (COOH) modified
surfaces were synthesized by oxidizing octyl modified surfaces with
5% acidified KMnO4 solution for 30 min at room temperature.41
Similarly, hybrid SAM was prepared by treating amine modified
surface with p-tolyl isocyanate solution (1%, v/v) for 4 h in the
presence of catalyst dibutyltin dilaurate. The reaction between amine
(NH2) head groups at surface and isocyanate (NCO) forms urea
linkage at surface.13,38−40
2.3. Characterization of Modified Surfaces. The modified
surfaces were characterized in terms of functional groups, wettability
and morphology using FTIR, contact angle Goniometer and AFM,
respectively. Surface silanization was confirmed using FTIR
(Spectrum TWO, PerkinElmer) instrument at a scanning rate of 15
scans per second with resolution 1 cm−1, taking unmodified surface as
background. Modified silicon surfaces were characterized for surface
topology and roughness. Surface silanization resulted in nanoscale
roughness as evidenced by AFM analysis and was compared with
unmodified surfaces. Innova AFM system (Bruker) fitted with silicon
nitride tip of <10 nm radius was used for analysis. Surface roughness
parameter (Ra) was determined using Gwyddion software obtained
from GNU General Public License, as described in our previous
reports.33,38,42 Contact angles of water and MI on different surfaces
were recorded for evaluating wettability and surface energies using
Holmarc instrument (India) with the sessile drop method reported
previously by our group.31,33 Contact angle was recorded for at least
seven different points on the same surface and analyzed using
compatible software provided by manufacturer. Formation of drop on
surface depends on the surface hydrophobicity and nature of solute
and is regulated by the interfacial tensions and is related through
Young’s equation. Surface energy (γSV) was evaluated based on
contact angles of both the liquids, using following geometric mean
expression.9,31,42
γij = γi + γj − 2θ[(γidγjd)1/2 + (γiPγjP)1/2 ]
(1)
Where, γdi and γPi are dispersive and polar component of liquid surface
energy while γdj and γPj are the dispersive and polar component of solid
surface energy, respectively. θ is the interaction parameter, whose
value is taken as 1 for most of the similar types of molecules.
2.4. Protein Adsorption on Modified Surfaces and Secon-
dary Structure Analysis. Physical and chemical properties of
surfaces such as roughness, wettability and functionality regulate
protein adsorption which further governs cell adhesion and
proliferation.27,43 FBS (10%, v/v) prepared in phosphate buffer
(PBS, pH 7.4) was used for protein adsorption studies. For
adsorption, modified surfaces were incubated in protein solutions
for 1 h at room temperature and were later washed to remove
unbound protein molecules. Adsorbed protein molecules were
desorbed by using reported protocol.31,40 Briefly, surfaces were
treated with 5% (w/v) sodium dodecyl sulfate (SDS) solution for 1 h
at 37 °C under agitation (120 rpm). Desorbed protein masses were
quantified using QuantiPro BCA assay kit (Sigma, India).40 For
secondary structure analysis, the modified surfaces were incubated in
FBS solution for 1 h and washed thrice with PBS followed by rinsing
with Mili-Q water to remove unbound protein molecules. Surfaces
were later dried for 30 min at 37 °C before recording FTIR spectra
under water and CO2 correction mode to avoid background
DOI: 10.1021/acsbiomaterials.8b00795
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interferences. The percentage distribution of secondary structures (α-

helix, β-sheet, β-turn, random and side chain) of adsorbed FBS on
different surfaces were evaluated and compared with the native
structure of the FBS (i.e., without adsorption). Amide I range (1600−
1700 cm−1) of the recorded FTIR spectra after FBS adsorption were
fitted with Gaussian curves in Origin 8.5 software to find the
percentage of each secondary structures.
