Journal of Biomaterials and Nanobiotechnology, 2011, 2, 125-133
doi:10.4236/jbnb.2011.22016 Published Online April 2011 (http://www.SciRP.org/journal/jbnb)
Preparation, Properties, and Cell Attachment/
Growth Behavior of Chitosan/Acellular Derm
Matrix Composite Materials
Tao Lu1, Ruixin Li2, Yan Zhang2, Yuxian Yan2, Yong Guo2, Jing Guan2, Jimin Wu2, Zhihong Li2,
Bo Ning2, Shujie Huang2, Xizheng Zhang2,*
1Affiliated Hospital of Chinese People’s Armed Police Force Medical College, Tianjin, China; 2Institute of Medical Equipment,
Tianjin, China.
Received December 3rd, 2010; revised January 1st, 2011; accepted January 9th, 2011.
ABSTRACT
Composite membranes and sponge scaffolds consisting chitosan (CS) and acellular derm matrix (ADM) in six ratios
were prepared by solvent evaporation technique and freeze-drying method, respectively. The composite materials were
characterized by water contact angle measurement, scanning electron microscopy (SEM), water absorption and HaCat
cells compatibility. The SEM result showed that CS/ADM three-dimensional (3D) micro-porous structures were suc-
cessfully produced. The water absorption value of all scaffolds was over 18 times of its initial weight, which is high
enough for skin regeneration scaffold, but there were no significant differences of water absorption ratio between de-
ionized water and PBS solution for same scaffold (P > 0.05). HaCat cells were distributed uniformly on the surfaces of
membrane 4 - 6, and an almost confluent monolayer was formed on membrane 6 on the fifth day, whereas cells main-
tained round and spherical in shape on the surface of membrane 1. The results showed that the cell compatibility of
pure CS membrane needed to be improved, and addition of ADM realized this purpose. The results of compatibility of
HaCat cells on scaffolds showed that the cell proliferated well on the scaffolds 3 and 4. In our study, the cell’s attach-
ment and growth on the composite membranes was mainly determined by the content of the membrane, whereas the
cell’s attachment and growth in the scaffolds was determined by both the content and structure of the scaffolds.
Keywords: Chitosan, Acellular Derm Matrix, Membrane, Scaffolds, HaCat Cell Compatibility
1. Introduction
Skin being the largest and most highly complex organ in
the human body is the most affected organ in injuries [1].
Every thing is done to reduce risks for health, especially
from the growing number of synthetic compounds and
new formulations [2]. Skin corrosivity testing in vivo
may cause severe discomfort and pain to test animals.
Therefore, many attempts have been made to replace the
in vivo test in laboratory animals. Skin corrosion is de-
fined as the production of irreversible tissue damage in
the skin following the application of a test material
(OECD, 2002) [3]. The new OECD Test Guideline 431
“In Vitro Skin Corrosion” (OECD, 2004) [4] defines the
requirements for in vitro skin models to be validated for
skin corrosivity testing and defines general and func-
tional model conditions that need to be evaluated before
the skin models will be routinely used. The most impor-
tant general conditions are a multi-layered, functional
stratum corneum with the necessary lipid profile, and
absence of any contamination. The most important func-
tional conditions specified in TG 431 are a stable and
sufficiently high cell viability (expressed as metabolic
conversion capacity), a sufficient resistance to a slowly
penetrating cytotoxic marker chemical, reproducibility of
data over time and between laboratories, and finally, ca-
pability to correctly classify twelve reference chemicals
specified in TG 431. But in China, only a few works have
been done about it, so we try to reconstruct a new human
epidermal model scaffold.
