ARTICLE IN PRESS

Biomaterials 25 (2004) 1453–1460

Preparation of acrylic grafted chitin for wound dressing application

Siriporn Tanodekaewa,*, Malinee Prasitsilpa, Somporn Swasdisonb,
Boonlom Thavornyutikarna, Thanawit Pothsreea, Rujiporn Pateepasenc
a
National Metal and Materials Technology Center, 114 Thailand Science Park, Paholyothin Rd, Klong 1, Klong Luang, Pathumthani 12120, Thailand
b
Department of Oral Pathology, Faculty of Dentistry, Henri Dunant Rd., Chulalongkorn University, Bangkok 10330, Thailand
c
Scientific and Technological Research Equipment Center, Chulalongkorn University, Bangkok 10330, Thailand
Received 19 February 2003; accepted 11 August 2003

Abstract

Chitin grafted with poly(acrylic acid) (chitin–PAA) was prepared with the aim of obtaining a hydrogel characteristic for wound
dressing application. The chitin–PAA films were synthesized at various acrylic acid feed contents to investigate its effect on water
sorption ability. Acrylic acid (AA) was first linked to chitin, acting as the active grafting sites on the chain that was further
polymerized to form a network structure. The evidences of grafting were found from FTIR and solid state 13C NMR spectra. The
TGA results exhibited the high degradation temperature of the grafted product suggesting the formation of a network structure.
The degree of swelling (DS) of chitin–PAA films was found in the range of 30–60 times of their original weights depending upon
the monomer feed content. The chitin–PAA film with 1:4 weight ratio of chitin:AA, possessed optimal physical properties. The
cytocompatibility of the film was investigated with a cell line of L929 mouse fibroblasts. The morphology and behavior of the cells
on the chitin–PAA film were determined after different time periods of culture up to 14 days. The L929 cells proliferated and
attached well onto the film. These results suggested that the 1:4 chitin–PAA has a potential to be used as a wound dressing.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Chitin; Poly(acrylic acid); Hydrogel; Biocompatibility; Scanning electron microscopy (SEM); Wound dressing

1. Introduction Among many attempts, grafting of various monomers

Chitin is one of the most abundant organic materials
in nature. It can be easily prepared from the shells of
crab, shrimp and squid pens. Because of its availability,
biodegradability as well as biocompatibility, chitin and
its derivatives have been used for a variety of applica-
tions such as water treatment, textile and paper,
cosmetic, food and health supplements, agriculture
and biotechnology [1]. In biomedical area, it was found
that chitin possessed high activity as a wound healing
accelerator [2]. Many types of chitin-based materials
adapted for uses in wound dressing application have
been patented [3–7]. However, low water sorption ability
of chitin which yields an inefficient exudate removal
from the wound surface limits its utility as a wound
dressing. The chemical structure of chitin has been
modified to overcome this undesirable characteristic.
*Corresponding author. Tel.: +66-2-564-6500; fax: +66-2-564-
6445.
E-mail address: siriporn@mtec.or.th (S. Tanodekaew).
containing hydrophilic groups onto chitin chains seems
to be a promising method to enhance its water sorption
ability. Grafted products gain a hydrogel characteristic
with a great water retention capacity whereas the
beneficial properties of chitin such as biodegradability
and bioactivity still remain. In fact, there have been very
few publications on the grafting of hydrophilic mono-
mers onto chitin, probably due to the insolubility of
chitin in water and common organic solvents. On the
contrary, a lot of research works have been studied on
chitosan, a principal derivative of chitin produced by
deacetylation process. Chitosan shows enhanced solu-
bility in dilute acids. Chemical modification to alter its
properties can be carried out under milder conditions
compared with that of the parent chitin. Consequently,
there have been many interesting graft copolymers of
chitosan reported [8–14]. Acrylic acid (AA), one of
well known hydrogel forming monomers, has been
widely applied in graft copolymerization of chitosan to
increase its hydrophilicity. The detail of hydrogel
forming of chitosan and poly(acrylic acid) (PAA) has

