ARTICLE IN PRESS

Biomaterials 27 (2006) 14521461

www.elsevier.com/locate/biomaterials

Electrospinning of collagen nanobers: Effects on the behavior of

normal human keratinocytes and early-stage wound healing
Kyong Su Rhoa, Lim Jeongb,c, Gene Leea, Byoung-Moo Seod, Yoon Jeong Parka,c,
Seong-Doo Honge, Sangho Roha, Jae Jin Choa, Won Ho Parkb,c,1, Byung-Moo Mina,c,
a

Department of Oral Biochemistry and Craniomaxillofacial Reconstructive Science, Dental Research Institute, and BK21 HLS,
Seoul National University College of Dentistry, Seoul 110-749, South Korea
b
Department of Textile Engineering, Chungnam National University, Daejeon 305-764, South Korea
c
IBEC, Seoul National University College of Dentistry, Seoul 110-749, South Korea
d
Department of Oral and Maxillofacial Surgery, Seoul National University College of Dentistry, Seoul 110-749, South Korea
e
Department of Oral Pathology, Seoul National University College of Dentistry, Seoul 110-749, South Korea
Received 12 May 2005; accepted 10 August 2005
Available online 6 September 2005

Abstract
Electrospinning of type I collagen in 1,1,1,3,3,3-hexauoro-2-propanol (HFIP) to fabricate a biomimetic nanobrous extracellular
matrix for tissue engineering was investigated. The average diameter of collagen nanobers electrospun from 8% collagen solution in
HFIP was 460 nm (range of 1001200 nm). The as-spun collagen nanobrous matrix was chemically cross-linked by glutaraldehyde
vapor with a saturated aqueous solution and then treated with aqueous 0.1 M glycine to block unreacted aldehyde groups. With vapor
phase cross-linking for 12 h, porosity of the collagen matrix decreased from 89% to 71%. The collagen nanobrous matrix showed good
tensile strength, even in aqueous solution. Effects on cytocompatibility, cell behavior, cell and collagen nanober interactions, and open
wound healing in rats were examined. Relatively low cell adhesion was observed on uncoated collagen nanobers, whereas collagen
nanobrous matrices treated with type I collagen or laminin were functionally active in responses in normal human keratinocytes.
Collagen nanobrous matrices were very effective as wound-healing accelerators in early-stage wound healing. Our results indicate that
cross-linked collagen nanobers coated with ECM proteins, particularly type I collagen, may be a good candidate for biomedical
applications, such as wound dressing and scaffolds for tissue engineering.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Electrospinning; Collagen nanobers; Extracellular matrix protein; Cell behavior; Wound healing

1. Introduction
Over the past decade, considerable effort has been
directed towards developing scaffolds for tissue engineering
using biodegradable and biocompatible synthetic or
Corresponding author. Department of Oral Biochemistry and
Craniomaxillofacial Reconstructive Science, Seoul National University
College of Dentistry, 28 Yeonkun-Dong, Chongno-Ku, Seoul 110-749,
South Korea. Tel.: +82 2 740 8661; fax: +82 2 740 8665.
E-mail addresses: parkwh@cnu.ac.kr (W.H. Park), bmmin@snu.ac.kr
(B.-M. Min).
1
Also correspondence to. Department of Textile Engineering, Chungnam National University, Daejeon 305-764, South Korea.
Tel.: +82 42 821 6613; fax: +82 42 823 3736.

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2005.08.004

natural polymers. Ideally, a scaffolding candidate should

mimick the structure and biological function of native
extracellular matrix (ECM) proteins, which provide
mechanical support and regulate cellular activities. In
addition, the scaffolding must support and dene the
three-dimensional organization of the tissue-engineered
space and maintain the normal state of differentiation within the cellular compartment [1]. To achieve
this objective, an engineered matrix must be biocompatible and must not induce adverse effects in
the surrounding tissue. In these regards, collagen
has been used in a variety of tissue-engineering applications.