Chitosan (CS) is an abundant, naturally occurring
polysaccharide obtained by deacetylation of natural chi-
tin [5]. It is biocompatible, biodegradable, easily formed
into structures under mild processing conditions and can
be chemically modified, so it is a natural choice as drug-
delivery carrier [6,7], cartilage/skin tissue engineering

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular
126
Derm Matrix Composite Materials
scaffolds [8-10], and regenerative membrane [11]. The
physical properties of a polymer can be altered by intro-
ducing a second polymer that improves the properties of
the original polymer in certain aspects, such as hydro-
phobility, cell compatibility. CS and some of its com-
plexes have been studied for a number of biomedical
applications, including wound dressings, drug delivery
systems and space-filling implants [12-18]. In a study
comparing purified collagen, naturally occurring ex-
tracellular matrix (ECM) scaffolds, and synthetic scaf-
fold materials for in vitro endothelial cell attachment [19],
it was found that ECM possessed the ability to recruit
circulating marrow-derived progenitor cells and attract
mature endothelial cells from selected organs such as the
heart and liver to promote successful vascularization of
engineered tissue structures. These studies reveal that
extracellular components in a cell-free or acellular derm
matrix (ADM) are critical for success in biomedical ap-
plications as scaffolds. HaCat cells, a human keratinocyte
line, are commonly utilized as an in vitro cell model for
toxicity testing and the discernment of process of chemi-
cally induced skin carcinogenesis.
In this study, we prepared CS/ADM composite mem-
branes and scaffolds by solvent evaporation technique
and freeze-drying method, respectively, and investigated
the characteristics of composite materials by water con-
tact angle measurement, scanning electron microscopy
(SEM) observation, water absorption ratio and HaCat
cells compatibility test.
2. Methods
2.1. Materials
CS (Mw ≈ 20 000, degree of deacetylation of 75% - 85%)
was purchased from the Sigma Chemical Company
(Sigma-Aldrich Co.Ltd., USA) and used without further
purification. Dulbecco’s modified eagle’s medium
(DMEM), fetal calf serum (FCS) and trypsin-EDTA (1×)
were purchased from Gibco Laboratories (Invitrogen
Corporation, CA, USA). All other chemicals were of
analytical grade and used without further purification.
2.2. Preparation of CS/ADM Composite
Membranes and Scaffolds
ADM was prepared as follows: Porcine skin of 0.3 - 0.4
mm in thickness was obtained by removing the epider-
mal. It was digested in 0.25% trypsin solution at 37℃
for 24 h to remove the epidermis and other cellular com-
ponents. The remaining dermis was then immersed in
0.5% Triton X-100 solution for 36 h with continuous shak-
ing to further remove cellular components, and subse-
quently washed with deionized water to obtain ADM.
ADM particles was prepared by milling the prepared ADM.
Acetic acid was used as a solvent. To avoid the ADM
particles aggregation, an alternative method was devel-
oped. First, ADM particles were scattered in water by
stirring, and then CS powder, not solution, was added
with strong stirring to ensure that the powder was uni-
formly mix with ADM particles. Finally, acetic acid was
added to the solution. Since the CS powder was already
uniformly dispersed, the addition of acetic acid caused
the CS powder to immediately dissolve, thus avoiding
the aggregation caused by ADM particles. Therefore, a
homogeneous solution was obtained. The proportions of
CS powder and ADM particles are listed in Tab.1. The
resulting solution was allowed to stand at 4℃ until all
air bubbles had disappeared.
Composite membranes were prepared by solvent
evaporation technique as follows: the mixture solution
was cast into cell culture plates and allowed to dry at
ambient temperature to form the composite membranes.
The prepared composite membranes were neutralized
with 1 wt.% aqueous NaOH solution for 30 min and
subsequently washed with deionized water to remove the
remaining NaOH. Composite porous scaffolds were pre-
pared by using the same mix solutions: the mixture solu-
tion was cast into cell culture plates and frozen at –20℃,
and the composite scaffolds were prepared by freeze-
drying method. The prepared composite scaffolds were
neutralized with 1 wt.% aqueous NaOH solution for 2 h
and subsequently washed with deionized water to remove
the remaining NaOH. All the membranes and scaffolds
were sterilized before seeding cells.
2.3. Water Contact Angles and Wettability of
Composite Membranes
The water contact angles of the surface of the composite
membranes were measured with HARKE SPCA contact
angle goniometer (Beijing Harke Instrument Company).
