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Radiation Physics and Chemistry 79 (2010) 606611

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Radiation Physics and Chemistry


journal homepage: www.elsevier.com/locate/radphyschem

Cytotoxicity and wound healing properties of PVA/ws-chitosan/glycerol


hydrogels made by irradiation followed by freezethawing
Xiaomin Yang a,b, Kang Yang a,b, Shengwei Wu a, Xiliang Chen a,b, Feng Yu a,b, Jungang Li a,
Mingwang Ma a,b, Zhiyong Zhu a,n
a
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P.O. Box 800-204, PR China
b
Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China

a r t i c l e in f o a b s t r a c t

Article history: Hydrogels based on poly(vinyl alcohol), water-soluble chitosan and glycerol made by irradiation
Received 5 August 2008 followed by freezethawing were evaluated as wound dressing. MTT assay suggested that the extract of
Accepted 21 December 2009 hydrogels was nontoxic towards L929 mouse broblasts. Compared to gauze dressing, the hydrogel can
accelerate the healing process of full-thickness wounds in a rat model. Wounds treated with hydrogel
Keywords: healed at 11th day postoperatively and histological observation showed that mature epidermal
Wound dressing architecture was formed. These indicate that it is a good wound dressing.
Water soluble chitosan & 2009 Elsevier Ltd. All rights reserved.
Poly(vinyl alcohol)
Biocompatibility

1. Introduction initiators or crosslinkers which may be harmful and difcult to


remove. The hydrogel formation and sterilization can be achieved
The pioneering work of Winter in the 1960s initiated the in one step. But this kind of hydrogel often has poor mechanical
concept that a wet environment can enhance the wound healing strength. Hydrogels, prepared by freezethawing without any
process (Winter, 1962; Winter and Scales, 1963). Since then, initiators or crosslinkers, too, from PVA aqueous solutions, have
many new types of wound dressings were developed (Atiyeh large mechanical strength, but bear limited swelling capacity and
et al., 2002; Balakrishnan et al., 2005; Denuziere et al., 1998; Jones thermal stability, and are opaque in appearance. Freeze-thawing
and Vaughan, 2005; Purna and Babu, 2000). Hydrogel is just one followed by irradiation was once used to enhance the mechanical
of them and has been widely studied as a wound dressing. It can strength of the hydrogels (Nho and Park, 2002; Park and Nho,
absorb excess of wound exudates, protect the wound from 2003).
secondary infection and effectively promote the healing process We previously found that hydrogels made by irradiation
by providing a moisturized wound healing environment (Winter, followed by freezethawing showed strong mechanical strength
1962). It can also be removed without causing trauma to the and high swelling capacity, and were more transparent than those
wound. made by freezethawing followed by irradiation (Yang et al.,
Chitosan, a derivative of the biopolymer chitin, has been 2008a, 2008b). In a previous study, hydrogels based on PVA/
extensively applied in biomedical and pharmaceutical research ws-chitosan/glycerol were developed by irradiation followed by
because of its low toxicity and good biocompatibility. It is able to freezethawing. The water absorption capacity, mechanical
accelerate the reepithelialization and normal skin regeneration strength, and thermal properties of these hydrogels were studied
(Usami et al., 1994a, 1994b), and to confer considerable (Yang et al., 2008c). The present work explored the potential
antibacterial activity against a broad spectrum of bacteria (No ability of the PVA/ws-chitosan/glycerol hydrogels for wound-
et al., 2002; Tsai and Su, 1999). But chitosan itself cannot dissolve dressing through investigation of in vitro cytotoxicity and in vivo
in water but in acidic solutions. Protonated chitosan, a kind of wound healing efcacy in the rat model.
water soluble chitosan, can simplify the hydrogel-making process.
Considerable research has been carried out on hydrogels used
as wound dressing. These hydrogels are often developed by
2. Materials and methods
irradiation or by freezethawing. Irradiation is a useful tool to
make hydrogels for biomedical applications. There are no
2.1. Materials

n
Corresponding author. Tel./fax: +86 21 59556904. Poly(vinyl alcohol) (PVA) and glycerol were bought from
E-mail address: zhuzhiyong@sinap.ac.cn (Z. Zhu). Sinopharm Chemical Reagent Co. Ltd., China. The degrees of

