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: bgupta@textile.iitd.ernet.in

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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