Growth of 3T3 fibroblast on collagen immobilized poly...
Transcript of Growth of 3T3 fibroblast on collagen immobilized poly...
Indian Journal of Fibre & Textile Research
Vol. 35, September 2010, pp. 228-236
Growth of 3T3 fibroblast on collagen immobilized poly(ethylene
terephthalate) fabric
Navdeep Grover & Harpal Singh
Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi 110 016, India
Nalini Vemuri
Department of Biotechnology, Jaypee Institute of Information Technology, Noida 201 307, India
and
Bhuvanesh Gupta
Bioengineering Laboratory, Department of Textile Technology, Indian Institute of Technology, New Delhi 110 016, India
Received 8 October 2009; revised received and accepted 9 December 2009
Radiation induced grafting of acrylic acid (AA) and binary mixture of acrylic acid/N-vinyl pyrrolidone (NVP) has been
carried out on poly(ethylene terephthalate) (PET) fabric. The grafted fabrics are immobilized with collagen via carbodiimide
coupling to make the fabric bioreceptive and biocompatible for cell seeding and grafts. Atomic force microscopy and
scanning electron microscopy observations suggest that the collagen has a very similar structure on PET-g-AA/NVP and
PET-g-AA. After immobilizing collagen, PET induces growth and proliferation of 3T3 mouse fibroblasts as compared to
virgin PET. The results indicate that collagen immobilized PET-g-AA fabric shows better adhesion and proliferation than
PET-g-AA/NVP fabric.
Keywords Acrylic acid, Collagen immobilization, Grafting, N-vinyl pyrrolidone, Poly(ethylene terepthalate), 3T3 fibroblast
1 Introduction
Polymers remain the most versatile class of
biomaterials, being extensively applied in medicine and
biotechnology, as well as in the food and cosmetics
industries1.
Applications include surgical devices,
implants and supporting materials (e.g. artificial
organs, prostheses and sutures), drug-delivery systems
with different routes of administration and design,
carriers of immobilized enzymes and cells, biosensors,
components of diagnostic assays, bioadhesives, ocular
devices, and materials for orthopaedic applications2-8
.
Among them, poly(ethylene terephthalate) (PET) in
different shapes and forms is used worldwide clinically
in cardiovascular devices9. However, PET has excellent
mechanical strength, good stability in the presence of
body fluids and high radiation resistance which makes
it suitable for sterilization, but its surface needs precise
modification for the immobilization of biomolecules10
.
The development of biomaterials that are capable of
directing cell behavior is a rapidly growing area of
research.
Modification of synthetic polymeric scaffolds by
immobilization of natural polymers [components of
extracellular matrix (ECM)] like collagen, gelatin,
fibronectin, and laminin improves their biological
behavior11-20
. These biopolymers have high affinity
for cell adhesion and their proliferation is due to the
presence of specific peptide sequence. There is
increasing evidence that the cell adhesion and
proliferation on polymeric materials depend on
surface characteristics, such as chemistry, wettability,
surface energy, charge and topography of the
materials21
. To optimize these properties, the
frequently used modification methodologies include
introducing functional groups, incorporating
amphiphilic moieties, creating positively charged
materials or surfaces, and treating polymer surfaces
by gas plasma and ion implantation.21-29
. Fu et al.30
showed that the increase in wettability of cholic acid
functionalized star poly(DL-lactide) improves
adhesion of 3T3 mouse fibroblasts and ECV304
endothelial cells. The adherence and proliferation of
NIH 3T3 fibroblast was also affected by fibre
diameter and orientation on electrospun poly(DL-
lactic-co-glycolic acid)31
. Analysis of the morphology
of adherent NIH 3T3 fibroblasts indicates that the
projected cell area and aspect ratio increase
systematically with both increasing fibre diameter and
degree of fibre orientation, whereas cell proliferation
______________________ aTo whom all the correspondence should be addressed.
E-mail: [email protected]
GROVER et al.: GROWTH OF 3T3 FIBROBLAST ON COLLAGEN IMMOBILIZED PET FABRIC
229
is not sensitive to fibre diameter or orientation.
