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Surface Modification of Electrospun PCL Fibers for Enhanced Cell Adhesion and
Proliferation
Ahmad Arabi, Emily Boggs, Manan Patel, LTU Biomedical Engineering Department
ABSTRACT: Polycaprolactone (PCL) is gaining popularity in the field of tissue engineering due to its
non-toxic degradation byproducts and low-cost manufacturing method. However, it is also a markedly
hydrophobic material, leading to suboptimal cell-material interactions. Our study aims to chemically
modify electrospun PCL fibers to promote cell attachment, proliferation and matrix formation. Our
research examines a two-step technique used to modify the surface of PCL to increase cell adhesion and
proliferation.
One method immobilizes arginine-glycine-aspartic acid (RGD) peptides by introducing functional
groups on the PCL surface. The other method utilizes sodium hydroxide (NaOH) treatment to create
surface nanotopography with improved hydrophilicity. Changes in the PCL fiber surface topography,
hydrophilicity and chemistry will be evaluated using scanning electron microscopy (SEM), contact angle
measurement, x-ray photoelectron spectroscopy (XPS), and infrared spectroscopy (IR). Human
periodontal ligament fibroblast (HPDLF) cells will be seeded on the PCL fibers and their growth and
proliferation will be characterized by DAPI (4', 6-diamidino-2-phenylindole) staining and a Live/Dead
Assay using confocal microscopy and, AlamarBlue® Assay. Our preliminary results have shown decreased
surface contact angle and increased surface roughness with NaOH-treated PCL fibers. Ongoing study is
focused on the RGD modification and cellular response to the NaOH and RGD treated fibers as compared
to buffer -treated controls. We anticipate increased fibroblast adhesion, proliferation, and matrix formation
on the treated PCL fibers. The results will have important implications for the use of PCL fibers as
scaffold materials in regenerating a variety of tissues.
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BACKGROUND
Polycaprolactone (PCL) is a synthetic aliphatic polymer. It has excellent biocompatibility in a
sense that when it degrades, it produces non-toxic byproducts but has poor biocompatibility in a sense that
it has poor cell-material interaction and thus cells will adhere on its surface. It also has low cost and easy
processability, making it useful for a number of biomedical applications such as drug delivery systems and
other tissue engineering applications. Electrospun PCL fibers are especially attractive as a scaffold
material because they mimic the fibrous structure of the extracellular matrix (ECM) of many tissues.
A scaffold used for tissue engineering can be considered an artificial extracellular matrix since it
provides a surface for the cells to grow on. However, electrospun PCL has a hydrophobic surface due to
the lack of functional groups and thus will not promote cell adhesion. Preliminary studies have shown that
the water contact angle, which is a measure of hydrophilicity, of unmodified PCL fibers is about 82○. This
is considered fairly hydrophobic and literature has shown that cell like hydrophilic surfaces, which is water
contact angle of 50○ or less. It is now generally accepted in biomaterial community that both very
hydrophilic and hydrophobic surfaces are not ideal for cell adhesion. Rather, a moderate hydrophilicity
enables the polymer to adsorb proteins [9].
Surface modification of PCL is necessary for its use in tissue engineering due to these challenges.
Various approaches have been proposed for modifying synthetic hydrophobic biomaterial polymer
surfaces. One of the methods is Sodium Hydroxide (NaOH) treatment which causes the base hydrolysis of
esters bonds in PCL fibers creating carboxylate ions (Figure 1). This creation of functional group will
increase hydrophilicity of the fibers as well as provide means to covalently attach a bioactive compound to
improve the cell-material interaction. NaOH treatment also creates nanoroughness which results in
increase of surface area which will result in enhanced cell adhesion. . Literature has shown that
nanotopography is beneficial for enhanced cellular attachment [2].
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Fig.1 Reaction of the base hydrolysis of esters
Another approach is to immobilize RGD peptide on biomaterial surface. Immobilization is
essential as it will create a strong anchoring or RGD peptide on surface which ultimately leads to enhanced
cell adhesion [6]. RGD peptide is a common motif present in the ligands of extracellular matrix proteins
like fibronectin. Immobilizing this peptide onto the PCL fibers will result in the binding of cells to the
surface via integrins, which are present on the cell surface. Integrins recognize the RGD motif and bind to
it, thus attaching the cell to the surface. RGD will be immobilized on the PCL surface using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) chemistry. Other surface modification methods include
polyethylene glycol (PEG) treatment, ionized gas (plasma) treatments, chemical treatments as well as
various mechanical modification approaches to creating nanotopography [1] but these methods are not
feasible for our project and therefore were not considered.