Distilled water was deposited on the surface of the sam-
ples with an automatic pipette. After being deposited, all
contact angle measurements were taken within 20 s. With
a digital camera a computer image of the drop was de-
termined, and the water contact angle θ was calculated
according this formula: tg (θ/2) = h/r, where h is the high
of the water drop and r is the contact radius between wa-
ter drop and the bottom. Figure 1 shows the schematic
diagram of water contact angle. All measurements were
r
h
θ/2
Figure 1. Schematic diagram of water contact angle.

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular 127
Derm Matrix Composite Materials
taken at an ambient temperature (25℃) and they were
repeated four times for each sample.
2.4. H.E staining of Sponge Scaffold
Scaffold samples were first dehydrated with an increas-
ing series of alcohol concentrations (30%, 50%, 70%,
90%, 100%) and then embedded in paraffin. Paraffin-
embedded samples were sectioned at a thickness of 5 mm.
After removing the paraffin, samples were stained with
hematoxylin and eosin. After sealing, samples were ex-
amined by light microscopy to inspect the degree of
acelluar.
2.5. SEM Observation
For SEM observation, the specimens were fixed with
1.5% glutaraldehyde in 0.14 m sodium cacodylate buffer
(pH 7.3), then dehydrated in graded alcohols, critical-
point dried, sputter-coated with gold and analyzed in a
SEM equipped (HITACHI S-3400N) at an accelerating
voltage of 30 KV and current of 119 µA.
2.6. Water Absorption Ratio of Sponge Scaffold
The CS/ADM composite scaffolds were incubated in
deionized water and PBS solution at 37℃ for 24 h, and
swelling continued to reach constant weight of the sam-
ple. Before weighing the sample, surface water was re-
moved with filter paper. The water absorption ratio (R)
was calculated by the following equation:
R = (Ws − Wi) / Wi
where Ws is the weights of swollen state and Wi is the
weight of initial sample (before being immersed in water
or PBS).
Each value was averaged from three parallel meas-
urements.
2.7. Cell Compatibility
HaCat cells, an immortalized but non-tumorigenic human
epidermal keratinocyte cell-line which retain their dif-
ferentiation potential [20], were routinely cultured in
high glucose DMEM medium supplemented with 10%
(v/v) fetal calf serum, 10 µg/ml of streptomycin and 10
U/ml of penicillin, and incubated at 37℃ in a 5% CO2,
95% air humidified atmosphere.
2.7.1. Cell Compatibility of Membranes
HaCat cells were detached from the culture plated with
0.25% Trypsin, centrifuged, and then suspended in me-
dium. The cell concentration was determined by manual
count with a hemocytometer. 350 µl cells suspension at
density of 12 × 104/ml was seeded to each well (6-well
culture plate) with composite membranes covered bottom
and four wells for each sample. Cell proliferation was
measured at 1, 3, 5, 7 days using the 3-(4, 5-dimethyl-
thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)
assay (Tianjin Runtai Reagent Company). Each sample
was measured at 8 times. H.E staining also applied to the
cultural plates with cells.
2.7.2. Cell Compatibility of Scaffolds
Scaffolds were sliced into （0.0040 ± 0.0001）gram
pieces, and sterilized before use. 100 µl cells suspension
at density of 10 × 104/ml were seeded to each small pre-
wetted scaffold, and four scaffolds for each kind of sam-
ple. Cell proliferation was measured at 1, 3, 5, 7 days
using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT) assay in TECAN Elisa
Reader.
2.8 Statistical Analysis
Differences between groups were analyzed by the SARS
(version 11.0, http://www.pinggu.org/bbs/a-385796.html).
Differences were considered to be significant at P < 0.05.
3. Results and Discussion
3.1. Water Contact Angles of CS/ADM
Composite Membranes
The material–cell interaction is affected by many factors,
such as wettability (hydrophilicity/hydrophobility), sur-
face free energy, chemistry, charge, roughness, and ri-
gidity. The surface properties of scaffold are critical for
its application because the surface of the scaffold is the
place where the material interacts with the bioenviron-
ment and where the cells attach and proliferate. Hydro-
philicity is an important characteristic property for bio-
materials. To determine the hydrophilicity of the mem-
branes, their contact angles were investigated.