0969-806X/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.radphyschem.2009.12.017
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X. Yang et al. / Radiation Physics and Chemistry 79 (2010) 606611 607

polymerization and hydrolysis of PVA were 1750750 and 98%, intracellular blue formazan product. After overnight incubation,
respectively. Water soluble chitosan (ws-chitosan), manufactured the optical density of each well was read using a microplate reader
by protonating chitosan in HCl/CH3CH2OH solution, was obtained (Bio-Rad, Model 680) at 570 nm with a reference wavelength of
from Jinhu Chitin Co. Ltd., China. The molecular weight and 655 nm. The percentage cell viability of samples compared to the
deacetylation degree of chitosan before protonation were about control was calculated from the following equation:
200,000 and 91.7%, respectively. 3-[4,5-dimethylthiazolyl-2]-2,5-
ODsampleODpositive
diphenyl tetrazolium bromide (MTT) (Amresco, USA) was dis- Cell viability %  100
ODnegativeODpositive
solved in phosphate-buffered saline (pH= 7.4, 0.01 mol/L) at 5 mg/
ml, sterilized by ltration (0.22 mm, Millipore, USA) and stocked at
20 1C. Sodium dodecyl sulfate (SDS) (Amresco, USA) was 2.3.2. Microscopic observations
dissolved in 0.01 mol/L HCl solution at 10% concentration and The morphology of the cells was assessed in comparison with
stocked at room temperature. negative and positive controls. After incubation for 24 and 48 h,
the morphology of L929 broblasts from the 96-well micro-
2.2. Preparation of PVA/ws-chitosan/glycerol hydrogels culture plates were observed using an Olympus inverse phase
contrast microscope equipped with an objective of  10 magni-
The hydrogels were prepared as previously described. Briey, cation.
PVA was dissolved in distilled water at 96 1C for 3 h under
reuxing. The homogenous solution containing 9 wt% PVA, 2 wt% 2.4. In vivo wound healing study
ws-chitosan, and 1 wt% glycerol was obtained by stirring the
mixtures of PVA, ws-chitosan, and glycerol solutions at 40 1C for The wound healing characteristics of the hydrogel were
30 min before placing them in an ultrasonic water bath at 40 1C evaluated using a rat model. The animal study was carried out
for 15 min to remove bubbles. The mixed solution was irradiated abiding by the national regulations related to the conduct of
to 40 kGy in N2 atmosphere with 60Co g-ray at a dose rate of experimentation. Sprague-Dawley rats weighing about 220 g
0.76 kGy/h. The obtained hydrogels were further freeze-thawed obtained from Shanghai Slack Experimental Animal Center were
for two cycles. Each cycle of freezethawing was performed at fed in separate cages for a week before experiment. The rats were
20 1C for 1.5 h and then 25 1C for 1 h. anesthetized with 5% pentobarbital sodium by intraperitoneal
injection (1 ml/kg of weight of rats). The dorsal hair of each rat
2.3. In vitro cytotoxicity studies was clipped at the operation site and a wound (about 2  2 cm2)
with a depth of all the skin layers was prepared. The wounds were
2.3.1. MTT assay cleansed with normal saline and disinfected by 75% ethanol. The
The cytotoxicity test was performed according to GB/T test wounds (n =6) were then covered with the sterile PVA/
16886.5-2003 (ISO 10993-5: 1999). To prevent the adsorption of ws-chitosan/glycerol hydrogel and xed with sterile gauze.
some substances of the culture medium into the hydrogel, Similarly, control wounds (n= 6) were covered with sterile gauze.
extracts were prepared by adding PVA/ws-chitosan/glycerol After experiment, rats were kept in separate cages. On 2nd, 5th,
hydrogel fragments to sterilized distilled water at a concentration 8th, 11th, 14th, and 16th postoperative days, the dressings were
of 0.4 g/ml and incubated at 37 1C for 24 h. After this period, the removed and the appearance of the wound was photographed.
hydrogel was discarded and the extract was added into equal The wound area was estimated by outlining the wound area onto
volume of double concentrated RPMI-1640 culture medium a transparent PET membrane. New dressings were applied to the
(Gibco, USA) containing 6% new born calf serum (Gibco, USA). wound sites after they were cleansed with normal saline. The rate
The mixture (100% extract) was sterilized by ltration (0.22 mm, of wound closure was determined by the following equation:
Millipore, USA). 50% extract was obtained by diluting the 100%  
Wound area % of original At =A0  100
extract with RPMI-1640 culture medium containing 3% new born
calf serum. L929 mouse broblasts used in the experiment were
cultured at 37 1C with 5% CO2 in plastic culture dishes containing
RPMI-1640 culture medium with 10% new born calf serum to
obtain a conuent monolayer of cells prior to use. 100 ml of the
cell suspension, 5000 cell/well, were seeded in at-bottomed
96-well micro-culture plates (Costar, USA). The 96-well micro-
culture plates were incubated for 12 h at 37 1C in a 5% CO2
humidied incubator. After this period, the medium was
discarded and replaced with 100 ml of 100% extract and 50%
diluted extract, respectively. The RPMI-1640 culture medium
with 3% new born calf serum was used as a negative control
and RPMI-1640 culture medium with 3% new born calf serum and
0.7% acrylamide was used as a positive control. Samples and
controls were tested in sextuplet. These plates were then
incubated for 24 and 48 h, respectively, before the viability of
broblasts was determined by MTT assay.
MTT reagent is a pale yellow substrate which produces a dark
blue formazan product when it is incubated with viable cells. The
method of Mosmann (1983) modied by Tada et al. (1986) was
used in this study. Briey, at the end of culturing time, 10 ml of MTT
solution (5 mg/ml) was added to each well. After 4 h incubation at
37 1C in a 5% CO2 humidied incubator, 100 ml of 10% SDS in Fig. 1. Effects of 100% and 50% hydrogel extracts on L929 mouse broblast cell
0.01 mol/L HCl was added into each well to dissolve the viability after 24 and 48 h incubation.
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608 X. Yang et al. / Radiation Physics and Chemistry 79 (2010) 606611