RESEARCH PLAN
The research objective of this project is to find an optimized set of parameters for each of the two
modification methods to achieve optimum cell adhesion, proliferation, and matrix formation.
In this project, we will be modifying PCL fibers via two step modification method in order to
achieve a more controlled cell-scaffold interaction. The first step is involves NaOH treatment which nano
scale roughness by making the individual fibers rougher. The second approach involves chemical
immobilization of RGD on the PCL surface. The carboxylate ion produced in step one will be treated first
with EDC and then with sulfo-NHS to create a semi-stable amine reactive ester. Further reaction of this
NHS ester with the amine group of RGD peptide results in a stable amide bond, which is strong enough to
covalently bond RGD on PCL surface.
LAWRENCE TECHNOLOGICAL UNIVERSITY, DECEMBER 2012
The modified PCL fibers will be characterized for change in topography, surface chemistry, and
hydrophilicity using SEM, FT-IR and XPS, and Goniometer respectively.
We will also conduct a systematic study of cellular response to the modified fibers. Human
periodontal ligament fibroblasts will be used to perform this study. The cell attachment, proliferation, and
protein synthesis will be evaluated by DAPI staining and a Live/Dead Assay using confocal microscopy
and cellular metabolism studies will be done using AlamarBlue® Assay.
Fig. 2 Research plan flowchart
METHODS
Surface modification techniques and protocols:
PCL Fibers were treated with 0.5M and 1M concentrations of NaOH for 1 and 3 hour time
periods. Multiple samples for each trial were used. Trials were done to find an optimal NaOH
concentration and time to be 1M NaOH and 3 hours. These parameters the best combination creating a
contact angle of 46o, as shown in the figure below (Figure 3). We were expecting a contact angle of 50 o
and our preliminary study provided good results.
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Fig. 3 PCL fiber treated w/ 1M NaOH for 3 hours
We have investigated two methods of RGD immobilization. One involves the introduction of free
amine groups by Aminolysis [3], and the other uses 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) chemistry. The second method, though more complicated than the first, is a low-cost, highefficiency, and high-strength immobilization method which allows for the immobilization of RGD along
with attachment of important homing or differentiation factors in future studies.
The EDC method requires a carboxylate ion, as shown in the figure below (Figure 4). This means
that the fiber must initially be treated with NaOH to create the carboxylates. The method then involves the
reaction of carboxylate with EDC, creating an unstable reactive O-acylisourea ester. Sulfo-NHS is then
added to stabilize the intermediate which converts the unstable O-acylisourea into an amine-reactive NHS
ester. This ester will react with the amino end of the RGD peptide to form a stable covalent amide bond
between the PCL fiber and the RGD peptide.
Fig. 4 Reaction of the immobilization of RGD on PCL surface by EDC chemistry
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Surface characterization techniques:
The following techniques enable the evaluation of changes in hydrophilicity, surface topography,
and surface chemistry as a result of the two step modifications.
Water contact angle measurement will be done on buffered control fibers and modified fibers to
evaluate the hydrophilicity with help of a goniometer. Previous literature [9] states that a decrease in water
contact angle to 50o is an optimal improvement in hydrophilicity. For our pilot study, the ImageJ program
was used to find the water contact angle on high resolution photos of the PCL fibers. In January, a
goniometer at GM will be used for more accurate contact angle measurement.
SEM characterization provides detailed visual information regarding the morphology of the PCL
fibers. The images will be analyzed for change in fiber diameter and roughness.
An infrared spectrometer will be used to evaluate the modified film surface chemical composition
by recognizing the functional groups that are present on the surface. On the IR spectrometer, the lens will
be covered with the sample, and the spectrometer will pass infrared radiation through the sample at
different wavelengths (2.5-15 µm). The computer composes a spectrum using the absorption of specific
wavelength energies of the functional groups of the compound. We expect to see additional peaks in the
functionalized PCL at 1650 and 1540 cm-1 indicating the presence of amide bonds.
XPS determines the chemical composition of a surfaces top several nanometers. An electron
energy analyzer determines the energy of emitted photoelectrons when the sample is exposed to a beam of
x-rays. A photoelectron peak is then created with a unique binding energy and intensity that can quantify
the functional groups on the polymer surface. Detection of nitrogen and carbons from C=O bonds means
that RGD is present on the surface. XPS analysis will be done before and after treatment of PCL at
Oakland University.