2.5. Cell Adhesion on Modified Surfaces. L929 mouse
fibroblast cell line was maintained in CO2 incubator at 37 °C and
5% CO2 and cultured in Dulbecco’s modified Eagle’s medium
(DMEM, HiMedia, India) supplemented with 10% (v/v) FBS and 1%
(v/v) antibiotic (Pen Strep, Invitrogen). Modified surfaces were
sterilized by placing them under UV for 45 min prior to experiment.
Cells were grown in 25 cm2 cell culture flasks (Corning) to 80−90%
confluency. After trypsin treatment, cells were washed with excess
media, centrifuged and counted using hemocytometer. One ×105 cells
per mL were added to each surface and incubated for 6 h under three
different conditions: (i) incomplete media, i.e., without FBS, (ii)
complete media, i.e., supplemented with 10% FBS, and (iii) surfaces
preadsorbed with FBS. Two set of experiments were established; in Figure 1. Effect of surface modification on surface’s wettability and
one experiment, cells were counted using hemocytometer while in adsorbed protein mass from 10% FBS solution (PBS, pH 7.4) on
another experiment their images were recorded after 15, 30 45, 60 various modified surfaces.
120, and 360 min of seeding. For cell imaging experiment, cell
adhered surfaces were washed thrice with filtered PBS and fixed with Table 1. Contact Angles and Surface Energies of Modified
4% (v/v) paraformaldehyde solution overnight at 4 °C. Surfaces
Cells were further treated with 2% (w/v) BSA and 0.2% (v/v)
Triton X-100 for 6 h followed by actin filaments staining with FITC- static contact angle
Phalloidin for 12 h. Vinculin protein was first targeted with (deg)
antivinculin 1° antibody for 6 h, followed by Alexa fluor-350 labeled surface energy surface roughness
2° antibody for another 6 h. Cells nuclei were stained by incubating surfaces water MI (mJ.m−2) (Ra, nm)
substrates in 20 μg/mL of PI for 2 h at room temperature under dark unmodified 15 ± 1 22 ± 1 71 ± 1 0.10
conditions. Post staining, substrates were washed thrice with PBS and COOH 45 ± 1 38 ± 1 55 ± 1 1.14
imaged using Nikon fluorescent microscope (Model: Nikon Eclipse amine 60 ± 1 39 ± 1 47 ± 1 0.95
Ti−S). ImageJ software (developed at the National Institutes of hybrid 79 ± 2 43 ± 1 39 ± 1 0.46
Health) was used for analyzing cells and nuclei area and cell circularity
mixed 81 ± 1 42 ± 1 39 ± 1 1.12
of the adhered cells on different modified surfaces.
2.6. Statistical Analysis. Experiments were carried out in octyl 103 ± 2 60 ± 1 29 ± 1 0.51
triplicate, and results are expressed as mean ± standard deviation.
Statistically significant differences (p < 0.001 (#) and p < 0.05 (##)) stretching of Si-CH2R groups due to attachment of silane
between the means of different groups were determined by software molecules. Siloxane (Si−O−Si) bond formation between glass
SigmaPlot version 14.0 using one-way analysis of variance (ANOVA) and silane molecule after condensation can be seen at 1010
with the Tukey test.
cm−1 wavenumber, further confirming the silanization process.
