A water-soluble photocrosslinkable chitosan derivative prepared by Michael-addition reaction as a precursor for injectable hydrogel

Article abstract of DOI:10.1016/j.carbpol.2009.08.033

With the goal of obtaining photopolymerized hydrogels for use as tissue engineering scaffolds, a water-soluble (methacryloyloxy) ethyl carboxyethyl chitosan was prepared as a photopolymerizable prepolymer through Michael-addition reaction between chitosan and ethylene glycol acrylate methacrylate. N-substitution of chitosan was verified by both ¹H NMR and ¹³C NMR The degree of N-substitution, measured via ¹H NMR, was easily varied from 0.10 to 0.35 by varying the molar ratio of ethylene glycol acrylate methacrylate to chitosan. Using a Vero cell line, the water-soluble photocrosslinkable chitosan derivative was found to be noncytotoxic up to a concentration of 1.0 mg/mL. The precursor was blended with D-2959 photoinitiator in solution, and UV-irradiated to create hydrogels. FTIR verified the nearly complete conversion of the double bonds in the gel. Indirect cytotoxicity assessment of the hydrogel indicated that the hydrogel was non-toxic to Vero cells.

Full text of DOI:10.1016/j.carbpol.2009.08.033

Carbohydrate Polymers 79 (2010) 507–512
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
A water-soluble photocrosslinkable chitosan derivative prepared
by Michael-addition reaction as a precursor for injectable hydrogel
Xuanyue Gao a,1, Yingshan Zhou a,1, Guiping Ma , Suqing Shi , Dongzhi Yang , Fengmin Lu , Jun Nie
a
b
a
c
a,*
a
State Key Laboratory of Chemical Resource Engineering and Key Laboratory of Beijing City on Preparation and Processing of Novel Polymer Materials,
Beijing University of Chemical Technology, Beijing 100029, PR China
b
Department of Chemistry, Northwest University, Xi’an 710069, PR China
Department of Microbiology, Peking University Health Science Center, Beijing 100083, PR China
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2009
Received in revised form 23 August 2009
Accepted 25 August 2009
Available online 31 August 2009
With the goal of obtaining photopolymerized hydrogels for use as tissue engineering scaffolds, a water-
soluble (methacryloyloxy) ethyl carboxyethyl chitosan was prepared as a photopolymerizable prepoly-
mer through Michael-addition reaction between chitosan and ethylene glycol acrylate methacrylate.
1
13
N-substitution of chitosan was verified by both H NMR and C NMR The degree of N-substitution, mea-
1
sured via H NMR, was easily varied from 0.10 to 0.35 by varying the molar ratio of ethylene glycol acry-
late methacrylate to chitosan. Using a Vero cell line, the water-soluble photocrosslinkable chitosan
derivative was found to be noncytotoxic up to a concentration of 1.0 mg/mL. The precursor was blended
with D-2959 photoinitiator in solution, and UV-irradiated to create hydrogels. FTIR verified the nearly
complete conversion of the double bonds in the gel. Indirect cytotoxicity assessment of the hydrogel indi-
cated that the hydrogel was non-toxic to Vero cells.
Keywords:
Chitosan
Hydrogel
Photopolymerization
Water-solubility
Ó 2009 Published by Elsevier Ltd.
1
. Introduction
From a clinical perspective, the use of injectable hydrogels is
the ability to place the gel in vivo without surgical intervention
and minimal heat production (Poon, Zhu, Shen, Chan-Park, & Ng,
2007).
Chitosan has excellent biological properties such as biodegrad-
more attractive than the implantation of a preformed hydrogel as
it can minimize patient’s discomfort, risk of infection, scar forma-
tion, and the cost of treatment (Hou, Bank, & Shakesheff, 2004).
Injectable hydrogel can be used as tissue regeneration scaffolds
for cartilage and soft tissues (Fellah et al., 2006; Shin, Ruhe, Mikos,
ability, biocompatibility, antibacterial and wound-healing activity
(Khor & Lim, 2003; No, Park, Lee, & Meyers, 2002; Ueno, Mori, &
Fujinaga, 2001). Additionally, chitosan could achieve hemostasis
and promote normal tissue regeneration (Malette & Quigley,
1985). For these reasons, chitosan has been considered to be one
of the most promising biomacromolecules for tissue engineered
scaffolds.