Cell Culture and Seeding:
The cells being used for this project are human periodontal ligament fibroblasts (HPDLFs) –
ligament cells found in the jaw muscle. These cells were chosen for their hardiness and nearindestructability. In addition, fibroblasts are the main type of cell found on the ACL. They are responsible
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for secretion of the ligament’s extracellular matrix; a measure of success for the scaffold would be if the
fibroblasts seeded onto it also began secreting an extracellular matrix. The cells are first grown on a
culture plate and later seeded onto the modified PCL fibers (a control groups will also be seeded onto an
unmodified scaffold). Analysis will be done at one and two weeks for the extent of cell growth and
proliferation.
Cell characterization techniques:
For quantifying cell growth and migration, confocal fluorescent microscopy and DAPI (4',6diamidino-2-phenylindole) will be used. DAPI is a fluorescent stain that binds to adenine and thymine
molecules in DNA. As a result, the application of DAPI to a cell culture will cause the cells’ nuclei to
glow in fluorescent light. A confocal microscope will be used for viewing seeded cells as the three
dimensional nature of the images produced can lend to quantitatively measuring the depth of cell
penetration into the scaffold.
A Live/Dead Assay makes use of fluorescent dyes; viable cells are stained green and dead cells are
stained red (Figure 5). With the use of a confocal microscope, the dyed cells can be studied in situ for
overall viability, as well as distribution and density on the scaffold. The assay will also lend to a
quantitative measure of cell concentration.
An AlamarBlue® assay will be performed to study the metabolism and growth kinetics of the cellseeded scaffolds vs. controls. The assay consists of a reagent that, when taken up by metabolically active
cells, forms a fluorescent product. The amount of metabolic activity can thus be correlated with the
intensity of the fluorescence, which is measured via spectroscopy. The measured metabolic activity can
also be correlated to cell concentrations; by comparing the results at the 1 week and 2 week analysis time
points, a rough estimation of cell growth on the samples can be inferred.
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Fig. 5 Live/Dead Assay of PCL fiber seeded with cells
Controls:
Unmodified buffer-treated PCL fibers will also be done with every test and characterization
technique for comparison studies with the NaOH and RGD modified fibers. Surface characterization
methods will be done on the buffer-treated fibers. They will also be seeded with cells and characterized
with the same cell characterization assays done on the treated fibers.
Design:
72 PCL fibers will be needed for this experiment. 24 buffer treated (controls), 24 NaOH modified,
and 24 NaOH and RGD modified will be used. From each scaffold type, four will be used for surface
characterization studies; two for the contact angle measurement and FT-IR, and the other two for XPS.
This is necessary since these techniques are destructive. The other 20 will be used for cell seeding; 10 for
DAPI staining and Live/Dead assay, and 10 for AlamarBlue® Assay. Since we are analyzing cell growth at
1 and 2 week analysis, 5 scaffolds will be used for week 1 and 5 for week 2. These fibers will be used to
complete the datasheet below summarizing the final results (Figure 6).
LAWRENCE TECHNOLOGICAL UNIVERSITY, DECEMBER 2012
SCAFFOLD
ONLY
Buffer
treated
Trial #1
Water contact
angle
#1
#2
Infrared
Spectroscopy (IR)
#1
#2
9
XPS
#1
#2
Trial #2
Trial #3
NaOH
#1
#2
#1
#2
#1
#2
#1
#2
#1
#2
#1
#2
Trial #1
Trial #2
Trial #3
NaOH + RGD
Trial #1
Trial #2
Trial #3
CELLS ON
SCAFFOLD
Buffer
treated
Trial #1
Trial #2
Trial #3
Confocal Microscopy: DAPI/LiveDead/Alamar Blue Assay
#1
#2
#3
#4
#5
NaOH
Trial #1
Trial #2
Trial #3
#1
#2
#3
#4
#5
NaOH +
RGD
Trial #1
Trial #2
Trial #3
#1
#2
#3
#4
#5
Fig. 6 Table to be used in data collection and analysis.
In January, contact angle measurements will be done using a Goniometer, as well as a pilot study
for the RGD to create a protocol. Cell culture on the culture plate will be practiced to establish standard
operating procedure since we have not performed these techniques before. In February, cell-seeding
studied on a new set of fibers will be done and data will be collected from the surface characterization
studies. In March, cell characterization studies will be performed with simultaneous data collection. In
April, the data will be analyzed for statistical significance. We will also prepare posters for conferences
and a manuscript to be submitted to a biomaterials journal.
