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Preparation of composite hydroxybutyl chitosan sponge and its role in promoting wound healing
Accepted Manuscript Title: Preparation of Composite Hydroxybutyl Chitosan Sponge and Its Role in Promoting Wound Healing Authors: Shihao Hu, Shichao Bi, Dong Yan, Zhongzheng Zhou, Guohui Sun, Xiaojie Cheng, Xiguang Chen PII: DOI: Reference:
S0144-8617(17)31440-6 https://doi.org/10.1016/j.carbpol.2017.12.033 CARP 13095
To appear in: Received date: Revised date: Accepted date:
29-6-2017 4-12-2017 13-12-2017
Please cite this article as: Hu S, Bi S, Yan D, Zhou Z, Sun G, Cheng X, Chen X, Preparation of Composite Hydroxybutyl Chitosan Sponge and Its Role in Promoting Wound Healing, Carbohydrate Polymers (2010), https://doi.org/10.1016/j.carbpol.2017.12.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of Composite Hydroxybutyl Chitosan Sponge and Its Role in Promoting Wound Healing. Shihao Hu, Shichao Bi, Dong Yan, Zhongzheng Zhou, Guohui Sun, Xiaojie Cheng*, Xiguang Chen *. a
College of Marine Life Science, Ocean University of China, 5 Yushan Road,
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266003 Qingdao, PR China Tel.86-532-85712511
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E-mail address: [email protected]
*Send Correspondence to: Dr. Xiguang Chen (E-mail: [email protected]) and Dr. Xiaojie Cheng (E-mail: [email protected])
5# Yushan Road, Qingdao, P.R.China, 266003.
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Tel: 86-0532-82032586
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Fax: 86-0532-82032586
Graphical abstract
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College of Marine Life Science, Ocean University of China
Highlights The composite sponge emerged a special porous structure with a large specific surface area and permeability. The composite sponge had a better capacity of water retention.
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The composite sponge itself had a good antibacterial effect without any antimicrobials.
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The epithelial cells could proliferate inside the composite sponge which
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as a wound dressing.
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Abstract
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In this work, a composite sponge was produced by physically mixing
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hydroxybutyl chitosan with chitosan to form a porous spongy material
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through vacuum freeze-drying. Hydrophilic and macroporous composite hydroxybutyl chitosan sponge was developed via the incorporation of
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chitosan into hydroxybutyl chitosan. The composite sponge showed
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higher porosity (about 85%), greater water absorption (about 25 times), better softness and lower blood-clotting index (BCI) than those of chi-
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tosan sponge and hydroxybutyl chitosan sponge. The composite sponge with good hydrophilic could absorb the moisture in the blood to increase blood concentration and viscosity, and become a semi-swelling viscous colloid to clog the capillaries. Cytocompatibility tests with L929 cells
and HUVEC cells demonstrated that composite sponge were no cytotoxicity, and could promote the growth of fibroblasts. It made up for the shortcomings of hydroxybutyl chitosan with unfavorable antibacterial effect to achieve a higher level of antibacterial（>99.99% reduction）.
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Eventually, the vivo evaluations in Sprague−Dawley rats revealed that epithelial cells attached to the composite sponge and penetrated into the
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interior, in addition to this, it was also proved that the composite sponge (HC-1) had a better ability to promote wound healing and helped for
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faster formation of skin glands and re-epithelialization. The obtained data
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encourage the use of this composite sponge for wound dressings.
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Keywords: Chitosan, hydroxybutyl chitosan, antibacterial, wound heal-
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1. Introduction
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ing
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Skin, the largest organ of human body, covers the entire external surface and plays an important role in homeostasis and prevents the invasion
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by the microorganisms. Every year, millions of people suffered from the damage of skin losing caused by excessive physical and chemical factors or diseases. Subsequently, followed by a wound infection and serious tissue necrosis, endanger human's lives (Behrens, Sikorski & Kofinas, 2014). Accordingly, a dressing is required to protect loss of fluids and
proteins from the wound area and prevents any bacterial invasion replacing the function of skin temporarily. Additionally, it also enlarges the body's regeneration capacity by providing support for proliferation of cells (Ong, Wu, Moochhala, Tan & Lu, 2008).
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It is proverbial that chitosan-based sponge (a linear aminopolysaccharide obtained by deacetylation of the chitin) is an excellent
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bacteriostatic hemostatic material (Sharma*, 1997). A lot of chitosanbased dressings have been produced such as Syvek-Patch, Chitopack C,
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Tegasorb, HemCon Bandage, and KytoCel (Chen, Chang & Chen, 2008;
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Han, Dong, Su, Yin, Song & Li, 2014; Huang et al., 2015; Zakhem,
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Raghavan, Gilmont & Bitar, 2012). As the main component of these
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dressings, chitosan sponge is an effective hemostatic dressing with
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interconnected porous structures, high swelling capacity, good antibacterial activity and quick hemostaticability (Chan, Kim, Wang, Pun,
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White & Kim, 2016; Chen, Chang & Chen, 2008; Han, Dong, Su, Yin,
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Song & Li, 2014; Huang et al., 2015; Zakhem, Raghavan, Gilmont & Bitar, 2012). However, the poor hydrophilicity of sponges could not maintain the wound moist and cause secondary damage when applied and removed
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as a wound healing dressing (Patrulea, Ostafe, Borchard & Jordan, 2015). Up to now, there is almost no clinically used wound dressing satisfying with all the requirements of the ideal skin wound dressing (Patrulea, Ostafe, Borchard & Jordan, 2015).
Hydroxybutyl modified chitosan (HBC) is synthesized by conjugating hydroxybutyl groups to the C-6 hydroxyl (alkaline conditions) and C-2 amino (acidic conditions) groups of chitosan. Thus, the chemical modification endows chitosan with water solubility and controlled tem-
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perature-sensitive properties, and the aqueous to hydrogel phase transition process is reversible (Wang et al., 2013). Meanwhile, the freeze
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dried sponge still retains favorable biological properties, such as high
porosity, good hydrophilicity, non-toxicity, tissue adhesion and the
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ability to promote wound healing. Nevertheless, the antibacterial effect
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of the sponges is not obvious due to the hydroxybutyl modification,
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and the poor mechanical property in hydrated states lacked enough bar-
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rier function against wound infection (Xia et al., 2017).
