Hydrogel Wound Dressing of Chitosan Derivative
Content
Wound Repair and Regeneration
A novel in situ-formed hydrogel wound dressing by the photocross-linking of a chitosan derivative
Guozhong Lu, MD1n; Kai Ling, MS2n; Peng Zhao, BS2; Zhenghong Xu, PhD3; Cao Deng, MS3; Hua Zheng, PhD2; Jin Huang, PhD2; Jinghua Chen, PhD3
1. Department of Burn and Plastic Surgery, Wuxi third People’s Hospital, Wuxi, China, 2. College of Chemical Engineering, Wuhan University of Technology, Wuhan, China, and 3. School of Medicine and Pharmaceutics, Jiangnan University, Wuxi, China
Reprint requests: Jinghua Chen, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China. Tel: 186 510 85327353; Fax: 186 510 85329042; Email: [email protected]
n
ABSTRACT In situ photopolymerized hydrogel dressings create minimally invasive methods that offer advantages over the use of preformed dressings such as conformability in any wound bed, convenience of application, and improved patient compliance and comfort. Here, we report an in situ-formed hydrogel membrane through ultraviolet cross-linking of a photocross-linkable azidobenzoic hydroxypropyl chitosan aqueous solution. The hydrogel membrane is stable, flexible, and transparent, with a bulk network structure of smoothness, integrity, and density. Fluid uptake ability, water vapor transmission rate, water retention, and bioadhesion of the thus resulted hydrogel membranes (0.1 mm thick) were determined to range from 97.0–96.3%, 2,934–2,561 g/m2/day, 36.69–22.94% (after 6 days), and 4.8–12.3 N/cm2, respectively. These data indicate that the hydrogel membrane can maintain a long period of moist environment over the wound bed for enhancing reepithelialization. Specifically, these properties of the hydrogel membrane were controllable to some extent, by adjusting the substitution degree of the photoreactive azide groups. The hydrogel membrane also exhibited barrier function, as it was impermeable to bacteria but permeable to oxygen. In vitro experiments using two major skin cell types (dermal fibroblast and epidermal keratinocyte) revealed the hydrogel membrane have neither cytotoxicity nor an effect on cell proliferation. Taken together, the in situ photocross-linked azidobenzoic hydroxypropyl chitosan hydrogel membrane has a great potential in the management of wound healing and skin burn. Recently, Balakrishnan et al.8,9 reported that an in situforming hydrogel wound dressing can be prepared from gelatin and oxidized alginate in the presence of small concentrations of borax. Fabrication of hydrogel in situ will have advantages over the use of preformed hydrogel dressings because it allows the hydrogel to form complex shapes that adhere and conform to the wound defect.5,8 Most commercially available dressings in the form of membranes and sheets are problematic as far as the conformability is concerned and the in situ-formed dressings will, therefore, be superior to preformed dressings. However, the in situ-forming hydrogel was cross-linked by a reaction between a resulting alginate dialdehyde (ADA) and gelatin in the presence of 0.1 M borax. According to Draye et al.,10 a possible release of trace amounts of dialdehyde might induce toxicity of the hydrogel, similar to the cytotoxicity associated with the release of glutaraldehyde from glutaraldehyde cross-linked bioprotheses.11 Photopolymerization allows in situ hydrogel formation in a minimally invasive manner.12 It provides a fast and efficient method to cross-link polymers to form a hydrogel with significant temporal and spatial control. There are some reports of a photocross-linked chitosan hydrogel that is prepared by applying ultraviolet (UV) light irradiation to a photocross-linkable chitosan aqueous solution.13–15 This chitosan hydrogel could effectively stop
c Wound Rep Reg (2010) 18 70–79 2010 by the Wound Healing Society
These authors contributed equally to this work. Manuscript received: April 2, 2009 Accepted in final form: September 29, 2009 DOI:10.1111/j.1524-475X.2009.00557.x
The development of novel dressings to promote wound healing in humans has drawn increased attention during the last decades.1 Researchers have believed that an ideal wound dressing should protect the wound from bacterial infection, provide a moist and healing environment, and remain biocompatible.2 Although traditional (nonocclusive) wound dressings, which offer dry wound healing conditions, still account for the largest segment of the dressing market, the use of occlusive dressings, i.e., hydrocolloid and hydrogel dressings, which offer moist wound healing conditions, has increased significantly.1 Hydrogels, cross-linked hydrophilic polymers, exhibit excellent biocompatibility, causing minimal inflammatory responses, thrombosis, and tissue damage.3,4 Hydrogels can also absorb large quantities of water and well without the dissolution of the polymer because of their hydrophilic but cross-linked structure, thus giving them physical characteristics similar to soft tissues. In addition, hydrogels have high permeability for oxygen, nutrients, and other water-soluble metabolites.5 Thus, hydrogel materials possess the potential to be ideal wound dressings. For example, a commercial hydrogel dressing, ‘‘Geliperms,’’ was established to have the ability of providing optimal physiological conditions for wound healing.6 However, this material is limited to be used as a temporary cover, only because of its lack of adherence to the wound and its fragile nature.7
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Photocross-linked hydroxypropyl chitosan hydrogel membrane
bleeding, induce significant wound contraction, accelerate wound closure and healing, and showed nontoxicity and good biocompatibility.13 Inspite of intensive researches using photocross-linkable chitosan as wound dressing, there is a lack of detailed information about the properties of the membrane of the in situ photocross-linked chitosan hydrogel, such as water vapor transmission rate (WVTR), water retention, and bioadhesion, which limit the applications of photocross-linkable chitosan. In this article, we utilized our prepared photocross-linkable hydroxypropyl chitosan (HPCS),16 4-azidobenzoic hydroxypropyl chitosan (Az-HPCS), with different degree of substitution (DS) of Az- groups, to in situ-formed crosslinked bioadhesive hydrogel membrane and evaluated its potential as wound dressing. We characterized the basic properties, such as FUA, WVTR, water retention, and bioadhesion of photocross-linked Az-HPCS hydrogel membranes, to evaluate its potential as wound dressing. The morphologies of the hydrogel membrane in hydrated and a dehydrated states were observed using environmental scanning electron microscopy (ESEM) and scanning electron microscopy (SEM), respectively. Bacterial and oxygen penetration properties of the hydrogel membrane were also assessed. Cell culture experiments of dermal fibroblast (L929 mouse fibroblast) and epidermal keratinocyte (HaCaT human keratinocyte) were conducted to test the toxicity and biocompatibility of the hydrogel membrane.
MATERIALS AND METHODS
Materials
Chitosan was supplied by Zhejiang Yuhuan Ocean Biochemistry Co. Ltd., China. Its viscosity average molecular weight was 830 kDa, with an 87% degree of deacetylation. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED), and dialysis membrane (10 kDa MWCO) were purchased from Sigma-Aldrich (St. Louis, MO). 4-azidobenzoic acid was purchased from TCI (Tokyo, Japan). All other chemicals were purchased from SCRC (Shanghai, China), unless otherwise indicated.
Preparation of Az-HPCS
The method of the preparation of Az-HPCS is from our previous report.16 The typical procedure is shown in Figure 1A. Briefly, HPCS was firstly synthesized from chitosan and propylene oxide under alkali condition, according to Xie and colleague’s method.17 Subsequently, HPCS (10 g), EDC (0.350, 0.525, and 0.700 g), and 4-azidobenzoic acid (0.20, 0.30, and 0.40 g) were added to 150 mL of 50 mM TEMED aqueous solution. The mixture was stirred at room temperature for 72 hours and dialyzed against deionized water for 72 hours. The resulting product was obtained by freeze drying. The DS of Az- groups of the Az-HPCS prepared were 2.8%, 4.2%, and 5.6%, respectively. The constituents and structures of HPCS and Az-HPCS were examined and measured using an Elemental Analyzer–VarioEL III (ELEMENTAR, Hanau, Germany) and a Mercury-300 NMR Spectrometer (Varian Inc, Palo Alto, CA.) at 300 MHz at 25 1C, using D2O as the solvent.
c Wound Rep Reg (2010) 18 70–79 2010 by the Wound Healing Society
Figure 1. Scheme of the preparation of azidobenzoic hydroxypropyl chitosan (Az-HPCS) hydrogel.
Preparation of Az-HPCS hydrogel membranes
Az-HPCS hydrogel membranes were prepared by UV irradiation. Firstly, 363 mL of Az-HPCS aqueous solution (25 mg/mL) was spread uniformly on the surfaces of tissue culture dishes (7 cm diameter, JET BIOFILs , JET Bio-filtration Products Co. Ltd., Guangzhou, China). Then, a mercury UV light (4 W, 254 nm, Shanghai Jiapeng Scientific Apparatus Co. Ltd., Shanghai, China) was used to irradiate the dish surfaces for 90 seconds. Az-HPCS solutions were gelatinized and flexible, stable, and
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transparent hydrogel membranes (0.1 mm thick), were formed in situ eventually. The entire procedure is illustrated briefly in Figure 1B.