where At and A0 were the wound areas on the specied day and 3. Results and discussion
the day of operation, respectively. On the 16th day, the rats were
anesthetized and the full-thickness skin around the wound sites An ideal dressing should meet requirements such as absorbing
was dissected, washed with normal saline solution, xed in 4% uids effectively, pleasant in touch and painless in removal, high
phosphate-buffered polyformaldehyde solution, embedded in elasticity but also good mechanical strength, good transparency,
parafn and sectioned in 5-mm increments. The sections were and should act as a barrier against the microbes (Ajji et al., 2005).
made perpendicular to the surface of the wound and were xed In a former study, it was found that the PVA/ws-chitosan/glycerol
on a slide, and stained with hematoxylineosin (HE) reagents for hydrogel made in this experiment was exible and transparent,
histological study. and had good mechanical strength with high water absorption

Fig. 2. Photomicrographs of L929 mouse broblasts cultured for 24 h, magnied 100 times.

Fig. 3. Photomicrographs of L929 mouse broblasts cultured for 48 h, magnied 100 times.
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X. Yang et al. / Radiation Physics and Chemistry 79 (2010) 606611 609

capacity (Yang et al., 2008c). Ws-chitosan in the hydrogel has treated with gauze and hydrogels can be seen in Fig. 4. A healthy
good antibacterial property which can protect the wound from clean wound, pinkish red in color, was observed during the
secondary infection (Yang, et al., 2008a). healing process for wounds treated with hydrogel. However, for
the control wounds treated with gauze, there was somewhat
haemorrhagic. Hydrogel dressings adhered slightly to the wound
3.1. In vitro cytotoxicity
and could be removed from the wound surface without causing
further trauma. Nevertheless, for gauze dressings, the adherence
The evaluation of cytotoxicity is very important for hydrogels
of the gauze bers to the healing tissues is often occurred,
used for wound dressing applications. MTT assay was often used to
especially in the rst few days after operation. When these bers
evaluate the in vitro cytotoxicity of polymeric components. It is a
were enclosed by newly grown tissue, it was difcult to separate
quick effective method for testing mitochondrial impairment and
the dressing material from the tissue, which resulted in
correlates quite well with cell proliferation. It is based on the use of
considerable tissue trauma and bleeding during removal of the
tetrazolium salt 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazo-
dressings. Thus, the wound healing process was prolonged.
lium bromide (MTT), which can be converted to an insoluble blue
The rate of wound healing covered with hydrogel was
formazan product by mitochondrial enzymes in viable cells. The
compared to those dressed with conventional gauze, which was
effects of 100% and 50% hydrogel extracts on L929 mouse broblast
evaluated by determination of the wound area as a function of
cell viability after 24 and 48 h incubation are shown in Fig. 1.
time as shown in Fig. 5. In the case of wounds treated with
According to GB/T 16886.5-2003 (ISO 10993-5: 1999), samples with
hydrogel, the wounds contracted rapidly in the rst 8 days. More
cell viability larger than 75% can be considered as noncytotoxic. Fig. 1
precisely, there was almost 40% reduction in wound defect area at
shows that the remaining cell viability is larger than 80% for both 50%
the rst 2 days and about 90% wound contraction was achieved
and 100% hydrogel extracts incubated for 24 and 48 h, respectively. It
indicates that there is no cytotoxicity for the hydrogel extracts.