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461

Collagen is a natural ECM component of many tissues,

such as skin, bone, tendon, ligament, and other connective
tissues [2]. Among the isotypes of collagen, type I is the
principal structural and functional protein and is composed of two a1 chains and one a2 chain. The underlying a
chains that form these natural polymers are arranged
into a repeating motif that forms a coiled structure. The
specic complement of a subunits present within the bril
denes the material properties of the polymer [1]. In native
tissues, type I collagen brils range from 50 to 500 nm in
diameter and are very uniform in size [1]. The brillar
structure of type I collagen has long been known to be
important for cell attachment, proliferation, and differentiated function in tissue culture [35]. In native ECM,
collagen exists in a three-dimensional network structure
composed of multi-brils in the nanober scale
(50500 nm) [6]. Collagen can be isolated from a variety
of sources and is highly conserved and relatively nonimmunogenic. A nonwoven-type matrix, which is composed of nanobers, is easily produced via electrospinning
and is architecturally similar to the collagen structure of
ECM. Studies of adaptations in electrospinning to produce
tissue-engineering scaffolds composed of collagen nanobers (a matrix composed of 100 nm ber) have found that
the structural properties of electrospun collagen varies with
the tissue of origin, the isotope, and the concentration of
the collagen solution [1].
ECM proteins, such as type I and type IV collagen,
laminin, bronectin, and vitronectin, are effective in
promoting cellular adhesion and spreading. These proteins possess a arginineglycineaspartic (RGD) acid
sequence, that is recognized by integrins [7]. Integrins,
which are expressed in a variety of cell types, are a large
family of cell surface receptors that mediate cellcell and
cellECM adhesion [8]. Each integrin is a heterodimer
containing an a and a b subunit, both of which are
transmembrane glycoproteins that link ECM components
to the intracellular cytoskeletal and signaling networks.
The a and b subunits act in concert to regulate cell
adhesion, migration, proliferation, and survival [912].
Collagen binds mainly to the integrins a1b1 and a2b1 and
affects the attachment and differentiation of osteoblastic
cells [13].
In the present study, a nanobrous matrix of type I
collagen was produced via electrospinning to develop
biodegradable and biomimetic scaffolds. To assay the
cytocompatibility and cell behavior of electrospun collagen nanobers, cell attachment and spreading of
normal human keratinocytes seeded on the collagen
nanobrous matrix and the interaction between cells
and collagen nanobers were studied. To further evaluate the effect of ECM proteins, cellular responses
to a collagen matrix were investigated. Type I collagen, bronectin, and laminin were adsorbed onto
the matrices as substrates. Additionally, the effect of
collagen nanobers on open wound healing in rats was
examined.

1453

2. Materials and methods

2.1. Reagents
Type I collagen from calfskin was obtained from Regenmed Co. (Seoul,
Korea). 1,1,1,2,2,2-hexauoro-2-propanol (HFIP), glutaraldehyde, glycine, human plasma bronectin, and rat-tail type I collagen were
purchased from Sigma-Aldrich (Saint Louis, MO). Human placental
laminin was purchased from Invitrogen (Carlsbad, CA).

2.2. Electrospinning
The electrospinning setup utilized in this study consisted of a syringe
and needle (ID 0.84 mm), a ground electrode (d 21:5 cm, a stainlesssteel sheet on a drum whose rotation speed could be varied), and a high
voltage supply (Chungpa EMT; CPS-40K03, Seoul, Korea). The needle
was connected to the high voltage supply, which could generate positive
DC voltages up to 40 kV [14]. For the electrospinning of collagen bers,
collagen was rst dissolved in HFIP at a concentration of 8% (W/V) and
then delivered by a syringe pump (Model 100, KD Scientic, Incheon,
Korea) with a mass ow rate of 0.02 ml/min. The distance between the
needle tip and the ground electrode was 8 cm, and the positive voltage
applied to polymer solutions was in the range of 1520 kV. All
experiments were carried out at room temperature.

2.3. Cross-linking of the electrospun collagen matrix

The electrospun collagen nanobrous matrix was cross-linked by
treatment with glutaraldehyde vapor, saturated with 25% glutaraldehyde
aqueous solution at room temperature for various time periods, followed
by treatment with 0.1 M glycine aqueous solution to block unreacted
aldehyde groups.