Morphology observation
where W0 is the initial weight and Wt is the weight at time t, respectively.
Adhesion strength (AS)
The hydrated Az-HPCS hydrogel membranes were placed on a Peltier cooling stage inside an ESEM (FEI Quanta-200, FEI, Hillsboro, OR) and maintained at 4 1C. The hydrogel membranes were imaged using a voltage of 20 kV and a water vapor pressure between 4.0 and 4.7 Torr. The dehydrated Az-HPCS hydrogel membranes prepared by a lyophilization process, were gold coated to about 500Â10À8 cm thickness using an Hitachi (Tokyo, Japan) coating unit, IB-2 coater under a high vacuum, 0.1 Torr, and high voltage, 1.2 kV and 50 mA. Coated samples were examined using a Hitachi S4800 (Tokyo, Japan) electron scanning microscopy (SEM).
Fluid uptake ability (FUA)
The FUA of the hydrogel membranes were determined as described below. Completely dried membrane samples were cut into square-shaped specimens (1 cmÂ1 cm) and weighed. Then, the specimens were immersed in phosphate-buffered saline (PBS; pH 7.4) buffer, and taken out quickly at specific time intervals, i.e., 6, 12, 24, and 48 hours, respectively, and weighed after wiping with paper towels. The FUA of the hydrogel membranes was calculated as follows: FUA ð%Þ ¼ ðWs À Wd Þ100=Ws ð1Þ
Porcine skin was excised from the back of the ‘‘Yorkshire pig’’ breed at the animal center of Wuxi third people’s hospital in accordance with ethical guidelines stated in the Guide for the use and care of laboratory animals, National Research Council, China (1985). Fresh porcine skin was cut into strips (4.0 cmÂ2.0 cm and 1.5 mm thick). Two strips were placed flat, 2.0 cm on each side closely touching. 30 mL of AzHPCS aqueous solution (25 mg/mL) was then added between the two skin strips along the 2.0 cm side. After 90 seconds of UV-irradiation, a mechanical tester (Instron 3365, Illinois Tool Works, Glenview, IL) was utilized to measure the bioadhesion strength. One side of the adhered skin preparation was fixed on the base and the other side was attached vertically with a pair of grippers. And then the pair of grippers drew the adhered strips until they separated into two pieces. The tensile force (TF) was recorded by the equipment. The AS was calculated as follows: ASðN=cm2 Þ ¼ TF=C
2
ð4Þ
where C is the cross-sectional area (cm ) of the porcine skin tissue strip.
Bacterial penetration
where Wd is the weight of the initial dry specimens and Ws is the weight of the swollen specimens.
WVTR
The moisture permeability of the hydrogel membrane was determined by measuring the WVTR, according to the ASTM method E96-00.18 The hydrogel membranes prepared were mounted at the mouth of cylindrical plastic cups (36 mm diameter) containing 10 mL water with negligible water vapor transmittance. The material was fastened using Teflon tape across the edge to prevent any water vapor loss through the boundary and placed in a desiccator containing a saturated solution of ammonium sulfate at 37 1C and under 79% relative humidity (RH). The assembly was weighed at regular intervals of time and a weight loss vs. time plot was constructed. From the slope of the plot, WVTR was calculated as follows:8 WVTRðg=m2 =dayÞ ¼ slope  24=A where A is the test area of the sample in m .
Water loss
2
ð2Þ
To determine the water retention of the hydrogel membrane, the membrane samples were placed in a desiccator containing a saturated solution of ammonium sulfate at 37 1C under 79% RH, and the membranes were weighed at different time intervals. Weight remaining (WR) percentage was calculated as follows: WRð%Þ ¼ Wt Â100=W0
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Staphylococcus aureus and Escherichia coli, supplied by the Microbiology Laboratory of Huazhong Agricultural University (National Key Lab., China), were selected as test cells because they are the most frequent bacteria in the wound infection and representative of Gram-positive and Gram-negative bacteria, respectively. The two bacteria were incubated at 37 1C for 24 hours in nutrient broth (bacto-peptone 10 g, beef extract 3 g, NaCl 3 g in 1,000 mL distilled water; pH 7.0) before use. By diluting appropriately with the culture medium, each culture containing $ 108 cells/mL was prepared, which was used for a bacterial penetration test. AH-5.6 solution (25 mg/mL) was prepared in distilled water and then sterilized through a 0.22 mm filter membrane. 550 mL sterilized AH-5.6 solution was spread on a nutrient agar plate (agar 15 g, bacto-peptone 10 g, beef extract 3 g, NaCl 3 g in 1,000 mL distilled water; pH 7.0, 10 cm diameter). Subsequently, the plates were exposed to UV light (90 seconds, 4 W) for in situ cross-linking a layer of AH-5.6 hydrogel membrane on the surface of nutrient agar. A portion (200 mL) of a bacterial culture (S. aureus or E. coli) prepared was then dropped and quickly spread on the treated nutrient agar plates. The plates were incubated at 37 1C and checked for bacterial penetration after 24 and 48 hours, respectively. Nutrient agar plates with and without AH-5.6 solution cover were used as controls.
Oxygen penetration
ð3Þ
Oxygen penetration through the AH-5.6 hydrogel membrane prepared was performed by placing the membrane prepared on top of open 250 mL flasks containing 200 mL of deionized water and held in place with a screw lid (25 mm diameter).
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Photocross-linked hydroxypropyl chitosan hydrogel membrane
The negative control was a closed flask with an airtight cap preventing oxygen from entering the flask whereas the positive control was an open flask allowing oxygen to enter the flask and dissolve in the water as a recipient. The test flasks were placed in an open environment under constant agitation for 24 hours. The water samples collected were then analyzed for dissolved oxygen, according to Winkler’s method.19,20
Cell culture
dium were added in each wells (2Â104 cells/well) and grown at 37 1C for 2 days. After the incubation, an MTT cell proliferation assay kit (Beijing Cellchip Co. Ltd., Beijing, China) was used to measure the absorption data spectrophotometrically at 570 nm. The cell growth status was observed using an optical microscope (CKX-31; Olympus, Tokyo, Japan).
Cell culture under the coverage of hydrogel membrane
Two cell lines of L929 and HaCaT (from fourth to eighth passages) were purchased from the China Center for Type Culture Collection (CCTCC). Mouse C3H/An connective tissue, fibroblast cell line (L929) was culture in RPMI medium 1640 (Gibco Invitrogen, Carlsbad, CA) with 2 mmol/ L L-glutamine and 24 mmol/L NaHCO3 supplemented with 10% heat-inactivated fetal bovine serum (FBS), together with penicillin G (100 U/mL) and streptomycin (100 mg/mL) at 37 1C in a 5% CO2 atmosphere. Spontaneously immortalized human keratinocyte cell line (HaCaT) was grown in Dulbecco’s modified Eagle’s medium (DMEM containing 5.6 mmol/L glucose, Gibco) with 4 mmol/L L-glutamine, 44 mmol/L NaHCO3, and 1 mmol/L sodium pyruvate supplemented with 10% heatinactivated FBS, together with penicillin G (100 U/mL) and streptomycin (100 mg/mL) at 37 1C in a 5% CO2 atmosphere. After the cells achieved confluent on a dish, we subcultured cells at a ratio of 1 : 10.
In vitro cytotoxicity test
Cells were seeded into 12-well tissue culture plates at a density of 1Â104 cells/well and left overnight for attachment. Each wells of the plates were then completely coated by AH-5.6 hydrogel membranes (0.1 mm thick) prepared and co-cultured for 2, 4, or 6 days with a change of medium every other day. At various time intervals, the membranes were taken out and the plates were washed twice by PBS and treated with 0.05% trypsin-EDTA (Sigma) solution at 37 1C for 15 minutes. The detached cells were counted by a hemocytometer. Blank cultures with the absence of biomaterial were used as controls.
Statistical analysis
All experiments were repeated three or more times. All values were expressed as mean Æ standard deviation. Statistical differences were determined by two-tailed unpaired t-test and analyzed using a significance level set at p < 0.05.
RESULTS
Preparation of Az-HPCS
In order to test the in vitro cytotoxicity of AH-5.6 hydrogel membrane, 5 mL of AH-5.6 aqueous solution (25 mg/mL) was spotted on each well of two 12-well tissue culture plates (Falcon, BD Biosciences, Franklin Lakes, NJ) and in situ hydrogel membranes were formed by 90 seconds of UV irradiation. Subsequently, cells suspended in the culture me-
Figure 1A shows the two-step reaction scheme for preparing Az-HPCS: (a) by a modification of chitosan with propylene oxide at the C-6 hydroxyl groups; and (b) by introducing the photoreactive Az- groups to the C-2
Figure 2. NMR spectra of hydroxypropyl chitosan and azidobenzoic hydroxypropyl chitosan. (A) 1H NMR, (B) 13C NMR.