The presence of NH groups in amine, mixed, and hybrid
3. RESULTS AND DISCUSSION surfaces corresponds to the peak at 1672 cm−1, confirming the
3.1. Characterization of Modified Surfaces. Surface attachment of aminosilanes that possess NH2 as headgroup.
functional groups mainly regulate surface wettability if the Furthermore, hybrid surface was confirmed because of the
surface roughness is significantly low,33 polar groups increase formation of urea linkage between NH2 and NCO groups,
wettability, whereas hydrophobic/nonpolar groups decrease it. which resulted in the appearance of a peak at 1640 cm−1.31,38
Figure 1 shows the effect of surface modification on change in Peaks at 2935 and 2850 cm−1 correspond to asymmetric and
surface wettability due to varying terminal functional moieties. symmetric stretching of CH2 groups of the corresponding
We observed high wettability (θ = 15 ± 1°) for unmodified silanes. Peak at 2965 cm−1 in octyl, mixed, and hybrid surfaces
surface due to generation of OH groups after piranha represent asymmetric (νa-CH3) stretching peak of CH3
treatment. COOH (θ = 45 ± 1°) and amine (NH2, θ = 60 group.33
± 1°) groups being polar in nature also resulted in hydrophilic AFM images as shown in Figure 3 exhibited nanorough
surfaces while mixed (θ = 79 ± 2°) and hybrid (θ = 81 ± 1°) surface morphology for all the modified surfaces. Higher Ra
surfaces exhibited moderate wettability due to presence of both values (mentioned in Table 1) as compared to unmodified
hydrophobic and hydrophilic groups. Octyl chain is hydro- surface (Ra = 0.1 nm) indicated surface modification via
phobic in nature and hence induced hydrophobicity (θ = 103 silanization. Mixed surface (1.12 nm) exhibited higher surface
± 2°) to octyl modified surface. Intermolecular forces that exist roughness as compared to amine (Ra = 0.95 nm) and octyl
on the interfaces determine the surface energy. Using surface (Ra = 0.51 nm) due to phases separation and randomly
expression 1, we evaluated the surface energies of all modified distributed amine and octyl fractions on the surface. Hybrid
surfaces as tabulated in Table 1. It was found to be linearly surface (Ra = 0.46 nm) resulted smoother surface due to
related to surface wettability, i.e., surface exhibiting higher monomolecular structure as compared to mixed surface.
wettability (unmodified and COOH) possess higher surface Similar surface roughness of such modified surfaces have also
energies. been previously reported.13,38
Figure 2 shows the FTIR spectra of modified surfaces. Peaks 3.2. Effect of Surface Modification on Protein
at 860 and 1110 cm−1 in all the spectra corresponds to Si−C Adsorption. Surface roughness and topology, wettability,
3226 DOI: 10.1021/acsbiomaterials.8b00795
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Figure 2. FTIR characterization spectra of modified surfaces.

Figure 3. AFM images showing surface topologies of (a) unmodified, (b) amine, (c) octyl, (d) mixed, (e) hybrid, and (f) COOH surfaces. Scale
bar is 100 nm.

surface potential, and surface energy are physicochemical

properties of a surface that can be regulated by surface
modification. We have previously shown that such surface
properties do regulate highly complex process of protein
(insulin, lysozyme, BSA, fibrinogen) adsorption, adsorbed
mass, and their secondary structures.13,39,44−46 In the present
work, we aimed to determine the effect of such modification on
serum protein adsorption and subsequently their effect on cell
adhesion and spreading. To mimic cell culture conditions, we
used 10% FBS solution for protein adsorption and determined
the adsorbed mass using BCA assay.40 Figure 1 shows the
adsorbed mass of proteins on different modified surface as a
function of surface hydrophobicity. We noticed linearly
increasing adsorbed protein mass with the increasing surface
hydrophobicity. The pattern of protein adsorption observed in
this study is similar to the pattern of serum albumin adsorption
reported by our group previously.13,39 Because serum contains
a very high concentration of albumin (∼66 kDa) i.e., 35−50
mg/mL, we considered that maximum adsorbed protein
Figure 4. Percentage distribution of secondary structures of FBS
molecules were of albumin, which was also evidenced by the proteins on different modified surfaces.