The higher contact angle indicates lower hydrophilic-
ity. The water contact angles on the membranes are
shown in Figure 2. As a consequence, the contact angle
0
10
20
30
40
50
60
70
80
123456
w ater c ontact angle(degr ee)
0s
3s
6s
9s
12s
15s
18s
*Significance is indicated: P < 0.05 vs. sample 1
Figure 2. Water contact angle of composite membranes (n =
4).

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular
128
Derm Matrix Composite Materials
of all membranes decreased from 0s to 18s. In addition,
the contact angle of membrane 1 to 5 exhibited no sig-
nificant difference, and membrane 6 exhibited the high-
est contact angle, which was primarily due to the surface
become rougher with small ADM fibers scattered in the
CS. Although the wettability of membrane was changed
by blending with ADM, the contact angles for all the
materials were less than 70°, indicating that all these ma-
terials had good hydrophilicity.
Additionally, by comparing the data at different meas-
uring times, we found that the contact angles decreased
with time, indicating that the hydrophilicity of the matri-
ces increased with the absorption of water.
The contact angle of membrane has been considered to
be one of the physical parameters which related the af-
finity between cells and the matrix membranes. However,
no correlation between the initial cell anchoring rate and
the wet contact angle was found for the HaCat cells in
the present study.
3.2. H.E Staining of Scaffold
H.E staining photograph of scaffold 4 is shown in Figure
3, in which whole cells are absent from the scaffolds.
This indicated the preparation of acellular derm matrix
was successful.
3.3. SEM Observation
CS is a crystalline polysaccharide and is normally in-
soluble in aqueous solutions above pH 7. However, in
dilute acids (pH < 6), the free amino groups are proto-
nated and the molecule becomes soluble. This pH-de-
pendent solubility provides a convenient mechanism for
processing under mild conditions [21]. Figure 4 shows
the SEM image of scaffold 1, 4 and 5. This image re-
vealed the freeze-drying process generated an open pore
microstructure with a high degree of interconnectivity.
But with the increasing of ADM content, the pore uni-
formity decreased. Pure chitosan scaffold had the best
pore structure.
Figure 3. H.E staining of scaffold 4 (× 100).
Figure 4. SEM image of scaffold 1, 4, 5.
3.4. Water Absorption Ratio of Composite
Scaffolds
The ability of a scaffold to preserve water is an important
property for skin regeneration. The water absorption ra-
tios of various scaffolds are shown in Table 2. The water
absorption ability of the CS/ADM scaffold could be at-
tributed to both of their hydrophilicity and the mainte-
nance of their three-dimensional structure. Differences
between sample 1 and other groups were analyzed by the
SARS and the result was P < 0.05. So, there were sig-

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular 129
Derm Matrix Composite Materials
nificant differences of water absorption ratio between
pure CS scaffold and composite scaffolds. The main
reason was the pore structure of pure CS was the best,
which could be proved by SEM images in Figure 4. In
addition, differences between deionized water and PBS
solution groups were analyzed by the SARS and the re-
sult was P > 0.05 for each sample. So, there were no sig-
nificant differences of water absorption ratio between
deionized water and PBS solution for same scaffold.
The scaffold provides a necessary template and physi-
cal support to guide the differentiation and proliferation
of cells into targeted functional tissues or organs. The
scaffold should contain its three-dimensional structures
in liquid culture medium. It should absorb body fluid for
transfer of cell nutrients and metabolites through the ma-
terial [22]. Transport issues such as nutrient delivery,
waste removal, protein transport, gaseous exchange, and
general vascularization and guided tissue regeneration
are governed by the pore structure of the scaffold [23]. In
our study, the absolute water absorption value of all
scaffolds was over 18 times of its initial weight, which is
high enough for skin regeneration scaffold. This result is
similar with other studies on CS scaffolds. Ma et al. [22]
fabricated freeze-dried CS-collagen scaffolds that were
crosslinked with glutaric dialdehyde (GTA). They found
that crosslinking decreased the swelling ratio from 16 to
8.