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Table 1. Elemental analysis results of Az-HPCS samples N (%) found (calculated) 6.21 (6.29) 6.50 (6.47) 6.64 (6.66) C (%) found (calculated) 42.93 (43.02) 43.21 (43.15) 43.35 (43.27) H (%) found (calculated) 7.88 (7.95) 7.82 (7.90) 7.79 (7.86)
Sample AH-2.8 AH-4.2 AH-5.6
Az-HPCS, azidobenzoic hydroxypropyl chitosan.
Clearly, the hydrogel membrane possesses a dense and stable structure in a hydrated state. The morphology of the membrane was quite smooth and rather entire, without holes and pores. The SEM micrograph of the hydrogel in a dehydrated state is shown in Figure 3(C), which shows a compact bulk, porous, network structure. In conclusion, Figure 3 reveals that UV exposure renders HPCS chains cross-linking to a stable bulk network structure. When water molecules take up all interspaces of the network, a dense hydrogel membrane is formed.
FUA
amino groups of HPCS. Structures of HPCS and AzHPCS samples were confirmed by an NMR spectrum (Figure 2). The 1H-NMR spectra of HPCS and Az-HPCS are shown in Figure 2A. The 1H-NMR assignments of HPCS were as follows:21,22 d 4.72 ppm (H1), d 3.04 ppm (H2), d 3.40–3.79 ppm (H3–H8), and d 1.10 ppm (H9). AzHPCS has new peaks at d 7.09 and 7.69 ppm, which were attributed to the aromatic hydrogen of the Az- groups. The signal at d 2.02 ppm, corresponding to N-acetamido group of the parent chitosan, was essentially absent showing that essentially complete deacetylation had occurred during the alkalization and alkylation of the chitosan. Figure 2B shows the 13C-NMR spectra of HPCS and AzHPCS.23 Similarly, the peak assignments for HPCS and Az-HPCS were d 96.52 ppm (C1), d 74.65 ppm (C4), d 72.82 ppm (C5), d 68.06 ppm (C3), d 64.29 ppm (C8), d 61.03 ppm (C7), d 58.28 ppm (C6), and d 53.91 ppm (C2). Az-HPCS again has new peaks at d 123.43 and 127.46 ppm, which were attributed to the aromatic carbon of the Azgroups. These results also indicated that the alkylation (hydroxypropyl group) occurs at the C-6 position and the substitution (Az- group) occurs at the amino group. Elemental analysis was used to calculate the decetylation and substitution degrees of HPCS and Az-HPCS samples.17,23 The data indicated that HPCS samples were completely deacetylated and the hydroxypropyl substitution degree was 82%., and Az- substitution degrees of AzHPCS samples varied, i.e., 2.8%, 4.2%, and 5.6%, because different amounts of EDC and 4-azidobenzoic acid were used in the synthesis, as shown in Table 1.
Preparation of Az-HPCS hydrogel membrane
The FUA of Az-HPCS hydrogel membranes formed as evaluated by incubating the membranes in PBS at 37 1C. At the first 6 hours, the FUA of the membranes were 96.79 Æ 0.13% (AH-4.2) and 96.32 Æ 0.11% (AH-5.6). Then, the FUA reached a maximum value of 97.02 Æ 0.07% (AH-4.2) and 96.62 Æ 0.10% (AH-5.6) in the next 6 hours and remained unchanged as the time extended. The swollen gel membranes retained their integrity for 1 week after immersion. The results indicated AzHPCS gel membranes swelled rapidly in PBS and reached equilibrium within 12 hours, and were sufficient for the removal of exudate from wounds. Owing to the lack of cross-linked density, AH-2.8 hydrogel swelled and then dissolved finally.
WVTR
Figure 4 shows the loss of water vapor with time through the hydrogel when placed in a moisture-rich environment. At an RH of 79% and temperature of 37 1C, the WVTR for AH-4.2 and AH-5.6 hydrogel membranes were 2,934 Æ 126 g/m2/day and 2,561 Æ 115 g/m2/day, respectively. Compared with the WVTR value of 3,369 Æ 143 g/ m2/day obtained for a water-free surface the AH-5.6 hydrogel membrane should be able to reduce water vapor transmission by approximately 25% as wound dressing. At this WVTR, the membrane is able to prevent the accumulation of fluid in heavily exuding burn wounds while ensuring that wound dehydration does not occur.
Water retention
Figure 1B shows the method to cross-link Az-HPCS hydrogel in situ. Upon UV irradiation, the azide groups are known to release N2 and get converted into highly reactive nitrene groups. Az-HPCS chains were cross-linked to form in situ hydrogel by the insertion reaction of the nitrene groups.24 As we expected, an insoluble, flexible, and transparent Az-HPCS hydrogel was formed from Az-HPCS aqueous solution after 90 seconds of UV irradiation. ESEM and SEM were used to directly observe the morphology of the membrane structure in the hydrated state and in the dehydrated state. Compared with traditional SEM, ESEM can produce micrographs of nonconductors for water-containing samples without an electrically conducting coat (e.g., gold); hence it is ideal for imaging hydrogels in a hydrated state. The ESEM micrographs of AH-5.6 hydrogel membrane in a hydrated state are shown in Figure 3A (surface) and Figure 3B (cross-section).
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The water retention of the hydrogel membranes at an RH of 79% and temperature of 37 1C are shown in Figure 5. Generally, the hydrogel membranes showed good water retention ability, as they still retained 20–40% of water after exposure to air under dry conditions for 6 days. Clearly, the DS of the Az- group influences the water retention of hydrogel membranes. As shown in Figure 5, the water loss of the AH-5.6 hydrogel membrane decreased slower (50.44 Æ 1.95% in 4 days) than that of the AH-2.8 membrane (65.02 Æ 2.21%). After 6 days, the AH-5.6 hydrogel membrane was able to retain more water (36.69 Æ 2.52%) than AH-2.8 membrane (22.94 Æ 2.45%). AH-4.2 hydrogel membrane showed a similar water-loss behavior but its water loss rate was slightly faster than the AH-5.6 membrane. Taken together, the results suggested increasing the DS of the Az- groups could prolong the period of a moist microenvironment on the wound bed, when applying an Az-HPCS hydrogel membrane to wounds.
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Photocross-linked hydroxypropyl chitosan hydrogel membrane
Figure 3. ESEM and SEM images of the morphologies of azidobenzoic hydroxypropyl chitosan hydrogel membrane. (A and B) Hydrated state, (C) dehydrated state.
AS
Figure 6 shows the AS of in situ-forming Az-HPCS hydrogel membranes with porcine skin. The in situ photocrosslinking hydrogel was able to chemically adhere with the skin tissue (wound) during the forming process. We
showed that the strength increased in a DS-dependent manner. When the DS increased from 2.8% to 5.6%, the bioadhesion strength of the Az-HPCS hydrogel membranes increased by more than 2.5-fold (from 4.8 Æ 1.3 N/ cm2 [AH-2.8] to 12.3 Æ 2.0 N/cm2 [AH-5.6]). The fact should be convinced that the increasing of Az- DS induced
Figure 4. Water vapor transmission loss from azidobenzoic hydroxypropyl chitosan hydrogel membranes at 6, 12, 24, and 48-hour intervals (values are the mean Æ SD, n55, p < 0.05).
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Figure 5. Evaporation water loss from azidobenzoic hydroxypropyl chitosan hydrogel membranes (values are the mean Æ SD, n53, p < 0.05).
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Figure 6. Adhesion strength of forming azidobenzoic hydroxypropyl chitosan hydrogel membranes (values are the mean Æ SD, n53, p < 0.05).
Figure 7. Cell viability in the presence of the immobilized AH5.6 hydrogel membrane (values are the mean Æ SD, n53, p < 0.05).
more reactive points (nitrene groups) by UV exposure, resulting in more joint points with the adjacent skin tissue.
Bacteria penetration
The protection effect of the hydrogel membrane was studied. Gram-negative or Gram-positive bacteria were grown on nutrient agar plates, which were coated by either AH-5.6 solution or a hydrogel membrane (figure not shown). Numerous visible colonies were found on the control agar plates, suggesting the rapid growth of the two bacterial strains on the unshielded nutrient agar surface. The bacterial strains showed a similar growth behavior on AH-5.6-solutioncoated agar plate, suggesting that AH-5.6-solution coating cannot prevent bacterial invasion. However, there was no visible bacterial colony on the nutrient agar plates when it was coated with an AH-5.6 hydrogel membrane. This difference between solution and hydrogel membrane coating may be contributed by the bulk network structure of the membrane, which serves as a barrier for bacterial penetration.