2.4. Measurements
The morphology of electrospun collagen nanobers was observed on a
scanning electron microscope (SEM; Hitachi S-2350, Japan) after gold
coating. The average diameter and diameter distribution were obtained by
analyzing SEM images using a custom code image analysis program
(Scope Eye II, Masan, Korea). Porosity and pore parameters in the
interber region of the collagen nanobrous matrix were determined by a
mercury intrusion technique using an AutoPore III mercury porosimeter
(Micromeritics Instrument, Norcross, GA). The tensile strength of
collagen nanobrous matrices was measured by Instrons 8511 (Instron,
Canton, MA). In accordance with American Standards for Testing
Methods (ASTM) D882-97, standard testing method for tensile properties
of thin plastic sheeting, the collagen nanobrous matrices (0.2 mm thick)
were cut into a dumbbell-shape of 50 mm
10 mm. A load cell of 500 N
was hammered vertically at a speed of 1 mm/min onto the specimen. The
collagen nanobrous matrix served as the test specimen, while two
commercial tissue regenerative membranes (Resoluts LT, Bioxs) [15]
and a wound-dressing material (Beschitins W; Unitica, Japan) served as
controls.

2.5. Cells and cell culture

Normal human oral keratinocytes (NHOK) were prepared and
maintained, as previously reported [14,16]. Briey, NHOK were isolated
from human gingival tissue specimens obtained from healthy volunteers
(age range 2030 years) undergoing oral surgery. Oral keratinocytes were
isolated from separated epithelial tissue by trypsinization, and primary
cultures were established in a keratinocyte growth medium containing
0.15 mM calcium and a supplementary growth-factor bullet kit (KGM;
Clonetics, San Diego, CA). Normal human epidermal keratinocytes
(NHEK), prepared in a manner similar to the NHOK, were obtained from

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461

human foreskins of patients (13 years of age) undergoing surgery. NHEK

were also cultured in KGM. Approximately 70%-conuent primary
NHOK and NHEK were plated at 1
105 cells per 60-mm culture dish
and were cultured until the cells reached 70% conuence. Second passage
keratinocytes were used in the described experiments.

2.6. Cell adhesion and spreading assays

Cell adhesion was assayed using a modication of the method of
Mould et al. [17]. Briey, nonwovens of electrospun collagen nanobers
were cut out with punch (14-mm in diameter) and put onto 24-well culture
plates (Nunc, Denmark). The 24-well culture plates containing collagen
nanobers were coated with 200 ml/well of ECM proteins, in this case type
I collagen (50 mg/ml), bronectin (1 mg/ml), or laminin (10 mg/ml) in
phosphate-buffered saline (PBS), by overnight adsorption at 4 1C. We
tested the effect of 0.150 mg/ml of type I collagen, bronectin, and
laminin on human oral keratinocyte adhesion and spreading to the culture
plate surface and found that 50 mg/ml type I collagen, 1 mg/ml bronectin,
and 10 mg/ml laminin showed the approximately maximal effect on
NHOK (data not shown). The wells were then washed with PBS and
unbound sites were blocked with PBS containing 1% heat-inactivated
bovine serum albumin (BSA). The plates were rinsed again with PBS. Cells
were detached by treatment with 0.05% trypsin and 0.53 mM ethylenediaminetetraacetic acid (EDTA) in PBS, resuspended in the culture media
(1
105 cells/500 ml), added to each plate, and incubated for 1 h at 37 1C.
Unattached cells were removed by rinsing twice with PBS. Attached cells
were xed with 10% formalin in PBS for 15 min and rinsed twice with
PBS. Cells attached to the collagen nanobers were stained with
hematoxylin and eosin. The wells were gently rinsed three times with
double distilled water (DDW). The electrospun collagen nanobers were
mounted, and cells attached onto the nanobers were photographed. To
ensure a representative count, each nanober sample was divided into
quarters, and two elds per quarter were photographed with an Olympus
BX51 microscope at 100
.
Cell spreading was analyzed using photographs of the cell adhesion
assay. To ensure a representative count, each nanober sample was
divided into quarters, and two elds per quarter were photographed with
an Olympus BX51 microscope at 100
. Cells that adopted a attened,
polygonal shape with lopodia- and lamellipodia-like extensions were
regarded as spreading cells. In contrast, cells that resisted washing
and remained tethered to the plate surface were regarded as nonspreading cells. The percentage of cells displaying spread morphology was
quantied by dividing the number of spread cells by the total number of
bound cells.