Photopolymerizable chitosan derivatives have been prepared
previously through styrenation (Matsuda & Magoshi, 2002) and
methacrylation (Renbutsu et al., 2005) using reaction aldehyde
intermediates. However, these photopolymerizable chitosan deriv-
atives are only soluble at acidic pH, while solubility at physiologi-
cal pH values is required for the synthesis of useful biocompatible
hydrogels for biological applications. Thus, a useful chitosan-based
photopolymerizable precursor would be solubility in aqueous
solution at physiological pH. A water-soluble photopolymerizable
chitosan has previously been prepared by grafting 4-azidobenzoic
acid to available free amine groups of lactose modified chitosan
&
Jansen, 2003). For effective clinical application, injectable sys-
tems should be able to gel at mild physiological conditions in a
clinically relevant time period. The gel should show good biocom-
patibility and biodegrade into non-toxic degradation products
(Gutowska, Jeong, & Jasionowski, 2001; Hatefi & Amsden, 2002).
Several synthetic and natural polymers have been investigated
for developing in situ gelling systems induced by stimuli such as
temperature, light or pH (Jeong, Bae, Lee, & Kim, 1997; Kim &
Chu, 2000; Martens & Anseth, 2000; Nair, Starnes, Ko, & Laurencin,
2
007; Paige, Cima, Yaremchuk, Vacanti, & Vacaoti, 1995; Sawhney,
Pathak, & Hubbell, 1993). Among these, photopolymerization is
one of the most promising ways of making injectable hydrogels,
due to fast curing rates at room or physiological temperatures,
(Ono et al., 2000). However, this approach has a possible disadvan-
tage that is exposure to UV irradiation may result in protein dena-
turation when protein drug was capsulated (Amsden, Sukarto,
Knight, & Shapka, 2007).
*
Corresponding author. Tel.: +86 10 64445930; fax: +86 10 64421310.
E-mail address: Niejun@mail.buct.edu.cn (J. Nie).
These authors contributed equally to this work.
1
0
144-8617/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.carbpol.2009.08.033

5
08
X. Gao et al. / Carbohydrate Polymers 79 (2010) 507–512
Hence, the objective of this study is the synthesis and character-
2.4. Photocrosslinking of MAOECECS
ization of photopolymerizable water-soluble (methacryloyloxy)
ethyl carboxyethyl chitosan (MAOECECS). Briefly, chitosan was re-
acted with ethylene glycol acrylate methacrylate by Michael-addi-
A concentrated solution of MAOECECS in deionized water (6 w/
v%) containing the photoinitiator D-2959 (0.1 w/v%) was poured
into a flat mold consisting of two glass plates separated by a
2 mm-thick spacer frame. D-2959 was used in this study, as it
has been demonstrated to be the least cytotoxic to various cells
(Williams, Malik, Kim, Manson, & Elisseeff, 2005). The solution
was then exposed to long wave ultraviolet light (320–480 nm,
EXFO lite, EFOS Corpration, Mississauga, Canada) at intensity of
2
tion reaction between –NH of chitosan and C@C of acrylate to
produce a photocrosslinkable MAOECECS hydrogel precursor. The
MAOECECS was blended with a photoinitiator and water, and
UV-irradiated to create hydrogels. A series of characterization of
MAOECECS including crystalline and thermal stability was studied.
The cytocompatibility and biodegradability behaviors of MAOE-
CECS hydrogel also were investigated.
2
10 mW/cm for up to 15 min to yield the photopolymerized
hydrogel.
2
. Materials and methods
.1. Materials
Chitosan (CS, MW = 1.2 Â 10 , degree of deacetylation = 84.4%)
2
2
.5. Characterization methods
2
.5.1. FTIR spectroscopy
The Fourier Transform Infrared spectra (FTIR) of CS, MAOECECS
5
and the hydrogels were recorded at room temperature by using a
Nicolet 5700 instrument (Nicolet Instrument, Thermo Company,
USA) over the wave number range of 4000–400 cm
was purchased from Zhejiang Golden-Shell Biochemical Co., Ltd.,
China. 2-Hydroxyethyl Methacrylate (HEMA) was from Beijing
Chemical Reagents Company, distilled under reduced pressure in
the presence of hydroquinone, and stored at 4 °C until use. Acryloyl
chloride was purchased from Shanghai Chemical Reagents Com-
pany, China. Triethylamine and toluene were obtained from Beijing
Chemical Reagents Company, distilled under reduced pressure and
stored until use. Cytocompatible UV photoinitiator Darocur 2959
À1
.