observation of the similar protein adsorption pattern as
obtained in the case of BSA adsorption.13,38
Figure 4 shows the distribution of the secondary structure of fibrinogen (FB) (1.5−4 mg/mL) proteins. Effect of surfaces
the adsorbed FBS on different surfaces. Percent secondary plays a major role during competitive protein adsorption from
structures (α-helix, β-sheet, β-turn, random coil, and side mixed protein solution, as reported previously.39 Hence, the
chains) were analyzed by deconvolution of the FTIR spectra in variation in the secondary structures of adsorbed FBS can be
the range 1600−1700 cm−1, as reported by our group attributed to the surface wettability and functionality. Native
previously.31,39 Deconvoluted FTIR spectral images on differ- FBS contains around 45% of α-helix, and approximately 25% of
ent surfaces are shown in Figure S1. FBS majorly contains BSA each β-sheet and β-turn, while remaining 5% comprised of
(35−50 mg/mL) followed by IgG (8−22 mg/mL) and random coil. α-helix content on amine, COOH and hybrid
3227 DOI: 10.1021/acsbiomaterials.8b00795
ACS Biomater. Sci. Eng. 2018, 4, 3224−3233
ACS Biomaterials Science & Engineering
Figure 5. Effect of surface modification on % cell adhesion at different time interval under (a) media without FBS, (b) media supplemented with
10% FBS, and (c) surface with preadsorbed FBS. Values represent the mean ± standard deviation (SD). # and ## denote p < 0.001 and p < 0.05,
respectively.
surfaces was found to be in the range 40−45%, which may
either be due to BSA or FB molecules as IgG (mainly contains
β-sheet, ∼70%) is nonhelical protein molecule. It was
comparable to the unmodified (blank) surface whereas it
reduces significantly on other surfaces. β-sheet content
significantly increased on COOH surface possibly indicating
higher content of IgG comparatively. Percent β-turn on
COOH surface also indicated more molar ration of IgG as
compared to BSA and FB, whereas turn (%) was higher on
other surfaces, indicating the presence of a majority of BSA
and FB molecules. Furthermore, a lower amount of β-turn and
a higher amount of β-sheet on COOH surface suggested the
presence of intramolecular interactions. On the other hand, the
higher amount of β-sheet and β-turn on hydrophobic octyl
surface suggested the presence of intermolecular interactions.40
3.3. Cell Adhesion and Spreading. Surface properties
widely regulate cell behavior such as adhesion, spreading,
migration, and proliferation at interface. Various researchers
argued that surface wettability directly relates to cell adhesion
and that hydrophilic surfaces support better cell adhesion than
hydrophobic surfaces.47 Although other researchers contra-
dicted this hypothesis and reported that hydrophobic SAM
surfaces offered better cell spreading and proliferation
comparatively to lesser hydrophobic surfaces.48 Arima and
Iwata reported several modified surfaces with different
chemistry and wettability and showed that surfaces with
contact angle range 40−50° exhibit better cell behavior.27,36
However, in our recent report, we contradicted and concluded
that surface wettability along with surface energy regulates
protein adsorption and results in their different secondary
structures that in turn regulate cell adhesion.31
Figure 5 shows the % cell adhesion on various modified
surfaces under three different conditions: (i) incomplete media
i.e. without FBS, (ii) complete media, i.e., supplemented with
10% FBS, and (iii) surfaces preadsorbed with FBS. We
observed a maximum no. of cells adhered (about 90%) on
hybrid, amine and COOH surfaces in case of 10% FBS
supplemented cell media, around 80% adhesion on amine and
hybrid surfaces with preadsorbed FBS on surfaces and
approximately 60% on hybrid surfaces in case of incomplete
media, after 360 min of cells seeding. Under all three
conditions, we observed better cell adhesion on hybrid and
amine surfaces, especially on hybrid surfaces which may be due
to (i) hybrid surfaces contains both hydrophilic and hydro-
phobic moieties on the same molecule that might be helping in
cell adhesion and spreading. (ii) Hybrid surfaces exhibit a
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contact angle (θ = 79°) that lies in the range (50−80°) that is
shown to be optimal for cell adhesion. (iii) Adsorbed protein
molecules form soft layer on hybrid surface38 and may be
helpful for cell adhesion. Amine surfaces exhibited better cell
adhesion because of their positive surface charge, which results
in cell adhesion with the negatively charged cell membrane via
ionic interactions. COOH surfaces also exhibited significantly
better cell adhesion as compared to unmodified, mixed, and
octyl surfaces. Surfaces treated with media without FBS
exhibited poor cell adhesion. Protein molecules present in the
serum adsorb quickly when comes in contact with surfaces and
serves as cushions for adhering cells. Furthermore, serum
contains cell adhesive proteins such as FN which contains
RGD tripeptide sequences and helps in integrin mediated cell
adhesion.49,50 Hence, we observed better cell spreading area
and morphology on surfaces treated with FBS as compared to
surfaces treated with cells in serum free media. It is noteworthy
that such surface modifications are biocompatible,31 and the
variation in the % cell adhesion in all three above stated
conditions are due to adsorbed serum proteins and their
secondary structures upon adsorption (as explained in section
3.3.3).