Risbud et al.32
modulated the growth of NIH 3T3
fibroblast by Chitosan-PVP (polyvinyl pyrrolidone)
hydrogel. The hydrogel is found to be non-toxic and
biocompatible with fibroblast but inhibits the growth
of fibroblast. These findings indicate the possible use
of Chitosan-PVP hydrogels in scar preventive wound
management since the early migration and
proliferation of fibroblasts in the wound area are
implicated in wound scarring. Neff et al.33
described a
method for coupling peptides to hydrophobic
materials for the purpose of simultaneously
preventing nonspecific protein adsorption and
controlling cell adhesion. Polystyrene (PS) modified
with polyethylene oxide (PEO)/polypropylene oxide
(PPO)/ polyethylene oxide (PEO) copolymers alone
was found to be inert to NIH 3T3 cell adhesion both
in the presence of serum proteins and when exposed
to activated RGD peptide. In contrast, PS conjugated
with RGD via end group-activated PEO/PPO/PEO
copolymers supported cell adhesion and spreading.
Inhibition of growth of human foreskin fibroblast on
poly(ethylene oxide) (PEO) modified poly(ethylene
terephthalates) (PET) was also observed by Desai et
al.34
PET films were covalently grafted by PEO of
molecular weights 5000, 10000, 18500, and 100000
g/mol. It has been shown that the higher-molecular-
weight PEO surfaces support cell growth to a much
lower extent than the two lower-molecular-weight
PEOs. Jou et al.35
modified the PET fibres by γ-ray
induced graft polymerization of acrylic acid. The
modified fibres were further grafted with chitosan
(CS) via esterification and subsequently hyaluronic
acid (HA) was immobilized. The results indicate that
PET fibres not only exhibit antimicrobial activity, but
also improve the cell proliferation for fibroblast. In
our previous work, the plasma induced graft
polymerization of acrylic acid has been carried out on
PET films followed by the collagen (type I and type
III) immobilization and human smooth muscle cell
expansion11,12
. Recently, the functional designing of
the PET knitting under various conditions using pre-
irradiation grafting of acrylic acid (AA) and binary
mixture of AA/NVP (N-vinyl pyrrolidone) has been
carried out36,37
. The grafted knittings were
immobilized with collagen type I and subsequently,
the growth of human msenchymal stem cell (hMSC)
was also studied.
With the rapid development of tissue engineering
and gene therapy, collagen-based biomaterials are
frequently used as cell transplant devices. They are
biodegradable, biocompatible and non-immunogenic,
and are widely used for wound dressing and related
surgical applications38
. Collagen has also found
application in tissue engineering, with collagen
scaffolds supporting the adherence and proliferation
of human cells which could subsequently be
implanted into the patient39
. The goal of this work was
to develop the bioreceptive material surface that
would enable cell-ligand interactions for achieving
better cell adhesion and proliferation for the cell
seeding and grafts. In previous work, it has been
observed that the grafted acrylic acid shows toxic
effects on cell growth due to a low pH environment
around PET matrix10-12
. The idea of this study is to
develop biocompatible features by co-grafting of N-
vinyl pyrrolidone (NVP) with acrylic acid and the
grafted PET fabrics are immobilized with collagen
and characterized by SEM and AFM. The growth and
proliferation of mouse 3T3 fibroblast has been
compared on these collagen immobilized surfaces, i.e.
PET grafted with acrylic acid and binary mixture of
acrylic acid & NVP (PET-g-AA-g-COL and PET-g-
AA/NVP-g-COL). The results indicate that the
collagen immobilized PET-g-AA shows better
adhesion and proliferation than PET-g-AA/NVP and
virgin PET. Moreover, the recent work is carried out
on PET knittings which give 3-dimensional porosity
for cell growth. In this study, it has been tried to
translate the results of PET film to PET knitted fabric,
as these knitted fabrics hold a promising future as an
efficient matrix for the scaffold applications in tissue
engineering, such as urinary bladder reconstruction.
2 Materials and Methods
Weft knitted textured PET fabric of denier 80/34
(mass of 9000m/number of filaments in yarn), used in
this study, was prepared from textured yarn supplied by
Reliance Industries Ltd. (Mumbai, India). Acrylic acid
(AA) (after vacuum distillation) was obtained from
Merck India Ltd. N-vinyl pyrrolidone (NVP) (used
after vacuum distillation) was received by Fluka.
Collagen type I (Rat Tail, Sigma, USA) and 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide (EDAC,
Sigma-Aldrich, USA) were used as received.
Deionised water was used for all experiments.
Single end weft knitting was carried out on Krenzl,
Switzerland, weft knitting machine of diameter 3.5
inch keeping the gauge as 14 needles/inch.
INDIAN J. FIBRE TEXT. RES., SEPTEMBER 2010
230
2.1 Heat Setting and Extraction of Spin Finish
For dimensional stability, heat setting was carried
out at 200°C in free shrink condition on EARNST
BENZAG, Switzerland heat setting machine.