CHALLENGES
The most difficult challenges that will be encountered will involve the technical intricacies of
certain cell modification and characterization methods. Already, we have had some difficulties obtaining
readable images for measuring the water contact angle. Also, no protocols exist for treating electrospun
PCL fibers; we anticipate the need to modify existing protocols and evaluation assays to make things work
for our study.
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DESIRED OUTCOMES
We expect to find an optimized set of parameters for NaOH and RGD peptide treatments to
achieve the greatest cell adhesion, proliferation, and matrix formation. We aim to find the optimum
concentration of NaOH and time of treatment needed for an effective modification which will keep the
bulk properties of the PCL intact. We also expect to find ideal time and concentration of RGD required for
optimum cell adhesion. We also expect to establish a complete set of protocols on surface modification,
characterization and evaluation of cellular response.
FUTURE DIRECTIONS / IMPLICATIONS
We aim to create a set of methods for surface modification to enhance cell adhesion and
proliferation. These results will have important implications for the use of these fibers for scaffold use in
the regeneration of tissues such as ligaments, since PCL fibers have greater tensile strength than their film
counterparts. Future studies, using these optimized modification protocols, may focus on the behavior of
cells on PCL fibers undergoing mechanical stimulation. The modified PCL fibers also provide model
surfaces for us to further investigate the behavior of other types of cells in response to nanotopography and
RGD ligand.
COST ANALYSIS
The PCL fibers will be provided by the University of Michigan free of cost. Equipment,
chemicals, and materials will be provided by Lawrence Tech. XPS will be done at Oakland University and
the goniometer will be provided by GM next year. The major cost will be for the chemicals for
modification and the various characterization assays. Specific costs for the chemicals are shown below:

RGD peptide - $118/25 mg

Sulfo NHS - $150/500 mg

AlamarBlue® Assay kit - $164.82/25 mL
The budget for this experiment is $2,000 which will be provided by Dr. Yawen Li.
LAWRENCE TECHNOLOGICAL UNIVERSITY, DECEMBER 2012
ACKNOWLEGMENTS
Faculty Advisor: Dr. Yawen Li. Technical Advisors: Dr. Julie Zweisler-Vollick and Tristan
Maerz. Acknowledgments to Dr. Hsiao-Ping Moore (Lawrence Technological University), Dr. Kevin
Baker (Beaumont Orthopedics Department), Sam Tuck, and Dr. Joseph Corey (University of Michigan).
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REFERENCES:
[1] Goddard, J.M. (07/2007). "Polymer surface modification for the attachment of bioactive
compounds". Progress in polymer science (0079-6700), 32 (7), p. 698.
[2] Chen, Weiqiang (05/2012). "Nanotopography Influences Adhesion, Spreading, and Self-Renewal of
Human Embryonic Stem Cells". ACS nano (1936-0851), 6 (5), p. 4094.
[3] Zhu, Yabin (11/2002). "Surface Modification of Polycaprolactone Membrane via Aminolysis and
Biomacromolecule Immobilization for Promoting Cytocompatibility of Human Endothelial Cells".
Biomacromolecules (1525-7797), 3 (6), p. 1312.
[4] Hartman, Olga (07/2010). "Biofunctionalization of electrospun PCL-based scaffolds with perlecan
domain IV peptide to create a 3-D pharmacokinetic cancer model". Biomaterials (01429612),31 (21), p. 5700.
[5] De Luca, A. C. (2012). "Immobilization of cell-binding peptides on poly-ε-caprolactone film surface
to biomimic the peripheral nervous system". Journal of biomedical materials research. Part
A(1549-3296), p. n.
[6] Hersel, Ulrich (11/2003). "RGD modified polymers: biomaterials for stimulated cell adhesion and
beyond". Biomaterials (0142-9612), 24 (24), p. 4385.
[7] Zhang, Huina (09/2009). "The interaction between bone marrow stromal cells and RGD-modified
three-dimensional porous polycaprolactone scaffolds". Biomaterials (0142-9612), 30 (25), p.4063.
[8] Zhu, Yang (2013). "Aminolysis-based surface modification of polyesters for biomedical
applications". RSC advances (2046-2069).
[9] Ma, Zuwei, Zhengwei Mao, and Changyou Gao. "Surface Modification and Property Analysis of
Biomedical Polymers Used for Tissue Engineering." Colloids and Surfaces B: Biointerfaces 60.2
(2007): 137-57. Print.
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