0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2003.08.020
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1454 S. Tanodekaew et al. / Biomaterials 25 (2004) 1453–1460
been previously described [11–14]. Some of chitosan–
PAA hydrogels involved the use of glutaraldehyde for
crosslinking purpose. Although, it strengthened the
gel, glutaraldehyde was considered as a toxic chemical.
The toxicity of the end products, therefore, is the
concerned issue when toxic chemicals have been
employed, especially in the preparation of medical
products.
In the present paper, a novel procedure to prepare
acrylic grafted chitin (chitin–PAA), to impart a hydrogel
characteristic for wound dressing, is reported. Since
acrylic acid was directly grafted onto chitin, a costly and
time consuming process to produce soluble chitin
derivatives such as chitosan, employed as a starting
material, was unnecessary. Chitin–PAA’s were prepared
with various acrylic acid feed contents without the use of
any toxic crosslinkers. The grafted products were then
analyzed by NMR, FTIR and TGA. Degree of swelling
as well as in vitro cytotoxicity of the synthesized gels
were studied. The cellular morphology and behavior of
L929 mouse fibroblasts on the film were also investi-
gated using cell culture and SEM.
2. Materials and methods
2.1. Materials
Squid chitin (Taming Enterprises, Thailand) and
acrylic acid (Aldrich) were used as starting materials
for the preparation of chitin–PAA hydrogels. The
degree of deacetylation of chitin was 0.16 as calculated
from solid state 13C NMR spectra. Potassium perox-
odisulfate (Fluka) was used as a redox initiator. All
these chemicals were used as received.
2.2. Preparation of chitin–PAA hydrogels
Chitin powder (1 g) was stirred in acrylic acid (4, 6
and 8 g) with a small amount of sulfuric acid (5 M, 2 ml)
at 70
C for 1 h. After cooling to room temperature, a
paste-like mixture of chitin–AA was obtained. A small
portion of chitin–AA paste was removed for its
structural analysis. It was precipitated and washed with
deionized water until a decanted solution reached pH 7.
The dried chitin–AA sample was then characterized by
solid state 13C NMR (Bruker DPX-300 spectrometer),
FTIR (Perkin Elmer system 2000) and TGA (Perkin
Elmer Thermogravimetric Analyzer TGA7, at a heating
rate of 20
C/min under nitrogen atmosphere).
To make a chitin–PAA film, the paste mixture of
chitin–AA added with potassium peroxodisulfate
(1.25 wt% to the monomer) and deionized water (4 ml)
was cast in a petri-dish and maintained at 65
C for 4 h.
After polymerization, the film was neutralized, washed
with deionized water to remove any soluble materials,
and dried. The film was then investigated by solid state
13
C NMR, FTIR and TGA.
2.3. Swelling test
The preweighed chitin–PAA films (140–200 mg) were
immersed in deionized water at room temperature. At
certain time, the swollen films were removed from the
water, quickly wiped to remove excess water on the
surface, and weighed. The degree of swelling was
calculated as follows:
DS ¼ ðWw  Wd Þ=Wd ;
where Ww and Wd are weights of wet and dry film,
respectively.
2.4. SEM observation
2.4.1. Surface morphology of the chitin–PAA film
The morphology of dry and hydrated chitin–PAA
films without cells was examined using SEM. The
preparation of the hydrated samples involved fixation,
dehydration and gold sputtering as described below,
while dry samples were only gold sputtered prior to
SEM observation.
2.4.2. Cell–material response
A cell line of L929 (ECACC No. 85011425), mouse
connective tissue, fibroblast-like cells was cultured in the
growth medium which was made up of Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
10% (v/v) fetal bovine serum (FBS), together with
penicillin (100 units/ml) and streptomycin (100 mg/ml) at
37
C in a 5% CO2 atmosphere. Once 80% confluence
was reached, the cells were subcultured for cytotoxicity
study. The hydrogel samples were sterilized by g-ray
irradiation.
The study of the cell response to the 1:4 chitin–PAA
film was performed by plating the cells onto the pre-wet
film. Initially, a 2  7 mm2 piece of 1:4 chitin–PAA
sample was allowed to wet and expand by absorbing
small amount of growth medium without leaving any
excess medium before being attached centrally onto a
35-mm tissue culture dish with non-toxic dental wax.
L929 cells were then seeded at a density of 2  105 cells/
dish. The cultures were incubated for 14 days. The
growth medium was changed every 2 days. The cell
response was observed everyday with an inverted phase
contrast microscope. The cells on materials were then
prepared for SEM at the day of 1, 2, 7 and 14 after
exposed to the chitin–PAA samples. At each time point,
the samples were washed with 0.1 m phosphate buffer
(PB) and fixed with 2% glutaraldehyde in 0.1 m PB for
4 h at 4
C. After thorough washing with PB, the samples
were dehydrated by graded ethanol changes and critical
point dry. The samples were then gold sputtered in
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S. Tanodekaew et al. / Biomaterials 25 (2004) 1453–1460