2
.5.2. NMR spectroscopy
1
13
H NMR and C NMR studies were performed by using a Bruker
1
3
AMX 600 MHz instrument. C NMR spectra were recorded using
0 mg/mL samples under ambient temperature.
5
(
2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone)
2
.5.3. Thermal stability
To examine the thermal properties of CS and MAOECECS,
was obtained from Ciba-Geigy Chemical Co. (Tom River, NJ). Vero
cells were obtained from Department of Microbiology, Peking
University Health Science Center.
Thermal gravimetric analysis (TGA) was performed with
NETZSCH TG 209 F1 analyzer (Germany) between 20 and 500 °C
with a 10 °C/min heating rate under a nitrogen flow rate of
a
2
.2. Synthesis of ethylene glycol acrylate methacrylate (EGAMA) (Muh,
2
0.0 mL/min.
Marquardt, Klee, Frey, & Mulhaupt, 2001)
2
.5.4. XRD
A mixture of 83.81 g of HEMA and 105 mL of triethylamine dis-
solved in 250 mL of toluene was added into a three-necked flask
equipped with stirrer, thermometer, and dropping funnel. Under
cooling (0–5 °C), 79.79 g of acryloyl chloride dissolved in 50 mL
of toluene was added over 4 h. Then, the mixture was allowed to
stand overnight, the precipitate filtered off and washed twice with
The XRD patterns of CS and MAOECECS were performed via X-
ray diffractometer (Rigaku, Damax2500) with CuKa characteristic
radiation (wavelength k = 0.154 nm at 40 kV, 50 mA, and scan
speed of 1°/min in the 2h range of 5–50°).
2.5.5. Cell culture
2
0 mL of toluene. Then, the reaction mixture was extracted twice
with 75 mL of water, 50 mL of 1 mol/L HCl and 50 mL of 1 mol/L
NaHCO and dried overnight with Na SO . Subsequently, the tolu-
Vero cells were cultured in RPMI1640 medium supplemented
with 10% fetal bovine serum, together with 1.0% penicillin–strepto-
mycin, and 1.2% glutamine. Culture was maintained at 37 °C in a
3
2
4
ene was removed by rotary evaporation. The yellow crude product
was purified by silica gel column chromatography. Yield: 80%. IR:
wet atmosphere containing 5% CO
confluence, they were trypsinized with 0.25% trypsin containing
mM ethylenediamine tetraacetic acid (EDTA).
2
. When the cells reached 80%
À1
À1
À1
1
1
725 cm (s, C@O), 1637 cm (m, C@C), 811 cm (m, C@C). H
1
NMR: d = 6.4 (d, @CH ), 6.1 (1b, 8, m), 5.8 (9b, d), 5.6(1a, s), 4.3
2
(
5, 6, s), 1.9 (3, s).
2.5.6. Cytotoxicity assays
In vitro cytotoxicity of the noncrosslinked MAOECECS in the ab-
2
.3. Synthesis of water-soluble (methacryloyloxy) ethyl carboxyethyl
sence of D-2959 was assessed through effect on cell proliferation in
monolayer cultures of Vero cells. Lyophilized MAOECECS were dis-
solved in serum supplemented tissue culture medium at varying
concentrations (0.5, 1, 2.5, 5 and 10 mg/mL) and sterilized by filtra-
chitosan (MAOECECS)
2
Briefly, 1.5 g of chitosan (corresponding to 7.5 mmol NH ) was
first dissolved in 50 mL of water containing 0.48 mL of acetic acid,
after which ethanol (40 mL) and a certain amount of EGAMA were
added in sequence and stirred for 2 days at 60 °C. Thereafter, a sat-
tion (0.45 lm) prior to placement inside the wells. For these exper-
iments, the Vero cells were seeded in wells of 96-well plate at a
3
density of 10 cells per well. After incubated for another 24 h,
urated NaHCO
3
solution was added to the reaction mixture to ad-
the culture medium was removed and replaced with the test solu-
just the pH to 7–8, and the mixture solution was then precipitated
in acetone. The precipitated solid was collected by filtration and
then dissolved in water and dialyzed (membrane molecular weight
tions and incubated for another 48 h, and then 100
tion was added to each well. After 3 h incubation at 37 °C, 200
of dimethyl sulfoxide was added to dissolve the formazan crystals.