2.7. Scanning electron microscopy

SEM was used to examine the morphological characteristics of cells
cultured onto the collagen nanobrous structure. Electrospun collagen
nanobers were cut out with punch (14-mm in diameter) and put onto the
24-well culture plates. NHOK were plated at 3
104 cells onto each
electrospun collagen nanober and cultured for 1, 3, or 7 days at 37 1C in
5% CO2. The medium was changed every 2 days during the culture period.
Loosely adherent or unbound cells were removed from the experimental
wells by aspiration, the wells were washed twice with a 0.1 M cacodylate
buffer, and the remaining bound cells were xed in 2.5% glutaraldehyde in
a 0.1 M cacodylate buffer (pH 7.4) for 10 min. The xative was then
aspirated. After being washed in the buffer, electrospun collagen
nanobers were dehydrated in a graded series of ethanol solutions. After
critical point drying (Polaron model 5400; Bio-Rad, Hercules, CA), the
samples were sputtered with gold, using an SEM coating system (BioRad), and the probes were examined by SEM (JEOL, JSM 840A, Japan).
To ensure a representative count, each electrospun collagen nanober was
divided into quarters, and one eld per quarter was photographed.
Spreading and nonspreading cells were distinguished as described in the
above section.

2.8. Open wound healing test

Twelve SpragueDawley rats weighing 240710 gm were used in this
study. After anesthetization, an intramuscular injection of a mixture of
ketamine (50 mg/kg) and xylazine (10 mg/kg), two full-thickness rectangular wounds of 1 cm
1 cm were prepared on each of the rats back,
parallel with the vertebral column. The electrospun collagen nanobers
without ECM protein coating were then applied to the wounds of each rat.
The same wound was treated with cotton gauze as a control. On the 7th or
10th postoperative day, macroscopic photographs of the wounds were
obtained, and the wound area was measured using a slide caliper.
Additionally, the wound was removed on the 7th or 28th postoperative
day after the rats are euthanized with an intramuscular injection, a
mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg), then with
intravenous injection of KCl (3 ml/kg), for histological examination of
epithelialization and granulation.

2.9. Statistics and data analysis

Cell adhesion and spreading of electrospun collagen nanobers and
ECM protein-coated electrospun collagen nanobers were compared by
analysis of variance (ANOVA), using the STATISTICA 6.0 software
package. When signicant differences were found, pairwise comparisons
were performed using Scheffes adjustment. Differences were considered
statistically signicant for P values o0.05.

3. Results and discussion

3.1. Electrospinning of type I collagen
The fabrication of a collagen nanobrous matrix via
electrospinning is very desirable because collagen nanobers are a principal structural component of the ECM
matrix. Huang et al. [18] electrospun collagen dissolved in
acetic acid aqueous solution, but failed to prepare the
continuous bers. Instead, collagen-poly(ethylene oxide)
(PEO) composite bers were produced by the electrospinning of a weak acid solution of collagen at room
temperature [18], but pure collagen bers could not be
obtained. Matthew et al. [1] rst electrospun type I and III
collagen using HFIP as a solvent and found that the
structural properties of electrospun collagen varied with
the isotype (type I vs. type III) and with the concentration
of the collagen solution. Although it was essential to
optimize the vapor cross-linking conditions, they did not
investigate in detail the effect of vapor cross-linking on the
collagen matrix.
In this study, continuous collagen nanobers were
obtained at a concentration of 8% by weight, a concentration that appears to correspond to the onset of signicant
chain entanglements. The solution concentration of 8% by
weight was chosen to fabricate a nonwoven matrix of
randomly arranged collagen bers with nanometer scale
diameters. Fig. 1A shows a SEM micrograph of the asspun (i.e., uncross-linked) collagen nanobers under
magnication of 5000
. The as-spun collagen nanobers
were a somewhat less uniform matrix of bers with a
smooth surface. From the image analysis, it was found that
the bers had an average diameter of 460 nm, with a range
of 1001200 nm, as shown in Fig. 1B.