Heat-set knitted fabric was soxhlet extracted in
methanol for 10 h for the removal of spin finish. The
fabric was then removed and boiled in distilled water
for 1 h followed by overnight drying at 60°C.
2.2 Irradiation and Graft Polymerization
Knitted PET fabrics were exposed to γ-rays from a 60
Co source (900 Curies supplied by Bhabha Atomic
Research Centre) in the presence of air. The dose rate
of radiation was 0.16 kGy/h.
Grafting was carried out in glass ampoules of 2×10
cm2 size with B-24 joints. A weighed amount (~500
mg) of fabric was placed in gamma chamber for
irradiation. After the irradiation of 40kGy, the fabric
was immediately placed into ampoules containing
monomer and the solvent (THF/water or
MEK/water)36,37
. Nitrogen was purged into the
ampoule to remove air trapped inside the reaction
mixture. The ampoule was subsequently placed in a
water bath maintained at required temperature. After a
desired period, the ampoule was removed and the
sample was washed with boiling distilled water for 10
h to remove any surface adhered homopoylmer. The
samples were then dried in an oven at 60°C under
vacuum and the degree of grafting was determined
using the following expression:
( )Degree of grafting % 100g iW W
W
−= × …. 1
where Wi and Wg are the weights of ungrafted and
grafted fabrics respectively. 2.3 Collagen Immobilization
PET fabrics (virgin and grafted) of the size 0.5×0.5
cm2 were washed in boiling distilled water for 2 h.
The samples were placed in EDAC (26mM) solution
for 24 h at 4°C to activate the carboxyl groups40
. After
activation, the samples were washed with distilled
water and subsequently dipped in collagen type I
solution at different pH (in acetate buffer) for 24 h at
4°C. The samples were then washed in deionized
water for 30 min at room temperature by stirring to
remove the unbound collagen and dried by freeze
drying at -80°C (ref. 41).
Collagen content at the fabric surfaces was
measured by a ninhydrin method42,43
. The fabrics
(0.5×0.5 cm2) were immersed in 2 mL of 6 M HCl for
12 h at 120°C to hydrolyze the protein. The resulting
solution was cooled, neutralized with 2 mL of 6 M
sodium hydroxide solution and then diluted 10 times
with deionized water. Further, 1 mL of this diluted
hydrolyzed solution was added to 2 mL of ninhydrin
and heated at 120°C for 15 min to give a violet-blue
color and cooled. The optical density of the solution
was measured using a spectrophotometer at a
wavelength of 570 nm. Collagen content was
calculated from a standard calibration plot. All the
surface densities were the averages of three
measurements. The standard curve was obtained using
a known amount of collagen as the reactant following
the same process as stated above.
2.4 Atomic Force Microscopy (AFM)
Topographical studies of the fibre surface were
carried out in air using atomic force microscope,
molecular imaging (MI), USA and was operated in
the contact mode using an etched silicon tip attached
to the end of a cantilever. Cantilevers used for this
mode NSC 12 (c) were obtained from MikroMasch
having force constant 4.5 N/m and frequency
150 kHz. 2.5 Cell Line and Maintenance
The cell line 3T3-L1 (Mouse, embryo fibroblasts)
was obtained from National Culture of Cell Science,
Pune. The cell line was maintained in Dulbecco’s
Modified Eagle’s medium (DMEM) containing 4mM
L-glutamine, 1.5gm/L sodium bicarbonate, 4.5gm/L
glucose, antibiotics (penicillin, 100U/ml; streptomycin,
100µg/ml; gentamycin, 100µg/ml) and supplemented
with10% Fetal Calf Serum (Sigma, Ma, USA). The
cells were grown in T-25 flasks (Polylab) at 37°C in
5% CO2 incubator (New Brunswick Scientific) for a
period of 3-5 days with replenishment of the medium
twice a week. On attaining confluence, the cells were
dislodged using 0.25% Trypsin and 0.03% EDTA.
Viability was determined using Neubauers’ chamber
and 0.4% Trypan Blue dye and viewed using Olympus
Microscope. The total number of viable cells was
determined using the following formula:
Number of = (% viability) × (Total number of viable
viable cells & non-viable cells)
2.6 Cell Growth on Fabrics
The sterilized (by gamma radiation) virgin and
modified PET fabrics of 1×1 cm2
size were rinsed
GROVER et al.: GROWTH OF 3T3 FIBROBLAST ON COLLAGEN IMMOBILIZED PET FABRIC
231
with DMEM medium and plated in 24-welled tissue
culture plates. The scaffolds were seeded with 3T3-L1
cells at a cell concentration of 1.2×105 cells/mL and
incubated at 37°C in 5% CO2 atmosphere. At various
time periods (1, 3 and 5 days) the scaffolds were
rinsed with Trypsin–EDTA solution and the dislodged
cells were centrifuged at 1800 rpm for 10 min.