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Thus, it is hypothesized that the combination of chitosan and hydroxylbutyl chitosan may make up for their own drawbacks, because
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there is the possibility to mix them physically without precipitation due
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to their same charge. In this study, the hydroxybutyl chitosan was synthesized and the
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neutral chitosan solution was prepared. Meanwhile, the composite hydroxybutyl chitosan sponge was prepared via mixing hydroxybutyl chitosan and neutral chitosan solution according to different ratio. The pure chitosan sponge and the hydroxybutyl chitosan sponge were also
prepared. As a wound healing material, the sponge features and physicochemical properties were characterized, including porosity, water absorption and retention ability, blood compatibility, cytotoxicity, and antibacterial activity. At the same time, the effectiveness of promoting
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wound healing in vitro rat model was also evaluated. 2. Experimental
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2.1 Materials
Chitosan (MW≈700kDa, DD＞75%), was purchased from Laizhou
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Haili Biological Product Co., Ltd (Shandong, China). 10% fetal bovine
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serum, Cell Counting Kit-8 was supplied by Solarbio (Beijing, China).
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1, 2 -Butene oxide were purchased from Sigma Co. Ltd. All other
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chemical reagents were A.R. grade.
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2.2 Preparation of novel composite wound dressings Preparation of nearly neutral chitosan acetate solution: A certain
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amount of chitosan was dissolved in 1 % acetic acid solution to obtain
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the 0.5% CS-Ac solution. The CS-Ac solution was placed in a dialysis bag with a rejection molecular weight of 8000-14000 replacing dis-
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tilled water twice a day until the pH reached about 6.2. The solution in the dialysis bag was then concentrated to a chitosan mass fraction of 1% collected to the beaker in reserve. Hydroxybutyl chitosan (HBC) was synthesized according to Wang’s method (Wang et al., 2013). Briefly, chitosan was mixed with NaOH
(50%, w/w) and stirred for 24 h. After the removal of additional NaOH solution, chitosan was dispersed in isopropanol–water and then mixed with 1, 2-butene oxide. After a reaction time of 24 h at 55 °C, the product was then neutralized, dialyzed with distilled water and lyophilized.
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HBC solution (3 wt %) was prepared at 4℃. As the main matrix of composite material is hydroxybutyl chitosan, the purpose for adding
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chitosan is to enhance its antibacterial effect and mechanical strength.
The composite material has not enough mechanical strength to main-
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tain certain shape after exudation absorption with too much hydroxy-
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butyl chitosan, and the bacteriostatic effect is also not ideal with low
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content chitosan. In contrast, the composite material has relatively poor
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hydrophilic and water-retention capacity with too much chitosan. So
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the final CS:HBC solution contained chitosan/hydroxybutyl-chitosan at the respective weight ratios of 1:3, 1:2 and 1:1 designated HC-1, HC-
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2, HC-3 respectively. Then, 5 mL solution (3 wt %) was poured into
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each well of polystyrene 6-well plates (Corning, Inc.; CS016-0092; USA), frozen at −20 ℃ in a freezer for 24 h and lyophilized in a freeze-
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dryer (SIM, Inc.; USA) under vacuum for 48 h. 2.3 Physicochemical Characterizations The structure of HBC was analyzed with the KBr pellet method on a NEXUE470 Fourier Transform Infrared Spectrophotometer (Nicolet,
Madison, USA). The powder samples were characterized by FTIR using a spectral width ranging from 4000 to 400 cm−1, with a 4 cm−1 resolution and an accumulation of 20 scans. And the degree of substitution (DS) of HBC was calculated by 1H NMR (Wang et al., 2017) on a
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Bruker ARX 400 MHz spectrometer (Germany). Then the samples were cut into cylindrical shape with a diameter of 13 mm and a thick-
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ness of 10 mm, and the surface morphology were elucidated by scanning electron microscope (SEM, JSM-810, JEOL Ltd, Japan).
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2.4 Swelling and water retention test
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The swelling test was performed by immersing a pre-weighed dry
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sample in SBF solution. The samples were weighed 15 min later as
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initial weight after wiping off excess water with filter paper. The swell-
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ing sponges were then incubated for 8 h at 37℃, 50% with humid atmosphere and the following weight was evaluated with completely
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dried sample. Same experiment was repeated for three times (Liang,
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Lu, Yang, Gao & Chen, 2016). The water uptake rate (𝑊𝑢 %) and water retention rate (𝑊𝑟 %) were
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determined as follows: 𝑊𝑢 % = (𝑊𝑑 − 𝑊𝑠 )⁄𝑊𝑠 × 100 𝑊𝑟 % = (𝑊𝑛 − 𝑊𝑠 )⁄(𝑊𝑑 − 𝑊𝑠 ) × 100 Where 𝑊𝑠 , 𝑊𝑑 and 𝑊𝑛 are the dry weight, the weight of swol-
len sample at equilibrium and the last weight of the sample, respectively. 2.5 Mechanical Testing Sponge samples were sliced into uniform parallel discs of diame-
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ter of 13mm and thickness of 5 mm. The mechanical stability of samples were investigated by applying uniaxial compression using
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Zwick/Roell Z010 machine (Germany). Samples were compressed up
to 80% of their original length at the displacement rate of 1 mm min −1.
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The compressive modulus of the sponge samples was calculated from
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Yang, Yang, Peng & Hu, 2016).
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the slope of the graph obtained by stress (MPa) versus strain (%) (Fan,
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2.6 Porosity of the sponge dressings
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The porosity of the prepared sheets was determined using the reported method (Tran & Mututuvari, 2016). Briefly, the sheets were im-
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mersed into absolute ethanol until it was saturated. The sheets were
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weighed before and after the immersion in alcohol. The porosity was cal-
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culated using the formula: P = (𝑊2 − 𝑊1 )⁄(𝜌𝑉2 − 𝜌𝑉1 ) × 100
In the equation, 𝑊1 and 𝑊2 indicate the weight of samples before
and after immersion in alcohol, respectively. 𝑉1 is the volume of alcohol before immersion, 𝑉2 is the volume of alcohol after immersion, 𝜌 is a constant (the density of alcohol at normal temperature). All samples
were triplicated in the experiment. 2.7 In Vitro Hemolysis Test The hemolysis ratios of samples at different concentration were tested in vitro (Feng et al., 2016). The sample was immersed in normal saline
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and incubated to 37 °C. Then, 20μL erythrocyte stock dispersions were added into sample suspensions (1 mL) and incubated at 37 °C for 1 h.