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461

1455

Weight loss (%)

0
40

10

15

20

25

Cross-linking time (h)

35
Number of fibers

Fig. 2. Weight loss of the collagen nanobrous matrix by cross-linking

time.

30
25
20
15
10
5
0
0

(B)

0.5

1.0

1.5

2.0

Diameter (m)

Fig. 1. SEM image (A) and ber diameter distribution (B) of as-spun type
I collagen nanobers. Bar, 10 mm; 5000
magnication.

3.2. Cross-linking of the collagen nanofibrous matrix

The as-spun collagen matrix needed to be cross-linked
because it was readily swellable or partially soluble in
water. Collagen is commonly cross-linked using water
soluble glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in an aqueous system [19,20]. In
the present study, the collagen nanobrous structure was
immediately changed into a dense membrane structure by
cross-linking in an aqueous system, even though the
aqueous medium contained a small amount (below 5%)
of water. Therefore, we chose vapor cross-linking by
glutaraldehyde to avoid the collapse of the collagen
nanobrous matrix during cross-linking in the aqueous
system. Additionally, the unreacted aldehyde groups were
treated with 0.1 N glycine aqueous solution after vapor
cross-linking. Fig. 2 shows the effect of cross-linking time
on the weight loss (%) of the collagen nanobrous matrix.
The weight loss (%) of collagen matrix was determined by
weighing the dried sample before and after the immersion
in distilled water for 1 h. The as-spun (uncross-linked)
collagen matrix showed a weight loss of 3.6% and was
highly swollen in water. As cross-linking time increased up
to 9 h, the weight loss values of collagen matrices gradually

Fig. 3. SEM images of collagen nanobrous matrices before (A) and after
(B) vapor phase cross-linking for 12 h. Bar, 10 mm; 5000
magnication.

decreased and they swelled less in water. Considering the

weight loss and dimensional stability of cross-linked
collagen matrices, we choose vapor cross-linking for 12 h
as an appropriate condition. Fig. 3 shows a SEM

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461

micrograph of a collagen nanobrous matrix after crosslinking for 12 h.

3.3. Characterization of the cross-linked collagen

nanofibrous matrix
Pore parameters of as-spun and cross-linked collagen
matrices determined by mercury porosimetry are summarized in Table 1. The porosity of the as-spun collagen
nanobrous matrix was 89%, indicating that it was highly
porous. The total pore volume was 9.328 ml/g, and the
total pore area was 60.856 m2/g. Whereas the bulk density
of the matrix composed of collagen nanobers increased
from 0.096 to 0.452 g/ml during cross-linking for 12 h, the
porosity and pore volume decreased from 89% to 71% and
from 9.328 to 1.566 ml/g, respectively, indicating that the
collagen matrix was distorted to form a denser structure by
cross-linkage. However, we believe that the cross-linked
collagen matrix had a higher dimensional stability in
aqueous medium due to cross-linking for 12 h. From the
deviation in average pore diameter in terms of volume (V),
area (A), and 4V/A, it can be concluded that the pore size
distribution was very broad.
Fig. 4 shows the inuence of cross-linking on the pore
size distribution in the as-spun collagen matrix. The
maximum pore size of the as-spun collagen matrix was
appropriately 180 mm, while that of the cross-linked
collagen matrix was approximately 210 mm. However, the
amount and distribution of pores in the cross-linked
collagen matrix were smaller and narrower, respectively.
The mechanical properties of the cross-linked collagen
nanobrous matrices were assessed using a tensile test. The
maximum load value of Instrons (Instron) on the collagen
matrix was evaluated. Table 2 shows that the tensile
strength of the collagen matrix (0.2 mm thick) was above
10 MPa. The tensile strength of the collagen nanobrous
matrix was comparable to that of two commercial tissue
regenerative membranes (Resoluts LT, Bioxs) [15] and a
wound-dressing material (Beschitins W; Unitica). This
indicates that the cross-linked collagen nanobrous matrix
provided a similar level of mechanical stability when
applied in the wound-healing procedure. Although the
cross-linked collagen nanobrous matrix had a lower