The dissolved solution was swirled homogeneously about 10 min
by the shaker. The optical density of the formazan solution was de-
tected by an ELISA reader (Multiscan MK3, Labsystem Co., Finland)
at 570 nm.
lL MTT solu-
l
L
À1
cut-off 12,000 g mol ) against water for 2 days and lyophilized to
1
13
obtain pure MAOECECS. H NMR and C NMR spectrum was per-
formed to confirm structure of the MAOECECS. The solubility of the
product in water was evaluated from the visual observation meth-
od where 50 mg of product was suspended in water (5 mL) for
The cytotoxicity of the photopolymerized hydrogel was as-
sessed by culture of Vero cells supplemented with the extractant
1
day.

X. Gao et al. / Carbohydrate Polymers 79 (2010) 507–512
509
of the hydrogel. The hydrogel, chitosan membrane and carboxyl-
3. Results and discussion
ethyl chitosan membrane were sterilized with highly compressed
steam for 15 min and placed in wells of 24-well culture plate
respectively. The samples were then incubated in 1 mL RPMI1640
medium at 37 °C for 24 h. The extraction ratio was 1.25 cm /mL.
At the end of this period, the hydrogel or the membranes were
3.1. Characterization of MAOECECS and its hydrogels
À1
In order to produce a photocrosslinkable product, methacryla-
tion was carried out on chitosan polymer chain so that they can
undergo free radical polymerization. The synthesis of MAOECECS
removed, and the so-called extracts were obtained. Vero cells were
3
seeded in wells of 96-well plate at a density of 10 cells per well.
was achieved via Michael addition of –NH of chitosan to the acry-
2
After incubated for another 24, 48 and 72 h, the culture medium
was removed and replaced with the as-prepared extraction med-
late group of ethylene glycol acrylate methacrylate (EGAMA). Four
MAOECECS samples (denoted MAOECECS1–4) were prepared by
using increasing amounts of EGAMA (1.41–5.62 g) in order to yield
products with different degrees of substitution (DS), the values for
which are also listed in Table 1. DS increases from MAOECECS1–4
indicating that DS is positively correlated to the ratio of EGAMA
used in the reaction.
ium and incubated for another 24 h, then 100
lL MTT solution
was added to each well. After 3 h incubation at 37 °C, 200
lL of
dimethyl sulfoxide was added to dissolve the formazan crystals.
The dissolved solution was swirled homogeneously about 10 min
by the shaker. The optical density of the formazan solution was
detected by an ELISA reader (Multiscan MK3, Labsystem Co., Fin-
land) at 570 nm.
FTIR spectra of chitosan, MAOECECS and photopolymerized
hydrogel are showed in Fig. 1. Fig. 1a shows the principal spectral
À1
À1
For the reference purpose, cells were seeded to medium con-
taining 0.64% phenol (positive control) and a fresh culture medium
features in chitosan: 3424 cm (O–H stretch), 2921 cm (C–H
À1
À1
stretch), 1650 cm (C@O stretch), 1597 cm (N–H stretch bend),
1155 cm (bridge-O stretch), 1081 cm (O–H stretch). After Mi-
chael addition with EGAMA, the FTIR spectrum for all four MAOE-
À1
À1
(
negative control) with same seeding condition, respectively.
À1
CECS samples showed absorptions at 1717 and 810 cm , which
2.6. Statistical analysis
are indicative of the carbonyl group and the double bond (vinyl)
of the methacrylate group, respectively (Fig. 1b–e). After photopo-
lymerization to form hydrogel, the peak originating from the vinyl
Results are depicted as mean ± standard deviation. Significance
between the mean values was calculated by using ANOVA one-way
analysis (Origin7.0 SRO, Northampton, MA, USA). Probability val-
ues p < 0.05 were considered significant (n = 6).
À1
bond (ꢀ810 cm ) disappeared, indicating almost full conversion
and photopolymerization of the vinyl group (Fig. 1f).
1
H NMR of MAOECECSs and chitosan are provided in Fig. 2.
Methacrylation by Michael addition is achieved, as evidenced by
peaks arsing at 5.7 and 6.1 ppm due to protons on the vinyl carbon
and a peak at 1.9 ppm due to the methyl group in methacrylate.