Viability count was determined as mentioned earlier. 2.7 Scanning Electron Microscope (SEM)
The surface morphology of collagen on
collagen immobilized PET fabrics was studied using
TM 1000 (tabletop microscope, HITACHI) without
any conductive coating at ×200 magnification in
backscattered electron detector mode.
PET fabrics seeded with 3T3-L1 cells and
terminated at various time periods (1, 3 and 5 days)
were rinsed with PBS (phosphate buffer saline) buffer
(pH 7.2) and fixed in 1% (v/v) glutaraldehyde and 4%
(v/v) formaldehyde. This was followed by serial
washing with 30%, 40%, 50% (v/v) ethanol/water and
then freeze drying. The surface characteristics were
studied using STEREO-SCAN 360 (Cambridge
Scientific Industries Ltd.) scanning electron
microscope (×1000) after coating them with gold.
3 Results and Discussion
3.1 Collagen Immobilization
In an earlier investigation11,12
, it has been found that
PET surfaces with low graft levels of PAA (0.4 µg/cm2)
as well as complexed collagen content of <1 µg/cm2 are
bound to be the most appropriate for human smooth
muscle cell culture. However, at higher graft levels of
PAA, there is a decrease in the growth of the cells,
indicating that with an increase in collagen content, large
areas of the noncomplexed collagen get detached from
the film surface during cell seeding, exposing the cells to
the PAA layer locally and hence to a low pH
environment leading to the cell degeneration. The idea
of grafting acrylic acid in conjunction with NVP is to
create hybrid network on the fabric surface. The
introduction of carbonyl functionality makes the surface
bio-interactive and biocompatible due to the presence of
NVP for the cell seeding and grafts. Since, the
hydrophilic polymers derived from NVP are useful
materials and promote endothelial cell growth without
protein pre-coating44-46
. In present study, PET-g-AA and
PET-g-AA/NVP fabrics with a graft densities of 5.3%
and 5.5% respectively (Table 1), modified with collagen
immobilization at pH 4.6, have been studied as matrices
for 3T3 mouse fibroblasts cells.
Collagen stability on the acrylic acid grafted PET
(PET-g-AA) surface is needed to achieve proper cell
growth. Although this problem could be avoided by
controlling the PAA graft level, higher levels of collagen
immobilization remain desirable because they would
provide more flexibility in controlling the long-term
properties of the surfaces. In a recent investigation,41
collagen has been covalently immobilized at pH 4.6 by
carbodiimide coupling reaction. The topography of fibre
surfaces, as observed by AFM, is shown in Fig. 1. It has
been clearly seen that the topographical changes on
PET-g-AA and PET-g-AA/NVP are different in
comparison to virgin surface. It has been shown earlier37
that the surface morphology of the grafted fabric is
strongly affected by the nature of the additives in
reaction medium. In case of PET-g-AA, the grafting has
been carried out in MEK/water medium and the grafting
of NVP/AA has been carried out in THF/water
medium36,37
. Due to the nonsolvent nature of MEK for
PAA, the grafted chains are precipitated which results
in the formation of isolated domains of PAA. On the
other hand, THF (which acts as a solvent for PAA) in
the reaction medium shows less surface
nonhomogenity as compared to MEK37
. When collagen
is immobilized on these grafted surfaces, the
topography looks almost similar. The SEM
observations show that collagen form web like
structure after freeze drying on PET fabric surface
(Fig. 2). These observations clearly indicate that the
grafted chains and immobilized collagen form their
own domains and morphologies at the surface.
3.2 Growth of 3T3 Mouse Fibroblast
The growth and proliferation of 3T3 mouse
fibroblasts are shown in Fig. 3. The cell count after 1
day of cultured fabrics shows that the adherence of
cells is better in collagen immobilized grafted fabrics.
