162 Biomacromolecules 2004, 5, 162-168

Novel Chitosan-Based Films Cross-Linked by Genipin with

Improved Physical Properties
J. Jin, M. Song,* and D. J. Hourston
Institute of Polymer Technology and Material Engineering, Loughborough University,
Loughborough LE11 3TU, United Kingdom

Received August 7, 2003; Revised Manuscript Received November 6, 2003

Novel cross-linked chitosan-based films were prepared using the solution casting technique. A naturally
occurring and nontoxic cross-linking agent, genipin, was used to form the chitosan and chitosan/poly(ethylene
oxide) (PEO) blend networks, where two types of PEO were used, one with a molecular weight of 20 000
g/mol (HPEO) and the other of 600 g/mol (LPEO). Genipin is used in traditional Chinese medicine and
extracted from gardenia fruit. Importantly, it overcomes the problem of physiological toxicity inherent in
the use of some common synthetic chemicals as cross-linking agents. The mechanical properties and the
stability in water of cross-linked and un-crosslinked chitosan and chitosan/PEO blend films were investigated.
It was shown that, compared to the transparent yellow, un-cross-linked chitosan/PEO blend films, the genipin-
cross-linked chitosan-based film, blue in color, was more elastic, was more stable, and had better mechanical
properties. Genipin-cross-linking produced chitosan networks that were insoluble in acidic and alkaline
solutions but were able to swell in these aqueous media. The swelling characteristics of the films exhibit
sensitivity to the environmental pH and temperature. The surface properties of the films were also examined
by contact angle measurements using water and mixtures of water/ethanol. The results showed that, with
the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and chitosan/PEO
blends were more hydrophobic than un-crosslinked ones.