The same groups in unreacted EGAMA appear at 6.1, 5.6 and
1.9 ppm, respectively. N-methacrylation can be discerned in the
spectrum, as a reduction in peak area at 3.1 ppm (H-2 proton for
the deacetylated residues) and the appearance of a small peak cen-
tered at 3.0 ppm corresponding to H-2 for the N-methacrylated
residue. The DS of methacrylate groups onto the chitosan backbone
was calculated from the relative integrations of the methyl protons
Table 1
Mole ratio of reactant and characteristic properties of MAOECECS.
DSa
Sample
name
EGAMA
(g)
EGAMA to –NH
(mole ratio)
2
Solubility in
deionized water
MAOECECS1
MAOECECS2
MAOECECS3
MAOECECS4
1.41
2.82
4.21
5.62
1.0
2.0
3.0
4.0
0.10
0.26
0.30
0.35
Full solubility
Full solubility
Full solubility
Full solubility
a
DS was calculated from 1H NMR studies.
3
at 1.9 ppm with respect to the methyl protons of –NHCOCH at
Fig. 1. FTIR spectra of (a) chitosan, (b) MAOECECS1, (c) MAOECECS2, (d) MAOECECS3, (e) MAOECECS4 and (f) MAOECECS hydrogel.

5
10
X. Gao et al. / Carbohydrate Polymers 79 (2010) 507–512
bond and the carbon of the methyl substituent in the methacrylate
group were seen at 175 and 13 ppm while the carbons in the dou-
ble bond (C@CH ) were found at 136 and 127 ppm, respectively.
Other peaks of MAOECECS2 were assigned in the region between
and 100 ppm by comparison with the spectrum recorded for
chitosan: 100(C1), 77.7(C4), 75.1(C5), 72.0(C3), 63.0(e,f, –OCH2-
CH O–), 60.5(C6), 55.5(C2), 44.9(i, –NHCH –), 34.9(h, –CH COO–),
and 17.4(j, NHCOCH ). No peak was located around 65 ppm where
2
0
2
2
2
3
the methacryloyl group is substituted, implying that the methacry-
loyl group is not substituted at the 6-O position of the polymer
chain. However, the peak at 44.9 ppm due to the substitution of
the methacryloyl groups located at the 2-N position implies that
substitutions just occurred at the amino groups.
3.2. Thermogravimetric analysis (TGA)
Thermographs of chitosan and MAOECECS with different DS are
shown in Fig. 4. The chitosan shows obvious loss of weight starting
from 280 to 330 °C and maximum thermal decomposition at
3
05 °C, which could be attributed to a complex process including
dehydration of the saccharide rings, depolymerization and decom-
position of the acetylated and deacetylated units of the polymer
(Peniche-Covas, Argüelles-Monal, & San Román, 1993). It could
be seen that, a slow process of weight loss appeared in the TG spec-
tra of MAOECECS starting from 180 to 220 °C, which was attributed
to the abolition of ester groups, similar with the results of Yao (Yao,
Zhang, Ping, & Yu, 2007). As observed in Fig. 5, all MAOECECSs
show obvious loss of weight starting from 240 to 320 °C, lower
than chitosan (280–330 °C). The initial thermal decomposition
temperature of MAOECECS dropped from 271 to 249 °C with
increasing of DS from 0.10 to 0.35. The results obtained from
TGA curves indicate a decrease of thermal stability after reaction
with EGAMA. Introduction of methacrylate groups into polysac-
charide structure would disrupt the crystalline structure of chito-
san, especially through the loss of the intra/intermolecular
hydrogen bonding, resulting in reduced interaction forces com-
pared to native chitosan.
Fig. 2. 1H NMR spectra of MAOECECSs in D
O and CS in CD COOD/D O.
2 3 2
2
.0 ppm: DS = 0.15ÃI1.9/I2.0. By calculation, DS increased from 0.10
to 0.35 when mole ratio of acrylate group in EGAMA to –NH of
chitosan. Fig. 2 also shows there is no peak between 4.90 and
.15 ppm, indicating substitution onto amino group of chitosan
was mono-substitution (Sashiwa, Shigemasa, & Roy, 2000; Sashiwa
et al., 2003). Other peaks were attributed as follows. 4.50(c, H-1 of
2
5
3.3. XRD
GlcNAc), 4.37 (d, –COO–CH
kyl group, H-3,4,5,6 of GlcN and GlcNAc), 3.0(f, H-2 of GlcNAc),
.8(g, H-2 of alkylated GlcN), 2.5(h, –CH COO–), 2.0(i, –NHCOCH ).