vacuum and examined by SEM. The experiments were
repeated three times with different batches of the chitin–
PAA film made.
3. Results and discussion
3.1. Preparation of chitin–PAA hydrogels
The chitin–PAA’s were prepared by a two-step
reaction, as shown in Fig. 1. Chitin was first mixed
with acrylic acid, yielding a heterogeneous mixture.
Under acid-catalyzed reaction, the esterification was
occurred to form ester linkages between carboxylic
groups of acrylic acid and hydroxyl groups of chitin.
Chitin–AA, the paste-like mixture containing active
grafting sites was obtained. In fact, the esterification by
reacting acrylic acid with chitin is not easily occurred.
Regardless of the heterogeneous aqueous reaction which
is a result of water insolubility of chitin, the chemical
structure of chitin itself hinders the accessibility of
reacting acrylic acid monomer. However, chitin used in
Fig. 1. Scheme of the preparation of chitin–PAA hydrogel.
1455
this experiment is in the b-form which chains arrange in
a parallel fashion with relatively weak interchain
hydrogen bond [15]. This kind of loosely packed
structure, thereby, facilitates the interaction between
acrylic acid and chitin to yield ester linkages.
In the second step, the casting solution of chitin–AA
was further polymerized by the addition of initiator.
The subsequent addition of acrylic acid molecules to the
active grafting sites propagated the growing of acrylic
side chains on chitin. Meanwhile, both interchain
crosslinking and PAA homopolymerization were con-
currently occurring. The former resulted in a forming of
network structure which strengthened the gels when
swelling in water while the latter affected the swelling
ability of the gels, which will be discussed later.
3.2. Structural analysis
The existence of ester linkages between acrylic acid
and chitin was evidenced by FTIR. The spectrum of
chitin–AA (Fig. 2b) shows a new peak at 1726 cm1
corresponding to the carbonyl absorption of acrylic
acid, in addition to the saccharide characteristic peaks of
chitin (Fig. 2a). This peak was found markedly broad as
acrylic side chains increased, as seen in the spectrum of
chitin–PAA (Fig. 2c). This broad band corresponded to
Fig. 2. FTIR spectra of (a) chitin, (b) chitin–AA, (c) chitin–PAA, and
(d) PAA.
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1456 S. Tanodekaew et al. / Biomaterials 25 (2004) 1453–1460
CQO stretching vibration of carboxylic acid groups
from acrylic chains as observed in the spectrum of PAA
(Fig. 2d).
The evidence of ester linkages, however, was not
observed by the solid state 13C NMR technique. The
spectrum of chitin–AA (Fig. 3b) was apparently
identical to that of pure chitin (Fig. 3a) with an
insignificant change in the degree of deacetylation. It
suggested that N-acetyl groups of chitin were well
protected and still remained after the esterification
process. The number of ester linkages existed in
chitin–AA was most probably too small. With the
limited sensitivity of the solid state technique, it was
unable to detect the resonance signals designated for the
links. As expected for the large amount of acrylic side
chains, the chitin–PAA spectrum (Fig. 3c) appears
Fig. 3. Solid state 13
C NMR spectra of (a) chitin, (b) chitin–AA, and
(c) chitin–PAA.
Fig. 4. Thermograms of (a) chitin, (b) chitin–AA, (c) chitin–PAA, (d) chitin/PAA blend, and (e) PAA.
characteristic peaks of chitin and also a peak at
183.5 ppm representing carbonyl groups and broad
peaks at 30–50 ppm representing methylene and methine
groups of acrylic chains.
3.3. Thermogravimetric analysis
The thermograms of chitin, chitin–AA, chitin–PAA,
chitin/PAA blend and PAA are shown in Fig. 4. The
thermogram of chitin (Fig. 4a) exhibits two decomposi-
tion stages. The one in the range of 50–110
C was due to
the loss of water, and the other in the range of 300–
380
C has been described to the degradation of
saccharide structure, including the dehydration of
saccharide rings and the depolymerization and decom-
position of acetylated and deacetylated units of chitin
[14]. The thermogram of PAA (Fig. 4e) shows three
decomposition stages. The first decomposition stage in
the range of 50–180
C was attributed to the loss of
bound water. The second one in the interval of 215–
300
C has been described to the dehydration and
decarboxylation of the polymer which leads to the
formation of inter- and intra-molecular anhydride
[14,16]. The third decomposition stage in the range of
365–470
C was a result of the degradation of the
residual polymer.
As seen from the thermogram of chitin–AA (Fig. 4b),
the degradation of its saccharide structure appears in the
range of 280–340
C which is approximately 50
C lower
than that of chitin. It suggested that chitin–AA was less
thermal stability than chitin. In other words, it implied
that the links of acrylic acid to chitin, as evidenced from
FTIR, possibly reduced the thermal stability of chitin.
The thermogram of chitin–PAA (weight ratio of
chitin:AA=1:4) (Fig. 4c) shows rather complicated
decomposition stages whereas a physical blending