Introduction critically important and the most difficult to satisfy.1,2 As a

Chitosan, a natural biomaterial, has recently attracted much
attention from scientists in different parts of the world.1-3 It
has been reexamined and found to be a useful resource as a
functional material.4,5 Because of the presence of amino
groups, chitosan is soluble in aqueous acidic media and forms
viscous solutions that can be used to produce gels in various
forms, e.g., beads, membranes, coatings, fibers, and sponges.6-8
The amino and hydroxyl groups of chitosan give rise to it
being easily chemically modified. Chitosan, as a functional
material, offers a special set of characteristics: biocompat-
ibility, biodegradability, and anti-bacterial properties.6,7 It is
also biologically inert, safe for human use, and stable in the
natural environment.5,9,10 The above characteristics make
chitosan suitable for use in a number of biomedical applica-
tions, including artificial skin, tissue regeneration, and drug
delivery systems.2,11-13 It is vital that in all of these
applications the material must be demonstrably nontoxic. In
other words, the polymer, its possible degradation products,
any residual monomer, and all additives must be free of
harmful effects. These requirements have not always been
met. Chitosan for tissue regeneration and similar medical
applications must be biocompatible and mechanically satis-
factory. In terms of mechanical performance, for example
in structural materials in hip reconstruction and replacement,
the biomaterial’s strength and other physical properties are
* To whom correspondence should be addressed.
10.1021/bm034286m CCC: $27.50
Published on Web 12/09/2003
result, the application of chitosan as a biomedical material
has been limited to a few specific areas,1,2 which include
wound dressings, sutures, and skin and tissue engineering.
Many recent attempts14-17 have tried to improve the
mechanical properties of chitosan. Modification of chitosan
through blending with other polymers18,19 and cross-link-
ing20,21 are both convenient and effective in improving its
physical properties for practical applications. There have been
some reports22-24 dealing with polyblends of chitosan with
other natural or synthetic polymers. Recently, chitosan and
poly(ethylene oxide) (PEO) blends have been reported for
the preparation of membranes for haemodialysis25 and semi-
IPNs for pH-sensitive drug delivery.26 PEO is a biologically
inert and flexible polymer. It has suitable uses for medical
purposes, and it can improve the mechanical properties of
blends. These properties of chitosan/PEO films were exam-
ined by Alexeev et al.,14 and it was observed that for the
chitosan/PEO blends containing 16.7 wt % of PEO there was
a six times improvement in the elongation at break and a
doubling in the tensile strength. It was shown that at 16.7
wt % of PEO the film structure is likely to be homogeneous,
and therefore, this gives rise to the improved mechanical
characteristics.14 The mechanical properties and the stability
of chitosan/PEO blends have also been investigated by Wang
et al.15 Their results showed that there were specific
interactions between PEO and chitosan and that the maxi-
mum interaction between them occurred at 20wt % PEO
content. The results also indicated that these chitosan blend
© 2004 American Chemical Society
Chitosan-Based Films Cross-Linked by Genipin
Scheme 1. Schematic of Genipin Chemical Structure
films were unstable, for the films partly dissolved in distilled
water after having been immersed for 8 h. These blend films
are, therefore, limited in their uses and applications.
Many other investigations20,21 have been carried out to
overcome such undesirable properties by the method of cross-
linking. The most common synthetic chemical used as a
cross-linking reagent is gluteraldehyde.20 Among others are
formaldehyde,27 epoxy compounds,28 and dialdehyde starch.29
However, all of these cross-linking agents are chemically
synthesized and are not free from the problems caused by
physiological toxicity.30,31
Genipin is found in traditional Chinese medicine and
extracted from gardenia fruit.32,33 It has been reported that
genipin, whose chemical structure is shown in Scheme 1, is
an effective naturally occurring-cross-linking agent and can
react spontaneously with amino acids or proteins to form
dark blue pigments.34
Sung and co-workers35-38 have undertaken studies to
investigate the cytotoxicity, feasibility, and biocompatibility
of genipin for tissue fixation. The results of these studies38
demonstrated that genipin was 10 000 times less cytotoxic
than glutaraldehyde, a finding that encouraged us to inves-
tigate the use of this agent to produce novel materials for