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After then, the mixtures were centrifuged at 2000 rpm/min for 5 min. The absorbance of the supernatant was determined at 545 nm by UV−vis
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spectrophotometer (UV-1200 MAPADA, China). In order to eliminate
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the influence of the absorbance of the samples, distilled water mixed with
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samples and normal saline mixed with samples were set as positive con-
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trol and negative control respectively. Same experiment was repeated for
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three times. The hemolysis rate (HR, %) was calculated using the formula： HR% = (𝐷𝑠 − 𝐷𝑛 )⁄(𝐷𝑝 − 𝐷𝑛 ) × 100
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where 𝐷𝑠 , 𝐷𝑛 , and 𝐷𝑝 are the absorbance of the sample, normal
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saline mixed with samples, and the distilled water mixed with samples respectively.
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2.8 Whole blood clotting and blood cell adhesion tests The blood clotting studies were done based on reported literature
(Lan et al., 2015; Ong, Wu, Moochhala, Tan & Lu, 2008). Blood was drawn from rabbit heart and mixed with anticoagulant agent acid citrate dextrose at a ratio of 85%:15%. Triplicate samples were used for this
study and blood without sample was used as negative control. Blood was added to each sample and placed in a 25-mL plastic Petri dish, which was followed by the addition of 10 μ L of 0.2 M CaCl2 solutions to initiate blood clotting. These samples then were incubated at 37 °C
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for 10 min. Fifteen milliliters (15 mL) of distilled water was then added dropwise without disturbing the clot. The sample sheets were then
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treated according to the above-mentioned method prior to SEM obser-
vation. Subsequently, 10 mL of solution was taken from the dishes and
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was centrifuged at 1000 rpm for 1 min. The supernatant was collected
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for each sample and kept at 37 °C for 1 h. Two hundred microliters
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(200 μ L) of this solution was transferred to a 96-well plate. The optical
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density was measured at 540 nm using a plate reader (BioTek
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PowerWave XS). The absorbance of citrated whole blood in deionized water was used as the reference (Abs of blank). Finally, the blood-clot-
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ting index (BCI) of the CG biomaterial was calculated from the follow-
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ing equation:
BCI = 𝐴𝑠 ⁄𝐴𝑏 × 100
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Where 𝐴𝑠 is the Abs of sample and 𝐴𝑏 is the Abs of blank. 2.9 In Vitro Cytotoxicity Test Cell viability was estimated by Cell Counting Kit-8 (CCK-8) based
on WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium), and assessed by contacting extracts of
the samples (Quan, Li, Luan, Yuan, Tao & Wang, 2015; Zhou, Yang, Liu, Mao, Gu & Xu, 2013). Briefly, L929 mouse fibroblast cells and HUVEC cells were seeded in 96-well plates at 1−3 × 105 cells/well and allowed to attach for 24 h, respectively. Then, the culture media was
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respectively replaced with media containing extracts of the samples at different concentration (0.3125, 0.625, 1.25, 2.5 and 5 mg/mL). The
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blank control without sample was also carried out under identical con-
ditions. After 24, 48, and 72 h incubation, the cell viability was deter-
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mined by measuring the absorbance at 450 nm on a microplate reader
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(Multiskan MK3). Measurements at each time-point were replicated
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five times. Finally, the toxicity grade was assessed based on the relative
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lowing formula:
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growth rate (RGR) of the cells which calculated according to the fol-
RGR = 𝐴𝑡 ⁄𝐴𝑐 × 100
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Where 𝐴𝑡 is the Abs of treatment group and 𝐴𝑐 is the Abs of con-
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trol group.
2.10 In Vitro Antibacterial Studies
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To evaluate the antimicrobial activity of the samples, Gram-positive
bacteria S. aureus and Gram-negative bacteria E. coli were used as the model microorganism. The samples were packed (0.3 g each) and placed under the UV light 48h for irradiation sterilization. S. aureus and E. coli were inoculated in sterilized LB broth overnight at 37 °C in
a shaking incubator, respectively. The concentration of bacteria was 106 colony-forming units per milliliter (CFU/mL). Each sample was mixed with 1 mL bacterial suspension containing ∼106 CFU/mL in conical flasks and added 9 mL PBS solutions(pH = 6.2). The blank without
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sample was as the control group. The test conical flasks were incubated at 37 ℃ in a shaking incubator operating at 130 rpm for 24 h.
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After the incubation period, the quantification of viable bacteria was done by serial dilution of the bacteria culture in PBS solutions followed
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by plating on LB agar plate. The colonies were calculated by the plate
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Lee, 2015; Xia et al., 2016).
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count method. Three parallels were done in the test (Kim, Kim, Ryu &
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2.11 Animal experiment for wound healing
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In vivo wound healing studies were performed on male Sprague−Dawley (SD) rats, weighing 200−250 g and 4−6 weeks of age. The
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twenty rats were divided into five groups and allowed to take normal rat
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feed and water without restriction (Loo et al., 2014). After hair removal and anesthesia, two full-thickness wounds of 1 cm × 1.5 cm were created
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on the dorsum of each rat. The right side was the test group fixed with radiation-sterilized samples (CS, HBC, HC-1 and HC-2), and the other side was the control group covered with medical gauze. Photographs were taken and the wound area was measured every three days. The skin wound tissue of the rat was excised, fixed with 10% formalin, and stained
with a hematoxylin−eosin (H&E) reagent for histological observations at days 3, 6, 9 and 12 (Singh & Dhiman, 2016). All above mentioned experimental rabbits were treated with the National Research Council’s Guide for the care and use of laboratory ani-
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mals. 2.12 Statistical analysis
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All the data were expressed as the mean ± standard deviation (SD),
and assessed by a one-way analysis of ANOVA to demonstrate differ-
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ences between groups. P values <0.05 were considered to be statistically
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significant.
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3. Results and Discussion
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3.1 Material Characterization
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In present research, hydroxybutyl chitosan was synthesized by introducing hydroxybutyl groups into the C-6 hydroxyl groups of chitosan
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in alkaline conditions. The FTIR spectrum of chitosan and HBC
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showed peaks assigned to the C–H stretching and bending of the CH3 group at 2965.48 cm-1 and 1460.15 cm-1 in Fig. 1A. These peaks indi-
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cated that -CH3 group was introduced into the chain of chitosan after reaction with 1, 2 -epoxy butane (Wang et al., 2013). In addition, the characteristic band of C-6 hydroxyl in chitosan at 1154.88 cm-1 became weaker and almost disappeared in HBC, suggested that the hydroxy-
butyl substitution mainly occurred at C-6 hydroxyl groups. Overall, hydroxybutyl was successfully conjugated onto the hydroxyl groups of chitosan. The 1H-NMR spectra of the HBC was shown in Fig. 1B. The peak-1 in the1H NMR spectra of HBC was the C-1 hydrogen of HBC,
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and the peak-2 was attributed to the appearance of -CH3 on the hy-
droxybutyl group (Wang et al., 2016). The degree of substitution of
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HBC was determined to be about 129% based on the integrated area
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rate of peak-2 and peak-1.