3.3.1. Fluorescence Imaging Analysis of Adhered Cells.
Phase contrast images of the adhered cells after each time
interval under all three experimental conditions were captured
and analyzed using ImageJ software (see Figures S2 and S3).
We also employed fluorescence microscopy for determining
vinculin distribution and actin cytoskeleton of adhered cells.
Vinculin is a actin binding focal adhesion protein that not only
helps in interactions of integrins with cytoskeleton but also
regulates cell spreading and migration.51 During cell adhesion,
vinculin binds to actin and stimulates polymerization and
engaging actin remodeling proteins. Hence, fluorescent
staining of vinculin protein indicates the formation of the
focal adhesion sites which further can be used to distinguish
between poor and good cell adhesion based on its distribution
inside the cells. Charged (i.e., amine and COOH surfaces) and
hybrid surfaces exhibited better vinculin distribution as
compared to unmodified, mixed, and octyl surfaces as shown
with arrow marks in Figure 6. Actin filaments form the
cytoskeleton and help the cells in mechanosensing and
spreading by linking to focal adhesions at cell−substrate
sites.52 By staining the actin filaments, we were able to observe
the actual morphology of cells on different modified surfaces.
Figure 6 shows adhered L929 cells exhibiting spreading and
morphological behavior on different modified surfaces with
DOI: 10.1021/acsbiomaterials.8b00795
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Figure 6. Fluorescent images of L929 cells cultured for 6 h on
different surfaces preadsorbed with FBS and stained for vinculin
protein (blue, 1° Ab followed by Alexafluor-350 labeled 2° Ab), actin
filaments (green, FITC-Phalloidin) and nuclei (red, PI dye). Arrow
marks indicate focal adhesion spots of bright blue color due to
vinculin staining by Alexa fluor-350. Scale bar is 25 μm.
Figure 7. Effects of different modified surfaces on (a) % cell adhesion; (b) average cell area; (c) average nuclei area; and (d) circularity after 6 h of
incubation from incomplete media, media with FBS, and surfaces preadsorbed with FBS. Values represent the mean ± SD.
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preadsorbed FBS after 360 min of incubation. See Figures S4
and S5 for fluorescent cell images for experiments carried out
in medium with and without serum. As shown in Figure 6,
adhered cells on hybrid and amine surfaces exhibited better cell
features such as area and morphology, vinculin distribution
(spreading) as compared to other surfaces under preadsorbed
FBS condition, indicating better integrin expression on these
surfaces. Cells adhered on surfaces with FBS in media also
exhibited better cell features on hybrid and amine surfaces as
compared to other surfaces. Under FBS free media, cells
showed poor adhesion (as evidenced by poor vinculin
localization inside the cells) and morphology (high cell
shape index) even after 360 min of seeding, indicating poor
integrins expression.