tensile strength after being hydrated with phosphate buffer

(pH 7.4), which is common for cross-linked hydrophilic
materials, the matrix presented enough stability to maintain its shape for in vivo procedure. Therefore, there is no
mechanical problem in using a collagen nanomembrane in
vivo as the wound-dressing material. As the collagen
nanobrous matrix possesses almost identical tensile
strength to commercial products, it was anticipated that
the collagen nanobrous matrix could be a suitable tissueengineering scaffolding for skin regeneration.
3.4. Effect of collagen nanofibers, alone or in conjunction
with ECM protein coating, on cell adhesion and spreading of
normal human keratinocytes
Tissue-engineering scaffolding must promote cell growth
and physiological function and must maintain normal
states of cell differentiation. We examined the biological
properties of an electrospun collagen matrix in cell culture
experiments and studied the cytocompatibility of electrospun collagen nanobers, alone or in conjunction with
ECM protein coating, as a possible wound-dressing
material and scaffolding for tissue engineering. Since the
absence of cytotoxicity of collagen had already been
1.8
As-spun

Incremental intrusion (mlg-1)

1456

Cross-linking time:12 h

1.5
1.2
0.9
0.6
0.3
0
400

300
200
Diameter (m)

100

Fig. 4. Effect of vapor phase cross-linking on the pore size distribution of

as-spun collagen matrix.

Table 1
Mercury porosimetry analysis of as-spun and cross-linked collagen nanobrous matrices
Samples

As-spun
Cross-linked (12 h)
a

TIV (ml/g)a

9.328
1.566

TPA (m2/g)b

60.856
39.452

TIV, total intrusion volume;

TPA, total pore area;
c
APD, average pore diameter; 4V/A, 4 volume/area;
d
D, bulk density;
e
P, porosity.
b

APD (mm)c
Volume (V)

Area (A)

4V/A

77.993
135.989

0.008
0.007

0.613
0.159

D (g/ml)d

P (%)e

0.096
0.452

89.21
70.83

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461
Table 2
Tensile strength of the cross-linked collagen nanobrous matrix and of
three commercial matrices
Groups

Tensile strength (MPa)

Reference

Collagen NF (0.2 mm)a

Collagen NF, hydrated form
Beschitins W (1 mm)
Resoluts LT
Bioxs

11.4471.20
7.4071.17*
15.8970.63*
11.72
14.50

[20]
[20]

Values in parentheses represent the thickness of matrices. Data are

expressed as mean7SD from three independent determinations.
ANOVA: Po0:05. Pairwise comparisons: *Po0.05 versus collagen
NF.
a
Collagen NF, cross-linked collagen nanobrous matrix.