medical purposes.
The goal of the current work is to develop novel chitosan/
PEO blend films, designed to be mechanically resilient,
chemically inert, and stable, by using genipin as the cross-
linker to prepare the chitosan networks. The aim of this paper
is to report on the mechanical properties and the stability in
water of these novel films.
Experimental Section
Materials. Chitosan was purchased from Fluka, U.K. The
molecular weight of chitosan was measured by means of gel
permeation chromatography (GPC). Mw ) 3.0 × 105, Mn )
5.8 × 104, and Mw/Mn ) 5.5. The degree of deacetylation
of the chitosan was 88%, which was determined by Fourier
transform infrared (FT-IR). Poly(ethylene oxide) with mo-
lecular weight of 20 000 g/mol (HPEO) was obtained from
BDH Laboratory Supplies, U.K. Poly(ethylene glycol)
powder with viscosity average molecular weight of 600 g/mol
(LPEO) was obtained from the Aldrich Chemicals, U.K.
Genipin was obtained from Challenge Bioproducts Co.,
Taiwan. Acetic acid was obtained from Aldrich Chemicals,
U.K. All chemicals were used without further purification.
Preparation of Chitosan /PEO Blend Films. Chitosan
was dissolved in 1 wt % aqueous acetic acid at room-
temperature overnight to obtain a concentration of 1.5 wt/
v%. The viscous chitosan solution was filtered through filter
paper to remove any undissolved gel. The clear, light yellow
chitosan solution was then mixed with a 5% aqueous solution
Biomacromolecules, Vol. 5, No. 1, 2004 163
of PEO in different weight percentage ratios and the mixtures
were stirred overnight at room temperature. The degassed
and well-mixed solutions were then cast onto glass plates,
evaporated at 35°C and dried to constant weight. The
thickness of the dried films ranged from 0.3 to 0.6 mm.
Preparation of Genipin-Cross-Linked Films. 0.5 wt %
of genipin (chitosan/genipin ) 100/0.5 by weight) was
dissolved in 3 mL of water and then was added to the
chitosan solutions, or to the chitosan/PEO mixed solutions,
under stirring for 30 min at room temperature. After 2 h,
the solutions started to turn light blue and became increas-
ingly viscous. The solutions were then immediately cast on
glass plates and dried to constant weight at ambient tem-
perature. The cross-linked chitosan and chitosan/PEO blends
become dark blue after 1 day. The thicknesses of the dried
films were measured to be 0.3-0.6 mm.
Mechanical Testing. Tensile strength and percentage
elongation were measured on film strips using a L1oyd
Instruments (U.K.) tensometer operated according to the
D638 ASTM standard. The tensometer was fitted with a
500N load cell. The extension rate was 10 mm/min. To
examine the physical properties of the films as a function of
moisture content, they were placed in a relative humidity
chamber over distilled water (pH 7). The moisture content
was determined by drying to constant weight. Each test was
repeated at least 3 times and the average is reported.
Solubility and Stability in Neutral Water. The cross-
linked/un-crosslinked chitosan and chitosan/LPEO blend
films were immersed in 100 mL of methanol for 24 h to
remove the acetic acid in the films and were then taken out
and dried for 12 h at 35 °C. The dried films were weighed
accurately. The dried films were then immersed in distilled
water (pH 7) for 8 h. They were then taken out and dried
for 24 h under vacuum at 35°C. A second weighing was
conducted to determine the amount of LPEO extracted from
the blends. All of the films used were from the same samples
as used for the tensile strength measurements. Stability in
water, therefore, can be expressed by the following equa-
tion:
S ) W2/W1 × 100
where S is the percentage of the films remaining after
immersion in neutral water. W1 and W2 are the weight of
dried films before and after immersion in water, respectively.
Each experiment was repeated three times, and the average
value is reported.
Swelling Studies. The swelling characteristics of the cross-
linked and the un-crosslinked chitosan and their blend films
were determined by swelling the films in media of different
pHs at both room temperature and 38°C. The samples
(approximately 0.05 g) were first treated as described above
to remove the residual acetic acid. Blends and networks were
immersed in buffer solutions ranging from pH 2 to 13. The
immersion time was 48 h. It was confirmed that after a 48-h
period the films had reached their swelling equilibrium. The
films were withdrawn from the buffer solutions, and their
wet weights were determined after first blotting with a filter
paper to remove surface absorbed water, followed im-
mediately by weighing. The specific solution content, or
164 Biomacromolecules, Vol. 5, No. 1, 2004 Jin et al.