Figure 7 presents the analysis of various cell behaviors on
different surfaces under all three stated conditions after 360
min of cell seeding. Interestingly, variation in % cell adhesion
(see Figure 7a) among different surfaces was found to follow
similar pattern in all three cases i.e. maximum on hybrid (θ =
79°) surface followed by amine (θ = 60°), COOH (θ = 45°)
and mixed (θ = 81°) surfaces while least on extremely
hydrophilic (unmodified, θ = 15°) and hydrophobic (octyl, θ =
102°) surfaces. The data obtained by us agreed well with the
previous reports that cell adhesion gets optimum at surfaces
exhibiting the wettability in the range 50−80°.27,53,54 The
maximum cell area was observed on surfaces preadsorbed with
FBS followed by surfaces treated with cells in media
supplemented with 10% FBS and least on surfaces with cells
in incomplete media, as shown in Figure 7b. Cell area was
found linearly increasing with the increasing contact angle and
was observed maximum on hybrid surface and then drastically
decreased on mixed and hydrophobic octyl surfaces. Although
mixed (θ = 81°) and hybrid (θ = 79°) surfaces exhibit almost
similar contact angle but cell behavior such as number and area
DOI: 10.1021/acsbiomaterials.8b00795
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ACS Biomaterials Science & Engineering Article

of adhered cells on them was entirely different. Hybrid are

monomolecular SAMs while mixed SAMs are mixture of amine
and octyl silanes and suffers from the phase separation issues
and hence results in poor cell adhesion and spreading.31,38 This
confirms that surface wettability is not the only factor that
regulates cell adhesion process.
Surfaces with preadsorbed FBS exhibited the maximum cell
area due to the fact that adsorbed protein molecules undergoes
reorientation and exposes cell binding sites present in cell
adhesive proteins (e.g., FN). On preadsorbed FBS surfaces,
cells adhered on hybrid (502 ± 68 μm2), amine (484 ± 55
μm2) and COOH (481 ± 54 μm2) surfaces exhibited better
spreading as compared to blank (465 ± 65 μm2), mixed (387
± 50 μm2) and octyl (330 ± 48 μm2) surfaces. Similar pattern
of cell area variation was observed for cells with FBS in media
while cells seeded in incomplete media showed better
spreading on amine (297 ± 21 μm2) surface but was Figure 8. Average numbre of cells adhered vs average cell area during
comparable to cell spreading on hybrid surface (289 ± 36 cell adhesion studied under three different conditions.
μm2). Cell area was significantly reduced in the absence of
serum proteins indicating that proteins molecules help in cell
spreading. Effect of surfaces wettability on nuclei size was also
evaluated as nuclei size of the adhered cells indicate the nuclear
functional activity occurring during proliferation.55 Effect of
surface modification on nuclei size is less reported and it is
predicted that the increase in nuclei area may result in the
higher proliferation rate. Surfaces preadsorbed with FBS
exhibited the maximum nuclei area and was followed by cell
with FBS and least on surfaces treated with media without
FBS. Average nuclei area of adhered cells was the maximum on
hybrid surface, followed by amine and COOH surfaces (shown
in Figure 7c). The least nuclei size was observed on mixed
surface due to phase separation issues as mentioned earlier.
Similar pattern of nuclei area variation was observed in other
two conditions.
Circularity of adhered cells can be explained based on the Figure 9. Correlation between % surface coverage and average cell
value of cell shape index (CSI), calculated using formula is area on surfaces studied under the effect of FBS proteins.
CSI = 4πA P 2 where A is area and P is perimeter. It is described
on the scale range of 0.0 (line) to 1.0 (circle) and is used to
define the shapes of the cell. Lower value of circularity signifies
better cell spreading having more focal points due to which cell
shape changes. Effect of seeding time on cell circularity has
been shown in Figure S7 for all modified surfaces with and
without FBS proteins. Hybrid surface showed the least and
similar CSI value (0.80 ± 0.08) with preadsorbed FBS and
FBS in media samples and was significantly lesser, indicating
better adhesion and spreading in comparison to other silanized
and unmodified surfaces, as presented in Figure 7d. Figure 8
shows the relationship between average cell spreaded area with
no. of adhered cells on different surfaces in the presence and
absence of FBS. The average area of adhered cells, however,
significantly increased on surfaces with preadsorbed FBS and
media with FBS but did not significantly enhance the number
of adhering cells after 6 h of cell seeding.