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Figure 1. A) FT-IR spectra of hydroxybutyl chitosan (spectrum pink) and chitosan control (spectrum red). B) 1H NMR spectra of HBC.
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Though dressing samples made from the pure chitosan sponge, the pure hydroxybutyl chitosan and the composite sponge were porous and
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no obvious difference on the macroscopic observation as shown in Fig. 2 (A1-E1). The composite sponges were named HC-1, HC-2, HC-3 ac-
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cording the weight of hydroxybutyl chitosan/chitosan ratios: 1/1, 2/1 and 3/1, respectively. The microscopic difference of the sample sheets illustrated by SEM shown as the Fig. 2 (A2-E2). Hydroxybutyl chitosan (named as HBC) sponge had the smallest pore size compared with chitosan (named as CS) sponge, which had the biggest pore size
(100±10μm vs 250±20μm). The pore size of the composite sponge (HC-1, HC-2 and HC-3) were about 200μm, and the pore wall was filled with a particular microporous (40μm) structure. The composite sponge blended with CS converted the pore size and structure, enlarged
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the specific surface area and permeability.
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Figure 2. A1-E1 were the photographs of the sponge samples for CS, HC-1, HC-2, HC-3 and HBC (the size of sponge: 13 mm diameter, 10 mm thickness), and A2-E2 were the SEM images of the interior porous structure of the CS, HC1, HC-2, HC-3 and HBC respectively.
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3.2 Swelling and water retention of the dressings
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Water absorption and retention capability of wound dressing is an important property to evaluate the efficacy of clearing the wound exu-
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date and keeping moist. The excellent swelling properties of the
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sponges are conduce to stop bleeding, and a moist environment can promote the healing of the wounds. The maintenance of a moist wound
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bed has been widely accepted as the most ideal environment for effective wound healing (Fan, Yang, Yang, Peng & Hu, 2016). CS sponge and composite sponge absorbed large amounts of SBF solution, and the order of swelling ratios for samples (CS＞HC-1＞HC-2＞HC-3＞ HBC) as shown in Fig. 3 (A). The swelling property decreased with the
increase of the weight ratio of HBC. It is maybe because HBC porous sponge is too dense to reach the swelling equilibrium within 15 minutes, and the composite sponge containing hydrophilic HBC delay further water absorption due to the formation of sticky spots after absorbing
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water. Despite CS sponge had the highest water absorption rate (Fig. 3 A),
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the capacity of water retention was not optimistic compared to compo-
site sponge as shown in Fig. 3 B, and the water content dropped to 20%
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within 2 hours. All the composite sponge (HC-1, HC-2 and HC-3
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sponge) had the better water retention compared to CS and HBC
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sponge, and HC-2 sponge had the best water retention. Therefore, the
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addition of a certain of HBC into chitosan sponge could significantly
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improve water retention, and the excessive HBC also reduced its water retention capacity. The main reason is that the water in pure chitosan
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sponge can evaporated easily as the form of free water. However, the
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most water in HC sponge groups can maintain effectively for a long time because the addition of HBC transfer free water into bound water
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by hydrophilicity. Anyway, chitosan also plays an important role of support when HBC is in contact with water to form a sticky colloid in the process of evaporation of water. In a word, HC-2 composite sponge have favorable water absorption and retention capability.
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Figure 3. Swelling ratios of various sponge samples including CS, HC-1, HC2, HC-3 and HBC in SBF solution after incubation at room temperature for 15 minutes (A) and the changes of water retention rate with time at 37 °C and humid atmosphere of 50% (B).
3.3 Porosity of the sponge dressings
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Porosity of samples was evaluated using an alcohol displacement
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method. It was found that the porosity of the CS sponge was the mini-
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mal (73 %), the HBC sponge showed the highest porosity (90 %), the
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porosity of HC-1, HC-2 and HC-3 composite sponge were about 85 %
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as shown in Fig. 4. The porosity of composite sponge decreased after mixing with CS, and there was little difference in porosity between the
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composite sponges. Because the pores of CS sponge is macroporous
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but less, however the pore of HBC sponge is microporous but more
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according to the previous image of the electron microscope (Fig. 2).
Figure 4. Porosity evaluation of CS, HC-1, HC-2, HC-3 and HBC sponge by
an alcohol displacement method.
3.4 Mechanical Testing As a wound dressing, which requires it to be in close contact with the skin surface, and its mechanical strength needed to be tested to de-
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termine the comfort of the materials (Singh & Dhiman, 2016). When rubbing against, it should not be easy to be torn out. Therefore, the
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compressive strength of sponges were tested by compressive testing
(Kumar et al., 2012). The compressive strength of CS, HBC, HC-1, HC-2 and HC-3 sponge appeared in Fig. 5. It could be seen that the
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curve of pure HBC sponge was almost a straight line indicating that
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HBC sponge alone was poor soft and fragile compared to the composite
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sponge. The HC-1 sponge had the best comfortable performance because of its lowest deformation pressure with the same strain compared
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to other samples at 40% & 80% as shown in Table 1. Furthermore, the
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curve of HBC sponge showed fit elastic curve and could be compressed
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repeatedly after the incorporation of CS.
Figure 5. Compressive strength (MPa) versus strain (%) of the sponge (CS, HBC,
HC-1. HC-2 and HC-3) at the displacement rate of 1 mm min−1. Table 1. The compression stress values (kPa) at 40% & 80% strain in compressive testing. CS
HBC
HC-1
HC-2
HC-3
40 % strain
53.60 ± 0.83
72.63 ± 1.21
44.47 ± 1.32
52.48 ± 1.09
54.16 ± 0.99
80 % strain
268.88 ± 1.24
137.89 ± 3.78
98.31 ± 2.25
145.34 ± 3.44
226.64 ± 2.11
3.5 In Vitro Hemolysis Test
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Hemolysis is a very simple and reliable way to evaluate the blood compatibility of wound dressings. Generally, the lower the hemolysis
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rate of wound dressings, the better the blood compatibility was (Feng et al., 2016). According to the national assessment criteria, the wound
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dressing could be safe which can be applied for clinic biomedical ma-
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terials in the condition of hemolysis rate lower than 5% (Archana,
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Singh, Dutta & Dutta, 2015). All the samples could absorb some blood
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cells, and the supernatant was clear and transparent as shown in the Fig.