C NMR was also performed on MAOECECS2 to confirm the Mi-
chael-addition reaction (Fig. 3). The carbonyl carbon of the ester
2 2 2
–CH OCO–), 3.6–4.0(N–CH – of N-al-
Fig. 5 presents XRD patterns of CS and MAOECECS with different
DS. It could be seen that CS powder exhibited three typical peaks at
2
2
3
1
3
2
1
h = 10.3°, 15.2° and 19.9°, agree to Samuels’ observation (Samuels,
981). According to Samuels, the reflection falling at 2h = 10.3° was
Fig. 3. 13C NMR spectrum of MAOECECS2 in D
O.
Fig. 4. TGA curves of CS and MAOECECS with different DS.
2

X. Gao et al. / Carbohydrate Polymers 79 (2010) 507–512
511
Fig. 7. Cytotoxicity test of CS, crosslinked CECS membrane, MAOECECS2 and
*
negative controls (p < 0.05). p < 0.05 when compared to the negative control of
indirect cytotoxicity. The data represented mean and standard deviations of six
samples.
Fig. 5. XRD patterns of CS and MAOECECS with different DS.
Injectable materials are advantageous in that they can be ap-
plied to tissue defects with irregular shapes and form tight inter-
faces with surrounding tissues. However, it is hard to remove
unreacted residues after curing. Therefore, cytotoxicity of leach-
able products after cross-linking should be examined. Fig. 7 shows
the absorbance obtained from an MTT assay of Vero cells which
were cultured with the extraction medium from various types of
specimens in comparison with control. It could be seen that, no
statistically significant differences (p < 0.05) were observed in the
cell activity of Vero culture within 72 h in the presence of MAOE-
CECS hydrogels extracts in comparison with control, although the
average absorbance values were lower than that of the control con-
dition. However, when crosslinked carboxyethyl chitosan (CECS)
membrane or CS membrane extract was used, statistically signifi-
cant differences (p < 0.05) were observed in the cell activity in
comparison with control within 72 h, but the viability of the cell
still reached 80% of the negative control. This indicates that the
crosslinked CECS membrane or CS membrane were less toxic to
Vero cells. A similar result was observed in the CS membrane when
culture time was 48, 72 h. The obtained results clearly suggest that
MAOECECS hydrogel is non-toxic to Vero cells and are good candi-
dates to be used as injectable materials.
assigned to crystal form I. The strongest falling at 2h = 19.9° corre-
sponded to crystal form II. However, for all MAOECECSs, the crys-
talline peak at 2h = 10.3° and 15.2° disappeared and strong peak
at 2h = 19.9° became a relative obtuse and broad. It suggested that
the large number of hydrogen bonding in the CS powder was de-
stroyed through the N-acylation, thus forming a smaller fraction
of crystalline phase and a larger fraction of amorphous phase.
3.4. MTT assay
An ideal biomedical material should not release toxic products
or produce adverse reactions, which could be evaluated through
in vitro cytotoxic tests. The cytotoxicity of the MAOECECS with dif-
ferent concentration was assessed by using Vero cells line as refer-
ence (Fig. 6). It could be seen that, no statistically significant
differences were observed in the cell activity of Vero cells culture
for 48 h in the presence of MAOECECS culture solution with a con-
centration up to 1 mg/mL in comparison with negative control
(
NC), although the average absorbance values were lower than that
of the control condition. However, when a concentration of MAOE-
CECS was up to 2.5 mg/mL, statistically significant differences
(
p < 0.05) were observed in the cell activity in comparison with
4
. Conclusions
control. The obtained results clearly suggest that MAOECECS is
non-toxic to Vero cells at the low concentration.
Chitosan can be reacted with ethylene glycol acrylate methacry-
late to yield a water-soluble, photopolymerizable precursor MAOE-
CECS that can be used to prepare hydrogels. The degree of DS can
be readily manipulated by adjusting the molar excess of EGAMA to
chitosan reactive amine ratio and the reaction time. The MAOE-
CECS shows noncytotoxicity toward Vero cells. Moreover, the gels
prepared through photopolymerization of their precursors did not
possess unreacted C@C and supported the growth of Vero cells.
MAOECECS thus appears to be a promising biomaterial for use in
tissue engineering.
Acknowledgment
Program for Changjiang Scholars and Innovative Research Team
in University is appreciated.
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