3.5. Morphology of HaCat Cell
Figure 5 shows the morphology of HaCat cells on cul-
ture plate photographed by a phase contrast microscope.
Cells exhibited flagstone shape and form a confluent
after being cultured for 3 days.
3.6. Morphology and Proliferation of HaCat Cell
on the Surface of Composite Membranes
3.6.1. Morphology of HaCat Cell Adhered on the
Surface of Composite Membranes
Prepared pure CS membrane was transparent, whereas
composite membranes were opaque with white small
fibers scattered in the membranes. In our study, the pre-
pared membranes stick to the culture plates tightly, so
that the cells could adhere on the surface of the mem-
brane instead of on the culture plates.
The cytocompatibility of the matrices is very impor-
tant for their applications. Since it is the biomaterial sur-
face that first comes into contact with the living tissue
when the biomaterial is planted in the body, the initial
response of the body to the biomaterial depends on its
surface properties. Surface properties that can influence
biocompatibility include surface charge, surface topog-
raphy, etc [24-29].
1 d × 40
3 d × 40
3 d × 100
Figure 5. Phase contrast images of HaCat cell on culture
plates.
To examine the cell-matrix interactions, HaCat cells
were cultivated on the surface of the composite mem-
branes, their morphology and distribution were observed
and photographed by a phase contrast microscope on day
1, 3, 5, 7 d. Figure 6 and Figure 7 show the morphology
of HaCat cell on the composite membrane on day 1 and
day 7, respectively. The micrographs of a 1-day culture
reflect the status of HaCat cell attachment and spreading.
Figure 6 shows that the attached cells on membrane 1
(pure CS membrane) were round and spherical in shape.
Whereas the cells on the other membranes were flat, po-
lygonal, and the difference of membrane 2 to 6 in spread-
ing was not distinct. Chen et al. [30] concluded that cells

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular
Derm Matrix Composite Materials
130
with a flat shape survive better than cells with a more
rounded shape. This result revealed that introduction of
ADM to the CS membrane surfaces increased the num-
ber of cells attached on day 1. This means that most cells
had finished attachment and were in the process of
spreading. Since spreading is an essential step in cell
adhesion prior to exponential growth phase [31], a
greater extent of cell spreading can have a profound elect
on cell adhesion and growth. The distributions of HaCat
cells cultured for 7 days are shown in Figure 7. It can be
seen that the viable HaCat cells on composite mem-
branes was much more than on CS membrane, indicat-
ing that the composite membranes could accelerate the
differentiation of HaCat cells. It is known that the sur-
face morphology of the substrate can exhibit a signifi-
cant influence on the attachment, proliferation and
function of cells in addition to the surface chemistry
[32]. The surface of pure CS membrane showed a
smooth photography. After addition of ADM, the
roughness of the composite membrane surface in-
creased. From Figure 6 2 to 6 it can be observed that
the small ADM fibers scattered in the CS.
1
6 5 4
3 2
Figure 6. Phase contrast images of HaCat cells on the composite membranes on day 1 (× 100).
1
654
32
Figure 7. Phase contrast images of HaCat cells on the composite membranes on day 7 (× 100).

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular 131
Derm Matrix Composite Materials
In order to visualize cell morphologies on different
membrane surfaces, cells and membranes were stained
using Hematoxylin and Eosin Staining Kit (H.E) and
observed with a microscope (BX51, Olympus, Japan).
Figure 8 shows a series of images of HaCat cells ad-
hered onto the membranes which was stained red. HaCat
cells were distributed uniformly on the surfaces of mem-
brane 4-6, and an almost confluent monolayer was
formed on membrane 6 on the fifth day, whereas cells
maintained round and spherical in shape on the surface of
membrane 1. The results showed that the cell compatibil-
ity of pure CS membrane needed to be improved, and
addition of ADM realized this purpose.