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6 (A). The hemolysis rate of CS, HBC, HC-2, HC-2 and HC-3 sponges were all lower than 5% and hardly cause the hemolysis shown as Fig.
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6 (B). The evidence suggests that the composite sponge is a material
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with good blood compatibility.
Figure 6. (A) Photographs of RBCs treated with CS, HBC, HC-1, HC-2 and HC-3 sponges. P-C group and N-C group were the positive control and negative control respectively. (B) Hemolysis ratio of CS, HBC, HC-1, HC-2 and HC-3 sponges. Data represents the mean ± SD (n = 5).
3.6 Whole blood clotting and blood cells adhesion tests
The study on blood clotting mainly evaluates the performance of the hemostatic material to induce thrombosis in blood using a performance evaluation parameter called blood-clotting index (BCI). The smaller the BCI is, the stronger the hemostatic potential of the material is (Gu,
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Park, Kim, Kang, Kim & Kim, 2013; Gu, Park, Kim, Lee, Kim & Kim, 2016). The BCI among the different materials showed that the compo-
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site sponges had smaller blood clotting index than the single CS sponge or HBC sponge (Table 2). The BCI of composite sponge varied with
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the content of CS, and the best hemostatic activity was observed in HC-
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2 sponge. The composite sponge has a greater porosity and higher me-
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chanical strength being favor to maintain the three-dimensional porous
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structure compared to HBC sponge. The result was consist with hemad-
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sorption analysis by SEM, which composite sponge could absorb more red cells permeated all over the porous structure (Figure 7B-F). Mean-
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while, the hydrophilic component of composite sponge could make the
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blood form viscous gel conducive to promote blood coagulation. The reason was also due to the interaction between cationic CS and nega-
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tively charged blood cells (Tseng, Tsou, Wang & Hsu, 2013). Table 2. The effects of different samples on blood clotting index (BCI), Control—Negative control without hemostatic material. BCI (%)
HBC
CS
HC-1
HC-2
HC-3
96.8 ± 1.3
56.1 ± 0.4
16.1 ± 0.5
14.4 ± 1.2
32.8 ± 1.4
s
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Figure 7. A was the photo of the composite sponge with blood; B-F were the SEM images of blood cells adhesion of the CS, HBC, HC-1, HC-2 and HC-3 respectively.
3.7 In Vitro Cytotoxicity Test
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The cytotoxicity of HBC, HC-1, HC-2 and HC-3 sponges were eval-
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uated by CCK-8. The cytocompatibility results indicated that all sam-
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ples were no toxic with up to 5 mg/mL extraction concentrations as
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shown in Fig. S1. The relative growth rate of HUVEC cells and the
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L929 cells reached 110% -120% after composite sponge group incubation within 48 hours as depicted in Table 3 and Table 4. The results
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demonstrated the composite sponge don’t only have no cytotoxicity but
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also promoted the growth of L929 cells and HUVEC to a great extent, which indicated that composite sponge was a good candidate to be used as wound dressing.
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Table 3. The relative growth rate (RGR) of different concentration samples in L929 fibroblasts cells after incubation for 24h, 48h and 72h. 24 h
48 h
72 h
0.312
0.625
1.25
2.5
5
0.312
0.625
1.25
2.5
5
0.312
0.625
1.25
2.5
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
119.2 ±
115.8 ±
117.3 ±
118.7 ±
106.5 ±
101.1 ±
106.5 ±
121.1 ±
97.8 ±
102.3 ±
107.1 ±
113.5 ±
110.1 ±
101.3 ±
1.0
7.9
6.6
4.3
2.9
3.9
5.4
2.0
4.8
3.8
4.3
4.2
3.6
2.2
%)
%)
%)
)
110.6 ±
108.8 ±
101.3 ±
100.2 ±
106.6 ±
114.2 ±
109.5 ±
112.4 ±
104.6 ±
3.0
4.4
4.3
3.1
1.3
6.4
51.9
4.9
3.9
5.5
2.2
2.5
2.5
3.3
111.3 ±
111.6 ±
108.0 ±
102.0 ±
100.0 ±
104.3 ±
111.2 ±
99.1 ±
104.1 ±
108.8 ±
122.7 ±
121.4 ±
112.1 ±
108.1 ±
5.3
7.6
6.2
5.0
3.2
3.1
1.7
5.0
2.5
6.9
3.8
3.8
5.1
4.4
103.1 ±
119.6 ±
120.9 ±
102.1 ±
96.9 ±
113.1 ±
102.1 ±
117.8 ±
102.9 ±
109.3 ±
116.2 ±
113.2 ±
101.2 ±
101.3 ±
3.9
3.2
5.5
5.3
4.4
5.0
1.8
3.0
4.3
2.3
4.7
2.6
5.3
4.3
114.1 ±
115.9 ±
107.2 ±
105.6 ±
103.7 ±
102.3 ±
102.4 ±
107.7 ±
101.9 ±
111.8 ±
105.9 ±
114.0 ±
105.0 ±
103.4 ±
6.5
3.8
5.7
5.3
5.6
3.7
2.8
3.7
7.4
7.2
4.0
1.8
4.0
5.7
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103.4 ±
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Table 4. The relative growth rate (RGR) of different concentration samples in HUVEC cells after incubation for 24h, 48h and 72h. 24 h
48 h
0.312
0.625
1.25
2.5
5
0.312
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
111.2 ±
109.8 ±
117.6 ±
117.0 ±
4.1
5.3
2.9
109.8 ±
100.0 ±
6.3
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s
102.7 ±
72 h
0.625
1.25
2.5
5
0.312
0.625
1.25
2.5
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
102.1 ±
111.5 ±
104.7 ±
105.0 ±
106.0 ±
105.9 ±
114.9 ±
113.2 ±
116.2 ±
115.0 ±
6.7
2.2
5.7
2.9
5.7
3.7
3.4
2.1
3.6
3.0
2.6
105.3 ±
108.3 ±
103.5 ±
110.2 ±
106.9 ±
104.7 ±
108.7 ±
111.3 ±
118.0 ±
115.7 ±
114.2 ±
121.7 ±
5.3
1.8
2.3
1.5
3.9
6.2
3.1
6.0
4.7
2.6
6.4
6.4
6.4
99.8 ±
103.1 ±
103.0 ±
111.3 ±
100.6 ±
113.5 ±
107.5 ±
106.8 ±
111.0 ±
105.9 ±
119.4 ±
123.0 ±
117.4 ±
116.7 ±
3.3
0.4
6.0
6.7
4.1
1.6
6.1
1.7
5.4
5.1
2.4
3.9
3.2
0.9
102.2 ±
103.8 ±
105.5 ±
107.6 ±
104.5 ±
115.2 ±
118.3 ±
111.3 ±
112.4 ±
110.3 ±
120.3 ±
113.0 ±
115.7 ±
114.6 ±
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)
102.1 ±
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%)
107.1 ±
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%)
101.4 ±
1.9
5.5
5.7
3.1
6.2
5.4
3.9
3.4
4.8
2.1
2.5
1.5
4.7
2.4
112.7 ±
109.6 ±
109.3 ±
104.6 ±
103.6 ±
121.7 ±
111.3 ±
113.8 ±
112.5 ±
109.9 ±
113.2 ±
113.9 ±
110.1 ±
113.7 ±
5.4
2.1
2.3
6.2
3.3
3.2
5.1
1.5
4.9
3.4
1.4
3.0
3.6
3.0
3.8 In Vitro Antibacterial Studies
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The antibacterial activity of samples against S. aureus and E. coli were tested as shown in Fig. 8. The CS, HC-1, HC-2 and HC-3 sponges had significant antibacterial activity (>99.9% reduction), compared with the control group. HC-1 sponge had the better antibacterial activity among
composite sponge, and the poor bacteriostatic activity showed in HBC sponge (Fig. 8). Meanwhile, the pH of the test medium maintained 6.206.89 after co-cultivation of bacteria and samples (Table 5), it eliminated the impact of low pH on bacterial growth. The results showed that the
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sponge mixed with CS could significantly improve the antibacterial ef-
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fect, and the higher the CS content, the better the antibacterial effect was.