It was important to establish the relationship between FBS Figure 10. Representation of cell adhesion phenomenon from bulk
proteins and cell adhesion and spreading. We found cell area suspension onto the surface.
and % surface coverage linearly related to each other with R2
ranging between 0.80 to 0.99, as shown in Figure 9. Percentage
coverage data can be seen in the supplementary file (see Figure 3.3.2. Cell Adhesion Kinetics. The adhesion of cells on
S6). Percent coverage depends on no. of adhered cells and surfaces was modeled theoretically as shown in Figure 10. The
average cell area in totality. Surfaces treated with media simplest kinetics of cell adhesion is explained as follows:
supplemented with FBS exhibited better correlation (R2 = kf ka
0.99) as compared to preadsorbed (R2 = 0.80) and media NB → NI + S ↔ NS
without FBS (R2 = 0.82). kd (2)

3230 DOI: 10.1021/acsbiomaterials.8b00795

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ACS Biomaterials Science & Engineering Article

Where N is the number of cells and B, I, and S refer bulk proteins molecules undergoes reorientation resulting in
suspension, interface and surface, respectively. kf is film mass exposure of cell binding sites, which in turn promotes cell
transfer coefficient, ka is rate constant for cell adhesion and kd adhesion.
is rate constant for cell detachment. 3.3.3. Relation between Secondary Structure of Protein
We assumed the absence of film mass transfer resistance due and Cell Adhesion. Rearrangement or reorientation of
to higher concentration of cells in bulk and kd was considered secondary structures of protein post adsorption influences
as negligible under present experimental time duration. Thus, the cell adhesion. Grohmann et al. reported that β-sheet
the simple kinetic equation describing the cell adhesion is as secondary structure of biomimetic polypeptide enhanced cell
follows: adhesion and proliferation over random coil structures.56 β-
dNS sheet provide more rigidity and space for cells to spread and
= ka[NI][S ] = ka[NB − NS][S ] {NI + NS = NB} hence, proliferation rates are higher on them as compared to
dt random coils. Apart from structural rigidity, exposure of cell
(3)
binding motifs (e.g., RGD tripeptide) upon adsorption of cell
The above equation can be written in terms of surface binding proteins (e.g., FN) mainly regulates the cell adhesion
coverage, θ(NSAC/AS) as follows: and spreading process.31
dθ For better realizing the role of the secondary structure of
= ka[(NBA C /AS) − θ ][Sf ] adsorbed FBS proteins on surfaces, we analyzed the effect of all
dt (4)
secondary structures on cell adhesion rate, individually. We
Where AS refers the total surface area, AC denotes the average found that the change in the α-helix content of adsorbed FBS
area of adhered cells and [Sf] refers the surface fraction with respect to unmodified surface exhibited linear relationship
available for cell adhesion. The solution of the above equation, with change in % adhered cells (Figure 11a) as well as with the
θ(%) = 100(1−exp(−[S f ]k a t)), was used to fit the initial surface coverage rate (Figure 10b) of L929 cells. The cell
experimental data and the ka values for different surfaces adhesion data in the presence of FBS in media (R2 = 0.65) and
were determined and are listed in Table 2. The highest rate of preadsorbed FBS (R2 = 0.65) showed better correlation with
change in α-helix content as compared to cells adhered on
Table 2. Rate of Surface Coverage on Different Modified surface in absence of FBS (R2 = 0 .45). Moreover, the
Surfaces by Adhering Cells in Three Different Conditions correlation between the change in α-helix content and initial
without FBS preadsorbed FBS
surface coverage rate was better on preadsorbed FBS (R2 =
surfaces (h−1) with FBS (h−1) (h−1) 0.76) and FBS in media (R2 = 0.72) as compared to control,
unmodified 0.014 0.037 0.042 i.e., without FBS in media ( R2 = 0.61). Hence, this clearly
COOH 0.021 0.060 0.062 indicates that the α-helix content of the adsorbed protein
amine 0.026 0.064 0.072 molecules in case of FBS plays key role in regulating cell
hybrid 0.028 0.065 0.073 adhesion. These findings will enable researcher’s working in
mixed 0.019 0.040 0.047 this domain to predict cell adhesion behavior based on surface
octyl 0.012 0.028 0.033 properties and the secondary structure of adsorbed proteins,
which will ultimately facilitate the design of biocompatible
surfaces.