weight uptake percentage, of the films is expressed by the

following equation:

Esw ) [(Ws - Wd)/Wd] × 100

where Esw is the percentage water adsorption of the film at

equilibrium. Wd and Ws are the weights of the samples in
the dry and swollen states, respectively. Each swelling
experiment was repeated three times, and the average values
are reported.
Contact Angle Analysis. Contact angle measurements
were carried out using a Data Physics OCA20 instrument,
fitted with an automatic image capture system. The liquids
used were pure water, mixtures of 70% water and 30%
ethanol, and 50% water and 50% ethanol. Because of the
rapid evaporation of ethanol and the need to maintain
consistency of results, dynamic tracking was used to capture
the contact angle of the drop every second for up to 1 min.
All films were neutralized with NaOH before use.

Results and Discussion

Tensile Strength and Percentage Elongation. The tensile

strength and the elongation at break plotted against the
%LPEO in the dry films of both cross-linked/un-crosslinked
chitosan and chitosan-LPEO blends are shown in parts a and
b of Figure 1, respectively. It was observed that the film
prepared from pure chitosan was rather brittle. The highest
tensile strength was found in the blend with 20 wt % of
LPEO, and the greatest enhancement of elongation at the
break for the blend films occurred at 65% LPEO content,
the highpoint of a general trend of elongation increase as
the LPEO content increased. Cross-linking with naturally
occurring genipin gave rise to improvements in both me-
chanical properties: the tensile strength and the elongation
at break for LPEO systems. Figure 1. (a) Tensile strength of cross-linked and un-crosslinked
dried chitosan/LPEO blends films versus blend composition. (b)
Table 1 shows the results of tensile strength and percentage Elongation at break of cross-linked and un-crosslinked dried chitosan/
elongation for HPEO systems. It is clearly seen that cross- LPEO blend films versus blend composition.
linking with naturally occurring genipin did not give rise to
Table 1. Tensile Strength and Percentage Elongation for HPEO
improvements in both mechanical properties: the tensile Systems
strength and the elongation at break for the HPEO systems
elongation stress
which results from the lower miscibility of chitosan with
at break at break
HPEO.39
PEO (%) (%) (MPa)
Figure 2, parts a and b, presents the stress-strain curves
Un-Cross-Linked HPEO System
for un-cross-linked and cross-linked chitosan blend films
10 19 60
(with 0.5% of genipin) with 50 wt % LPEO content after 20 25 63
their conditioning in environments of various relative 30 14 50
humidities. It can be seen in both cases that, as the water
Cross-Linked HPEO System
content in the films increased, the elongation at break 10 15 55
increased, whereas the tensile strength decreased. However, 20 17 70
cross-linking gave rise to greater increases in both tensile 30 15 58
strength and elongation at break than the equivalent dry
samples. At 15% water content, the increase in the tensile from disintegrating when the water content of its surrounding
strength of the cross-linked film was more than twice that environment increases, the greater improvements in mechan-
of the un-crosslinked one. At 25% water content, the ical properties due to cross-linking are of real significance.
elongation at break of the cross-linked film reached 80% Figure 3, parts a and b, shows the stress-strain plots of
and its tensile strength was found to be 15MPa, and the the cross-linked chitosan blend films (50 wt % LPEO) with
elongation at break was 35% and its tensile strength was different genipin contents at two water contents. Both results
found to be 6 MPa for the un-crosslinked film. Because it is indicated that 0.1% of genipin content gave the greatest
important to prevent the carrier in a drug delivery system increase in elongation at break. When the genipin content
Chitosan-Based Films Cross-Linked by Genipin
Figure 2. (a) Stress of un-crosslinked chitosan-LPEO(50%) blend
films versus strain with different water contents. (b) Stress of cross-
linked chitosan-LPEO(50%) blend films versus strain with different
water contents.
was increased further, to 0.5% and 0.8%, the improvements
in elongation at break were not as significant as that for 0.1%
genipin. The reasons for this are not yet clear. In future
research, the matter will be investigated further.
Chitosan can be described in terms of the degree of
deacetylation and average molecular weight in conjunction
with their film-forming properties.40 It has been observed
that the film formation of un-crosslinked blends is closely
related to their composition. Above 50% (w/w) and 65%
(w/w) of LPEO content, the blends are phase separated, and
no useful films could be formed. Similar results have been
reported by Zhao.41 However, cross-linked blends at 80 wt
% or above of LPEO overcame this problem. The use of
genipin as a cross-linker clearly improves the properties of
chitosan-based films.
Stability of Cross-Linked Blend Films in Water. Know-
ing that chitosan is naturally insoluble in water (pH 7),
whereas both LPEO and HPEO are soluble, the stability of
both cross-linked and un-crosslinked blend films in neutral
water was examined. Figure 4, parts a and b, shows the
degrees of stability of the films plotted against wt % of LPEO
(HPEO). A comparison shows that the un-cross-linked
chitosan-LPEO film is more stable than the un-cross-linked
chitosan-HPEO film, with the latter being very unstable
Biomacromolecules, Vol. 5, No. 1, 2004 165
Figure 3. (a) Stress-strain curves for chitosan-LPEO(50%) blend
films with a 5% water content and a range of cross-linking content.
(b) Stress-strain curves for chitosan-LPEO(50%) blend films with a
13% water content and a range of cross-linking levels.
which is because the miscibility of LPEO with chitosan is
higher than that of HPEO with chitosan.39 Cross-linking,
however, produced noticeable increases in the stability of
both blends, in particular these of the chitosan/HPEO blends.
This effect of cross-linking is of great importance to the bio-
medical applications of such materials since it is linked to
their stability during fabrication and storage and while in
service.
Swelling of the Cross-Linked Blend Films. Genipin-
cross-linked chitosan networks have the advantages of being
insoluble in acidic and alkaline solutions and the ability to
swell in these aqueous media. It is clear that chitosan is
soluble at pH e 6.5.7 Figure 5a shows that the swellability
of pure chitosan films was affected by the genipin content.
From the figure, it can be seen that films with low genipin
contents (0.1%) dissolved at pH ) 2, whereas films with
0.5% and 0.8% of genipin were insoluble in acidic solution.
Furthermore, for insoluble films, an increase in the cross-
linker content meant a decrease in the extent of swelling.
The degree of swelling of a series of films in different pH
environments at 23 and 38 °C is shown in Figure 5, parts b
and c, respectively. It can be seen that the swelling behavior
of the films was strongly dependent on the pH value of the
swelling medium, the wt % of LPEO in the film, and the
166 Biomacromolecules, Vol. 5, No. 1, 2004 Jin et al.