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Figure 8. The antibacterial activity of HBC, CS, HC-1, HC-2, and HC-3 sponges against S. aureus and E. coli. (Star symbols (*) represents the p < 0.05 level, indicating that the means are significantly different compared with the control.)
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The reason was supposed that the amino group reduced due to it partially substituted by hydroxybutyl group in HBC sponge, partially, and
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composite sponge made up for this drawback. It has been reported earlier that chitosan does not show antibacterial activity at neutral pH, because
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the amino group will not dissociate the positive charge (Jayakumar, Prabaharan, Kumar, Nair & Tamura, 2011). However, the CS sponge had a good antibacterial activity, which indicating that the synergistic effect of chitosan and weak acidity (pH=6.3) could enhance the bacteriostatic
effect, albeit in small amounts. The composite sponge showed higher activity against E. coli, compared to S. aureus, which can be attributed to the presence of a thick layer of peptide glycans in the cell wall of S. aureus, compared to E. coli.(Kumar et al., 2012) Table 5. The pH of the test medium after co-cultivation of bacteria and samples. HBC
HC-1
HC-2
E. coli
6.51 ± 0.12
6.21 ± 0.09
6.66 ± 0.15
6.20 ± 0.08
6.34 ± 0.12
S. aureus
6.45 ± 0.09
6.37 ± 0.10
6.89 ± 0.12
6.59 ± 0.13
6.66 ± 0.14
HC-3
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CS
6.47 ± 0.11
6.78 ± 0.18
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Control
3.9 Wound Healing Efficiency and Histological Observation In vivo wound healing study conducted in Sprague−Dawley (SD)
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rats was to evaluate the effect of the skin tissue reconstruction. HBC,
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CS sponge as well as composite sponge showed faster healing capacity
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after 6 days compared to that of a commercial dressing of gauze, HC1 and HC-2 sponges had the faster wound healing rate visually as
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shown in Fig. 9A. In the wound healing process, the surface of wound
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treated with CS sponge and gauze formed a scab over the wound, while the composite sponge group had smooth wound surface with no scab.
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The extent of wound closure was evaluated macroscopically as shown in Fig. 9B. After 12 days, the wounds treated with the composite
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sponge achieved significant closure to ∼90%, compared to the gauze, which showed ∼80% wound closure. It was also noted that the composite sponge (HC-1 and HC-2) dressing attached to the wounds with infiltration growth of epithelial cell (Fig. 9C). This was due to the higher water absorption and stability of the three-dimensional porous structure
are conducive to preserve nutrients and keep the wound moist, which could promote epithelial cells crawling growth in the composite sponge,
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and accelerate wound healing (Xu et al., 2015).
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Figure 9. In vivo study of rats healed wounds with the sponge samples. (A) Representative wound images of visual appearance; (B) wound size (%), at days 3, 6, 9 and 12 for each treatment regimen, (C) tissue adhesion of composite sponge. The wound of control group is treated with gauze.
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Results of histological analysis are depicted in Fig. 10. Several his-
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tological indicators can be used to evaluate the efficiency of wound dressings, including the angiogenesis, the presence of fibroblasts, se-
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baceous gland and the hair follicle development (Lih, Lee, Park & Park,
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2012). HBC sponge and composite sponge absorbed wound exudate to form a layer of gel on the surface of wound to lock the nutrients and
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kept the wound moist before 3 days. After 6 days, wounds covered with composite sponge presented significant development of sebaceous glands and fibroblasts with a layer of jelly, on the other hand, wounds covered with gauze were still in the early stage of healing without any glands, drying wound seemed to delay the wound recovery (Farrugia
et al., 2014). Therefore, granulation cells had infiltrated into the interior of the gel indicating the three-dimensional pore structure of the composite sponge dressing was suitable for supporting epithelial cell infiltration growth (Chang et al., 2010). After 12 days of contact with com-
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posite sponge, thickness of the granulation layer was similar to that of the unwounded skin, indicating optimal healing. The granulation layer
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was neither thinner nor thicker, suggesting that the healing was almost complete with no visible differences between new and old tissues (thus
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no scar). HC-1 sponge dressings exhibited either complete formation
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of the granulation layer or presence of numerous blood capillaries, in-
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dicating that the healing process was done (Gong et al., 2013). How-
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ever, fewer blood vessels or glands and the thinner granulation layer
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were observed in the dermis of the wound and deeper layers of epidermis covered with chitosan or gauze dressing. Given all that, composite
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sponge especially HC-1 sponge present an excellent ability to promote
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wound healing.
Figure 10.The H&E-stained images of rat epithelial wound tissue with control (gauze), CS, HBC, HC-1 and HC-2 sponges at 3 day, 6 day and 12 day respectively.