Oxygen penetration
toxicity of the hydrogel membranes. MTT assays were performed in vitro to evaluate the cytotoxicity affecting the viability of L929 cells and HaCaT cells. Figure 7 shows no reduction in the viability of L929 cells and HaCaT cells when exposed to the AH-5.6 hydrogel membrane. These cells did not adhere to and grow on the membrane, probably because of its hyperhydrous structure.13 However, these cells did grow normally beside the immobilized hydrogel membranes (Figure 8). These results suggest that the in situ-formed AH-5.6 hydrogel membrane does not cause cytotoxicity to skin cells.
Cell culture under the coverage of hydrogel membrane
The oxygen penetration across the AH-5.6 hydrogel membrane was carried out by measuring the dissolved oxygen in the purified water as a recipient using Winkler’s method.20 Under normal circumstance, purified water has a dissolved oxygen value in the range of 7.0–14.6 mg/mL at 0–35 1C.20 The solutions tested from the airtight flask (negative control) and opened flask (positive control) had dissolved oxygen values of 7.92 Æ 0.35 and 9.26 Æ 0.51 mg/ mL, respectively, whereas those from flasks covered with AH-5.6 hydrogel membrane had dissolved oxygen values of 8.77 Æ 0.31 mg/mL. The result suggested that sufficient oxygen was able to penetrate through the compact structure of AH-5.6 hydrogel membrane.
Cytotoxicity test
Figure 9 shows the increased cell numbers per well of L929 cell and HaCaT cell under a coating of AH-5.6 hydrogel membrane. At the beginning of the culture (day 1), there were a few, separated and single cells under the coverage of the AH-5.6 hydrogel membrane (Figure 10A and C). As time extended, most cells on the covered part of the culture plates began to attach, proliferate and grow. Finally, after 6 days of incubation, the cells clearly proliferated and migrated to the areas where the cells had not attached previoulsy (Figure 10B and D). This observation indicated that the coverage of AH-5.6 hydrogel membrane did not affect or inhibit the proliferation and migration of the dermal cell (L929 cell) and the epidermal cell (HaCaT cell).
DISCUSSION
The in situ photocross-linked Az-HPCS hydrogel membrane can bring some benefits to patients, such as convenience of use, conformability without wrinkling or fluting in the wound bed, ease of application, and improved patient compliance and comfort. However, there are some basic requirements, such as maintaining an optimal moist environment, oxygen permeation, bioadhesion, blocking bacterial infection, and biocompatibility, for this hydrogel membrane to be a practical wound dressing. An ideal wound dressing should be able to provide a optimal moisture environment for wound repair, i.e., it
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The cells of L929 and HaCaT stand for a typical dermal cell and epidermal cell and were used to assess the cyto76
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Figure 8. Light microphotographs of cells grown around the immobilized AH-5.6 hydrogel membrane. (A) L929 cell, (B) HaCaT cell. Original magnification Â100.
can rapidly absorb the excessive secreted exudates from the wound25 (FUA), and prevent the wound from dehydration.26 In this work, the AH-5.6 hydrogel membrane swelled rapidly and reached a maximum FUA value of 97.02 Æ 0.07% in 12 hours, suggesting that it is sufficient to absorb the exudates for a period of time. We also found that AH-5.6 hydrogel membrane has a WVTR of 2,561 g/ m2/day, which reduces the water vapor transmission about 25% and is very close to the optimal WVTR value, and comparable with the recommended WVTR value (2,000– 2,500 g/m2/day) for providing an adequate level of moisture without risking wound dehydration,26 suggesting it could prevent the wound from excess dehydration. The water retention property of a hydrogel is essential for a hydrogel to be used as wound dressing. Kickhofen et ¨ al.6 reported that water retention enables the gel to take up exudates and edema fluid from the wound into the dressing by an active upward-directed process when used in exudating wounds. Our results suggested that the AH-5.6 hydrogel membrane retained about 36.69% water after 6 days of exposure to air. Thus, these dressings will be more beneficial for wounds with moderate exudates rather than for dry wounds. In summary, the AH-5.6 hydrogel membrane could build a balance between the absorption of exudates and water loss, and thus facilitate cellular migration and enhance reepithelialization. More interestingly, the WVTR and water retention showed an Az-DS-dependent behavior, i.e., the higher the DS, the lesser the WVTR and water loss. This could be attributed to two reasons: (1) the AH-5.6 hydrogel membrane has a more compact structure; and (2) the higher cross-linked structure promotes stronger intermolecular and intramolecular hydrogen bondings, which results in
the increase of hydrophobicity in the polymer chain.27,28 In the future, WVTR and water loss of the membrane could be adjustable by controlling the Az-DS to favor wound repair and regeneration. Skin replacement must conform to the wound surface in all areas, and adhere in all areas. Without these properties, small air pockets exist where exudative fluid accumulates and may caused secondary bacterial infection. The graft must chemically adhere to the wound bed to avoid proliferation of the granulating layer and therefore control the subsequent contraction and deformity of the wound.7 The adhesion strength of the AH-5.6 hydrogel membrane to porcine skin was 12.3 Æ 2.0 N/cm2, suggesting that the hydrogel was chemically linked to the skin and could prevent it from separating from the skin. More importantly, both Gram-negative and Gram-positive bacteria could not penetrate the membrane, suggesting that the hydrogel membrane could serve as a barrier for bacterial infection. Oxygen penetration through a wound dressing is important because low oxygen concentration decreases the regeneration of tissue cell and slows down the healing process.26 Our hydrogel membrane showed a relatively high oxygen permeation ability, i.e., 8.77 Æ 0.31 mg/mL, suggesting it could allow enough oxygen entering for wound repair. The hydrogel membrane also showed good biocompatibility. The MTT assay results indicated that this material has no cytotoxicity against dermal and epidermal cells. Further study indicated that the coverage of the hydrogel membrane on the cell culture system did not affect cell growth, suggesting that Az-HPCS hydrogel membrane as a wound dressing will not affect the skin regeneration process.
Figure 9. Cell proliferation under the coverage of AH-5.6 hydrogel membrane. (A) L929 cell, (B) HaCaT cell.
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Figure 10. Light microphotographs of cells grown under the coverage of AH-5.6 hydrogel membrane. (A) Day 1 of L929 cell, (B) day 1 of HaCaT cell, (C) day 6 of L929 cell, (D) day 6 of HaCaT cell; original magnification Â100.
ACKNOWLEDGMENTS
This work was financially supported by the State Educational Ministry, China (the Project sponsored by SRF for ROCS; No. 2006-331) and by the National Natural Science Foundation of China (No 50873080 to J. Chen).
10.
11.
REFERENCES
1. Barlow YM. Developments in wound care: international trends and opportunities, Clinica reports. CBS 429. Richmond, UK: PJB Publications Ltd., 1994: 140. 2. Purna SK, Babu M. Collagen based dressing—a review. Burns 2000; 26: 54–62. 3. Graham NB. Hydrogels: their future, part I. Med Device Technol 1998; 9: 18–22. 4. Graham NB. Hydrogels: their future, part II. Med Device Technol 1998; 9: 22–5. 5. Nguyen KT, West JL. Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 2002; 23: 4307–14. 6. Kickhofen B, Wokalek H, Scheel D, Ruh H. Chemical and ¨ physical properties of a hydrogel wound dressing. Biomaterials 1986; 7: 67–72. 7. Kane JB, Tompkins RG, Yarmush ML, Burke JF. Burn dressings. In: Ratner BD, Hoffman AS, Schoen FJ, Lemons JE, editors. An introduction to materials in medicine. San Diego: Academic Press, 1996: 360–7. 8. Balakrishnan B, Mohanty M, Umashankar PR, Jayakrishnan A. Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 2005; 26: 6335–42. 9. Balakrishnan B, Mohanty M, Fernandez AC, Mohanan PV, Jayakrishnan A. Evaluation of the effect of incorporation of dibutyryl cyclic adenosine monophosphate in an in situ12.
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forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 2006; 27: 1355–61. Draye JP, Delaey B, Van de Voorde A, Van Den Bulcke A, De Reu B, Schacht E. In vitro and in vivo biocompatibility of dextran dialdehyde cross-linked gelatin hydrogel films. Biomaterials 1998; 19: 1677–8. Huang-Lee LLH, Cheung DT, Nimni ME. Biochemical changes and cytotoxicity associated with the degradation of polymeric glutaraldehyde derived crosslinks. J Biomed Mater Res 1990; 24: 1185–201. Li Q, Williams CG, Sun DD, Wang J, Leong K, Elisseeff JH. Photocrosslinkable polysaccharides based on chondroitin sulfate. J Biomed Mater Res A 2004; 68: 28–33. Ono K, Saito Y, Yura H, Ishikawa K, Kurita A, Ishihara M. Photocrosslinkable chitosan as a biological adhesive. J Biomed Mater Res 2000; 49: 289–95. Ishihara M, Nakanishi K, Ono K, Sato M, Saito Y, Yura H, Matsui T, Hattori H, Uenoyama M, Kikuchi M, Kurita A. Photocross linkable chitosan as a dressing for wound occlusion and accelerator in healing process. Biomaterials 2002; 23: 833–40. Obara K, Ishihara M, Ishizuka T, Fujita M, Ozeki Y, Maehara T, Saito Y, Yura H, Matsui T, Hattori H, Kikuchi M, Kurita A. Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing impaired db/db mice. Biomaterials 2003; 24: 3437–44. Ling K, Zheng F, Li J, Tang R, Huang J, Xu Y, Zheng H, Chen J. Effects of substitution degree of photoreactive groups on the properties of UV-fabricated chitosan scaffold. J Biomed Mater Res A 2008; 87: 52–61. Xie W, Xu P, Wang W, Liu Q. Preparation and antibacterial activity of a water-soluble chitosan derivative. Carbohydr Polym 2002; 50: 35–40.