3.6.2. Proliferation of HaCat Cells on the Surface of
Composite Membrane
MTT assay was used as a measure of relative cell viabil-
ity. HaCat cells were cultured on the membranes for 1, 3,
5, 7 d, and cell proliferation was determined by the MTT
method. Figure 9 shows the viability of HaCat cell on
different surfaces with different cultural time. There was
significant enhancement of cell proliferation and viability
on the composite membranes compared with that on pure
CS membrane (p < 0.05). Also, cells proliferation rate on
membrane 6 was the highest, the reason perhaps was that
the content of ADM was similar to the ECM environ-
ment in vivo. In addition, cell adhesion on the surfaces
increased and then decreased with a prolonged time pe-
riod of culture. The cells formed a confluent monolayer
on day 5, and their proliferation were inhibited in the
following day resulted in a decrease of viability. From
the results of cell culture, we can conclude that compos-
ite film is bioactive to HaCat cells by increasing the at-
tachment, spreading and proliferation of the cells. This
trend was not consistent with their surface hydrophilicity
indicated by the water contact angle. The present works
revealed the attachment and growth behavior of HaCat
mainly depend on the content of the membrane, not the
contact angle of the membrane.
3.7. Proliferation of HaCat Cells in the
Composite Scaffolds
HaCat cells were cultured in the sponge scaffolds for 1, 3,
5, 7 d, and cell proliferation was determined by MTT
method. The result of MTT test is shown in Figure 10. It
was clear that HaCat cells proliferated well on the scaf-
folds 3 and 4. On the other hand, the increasing ADM of
Figure 8. H.E Staining images of HaCat on composite membranes on day 5.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1d 3
d
5
d
7d
1
2
3
4
5
6
Figure 9. Proliferation of HaCat cells on the surface of
composite membrane.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
1
d
3
d
5
d
7
d
1
2
3
4
5
6
Figure 10. Proliferation of HaCat cells in the composite
scaffolds.

Preparation, Properties, and Cell Attachment/Growth Behavior of Chitosan/Acellular 132
Derm Matrix Composite Materials
sample 5 and 6 scaffolds resulted in lower numbers of
HaCat cells. This trend was not consistent with the statis-
tical results of cell proliferation on membranes. The rea-
son was mainly that the cell growth depended on both
material content and structure of the sponge scaffolds.
Control of scaffold pore morphology is critical for con-
trolling cellular colonization rates within cell scaffold
co-culture in vitro.
4. Conclusions
In this work, six composite membranes were prepared by
solvent evaporation technique and six composite scaf-
folds were prepared by freeze-drying method. The effect
of ADM on the characteristics of composite membranes
and scaffolds were investigated by static water contact
angle measurements and water absorption. The water
absorption value of all scaffolds was over 18 times of its
initial weight, which is high enough for skin regeneration
scaffold, but there were no significant differences of wa-
ter absorption ratio between deionized water and PBS
solution for same scaffold (P > 0.05). The scaffolds
morphology were observed by SEM, and the results
showed that CS/ADM three-dimensional (3D) micro-
porous structures were successfully produced. Cell com-
patibility of the composite membranes and scaffolds
were analyzed by HaCat cells co-culture in vitro. The
cell compatibility results revealed that composite mem-
branes with higher levels of ADM content provided
much better substrates in terms of cell attachment,
growth and morphology. But in the scaffolds, the cell
proliferation was determined not only by the hydrophil-
city and content, but also by the structure of the scaffolds.
As a consequence, a composite material includes mem-
brane 6 and scaffolds 4 will be the best materilas for
HaCat prolifferation. This study was the basic work for
further investigation, a new human epidermal model
composite material includes membrane and sponge scaf-
fold will be reconstructed and the effect of 12 reference
chemical substances in OECD TG 43 on the HaCat vi-
ability in composite material will be investigated in our
future research.
5. Acknowledgements
The research was supported by Key Project of AMMS
Innovation Fund (2008ZD09) and General Program of
Natural Science Foundation of Tianjin (07JCYBJC05100).
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