mixture of chitin and PAA (weight ratio of chitin:
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S. Tanodekaew et al. / Biomaterials 25 (2004) 1453–1460
PAA=1:4) (Fig. 4d) shows straightforward three
decomposition stages overlaying on those of chitin and
PAA individually. The complex degradation of chitin–
PAA began with the loss of water below 200
C. At the
temperature above 200
C, a distinct decomposition
stage at the temperature range of 440–510
C was
observed, besides the normal degradation stages of
chitin and PAA. This high decomposition temperature
together with the high residue content, up to 40 wt%, at
600
C observed in the chitin–PAA thermogram sug-
gested the presence of rigid structure which supported
the three-dimensional network formation of chitin–
PAA.
3.4. Swelling test
The influence of the chitin:AA feed ratio on the
degree of swelling of chitin–PAA hydrogels is shown in
Fig. 5. It was expected to observe the greater swelling of
the chitin–PAA film with a higher AA feed content. It
was, however, apparent that the swelling degrees
increased as a function of monomer feed only at the
initial period of immersion. After prolonged swelling in
water, chitin–PAA 1:6 was found to imbibe the greater
amount of water than chitin–PAA 1:8. In addition, tiny
pieces were found to detach from both gels whereas the
gel with a lower monomer feed content (chitin–PAA 1:4)
contained no debris and still remained intact throughout
the swelling test. Accordingly, there was no change in
the dried weight of the chitin–PAA 1:4 film observed
after being in water for a week whereas the weight loss
of 6% and 20% were found for the chitin–PAA 1:6 and
1:8 films, respectively. Consequently, the significant
errors in the measurements of the degrees of swelling
in these two samples existed. The loss of weight might be
associated with the presence of PAA homopolymer as
mentioned earlier. During polymerization, only certain
amount of acrylic acid monomer underwent graft
copolymerization while the rest was homopolymerized.
Fig. 5. Degrees of swelling of chitin–PAA at various monomer feed
contents: (a) chitin–PAA 1:4, (b) chitin–PAA 1:6, and (c) chitin–PAA
1:8.
1457
At a higher monomer feed content, the homopolymer-
ization was more favorable. Although, the PAA
homopolymer could be washed out during a film
washing process, some residue was strongly entrapped
in the film, enhancing swelling of the gel. The longer the
gel was in water, the greater the amount of water it
absorbed. Bearing such a great amount of water,
thereby, weakened the strength of the gels. As observed
in a very high monomer feed content (chitin–PAA 1:10),
the gel was very soft and difficult to be handled without
breaking. It was also worth mentioning that the actual
amount of acrylic acid incorporated in the chitin–PAA
film was governed by the nature of the heterogeneous
reaction. Consequently, some variations in swelling
ability were expected for each chitin–PAA batch. For
the strength aspect, chitin–PAA 1:4 seemed to be the
best candidate for wound dressing. The amount of water
absorbed in three different batches of chitin:PAA 1:4
were found in the range of 25–35 times of their dry
weights. This water sorption ability should be sufficient
for the removal of exudate from wounds. The water
sorption ability is an essential property for wound
dressing application. Large open wounds usually secrete
a lot of exudate and this excess fluid may cause
complications particularly infections. The swollen gels
showed integrity with a negligible loss of weight for one-
week immersion. Chitin–PAA 1:4 was then selected for
the investigation of its compatibility with cells.
3.5. SEM observation
Surface morphology of the chitin–PAA film is shown
in Fig. 6. Ultrastructurally, the surface of the dry film
appeared to be irregularly rough or cobble stone-like
and rather dense (Fig. 6a) while the hydrated film
swelled in the growth medium resulting in more
stretched pattern (Fig. 6b).
The L929 cells cultured in contact with the chitin–
PAA 1:4 hydrogel for 24 h observed under phase-
contrast light microscope was found intact and prolif-
erated without an inhibition zone (figures not shown).
The results suggested that this chitin–PAA 1:4 hydrogel
was non-cytotoxic and absent in unreacted AA mono-
mer molecules or degradation products that may
leach into the culture medium and possibly damage
the cells.
The L929 cells cultured on the chitin–PAA film were
closely investigated at different time points of culture
using SEM. The cellular behavior on a biomaterial is an
important factor determining its biocompatibility. The
cell attachment with different cell shapes was found
within 24 h after plating the cells. These included
spherical cells with numerous small cytoplasmic
processes, rounded cells with some blebs and attaching
filopodia and flattening, spreading cell with extended
lamellipodia (Fig. 7a–d). These different morphologies
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1458 S. Tanodekaew et al. / Biomaterials 25 (2004) 1453–1460