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18. ASTM Standard E96-00. Standard test methods for water vapour transmission of materials. Annual book of ASTM standards, Vol. 4.06. Philadelphia: ASTM, 2000. 19. Glazer BT, Marsh AG, Stierhoff KL, Luther GW. The dynamic response of optical oxygen sensors and voltammetric electrodes to temporal changes in dissolved oxygen concentrations. Anal Chim Acta 2004; 518: 93–100. 20. Wittaya-areekul S, Prahsarn C. Development and in vitro evaluation of chitosan–polysaccharides composite wound dressings. Int J Pharm 2006; 313: 123–8. 21. Peng Y, Han B, Liu W, Xu X. Preparation and antimicrobial activity of hydroxypropyl chitosan. Carbohydr Res 2005; 340: 1846–51. 22. Dong Y, Wu Y, Wang J, Wang M. Influence of degree of molar etherification on critical liquid crystal behavior of hydroxypropyl chitosan. Eur Polym J 2001; 37: 1713–20.
23. Zhang C, Ping Q, Zhang H, Shen J. Synthesis and characterization of water-soluble O-succinyl-chitosan. Eur Polym J 2003; 39: 1629–34. 24. Scriven EFV editor. Azides and nitrenes: reactivity and utility. Orlando: Academic Press, 1984: 1–291. 25. Tanodekaew S, Prasitsilp M, Swasdison S, Thavornyutikarn B, Pothsree T, Pateepasen R. Preparation of acrylic grafted chitin for wound dressing application. Biomaterials 2004; 25: 1453–60. 26. Queen D, Gaylor JD, Evans JH, Courtney JM, Reid WH. The preclinical evaluation of the water vapour transmission rate through burn wound dressings. Biomaterials 1987; 8: 367–71. 27. Liu XF, Guan YL, Yang DZ, Li Z, Yao KD. Antibacterial action of chitosan and carboxymethylated chitosan. J Appl Polym Sci 2001; 79: 1324–35. 28. Chen T, Zhang XH, Guo R. Surface active and aggregation of chitosan. Acta Phys-Chim Sin 2000; 16: 1039–42.
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A novel in situ-formed hydrogel wound dressing by the photocross-linking of a chitosan derivative
Guozhong Lu, MD1n; Kai Ling, MS2n; Peng Zhao, BS2; Zhenghong Xu, PhD3; Cao Deng, MS3; Hua Zheng, PhD2; Jin Huang, PhD2; Jinghua Chen, PhD3
1. Department of Burn and Plastic Surgery, Wuxi third People’s Hospital, Wuxi, China, 2. College of Chemical Engineering, Wuhan University of Technology, Wuhan, China, and 3. School of Medicine and Pharmaceutics, Jiangnan University, Wuxi, China
Reprint requests: Jinghua Chen, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China. Tel: 186 510 85327353; Fax: 186 510 85329042; Email: [email protected]
n
ABSTRACT In situ photopolymerized hydrogel dressings create minimally invasive methods that offer advantages over the use of preformed dressings such as conformability in any wound bed, convenience of application, and improved patient compliance and comfort. Here, we report an in situ-formed hydrogel membrane through ultraviolet cross-linking of a photocross-linkable azidobenzoic hydroxypropyl chitosan aqueous solution. The hydrogel membrane is stable, flexible, and transparent, with a bulk network structure of smoothness, integrity, and density. Fluid uptake ability, water vapor transmission rate, water retention, and bioadhesion of the thus resulted hydrogel membranes (0.1 mm thick) were determined to range from 97.0–96.3%, 2,934–2,561 g/m2/day, 36.69–22.94% (after 6 days), and 4.8–12.3 N/cm2, respectively. These data indicate that the hydrogel membrane can maintain a long period of moist environment over the wound bed for enhancing reepithelialization. Specifically, these properties of the hydrogel membrane were controllable to some extent, by adjusting the substitution degree of the photoreactive azide groups. The hydrogel membrane also exhibited barrier function, as it was impermeable to bacteria but permeable to oxygen. In vitro experiments using two major skin cell types (dermal fibroblast and epidermal keratinocyte) revealed the hydrogel membrane have neither cytotoxicity nor an effect on cell proliferation. Taken together, the in situ photocross-linked azidobenzoic hydroxypropyl chitosan hydrogel membrane has a great potential in the management of wound healing and skin burn. Recently, Balakrishnan et al.8,9 reported that an in situforming hydrogel wound dressing can be prepared from gelatin and oxidized alginate in the presence of small concentrations of borax. Fabrication of hydrogel in situ will have advantages over the use of preformed hydrogel dressings because it allows the hydrogel to form complex shapes that adhere and conform to the wound defect.5,8 Most commercially available dressings in the form of membranes and sheets are problematic as far as the conformability is concerned and the in situ-formed dressings will, therefore, be superior to preformed dressings. However, the in situ-forming hydrogel was cross-linked by a reaction between a resulting alginate dialdehyde (ADA) and gelatin in the presence of 0.1 M borax. According to Draye et al.,10 a possible release of trace amounts of dialdehyde might induce toxicity of the hydrogel, similar to the cytotoxicity associated with the release of glutaraldehyde from glutaraldehyde cross-linked bioprotheses.11 Photopolymerization allows in situ hydrogel formation in a minimally invasive manner.12 It provides a fast and efficient method to cross-link polymers to form a hydrogel with significant temporal and spatial control. There are some reports of a photocross-linked chitosan hydrogel that is prepared by applying ultraviolet (UV) light irradiation to a photocross-linkable chitosan aqueous solution.13–15 This chitosan hydrogel could effectively stop
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These authors contributed equally to this work. Manuscript received: April 2, 2009 Accepted in final form: September 29, 2009 DOI:10.1111/j.1524-475X.2009.00557.x
The development of novel dressings to promote wound healing in humans has drawn increased attention during the last decades.1 Researchers have believed that an ideal wound dressing should protect the wound from bacterial infection, provide a moist and healing environment, and remain biocompatible.2 Although traditional (nonocclusive) wound dressings, which offer dry wound healing conditions, still account for the largest segment of the dressing market, the use of occlusive dressings, i.e., hydrocolloid and hydrogel dressings, which offer moist wound healing conditions, has increased significantly.1 Hydrogels, cross-linked hydrophilic polymers, exhibit excellent biocompatibility, causing minimal inflammatory responses, thrombosis, and tissue damage.3,4 Hydrogels can also absorb large quantities of water and well without the dissolution of the polymer because of their hydrophilic but cross-linked structure, thus giving them physical characteristics similar to soft tissues. In addition, hydrogels have high permeability for oxygen, nutrients, and other water-soluble metabolites.5 Thus, hydrogel materials possess the potential to be ideal wound dressings. For example, a commercial hydrogel dressing, ‘‘Geliperms,’’ was established to have the ability of providing optimal physiological conditions for wound healing.6 However, this material is limited to be used as a temporary cover, only because of its lack of adherence to the wound and its fragile nature.7
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bleeding, induce significant wound contraction, accelerate wound closure and healing, and showed nontoxicity and good biocompatibility.13 Inspite of intensive researches using photocross-linkable chitosan as wound dressing, there is a lack of detailed information about the properties of the membrane of the in situ photocross-linked chitosan hydrogel, such as water vapor transmission rate (WVTR), water retention, and bioadhesion, which limit the applications of photocross-linkable chitosan. In this article, we utilized our prepared photocross-linkable hydroxypropyl chitosan (HPCS),16 4-azidobenzoic hydroxypropyl chitosan (Az-HPCS), with different degree of substitution (DS) of Az- groups, to in situ-formed crosslinked bioadhesive hydrogel membrane and evaluated its potential as wound dressing. We characterized the basic properties, such as FUA, WVTR, water retention, and bioadhesion of photocross-linked Az-HPCS hydrogel membranes, to evaluate its potential as wound dressing. The morphologies of the hydrogel membrane in hydrated and a dehydrated states were observed using environmental scanning electron microscopy (ESEM) and scanning electron microscopy (SEM), respectively. Bacterial and oxygen penetration properties of the hydrogel membrane were also assessed. Cell culture experiments of dermal fibroblast (L929 mouse fibroblast) and epidermal keratinocyte (HaCaT human keratinocyte) were conducted to test the toxicity and biocompatibility of the hydrogel membrane.