4. Conclusions A composite sponge was produced by physically mixing hydroxy-
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butyl chitosan with chitosan to form a porous spongy material through vacuum freeze-drying. The prepared composite sponge that have inter-
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connected pores showed ∼85% porosity of the total volume and were helpful with regard to absorbing large volumes of blood cells and
wound exudate (15-20 times). In vitro Cytotoxicity studies revealed
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that the sponge showed enhanced cell viability and proliferation. Initial
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investigations showed that HC-2 composite sponge performed better in
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water retention and erythrocyte aggregation than chitosan sponge and hydroxybutyl chitosan sponge separately, which is likely due to the sta-
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ble three-dimensional-reticulated porous structure of the composite
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sponge. Cytotoxicity tests had shown that the composite sponge induced higher cell proliferation with high cell viability and no obvious
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cytotoxicity in the mouse fiber cell line L929. Excellent antibacterial activity of composite sponge was also proved. Rats wound healing ex-
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periments showed that the composite sponge could support the creeping growth of epithelial cells to promote wound healing compared to chitosan sponge and hydroxybutyl chitosan sponge alone, and HC-1 sponge had the best ability to promote wound healing. All these studies indicated that the composite sponge especially HC-1 sponge had great
potential to be applied for a wound dressing. Acknowledgments This work was supported by National Natural Science Foundation of China (81671828), Applied Basic Research Plan of Qingdao (no.16-5-
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1-70-jch), and the Taishan Scholar Program, China. References
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Behrens, A. M., Sikorski, M. J., & Kofinas, P. (2014). Hemostatic strategies for traumatic and surgical bleeding. J Biomed Mater Res A, 102(11), 4182-4194.
Chan, L. W., Kim, C. H., Wang, X., Pun, S. H., White, N. J., & Kim, T. H. (2016). PolySTAT-modified chitosan
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chitosan hemostatic sponges as a promising surgical dressing. Int J Biol Macromol, 75, 322-329. Jayakumar, R., Prabaharan, M., Kumar, P. S., Nair, S., & Tamura, H. (2011). Biomaterials based on chitin and chitosan in wound dressing applications. Biotechnol Adv, 29(3), 322-337. Kim, K., Kim, K., Ryu, J. H., & Lee, H. (2015). Chitosan-catechol: a polymer with long-lasting mucoadhesive properties. Biomaterials, 52, 161-170. Kumar, P. T., Lakshmanan, V. K., Anilkumar, T. V., Ramya, C., Reshmi, P., Unnikrishnan, A. G., Nair, S. V., & Jayakumar, R. (2012). Flexible and microporous chitosan hydrogel/nano ZnO composite bandages for wound dressing: in vitro and in vivo evaluation. ACS Appl Mater Interfaces, 4(5), 2618-2629. Lan, G., Lu, B., Wang, T., Wang, L., Chen, J., Yu, K., Liu, J., Dai, F., & Wu, D. (2015). Chitosan/gelatin 1034.
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composite sponge is an absorbable surgical hemostatic agent. Colloids Surf B Biointerfaces, 136, 1026Liang, D., Lu, Z., Yang, H., Gao, J., & Chen, R. (2016). Novel Asymmetric Wettable AgNPs/Chitosan Wound
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Lih, E., Lee, J. S., Park, K. M., & Park, K. D. (2012). Rapidly curable chitosan-PEG hydrogels as tissue adhesives for hemostasis and wound healing. Acta Biomater, 8(9), 3261-3269.
Loo, Y., Wong, Y. C., Cai, E. Z., Ang, C. H., Raju, A., Lakshmanan, A., Koh, A. G., Zhou, H. J., Lim, T. C., of healing of burn wounds. Biomaterials, 35(17), 4805-4814.
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Moochhala, S. M., & Hauser, C. A. (2014). Ultrashort peptide nanofibrous hydrogels for the acceleration Ong, S. Y., Wu, J., Moochhala, S. M., Tan, M. H., & Lu, J. (2008). Development of a chitosan-based wound
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Quan, K., Li, G., Luan, D., Yuan, Q., Tao, L., & Wang, X. (2015). Black hemostatic sponge based on facile prepared cross-linked graphene. Colloids Surf B Biointerfaces, 132, 27-33. Sharma*, S. B. R. a. C. P. (1997). Use of chitosan as a biomaterial: Studies on its safety and hemostatic
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Xia, G., Liu, Y., Tian, M., Gao, P., Bao, Z., Bai, X., Yu, X., Lang, X., Hu, S., & Chen, X. (2017). Nanoparticles/thermosensitive hydrogel reinforced with chitin whiskers as a wound dressing for treating chronic wounds. J. Mater. Chem. B, 5(17), 3172-3185. Xu, R., Luo, G., Xia, H., He, W., Zhao, J., Liu, B., Tan, J., Zhou, J., Liu, D., Wang, Y., Yao, Z., Zhan, R., Yang, S., & Wu, J. (2015). Novel bilayer wound dressing composed of silicone rubber with particular micropores enhanced wound re-epithelialization and contraction. Biomaterials, 40, 1-11. Zakhem, E., Raghavan, S., Gilmont, R. R., & Bitar, K. N. (2012). Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering. Biomaterials, 33(19), 4810-4817. Zhou, Y., Yang, H., Liu, X., Mao, J., Gu, S., & Xu, W. (2013). Electrospinning of carboxyethyl
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92.
S0144-8617(17)31440-6 https://doi.org/10.1016/j.carbpol.2017.12.033 CARP 13095
To appear in: Received date: Revised date: Accepted date:
29-6-2017 4-12-2017 13-12-2017
Please cite this article as: Hu S, Bi S, Yan D, Zhou Z, Sun G, Cheng X, Chen X, Preparation of Composite Hydroxybutyl Chitosan Sponge and Its Role in Promoting Wound Healing, Carbohydrate Polymers (2010), https://doi.org/10.1016/j.carbpol.2017.12.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of Composite Hydroxybutyl Chitosan Sponge and Its Role in Promoting Wound Healing. Shihao Hu, Shichao Bi, Dong Yan, Zhongzheng Zhou, Guohui Sun, Xiaojie Cheng*, Xiguang Chen *. a
College of Marine Life Science, Ocean University of China, 5 Yushan Road,
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266003 Qingdao, PR China Tel.86-532-85712511
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E-mail address: [email protected]
*Send Correspondence to: Dr. Xiguang Chen (E-mail: [email protected]) and Dr. Xiaojie Cheng (E-mail: [email protected])
5# Yushan Road, Qingdao, P.R.China, 266003.
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Tel: 86-0532-82032586
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Fax: 86-0532-82032586
Graphical abstract
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College of Marine Life Science, Ocean University of China
Highlights The composite sponge emerged a special porous structure with a large specific surface area and permeability. The composite sponge had a better capacity of water retention.