demonstrated [1] and initial cell adhesion and spreading

could be important factors in developing wound dressing
and scaffolds for tissue engineering, the initial cell
attachment and spreading of electrospun collagen nanobers were studied. To assess cell adhesion, cross-linked
collagen nanobrous matrices were seeded with normal
human keratinocytes, such as NHOK and NHEK. The
adhesion of cultured keratinocytes was evaluated using a
cell adhesion assay in serum-free medium. Exponentially
proliferating NHOK and NHEK adherent to the electrospun collagen nanobers were microphotographed in the
adhesion assay after washing, xing, and staining the cells
with hematoxylin and eosin. Unexpectedly, a relatively low
level of cell adhesion of normal human keratinocytes was
observed on collagen nanobers without ECM coating
compared to a polystyrene surface. Although the reason
for this low adhesion remains unknown, it may be a
consequence of denaturalization of the collagen conformation induced by electrospinning or cross-linking. Generally,
the procedures used to reprocess a natural protein into an
engineered material compromise many of its biological and
structural properties [21].
Because collagen nanobrous matrices showed relatively
low cell adhesion in normal human keratinocytes compared with that of a polystyrene surface, we examined
the adhesion of cells on collagen nanobrous matrices
treated with ECM proteins for their ability to restore
collagens biological and structural properties. The adhesion of cultured keratinocytes was evaluated with a cell
adhesion assay in serum-free medium, using type I collagen (50 mg/ml), bronectin (1 mg/ml), or laminin
(10 mg/ml), which were adsorbed onto the collagen
nanobers as substrates. Type I collagen and laminin
signicantly promoted the adhesion of proliferating
NHOK and NHEK compared with collagen nanobers
only (Figs. 5A and B). The adhesion activity prole of
bronectin-coated electrospun collagen nanobers on
NHOK was similar to that of uncoated collagen nanobers, but cell adhesion was slightly promoted in NHEK.
These results indicate that type I collagen and laminin,
which are integrin ligands, are functionally active in cell

1457

adhesion onto electrospun collagen nanobers in normal

human keratinocytes.
To further evaluate the adhesion of type I collagen,
bronectin, and laminin, we determined whether
adherent cells were tethered to the substrate or spread
over the substrate. Photographs of exponentially proliferating NHOK and NHEK adherent to integrin
ligands were taken in the adhesion assay. For type I
collagen, 22% of the proliferating NHOK and 55%
of the proliferating NHEK showed a spreading morphology (Fig. 5C), i.e., they adopted a attened, polygonal
shape, with lopodia- and lamellipodia-like extensions.
The remaining nonspreading cells on integrin ligands
resisted washing and remained tethered to the nanober
surface (data not shown). Laminin displayed functional
properties similar to type I collagen, as 18% of proliferating NHOK and 47% of proliferating NHEK displayed a
spreading morphology. In contrast, extremely low cell
spreading was observed on either BSA- or bronectincoated collagen nanobers (Fig. 5C). These results indicate
that type I collagen is similar to laminin in that it supports
cell adhesion and spreading, but that bronectin is
functionally inactive in terms of cell adhesion and
spreading.
3.5. Wound healing and histological examination
In open wound healing tests, twin full-thickness rectangular wounds were made on the back of each rat. On the
7th and 10th days, wound closure in the collagen
nanober-covered wounds was similar to the control
group that had wounds covered with gauze (data not
shown). In contrast, microscopic examination revealed
that early-stage healing in the collagen nanober group
was faster than that of the control group (Fig. 6). In the 1week control group, the wound surface was covered by
brinous tissue debris, and below that layer, dense
inltration of polymorphonuclear leukocytes and proliferation of broblasts were seen. In the 1-week collagen
nanober group, however, the surface tissue debris
disappeared, and there was prominent proliferation of
young capillaries and broblasts. Late-stage healing
processes in the control group were similar to that of the
collagen nanober group (Fig. 6). In both groups,
epithelization of the wound was complete after 4 weeks.
Additionally, inammatory cells disappeared and connective tissue was densely formed.
As described above, the fabrication of a collagen
nanobrous matrix via electrospinning and cross-linking
appeared to provide good mechanical strength, even in
aqueous solution. Furthermore, collagen nanobrous
matrices coated with type I collagen or laminin were
functionally active in terms of cell attachment and
spreading in normal human keratinocytes. Additionally,
the electrospun collagen nanober nonwovens potentially
provide a three-dimensional structure for cell attachment,
growth, and migration. Therefore, cell morphology and the

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461

1458

PS

Collagen only

BSA

COL I

LN

FN

NHOK

NHEK

(A)
1400
1200

PS

Collagen only

BSA

COL I

FN

LN
*

Cells/11.25 mm2

1000
*

800

600
400
200
0

(B)

NHOK

NHEK

90

Percent cell spreading (%)

80

PS

Collagen only

BSA

COL I

FN

LN
*

70
60

50
40
30

20

10
0
(C)