MATERIALS AND METHODS
Materials
Chitosan was supplied by Zhejiang Yuhuan Ocean Biochemistry Co. Ltd., China. Its viscosity average molecular weight was 830 kDa, with an 87% degree of deacetylation. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED), and dialysis membrane (10 kDa MWCO) were purchased from Sigma-Aldrich (St. Louis, MO). 4-azidobenzoic acid was purchased from TCI (Tokyo, Japan). All other chemicals were purchased from SCRC (Shanghai, China), unless otherwise indicated.
Preparation of Az-HPCS
The method of the preparation of Az-HPCS is from our previous report.16 The typical procedure is shown in Figure 1A. Briefly, HPCS was firstly synthesized from chitosan and propylene oxide under alkali condition, according to Xie and colleague’s method.17 Subsequently, HPCS (10 g), EDC (0.350, 0.525, and 0.700 g), and 4-azidobenzoic acid (0.20, 0.30, and 0.40 g) were added to 150 mL of 50 mM TEMED aqueous solution. The mixture was stirred at room temperature for 72 hours and dialyzed against deionized water for 72 hours. The resulting product was obtained by freeze drying. The DS of Az- groups of the Az-HPCS prepared were 2.8%, 4.2%, and 5.6%, respectively. The constituents and structures of HPCS and Az-HPCS were examined and measured using an Elemental Analyzer–VarioEL III (ELEMENTAR, Hanau, Germany) and a Mercury-300 NMR Spectrometer (Varian Inc, Palo Alto, CA.) at 300 MHz at 25 1C, using D2O as the solvent.
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Figure 1. Scheme of the preparation of azidobenzoic hydroxypropyl chitosan (Az-HPCS) hydrogel.
Preparation of Az-HPCS hydrogel membranes
Az-HPCS hydrogel membranes were prepared by UV irradiation. Firstly, 363 mL of Az-HPCS aqueous solution (25 mg/mL) was spread uniformly on the surfaces of tissue culture dishes (7 cm diameter, JET BIOFILs , JET Bio-filtration Products Co. Ltd., Guangzhou, China). Then, a mercury UV light (4 W, 254 nm, Shanghai Jiapeng Scientific Apparatus Co. Ltd., Shanghai, China) was used to irradiate the dish surfaces for 90 seconds. Az-HPCS solutions were gelatinized and flexible, stable, and
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transparent hydrogel membranes (0.1 mm thick), were formed in situ eventually. The entire procedure is illustrated briefly in Figure 1B.
Morphology observation
where W0 is the initial weight and Wt is the weight at time t, respectively.
Adhesion strength (AS)
The hydrated Az-HPCS hydrogel membranes were placed on a Peltier cooling stage inside an ESEM (FEI Quanta-200, FEI, Hillsboro, OR) and maintained at 4 1C. The hydrogel membranes were imaged using a voltage of 20 kV and a water vapor pressure between 4.0 and 4.7 Torr. The dehydrated Az-HPCS hydrogel membranes prepared by a lyophilization process, were gold coated to about 500Â10À8 cm thickness using an Hitachi (Tokyo, Japan) coating unit, IB-2 coater under a high vacuum, 0.1 Torr, and high voltage, 1.2 kV and 50 mA. Coated samples were examined using a Hitachi S4800 (Tokyo, Japan) electron scanning microscopy (SEM).
Fluid uptake ability (FUA)
The FUA of the hydrogel membranes were determined as described below. Completely dried membrane samples were cut into square-shaped specimens (1 cmÂ1 cm) and weighed. Then, the specimens were immersed in phosphate-buffered saline (PBS; pH 7.4) buffer, and taken out quickly at specific time intervals, i.e., 6, 12, 24, and 48 hours, respectively, and weighed after wiping with paper towels. The FUA of the hydrogel membranes was calculated as follows: FUA ð%Þ ¼ ðWs À Wd Þ100=Ws ð1Þ
Porcine skin was excised from the back of the ‘‘Yorkshire pig’’ breed at the animal center of Wuxi third people’s hospital in accordance with ethical guidelines stated in the Guide for the use and care of laboratory animals, National Research Council, China (1985). Fresh porcine skin was cut into strips (4.0 cmÂ2.0 cm and 1.5 mm thick). Two strips were placed flat, 2.0 cm on each side closely touching. 30 mL of AzHPCS aqueous solution (25 mg/mL) was then added between the two skin strips along the 2.0 cm side. After 90 seconds of UV-irradiation, a mechanical tester (Instron 3365, Illinois Tool Works, Glenview, IL) was utilized to measure the bioadhesion strength. One side of the adhered skin preparation was fixed on the base and the other side was attached vertically with a pair of grippers. And then the pair of grippers drew the adhered strips until they separated into two pieces. The tensile force (TF) was recorded by the equipment. The AS was calculated as follows: ASðN=cm2 Þ ¼ TF=C
2
ð4Þ
where C is the cross-sectional area (cm ) of the porcine skin tissue strip.
Bacterial penetration
where Wd is the weight of the initial dry specimens and Ws is the weight of the swollen specimens.
WVTR
The moisture permeability of the hydrogel membrane was determined by measuring the WVTR, according to the ASTM method E96-00.18 The hydrogel membranes prepared were mounted at the mouth of cylindrical plastic cups (36 mm diameter) containing 10 mL water with negligible water vapor transmittance. The material was fastened using Teflon tape across the edge to prevent any water vapor loss through the boundary and placed in a desiccator containing a saturated solution of ammonium sulfate at 37 1C and under 79% relative humidity (RH). The assembly was weighed at regular intervals of time and a weight loss vs. time plot was constructed. From the slope of the plot, WVTR was calculated as follows:8 WVTRðg=m2 =dayÞ ¼ slope  24=A where A is the test area of the sample in m .
Water loss
2
ð2Þ
To determine the water retention of the hydrogel membrane, the membrane samples were placed in a desiccator containing a saturated solution of ammonium sulfate at 37 1C under 79% RH, and the membranes were weighed at different time intervals. Weight remaining (WR) percentage was calculated as follows: WRð%Þ ¼ Wt Â100=W0
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Staphylococcus aureus and Escherichia coli, supplied by the Microbiology Laboratory of Huazhong Agricultural University (National Key Lab., China), were selected as test cells because they are the most frequent bacteria in the wound infection and representative of Gram-positive and Gram-negative bacteria, respectively. The two bacteria were incubated at 37 1C for 24 hours in nutrient broth (bacto-peptone 10 g, beef extract 3 g, NaCl 3 g in 1,000 mL distilled water; pH 7.0) before use. By diluting appropriately with the culture medium, each culture containing $ 108 cells/mL was prepared, which was used for a bacterial penetration test. AH-5.6 solution (25 mg/mL) was prepared in distilled water and then sterilized through a 0.22 mm filter membrane. 550 mL sterilized AH-5.6 solution was spread on a nutrient agar plate (agar 15 g, bacto-peptone 10 g, beef extract 3 g, NaCl 3 g in 1,000 mL distilled water; pH 7.0, 10 cm diameter). Subsequently, the plates were exposed to UV light (90 seconds, 4 W) for in situ cross-linking a layer of AH-5.6 hydrogel membrane on the surface of nutrient agar. A portion (200 mL) of a bacterial culture (S. aureus or E. coli) prepared was then dropped and quickly spread on the treated nutrient agar plates. The plates were incubated at 37 1C and checked for bacterial penetration after 24 and 48 hours, respectively. Nutrient agar plates with and without AH-5.6 solution cover were used as controls.
Oxygen penetration
ð3Þ
Oxygen penetration through the AH-5.6 hydrogel membrane prepared was performed by placing the membrane prepared on top of open 250 mL flasks containing 200 mL of deionized water and held in place with a screw lid (25 mm diameter).
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The negative control was a closed flask with an airtight cap preventing oxygen from entering the flask whereas the positive control was an open flask allowing oxygen to enter the flask and dissolve in the water as a recipient. The test flasks were placed in an open environment under constant agitation for 24 hours. The water samples collected were then analyzed for dissolved oxygen, according to Winkler’s method.19,20
Cell culture
dium were added in each wells (2Â104 cells/well) and grown at 37 1C for 2 days. After the incubation, an MTT cell proliferation assay kit (Beijing Cellchip Co. Ltd., Beijing, China) was used to measure the absorption data spectrophotometrically at 570 nm. The cell growth status was observed using an optical microscope (CKX-31; Olympus, Tokyo, Japan).