Fig. 6. Surface textures of the chitin–PAA film: (a) dry film and (b) hydrated film.

Fig. 7. SEM micrographs of L929 cells on the chitin–PAA film. (a) cell population at low magnification, (b–d) different cell shapes at high
magnification.

probably exhibited different stages of the cells respond-
ing to the substrate. A schematic presentation and SEM
micrographs of different stages in adhesion and spread-
ing of cells in vitro as seen with phase contrast light
microscope and with SEM was previously presented
[17]. The whole process of cell adhesion and spreading
consists of cell attachment, growth of filopodia,
cytoplasmic webbing, flattening of the cell mass and
the ruffling of peripheral cytoplasm progressing in a
sequential fashion. These results suggested that the
chitin–PAA film was free from residual monomer that
may cause any toxicity to cells and it was compatible to
the fibroblast cells. In addition, the film offered a
hospitable surface for the cells to attach.
On day 2 of culture, the cells exhibited more
fibroblast-like characteristic, i.e. spindle shape (Fig. 8a).
After that, they progressively proliferated and covered
most of the film surface on day 7 (Fig. 8b). The culture
was extended up to 14 days, the cells were over grown
and piling up on one another which is usually found in
over confluent cultures on tissue culture plastic. It is well
known that wettability of biomaterials influences cell
adhesion and proliferation [18]. It was reported that
cell attachment and growth of anchorage-dependent
cells were inhibited on non-wettable biomaterials
[19–21]. This was attributed to the possible conforma-
tional changes of adsorbed proteins [22–23]. Fibroblasts
have been shown to respond to the surface roughness of
various substrates. The alteration of cell size, shape and
orientation was observed when the cells were cultured
on grooved surfaces [24–27]. Interestingly, macrophages
and fibroblasts responded to the materials with different
hydrophilicity in a reversed preference. It was found
that macrophages migrated preferentially onto more
hydrophobic substrates while fibroblasts accumulated
on those that are hydrophilic. In addition, macrophages
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S. Tanodekaew et al. / Biomaterials 25 (2004) 1453–1460
Fig. 8. Cellular morphology and growth of L929 on the chitin–PAA film; (a) after 2 days showing healthy fibroblast-like cells and; (b) after 7 days
showing cell confluence.
were found accumulated on rough surface in preference
to smooth ones while fibroblasts did the reverse. These
opposite preferences exhibited by macrophages and
fibroblasts may reflect physical and functional differ-
ences in their surface [28]. Although the surface of the
chitin–PAA film was not smooth, it allowed the L929
cells to attach and adjust the cell surface together with
cell mass pliable to the film texture and become
confluent on the film.
The modification of chitin by incorporating hydro-
philic PAA offers at least two advantages; firstly
improved the water sorption of the dressing. Secondly,
the increased hydrophilicity may facilitate the attach-
ment, growth and migration of dermal fibroblasts at the
wound site, which is important in wound healing
process. We previously found that chitin did not well
support the adhesion of fibroblasts [29].
4. Conclusion
Novel chitin–PAA hydrogels were synthesized. Chitin
was first modified with acrylic acid via an esterification
process. The polymerization of acrylic acid monomer
then proceeded simultaneously with the film forming
process with a use of redox initiator. FTIR results were
taken as evidence of ester linkages forming between
chitin and acrylic acid. The films possessed a network
structure and exhibited an enhancement of water
sorption ability. The swelling behavior and gel strength
were found to depend upon monomer feed content.
Chitin–PAA 1:4 yielded optimal swelling and gel
strength. The overall results of the cellular behavior on
the chitin–PAA 1:4 film in this present study suggested
that the material has a potential for biomedical
applications particularly as temporary skin substitute.
Acknowledgements
The authors are grateful to National Metal and
Materials Technology Center, Thailand for financial
1459
support and the Office of Atomic Energy for Peace
(OAEP), Thailand for the g-sterilization. The authors
would also like to thank Dr. Patricia Watts and staff in
the Animal Cell Culture Laboratory of BIOTEC for
their general assistance.
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