cell adhesion was found to be 0.0725 h−1 on the hybrid surface
with preadsrobed FBS. The theoritical model proposed above
was verified by fitting the experimental data of the % surface 4. CONCLUSIONS
coverage. The fitted data agreed well with the experimental Nanoscaled modified surfaces were successfully prepared via
data (as shown in Figure S6) and hence justifies the validity of silanization as evidenced by FTIR, WCA, and AFM results. We
the model. We observed better/faster cell adhesion on surfaces created surfaces with varying wettability to demonstrate their
with preadsorbed FBS, which is due to the fact that adhered effect on protein adsorption and subsequently on cell adhesion

Figure 11. Relationship between change in α-helix content with (a) change in % adhered cells and (b) initial surface coverage rate by L929 cells on
modified surfaces under different experimental conditions.

3231 DOI: 10.1021/acsbiomaterials.8b00795

ACS Biomater. Sci. Eng. 2018, 4, 3224−3233
ACS Biomaterials Science & Engineering
during for tissue engineering applications. We employed three
different experimental conditions, (a) complete media
supplemented with 10% FBS, (b) surfaces with preadsorbed
FBS, and (c) incomplete media, i.e., without FBS, to determine
the effect of FBS proteins and surfaces on cell adhesion and
behavior. Surfaces treated with incomplete media exhibited the
least cell adhesion rate, poor morphology and smaller adhered
cell and nuclei area irrespective of surfaces, whereas surfaces in
the presence of FBS in media and preadsorbed FBS exhibited
excellent cell features on amine, hybrid, and COOH surfaces.
Surface coverage rate of adhering cells was highest on hybrid
surfaces under all three conditions. It is noteworthy that
orientation and secondary structure of adsorbed FBS protein
molecules helped in cell adhesion and spreading in case of
preadsorbed FBS. Especially, hybrid surface showed better cell
and nuclei area among all the surfaces indicating better surface
properties for cell adhesion and proliferation. Furthermore, we
found the initial surface coverage rate and Δ adhered cells (%)
linearly increased with the change in α-helix content of
adsorbed FBS proteins. On the basis of the results obtained, we
can conclude that protein adsorption and its orientation
regulates cell adhesion and spreading and hybrid surface can be
potentially used for biomedical application, especially for tissue
engineering because of its excellent cell-supporting properties.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsbiomater-
ials.8b00795.
Fitted amide I FTIR spectra, bright-field images of the
adhered cells on modified surfaces with preadsorbed
FBS and FBS in media, fluorescent images of L929 cells
on surfaces in presence and absence of FBS in media,
surface coverage by adhered cells and their cell shape
index on modified surfaces under various experimental
conditions (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: lalitpandey@iitg.ernet.in. Tel.: +91-361-258-3201.
Fax: +91-361-258-2249.
*E-mail: abshar@iitg.ernet.in.
*E-mail: sudip@chemical.iitd.ac.in.
ORCID
Lalit M. Pandey: 0000-0002-1664-5730
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
Department of Bioscences and Bioengineering and Cental
Instrumentation facility, Indian Institute of Technology
Guwahati (IITG), India, are acknowledged by authors for
providing all necessary facilities required for this work. The
authors thank IIT Guwahti for financial assistance (Sanction
BSBE/SUG/IITG/00993) and the Department of Science and
Technology, Government of India, for financial assistance
(Sanctions DST/INSPIRE/04/2014/002020, ECR/2016/
001027) for this work.
3232
Article
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