Cell culture under the coverage of hydrogel membrane
Two cell lines of L929 and HaCaT (from fourth to eighth passages) were purchased from the China Center for Type Culture Collection (CCTCC). Mouse C3H/An connective tissue, fibroblast cell line (L929) was culture in RPMI medium 1640 (Gibco Invitrogen, Carlsbad, CA) with 2 mmol/ L L-glutamine and 24 mmol/L NaHCO3 supplemented with 10% heat-inactivated fetal bovine serum (FBS), together with penicillin G (100 U/mL) and streptomycin (100 mg/mL) at 37 1C in a 5% CO2 atmosphere. Spontaneously immortalized human keratinocyte cell line (HaCaT) was grown in Dulbecco’s modified Eagle’s medium (DMEM containing 5.6 mmol/L glucose, Gibco) with 4 mmol/L L-glutamine, 44 mmol/L NaHCO3, and 1 mmol/L sodium pyruvate supplemented with 10% heatinactivated FBS, together with penicillin G (100 U/mL) and streptomycin (100 mg/mL) at 37 1C in a 5% CO2 atmosphere. After the cells achieved confluent on a dish, we subcultured cells at a ratio of 1 : 10.
In vitro cytotoxicity test
Cells were seeded into 12-well tissue culture plates at a density of 1Â104 cells/well and left overnight for attachment. Each wells of the plates were then completely coated by AH-5.6 hydrogel membranes (0.1 mm thick) prepared and co-cultured for 2, 4, or 6 days with a change of medium every other day. At various time intervals, the membranes were taken out and the plates were washed twice by PBS and treated with 0.05% trypsin-EDTA (Sigma) solution at 37 1C for 15 minutes. The detached cells were counted by a hemocytometer. Blank cultures with the absence of biomaterial were used as controls.
Statistical analysis
All experiments were repeated three or more times. All values were expressed as mean Æ standard deviation. Statistical differences were determined by two-tailed unpaired t-test and analyzed using a significance level set at p < 0.05.
RESULTS
Preparation of Az-HPCS
In order to test the in vitro cytotoxicity of AH-5.6 hydrogel membrane, 5 mL of AH-5.6 aqueous solution (25 mg/mL) was spotted on each well of two 12-well tissue culture plates (Falcon, BD Biosciences, Franklin Lakes, NJ) and in situ hydrogel membranes were formed by 90 seconds of UV irradiation. Subsequently, cells suspended in the culture me-
Figure 1A shows the two-step reaction scheme for preparing Az-HPCS: (a) by a modification of chitosan with propylene oxide at the C-6 hydroxyl groups; and (b) by introducing the photoreactive Az- groups to the C-2
Figure 2. NMR spectra of hydroxypropyl chitosan and azidobenzoic hydroxypropyl chitosan. (A) 1H NMR, (B) 13C NMR.
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Table 1. Elemental analysis results of Az-HPCS samples N (%) found (calculated) 6.21 (6.29) 6.50 (6.47) 6.64 (6.66) C (%) found (calculated) 42.93 (43.02) 43.21 (43.15) 43.35 (43.27) H (%) found (calculated) 7.88 (7.95) 7.82 (7.90) 7.79 (7.86)
Sample AH-2.8 AH-4.2 AH-5.6
Az-HPCS, azidobenzoic hydroxypropyl chitosan.
Clearly, the hydrogel membrane possesses a dense and stable structure in a hydrated state. The morphology of the membrane was quite smooth and rather entire, without holes and pores. The SEM micrograph of the hydrogel in a dehydrated state is shown in Figure 3(C), which shows a compact bulk, porous, network structure. In conclusion, Figure 3 reveals that UV exposure renders HPCS chains cross-linking to a stable bulk network structure. When water molecules take up all interspaces of the network, a dense hydrogel membrane is formed.
FUA
amino groups of HPCS. Structures of HPCS and AzHPCS samples were confirmed by an NMR spectrum (Figure 2). The 1H-NMR spectra of HPCS and Az-HPCS are shown in Figure 2A. The 1H-NMR assignments of HPCS were as follows:21,22 d 4.72 ppm (H1), d 3.04 ppm (H2), d 3.40–3.79 ppm (H3–H8), and d 1.10 ppm (H9). AzHPCS has new peaks at d 7.09 and 7.69 ppm, which were attributed to the aromatic hydrogen of the Az- groups. The signal at d 2.02 ppm, corresponding to N-acetamido group of the parent chitosan, was essentially absent showing that essentially complete deacetylation had occurred during the alkalization and alkylation of the chitosan. Figure 2B shows the 13C-NMR spectra of HPCS and AzHPCS.23 Similarly, the peak assignments for HPCS and Az-HPCS were d 96.52 ppm (C1), d 74.65 ppm (C4), d 72.82 ppm (C5), d 68.06 ppm (C3), d 64.29 ppm (C8), d 61.03 ppm (C7), d 58.28 ppm (C6), and d 53.91 ppm (C2). Az-HPCS again has new peaks at d 123.43 and 127.46 ppm, which were attributed to the aromatic carbon of the Azgroups. These results also indicated that the alkylation (hydroxypropyl group) occurs at the C-6 position and the substitution (Az- group) occurs at the amino group. Elemental analysis was used to calculate the decetylation and substitution degrees of HPCS and Az-HPCS samples.17,23 The data indicated that HPCS samples were completely deacetylated and the hydroxypropyl substitution degree was 82%., and Az- substitution degrees of AzHPCS samples varied, i.e., 2.8%, 4.2%, and 5.6%, because different amounts of EDC and 4-azidobenzoic acid were used in the synthesis, as shown in Table 1.
Preparation of Az-HPCS hydrogel membrane
The FUA of Az-HPCS hydrogel membranes formed as evaluated by incubating the membranes in PBS at 37 1C. At the first 6 hours, the FUA of the membranes were 96.79 Æ 0.13% (AH-4.2) and 96.32 Æ 0.11% (AH-5.6). Then, the FUA reached a maximum value of 97.02 Æ 0.07% (AH-4.2) and 96.62 Æ 0.10% (AH-5.6) in the next 6 hours and remained unchanged as the time extended. The swollen gel membranes retained their integrity for 1 week after immersion. The results indicated AzHPCS gel membranes swelled rapidly in PBS and reached equilibrium within 12 hours, and were sufficient for the removal of exudate from wounds. Owing to the lack of cross-linked density, AH-2.8 hydrogel swelled and then dissolved finally.
WVTR
Figure 4 shows the loss of water vapor with time through the hydrogel when placed in a moisture-rich environment. At an RH of 79% and temperature of 37 1C, the WVTR for AH-4.2 and AH-5.6 hydrogel membranes were 2,934 Æ 126 g/m2/day and 2,561 Æ 115 g/m2/day, respectively. Compared with the WVTR value of 3,369 Æ 143 g/ m2/day obtained for a water-free surface the AH-5.6 hydrogel membrane should be able to reduce water vapor transmission by approximately 25% as wound dressing. At this WVTR, the membrane is able to prevent the accumulation of fluid in heavily exuding burn wounds while ensuring that wound dehydration does not occur.
Water retention
Figure 1B shows the method to cross-link Az-HPCS hydrogel in situ. Upon UV irradiation, the azide groups are known to release N2 and get converted into highly reactive nitrene groups. Az-HPCS chains were cross-linked to form in situ hydrogel by the insertion reaction of the nitrene groups.24 As we expected, an insoluble, flexible, and transparent Az-HPCS hydrogel was formed from Az-HPCS aqueous solution after 90 seconds of UV irradiation. ESEM and SEM were used to directly observe the morphology of the membrane structure in the hydrated state and in the dehydrated state. Compared with traditional SEM, ESEM can produce micrographs of nonconductors for water-containing samples without an electrically conducting coat (e.g., gold); hence it is ideal for imaging hydrogels in a hydrated state. The ESEM micrographs of AH-5.6 hydrogel membrane in a hydrated state are shown in Figure 3A (surface) and Figure 3B (cross-section).
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The water retention of the hydrogel membranes at an RH of 79% and temperature of 37 1C are shown in Figure 5. Generally, the hydrogel membranes showed good water retention ability, as they still retained 20–40% of water after exposure to air under dry conditions for 6 days. Clearly, the DS of the Az- group influences the water retention of hydrogel membranes. As shown in Figure 5, the water loss of the AH-5.6 hydrogel membrane decreased slower (50.44 Æ 1.95% in 4 days) than that of the AH-2.8 membrane (65.02 Æ 2.21%). After 6 days, the AH-5.6 hydrogel membrane was able to retain more water (36.69 Æ 2.52%) than AH-2.8 membrane (22.94 Æ 2.45%). AH-4.2 hydrogel membrane showed a similar water-loss behavior but its water loss rate was slightly faster than the AH-5.6 membrane. Taken together, the results suggested increasing the DS of the Az- groups could prolong the period of a moist microenvironment on the wound bed, when applying an Az-HPCS hydrogel membrane to wounds.