L929 mouse broblasts are large, spindle-shaped, adherent
cells growing as a conuent monolayer. Figs. 2 and 3 are the
photomicrographs of L929 mouse broblasts incubated for 24 and
48 h, respectively. It can be found that the morphologies of L929
mouse broblasts were almost the same for those of the 100% and
50% hydrogel extracts and the negative samples. The number of
the cells becomes larger with increasing incubation time. For the
positive samples, the cells were all dead only cell debris and cell
lysis remaining.
Data from both the MTT assays and the microscopic observa-
tions reveal that no difference was observed in the L929 mouse
broblasts activity between the study and the negative control
groups. The cells grew very well after 48 h of exposure to the
hydrogel extracts. Therefore, the hydrogels can be considered to
be biocompatible.

3.2. In vivo study

In our wound healing rat model, a full thickness wound of skin Fig. 5. Wound contraction as a function of postoperative time for wounds treated
was made on the back of each rat. The appearance of the wounds with hydrogel and gauze dressings, indicated by the percentage wound size.

Fig. 4. Photographs of macroscopic appearance of wounds dressed with gauze and hydrogels at 0th, 5th, 8th, and 11th days after operation.
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610 X. Yang et al. / Radiation Physics and Chemistry 79 (2010) 606611

Fig. 6. Histology of wounds treated with gauze and hydrogels at 16th postoperative day. Stained with hematoxylin and eosin. Magnied 100 times.

within 8 days. In contrast with the hydrogel-treated wounds, dressing in the rat model. Wounds treated with hydrogel healed
control wounds treated with conventional gauze healed much at 11th day postoperatively and histological observations showed
slowly. No wound contraction was observed at the rst 2 days and that mature epidermal architecture was formed. On the basis of
only about 53% wound contraction was achieved at 8 days. All the its biocompatibility and wound-healing efcacy, the PVA/ws-
hydrogel-treated wounds were almost healed at 11 days. chitosan/glycerol hydrogel can be considered as good wound
However, 90% wound closure was achieved only after 14 days dressing.
for the control wounds. It is known that new epidermis grew out
from the margin to the center of the wounds, reducing the depth
and area of the wounds with time. The fast wound contraction for Acknowledgements
wounds treated with hydrogel may attribute to fast epidermis
migration due to the moist wound healing environment. Winter We gratefully acknowledge the help of professor Wenxin Li for
has reported that epithelization can be accelerated if the wound is providing the wound healing work facilities. This work is
kept moist (Winter, 1962; Winter and Scales, 1963). The nancially supported by Shanghai Institute of Applied Physics,
epidermal cells migrated more easily over a moist wound CAS (Project no.: 90120432).
surface than under a scab in dry wounds (Winter and Scales,
1963). Furthermore, chitosan molecules have been reported to
induce migration of inammatory cells and broblasts, being Reference
activated to produce multiple cytokines (Nishimura et al., 1986;
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using gamma radiation. Nucl. Instrum. Methods Phys. Res. Sect. BBeam
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wounds treated with gauze also delayed the wound healing and chronic open wounds: the importance of moist environment in optimal
process as mentioned before. wound healing. Curr. Pharm. Biotechnol. 3 (3), 179195.
Balakrishnan, B., Mohanty, M., Umashankar, P.R., Jayakrishnan, A., 2005. Evaluation
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