NHOK

NHEK

Fig. 5. Cell adhesion and spreading of normal human keratinocytes plated onto electrospun collagen nanobers, alone or in conjunction with ECM
proteins. (A) Examples of cell adhesion and spreading on type I collagen, bronectin, or laminin in proliferating NHOK and NHEK. (B) Level of cell
adhesion of cultured cells on ECM proteins. (C) Incidence of cultured cells spreading on ECM proteins. Data are expressed as mean7SD (n 4).
ANOVA: Po0:05. Pairwise comparisons: *Po0:05. PS, polystyrene surface; Collagen only, uncoated collagen nanobers; BSA, bovine serum albumincoated collagen nanobers; Col I, type I collagen-coated collagen nanobers; FN, bronectin-coated collagen nanobers; LN, laminin-coated collagen
nanobers.

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Fig. 6. Representative photomicrographs of wound healing of rat skin; control group at 1 week (A), control group at 4 weeks (B), collagen nanober
group at 1 week (C), and collagen nanober group at 4 weeks (D) (H&E, 100
). Insets: 400
magnication.

interaction between cells and collagen nanobers, alone or

in conjunction with an ECM protein coating, were studied
in vitro for 7 days. SEM micrographs showed that
NHOK adhered and spread on the surface of the collagen nanobrous network and had started to migrate
through the pores and to grow under layers of the bers
on day 7 (Fig. 7). This is supported by another study
that demonstrated that electrospun collagen nanobers
were densely populated with smooth muscle cells
within 7 days and that smooth muscle cells were observed
deep within the matrix and fully enmeshed within the
brils of the electrospun collagen [1]. However, type I
collagen- and laminin-coatings on collagen nanobers
signicantly promoted interactions between cultured
oral keratinocytes and collagen nanobrous matrices, in
the following order: type I collagen4laminin4uncoated
collagen nanobers. NHOK also adhered and spread on
the surface of the type I collagen- and laminin-coated
collagen nanobrous network and migrated through the
pores and grew under layers of the bers within 13 days.
These cells interacted and integrated well with the

surrounding bers and grew in the direction of ber

orientation, forming a three-dimensional network of
nanobrous structure (Fig. 7). Our observation is consistent with earlier reports demonstrating that electrospun biodegradable scaffolds are capable of supporting
cell attachment and proliferation of human bonemarrow-derived mesenchymal stem cells [22] and normal
human keratinocytes [14]. These results support our view
that type I collagen and laminin, the integrin ligands,
are functionally active in promoting cell adhesion and
spreading of normal human epithelial cells onto the
collagen nanobrous matrices. Additionally, surface
modication of biomaterials with ECM proteins or
peptides was extensively tested. Given that the nal
goal of the scaffold design is the production of an ideal
structure that can replace natural ECM proteins until
host cells can repopulate and resynthesize a new natural
matrix, cross-linked collagen nanobers coated with
ECM proteins, particularly type I collagen, may be a good
candidate for biomedical applications, such as wound
dressing and scaffolds for tissue engineering.

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K.S. Rho et al. / Biomaterials 27 (2006) 14521461

Fig. 7. SEM micrographs of the interaction between NHOK and an electrospun collagen nanobrous structure coated with type I collagen after 0, 1, 3,
and 7 days of culture. Bar, 10 mm.

4. Conclusion
In the present study, a collagen nanobrous matrix
produced by the electrospinning process was introduced for
the application of wound dressing. The collagen nanobers
were characterized by a wide range of pore size distribution, high porosity, excellent mechanical strength, and high
surface area-to-volume ratios, which are favorable parameters for cell attachment, growth, and proliferation. In
the cell activity assessment, electrospun collagen nanobers
coated with type I collagen or laminin were found to
promote cell adhesion and spreading of normal human
keratinocytes. This may be a consequence of the high
surface area available for cell attachment due to their threedimensional features and of the restoration of natural
ECM proteins biological and structural properties.
Acknowledgments
This work was supported by a grant from the Korean
Science and Engineering Foundation through the Intellectual Biointerface Engineering Center at the Seoul National
University.
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