The cultured cells are also examined after 3 and 5
days for assessing their proliferation. The cell count
after 3 and 5 days shows that the proliferation of cells
is found to be better on collagen immobilized grafted
fabrics and collagen immobilized PET-g-AA shows
better adhesion as well as proliferation. This may be
Table 1—Details of PET fabrics
Sample Amount of
grafting, %
Collagen
content, µg/cm2
Virgin PET -- --
Irradiated PET -- 0.8
PET-g-AA 5.3 30.7
PET-g-AA/NVP (FAA, mole
fraction of acrylic acid, 0.63)
5.5 20.1
INDIAN J. FIBRE TEXT. RES., SEPTEMBER 2010
232
Fig. 1—AFM of PET surfaces (a) virgin, (b) PET-g-AA/NVP (FAA=0.63), (c) PET-g-AA, (d) PET-g-AA/NVP-g-COL (FAA=0.63), and
(e) PET-g-AA-g-COL. [Degree of grafting: PET-g-AA (5.3%) and PET-g-AA/NVP (5.5%). FAA— mole fraction of acrylic acid (AA) in
PET-g-AA/NVP]
Fig. 2—SEM of PET surfaces [(a) virgin,
(b) PET-g-AA/NVP-g-COL (FAA=0.63), and
(c) PET-g-AA-g-COL]. Degree of grafting:
PET-g-AA (5.3%) and PET-g-AA/NVP
(5.5%). FAA— mole fraction of acrylic acid
(AA) in PET-g-AA/NVP
GROVER et al.: GROWTH OF 3T3 FIBROBLAST ON COLLAGEN IMMOBILIZED PET FABRIC
233
due to the higher collagen content on PET-g-AA in
comparison to other matrices. Moreover, the presence
of PVP functional group in PET-g-AA/NVP may not
support the cell growth32,35
. It can be suggested that
PVP is not cytotoxic in nature but may be cytostatic
toward fibroblast. Adherence of fibroblasts to
collagen immobilized PET fabric was observed by
light microscopy (Fig. 4).
3.3 Morphology of Cells Grown on PET Fabrics
The morphology of cells is also assessed by SEM
from 1 to 5 days of culture. The cells adhered better
on collagen immobilized surface in comparison to
virgin surface (Fig. 5). After growing and
proliferating, cells attain their original morphology
(Figs 6 and 7). Moreover, these cells show a better
cell morphology on collagen immobilized surfaces in
comparison to virgin surface. The surface
immobilized proteins can interact with integrins of the
cellular membrane which represents an interesting
way to achieve cell adhesion and proliferation47,48
.
Therefore, the immobilization of collagen on grafted
surfaces improves bioreceptivity as well as
cytocompatibility of the surfaces for adherence and
growth of 3T3 fibroblast. These observations clearly
indicate that collagen immobilized grafted fabrics are
excellent substrates for adherence and growth of
fibroblasts.
Fig. 4—Light microscopy pictures ( X40) of (a) 3T3 mouse fibroblast at confluence, (b) PET-g-AA-g-COL matrix, (c) extension of cell
processes on PET-g-AA-g-COL, and (d) adherence and proliferation of cells on PET-g-AA-g-COL
Fig. 3—Growth and proliferation of 3T3 fibroblast on different PET
matrices
INDIAN J. FIBRE TEXT. RES., SEPTEMBER 2010
234
Fig. 5—SEM of 3T3 fibroblast cultured PET fabrics (for 1 day) [(a) virgin, (b) irradiated-COL, (c) PET-g-AA/NVP-g-COL (5.5%
grafted, FAA=0.63), and (d) PET-g-AA-g-COL (5.3% grafted)]
Fig. 6—SEM of 3T3 fibroblast cultured PET fabrics (for 3 days) [(a) virgin, (b) irradiated-COL, (c) PET-g-AA/NVP-g-COL (5.5%
grafted, FAA=0.63), and (d) PET-g-AA-g-COL (5.3% grafted)]
GROVER et al.: GROWTH OF 3T3 FIBROBLAST ON COLLAGEN IMMOBILIZED PET FABRIC
235
4 Conclusions
The AFM and SEM analysis shows that the
immobilized collagen forms their own domains on
fibre surface. The introduction of collagen on grafted
surface improves cell adhesion and their proliferation.
Collagen immobilized PET-g-AA shows better cell
adhesion and proliferation in comparison to PET-g-
AA/NVP. This may be due to the higher collagen
content on PET-g-AA and the presence of PVP on
PET-g-AA/NVP; PVP is not cytotoxic in nature but
may be cytostatic towards fibroblast. The SEM
observations of cultured matrices also show that
collagen immobilized grafted fabrics are excellent
substrates for adherence and growth of fibroblasts.
Acknowledgement
The authors would like to thank Council of
Scientific and Industrial Research (CSIR), India for
providing financial support to one of the authors
(NG).
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