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Figure 3. ESEM and SEM images of the morphologies of azidobenzoic hydroxypropyl chitosan hydrogel membrane. (A and B) Hydrated state, (C) dehydrated state.
AS
Figure 6 shows the AS of in situ-forming Az-HPCS hydrogel membranes with porcine skin. The in situ photocrosslinking hydrogel was able to chemically adhere with the skin tissue (wound) during the forming process. We
showed that the strength increased in a DS-dependent manner. When the DS increased from 2.8% to 5.6%, the bioadhesion strength of the Az-HPCS hydrogel membranes increased by more than 2.5-fold (from 4.8 Æ 1.3 N/ cm2 [AH-2.8] to 12.3 Æ 2.0 N/cm2 [AH-5.6]). The fact should be convinced that the increasing of Az- DS induced
Figure 4. Water vapor transmission loss from azidobenzoic hydroxypropyl chitosan hydrogel membranes at 6, 12, 24, and 48-hour intervals (values are the mean Æ SD, n55, p < 0.05).
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Figure 5. Evaporation water loss from azidobenzoic hydroxypropyl chitosan hydrogel membranes (values are the mean Æ SD, n53, p < 0.05).
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Figure 6. Adhesion strength of forming azidobenzoic hydroxypropyl chitosan hydrogel membranes (values are the mean Æ SD, n53, p < 0.05).
Figure 7. Cell viability in the presence of the immobilized AH5.6 hydrogel membrane (values are the mean Æ SD, n53, p < 0.05).
more reactive points (nitrene groups) by UV exposure, resulting in more joint points with the adjacent skin tissue.
Bacteria penetration
The protection effect of the hydrogel membrane was studied. Gram-negative or Gram-positive bacteria were grown on nutrient agar plates, which were coated by either AH-5.6 solution or a hydrogel membrane (figure not shown). Numerous visible colonies were found on the control agar plates, suggesting the rapid growth of the two bacterial strains on the unshielded nutrient agar surface. The bacterial strains showed a similar growth behavior on AH-5.6-solutioncoated agar plate, suggesting that AH-5.6-solution coating cannot prevent bacterial invasion. However, there was no visible bacterial colony on the nutrient agar plates when it was coated with an AH-5.6 hydrogel membrane. This difference between solution and hydrogel membrane coating may be contributed by the bulk network structure of the membrane, which serves as a barrier for bacterial penetration.
Oxygen penetration
toxicity of the hydrogel membranes. MTT assays were performed in vitro to evaluate the cytotoxicity affecting the viability of L929 cells and HaCaT cells. Figure 7 shows no reduction in the viability of L929 cells and HaCaT cells when exposed to the AH-5.6 hydrogel membrane. These cells did not adhere to and grow on the membrane, probably because of its hyperhydrous structure.13 However, these cells did grow normally beside the immobilized hydrogel membranes (Figure 8). These results suggest that the in situ-formed AH-5.6 hydrogel membrane does not cause cytotoxicity to skin cells.
Cell culture under the coverage of hydrogel membrane
The oxygen penetration across the AH-5.6 hydrogel membrane was carried out by measuring the dissolved oxygen in the purified water as a recipient using Winkler’s method.20 Under normal circumstance, purified water has a dissolved oxygen value in the range of 7.0–14.6 mg/mL at 0–35 1C.20 The solutions tested from the airtight flask (negative control) and opened flask (positive control) had dissolved oxygen values of 7.92 Æ 0.35 and 9.26 Æ 0.51 mg/ mL, respectively, whereas those from flasks covered with AH-5.6 hydrogel membrane had dissolved oxygen values of 8.77 Æ 0.31 mg/mL. The result suggested that sufficient oxygen was able to penetrate through the compact structure of AH-5.6 hydrogel membrane.
Cytotoxicity test
Figure 9 shows the increased cell numbers per well of L929 cell and HaCaT cell under a coating of AH-5.6 hydrogel membrane. At the beginning of the culture (day 1), there were a few, separated and single cells under the coverage of the AH-5.6 hydrogel membrane (Figure 10A and C). As time extended, most cells on the covered part of the culture plates began to attach, proliferate and grow. Finally, after 6 days of incubation, the cells clearly proliferated and migrated to the areas where the cells had not attached previoulsy (Figure 10B and D). This observation indicated that the coverage of AH-5.6 hydrogel membrane did not affect or inhibit the proliferation and migration of the dermal cell (L929 cell) and the epidermal cell (HaCaT cell).
DISCUSSION
The in situ photocross-linked Az-HPCS hydrogel membrane can bring some benefits to patients, such as convenience of use, conformability without wrinkling or fluting in the wound bed, ease of application, and improved patient compliance and comfort. However, there are some basic requirements, such as maintaining an optimal moist environment, oxygen permeation, bioadhesion, blocking bacterial infection, and biocompatibility, for this hydrogel membrane to be a practical wound dressing. An ideal wound dressing should be able to provide a optimal moisture environment for wound repair, i.e., it
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The cells of L929 and HaCaT stand for a typical dermal cell and epidermal cell and were used to assess the cyto76
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Figure 8. Light microphotographs of cells grown around the immobilized AH-5.6 hydrogel membrane. (A) L929 cell, (B) HaCaT cell. Original magnification Â100.
can rapidly absorb the excessive secreted exudates from the wound25 (FUA), and prevent the wound from dehydration.26 In this work, the AH-5.6 hydrogel membrane swelled rapidly and reached a maximum FUA value of 97.02 Æ 0.07% in 12 hours, suggesting that it is sufficient to absorb the exudates for a period of time. We also found that AH-5.6 hydrogel membrane has a WVTR of 2,561 g/ m2/day, which reduces the water vapor transmission about 25% and is very close to the optimal WVTR value, and comparable with the recommended WVTR value (2,000– 2,500 g/m2/day) for providing an adequate level of moisture without risking wound dehydration,26 suggesting it could prevent the wound from excess dehydration. The water retention property of a hydrogel is essential for a hydrogel to be used as wound dressing. Kickhofen et ¨ al.6 reported that water retention enables the gel to take up exudates and edema fluid from the wound into the dressing by an active upward-directed process when used in exudating wounds. Our results suggested that the AH-5.6 hydrogel membrane retained about 36.69% water after 6 days of exposure to air. Thus, these dressings will be more beneficial for wounds with moderate exudates rather than for dry wounds. In summary, the AH-5.6 hydrogel membrane could build a balance between the absorption of exudates and water loss, and thus facilitate cellular migration and enhance reepithelialization. More interestingly, the WVTR and water retention showed an Az-DS-dependent behavior, i.e., the higher the DS, the lesser the WVTR and water loss. This could be attributed to two reasons: (1) the AH-5.6 hydrogel membrane has a more compact structure; and (2) the higher cross-linked structure promotes stronger intermolecular and intramolecular hydrogen bondings, which results in
the increase of hydrophobicity in the polymer chain.27,28 In the future, WVTR and water loss of the membrane could be adjustable by controlling the Az-DS to favor wound repair and regeneration. Skin replacement must conform to the wound surface in all areas, and adhere in all areas. Without these properties, small air pockets exist where exudative fluid accumulates and may caused secondary bacterial infection. The graft must chemically adhere to the wound bed to avoid proliferation of the granulating layer and therefore control the subsequent contraction and deformity of the wound.7 The adhesion strength of the AH-5.6 hydrogel membrane to porcine skin was 12.3 Æ 2.0 N/cm2, suggesting that the hydrogel was chemically linked to the skin and could prevent it from separating from the skin. More importantly, both Gram-negative and Gram-positive bacteria could not penetrate the membrane, suggesting that the hydrogel membrane could serve as a barrier for bacterial infection. Oxygen penetration through a wound dressing is important because low oxygen concentration decreases the regeneration of tissue cell and slows down the healing process.26 Our hydrogel membrane showed a relatively high oxygen permeation ability, i.e., 8.77 Æ 0.31 mg/mL, suggesting it could allow enough oxygen entering for wound repair. The hydrogel membrane also showed good biocompatibility. The MTT assay results indicated that this material has no cytotoxicity against dermal and epidermal cells. Further study indicated that the coverage of the hydrogel membrane on the cell culture system did not affect cell growth, suggesting that Az-HPCS hydrogel membrane as a wound dressing will not affect the skin regeneration process.
Figure 9. Cell proliferation under the coverage of AH-5.6 hydrogel membrane. (A) L929 cell, (B) HaCaT cell.
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Figure 10. Light microphotographs of cells grown under the coverage of AH-5.6 hydrogel membrane. (A) Day 1 of L929 cell, (B) day 1 of HaCaT cell, (C) day 6 of L929 cell, (D) day 6 of HaCaT cell; original magnification Â100.
ACKNOWLEDGMENTS
This work was financially supported by the State Educational Ministry, China (the Project sponsored by SRF for ROCS; No. 2006-331) and by the National Natural Science Foundation of China (No 50873080 to J. Chen).
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