Figure 4. (a) Comparison of the stabilities of cross-linked and un-

crosslinked chitosan/HPEO blend films (0.5% genipin). (b) A com-
parison of the stabilities of cross-linked and un-crosslinked chitosan/
LPEO blend films (0.5% genipin).
temperature. When the temperature was increased from 23
to 38 °C, swelling in pH e 7 buffer solutions increased by
50-100%. In alkaline buffer solutions, however, there were
no notable increases in swelling, although the greatest
swelling now occurred under the lower alkaline condition
of pH 10 rather than the previous pH 12. It can be concluded
that genipin-cross-linked chitosan networks resulted in
significantly different swelling characteristics in the various
pH buffer solutions. The increased swelling of the cross-
linked films at pHs lower than 7 may be ascribed to the Figure 5. (a) Swelling behavior of chitosan films at 23°C with different
hydrolysis of amide linkages in the cross-linked chitosan cross-linking levels. (b) The swelling behavior of cross-linked chitosan/
LPEO blend films at 23°C. (c) The swelling behavior of cross-linked
network by acid and the regeneration of amine groups in chitosan/LPEO blend films at 38°C.
networks.36 Because of the fact that the amino groups
reformed in the network could be protonated in acid, the
equilibrium ratio of the swelling of the chitosan film in acid crosslinked samples were investigated by contact angle
was larger than that in the neutral solution. It would be a analysis. Figure 6 shows the contact angles for these different
desirable characteristic for a controlled-release system with liquid media when a drop was placed on the surfaces of the
controllable swelling ability to be pH- and temperature- pure chitosan film and the network. The greatest contact
sensitive. These films are, therefore, of general interest for angles, chitosan and network films, were achieved at 100%
biomedical applications such as artificial muscles or switches, water, measuring at 78.5° and 62.6°, respectively. Cross-
biomedical separation systems, and drug controlled-release linking, therefore, resulted in an improvement in the wetta-
systems.1,29 bility of the chitosan films. However, this improvement was
Contact Angle Analysis. The surface properties of the only present with 100% water, because chitosan networks
chitosan films and the blends for both cross-linked and un- exhibited higher contact angles than the un-crosslinked films
Chitosan-Based Films Cross-Linked by Genipin Biomacromolecules, Vol. 5, No. 1, 2004 167

Figure 6. Contact angles of the cross-linked and un-crosslinked

chitosan films with 100% water, 70% water/30% ethanol, and 50%
water/50% ethanol.

in 70% water/30%ethanol and 50%water/50% ethanol sys-

tems.
Figure 7a-c shows the contact angles for all of the
chitosan/LPEO (HPEO) blends and networks in all three
liquid systems. As seen in most cases, the contact angles
for cross-linked chitosan and the blends were much higher
than for the un-crosslinked ones. The surface became more
hydrophobic because of cross-linking, but the angle decreased
gradually when 10, 20, and 30 wt % of LPEO (or HPEO)
were incorporated. The lower contact angles corresponded
well to the fact that the LPEO (or HPEO) has high chain
mobility and hydrophilicity. An increase in the LPEO (or
HPEO) content, thus, allowed a greater wettability of the
surface. A slight, but gradual, decrease in contact angles was
also observed when water was diluted with 30 and 50% of
ethanol. On the whole, the contact angle analysis was helpful
in aiding the understanding of the surface properties of the
blends and networks.
Although cross-linking resulted in decreases in the wet-
tability of the film surfaces in some cases, this unwanted
effect was negated to some extent by the addition of PEO.
Furthermore, the swelling ability of cross-linked chitosan and
blends in ethanol is of particular importance to its success
as a drug carrier. For example, the anti-cancer drug, Taxol,
is only soluble in ethanol.42

Conclusions

Using naturally occurring genipin, as a cross-linking agent,
gave rise to improvements in the mechanical properties and
stability in water of chitosan and chitosan blend films and
produced interesting results concerning their swelling proper-
ties and wettability. The increases in the elongation at break
and the tensile strength of the cross-linked films, especially
in those with high water content, would prevent them from
disintegrating in potential applications. With the use of cross-
linking, there were also noticeable increases in the stability
of the chitosan/LPEO (or HPEO) blends. The swelling
behavior of the films exhibited pH and temperature-depend-
ent characteristics. However, the swelling extent can be
reduced by an increase in the genipin content. In terms of
wettability, with the one exception of cross-linked pure
chitosan in 100% water, the cross-linked chitosan and
Figure 7. (a) Contact angles for the cross-linked and un-crosslinked
chitosan/PEO blend films for 100% water. (b) Contact angles for the
cross-linked and un-crosslinked chitosan/PEO blend films for 70%
water/30% ethanol. (c) Contact angles for the cross-linked and un-
crosslinked chitosan/PEO blend films for 50% water/50% ethanol.
chitosan/PEO both molecular weights blends were more
hydrophobic than un-crosslinked ones.
These novel films are sensitive to the environment and
should be investigated further for drug delivery and bioma-
terials applications.
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BM034286M