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The composite sponge itself had a good antibacterial effect without any antimicrobials.
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The epithelial cells could proliferate inside the composite sponge which
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as a wound dressing.
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Abstract
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In this work, a composite sponge was produced by physically mixing
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hydroxybutyl chitosan with chitosan to form a porous spongy material
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through vacuum freeze-drying. Hydrophilic and macroporous composite hydroxybutyl chitosan sponge was developed via the incorporation of
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chitosan into hydroxybutyl chitosan. The composite sponge showed
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higher porosity (about 85%), greater water absorption (about 25 times), better softness and lower blood-clotting index (BCI) than those of chi-
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tosan sponge and hydroxybutyl chitosan sponge. The composite sponge with good hydrophilic could absorb the moisture in the blood to increase blood concentration and viscosity, and become a semi-swelling viscous colloid to clog the capillaries. Cytocompatibility tests with L929 cells
and HUVEC cells demonstrated that composite sponge were no cytotoxicity, and could promote the growth of fibroblasts. It made up for the shortcomings of hydroxybutyl chitosan with unfavorable antibacterial effect to achieve a higher level of antibacterial（>99.99% reduction）.
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Eventually, the vivo evaluations in Sprague−Dawley rats revealed that epithelial cells attached to the composite sponge and penetrated into the
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interior, in addition to this, it was also proved that the composite sponge (HC-1) had a better ability to promote wound healing and helped for
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faster formation of skin glands and re-epithelialization. The obtained data
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encourage the use of this composite sponge for wound dressings.
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Keywords: Chitosan, hydroxybutyl chitosan, antibacterial, wound heal-
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1. Introduction
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ing
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Skin, the largest organ of human body, covers the entire external surface and plays an important role in homeostasis and prevents the invasion
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by the microorganisms. Every year, millions of people suffered from the damage of skin losing caused by excessive physical and chemical factors or diseases. Subsequently, followed by a wound infection and serious tissue necrosis, endanger human's lives (Behrens, Sikorski & Kofinas, 2014). Accordingly, a dressing is required to protect loss of fluids and
proteins from the wound area and prevents any bacterial invasion replacing the function of skin temporarily. Additionally, it also enlarges the body's regeneration capacity by providing support for proliferation of cells (Ong, Wu, Moochhala, Tan & Lu, 2008).
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It is proverbial that chitosan-based sponge (a linear aminopolysaccharide obtained by deacetylation of the chitin) is an excellent
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bacteriostatic hemostatic material (Sharma*, 1997). A lot of chitosanbased dressings have been produced such as Syvek-Patch, Chitopack C,
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Tegasorb, HemCon Bandage, and KytoCel (Chen, Chang & Chen, 2008;
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Han, Dong, Su, Yin, Song & Li, 2014; Huang et al., 2015; Zakhem,
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Raghavan, Gilmont & Bitar, 2012). As the main component of these
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dressings, chitosan sponge is an effective hemostatic dressing with
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interconnected porous structures, high swelling capacity, good antibacterial activity and quick hemostaticability (Chan, Kim, Wang, Pun,
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White & Kim, 2016; Chen, Chang & Chen, 2008; Han, Dong, Su, Yin,
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Song & Li, 2014; Huang et al., 2015; Zakhem, Raghavan, Gilmont & Bitar, 2012). However, the poor hydrophilicity of sponges could not maintain the wound moist and cause secondary damage when applied and removed
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as a wound healing dressing (Patrulea, Ostafe, Borchard & Jordan, 2015). Up to now, there is almost no clinically used wound dressing satisfying with all the requirements of the ideal skin wound dressing (Patrulea, Ostafe, Borchard & Jordan, 2015).
Hydroxybutyl modified chitosan (HBC) is synthesized by conjugating hydroxybutyl groups to the C-6 hydroxyl (alkaline conditions) and C-2 amino (acidic conditions) groups of chitosan. Thus, the chemical modification endows chitosan with water solubility and controlled tem-
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perature-sensitive properties, and the aqueous to hydrogel phase transition process is reversible (Wang et al., 2013). Meanwhile, the freeze
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dried sponge still retains favorable biological properties, such as high
porosity, good hydrophilicity, non-toxicity, tissue adhesion and the
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ability to promote wound healing. Nevertheless, the antibacterial effect
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of the sponges is not obvious due to the hydroxybutyl modification,
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and the poor mechanical property in hydrated states lacked enough bar-
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rier function against wound infection (Xia et al., 2017).
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Thus, it is hypothesized that the combination of chitosan and hydroxylbutyl chitosan may make up for their own drawbacks, because
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there is the possibility to mix them physically without precipitation due
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to their same charge. In this study, the hydroxybutyl chitosan was synthesized and the
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neutral chitosan solution was prepared. Meanwhile, the composite hydroxybutyl chitosan sponge was prepared via mixing hydroxybutyl chitosan and neutral chitosan solution according to different ratio. The pure chitosan sponge and the hydroxybutyl chitosan sponge were also
prepared. As a wound healing material, the sponge features and physicochemical properties were characterized, including porosity, water absorption and retention ability, blood compatibility, cytotoxicity, and antibacterial activity. At the same time, the effectiveness of promoting
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wound healing in vitro rat model was also evaluated. 2. Experimental
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2.1 Materials
Chitosan (MW≈700kDa, DD＞75%), was purchased from Laizhou
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Haili Biological Product Co., Ltd (Shandong, China). 10% fetal bovine
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serum, Cell Counting Kit-8 was supplied by Solarbio (Beijing, China).
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1, 2 -Butene oxide were purchased from Sigma Co. Ltd. All other
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chemical reagents were A.R. grade.
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2.2 Preparation of novel composite wound dressings Preparation of nearly neutral chitosan acetate solution: A certain
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amount of chitosan was dissolved in 1 % acetic acid solution to obtain
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the 0.5% CS-Ac solution. The CS-Ac solution was placed in a dialysis bag with a rejection molecular weight of 8000-14000 replacing dis-
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tilled water twice a day until the pH reached about 6.2. The solution in the dialysis bag was then concentrated to a chitosan mass fraction of 1% collected to the beaker in reserve. Hydroxybutyl chitosan (HBC) was synthesized according to Wang’s method (Wang et al., 2013). Briefly, chitosan was mixed with NaOH
(50%, w/w) and stirred for 24 h. After the removal of additional NaOH solution, chitosan was dispersed in isopropanol–water and then mixed with 1, 2-butene oxide. After a reaction time of 24 h at 55 °C, the product was then neutralized, dialyzed with distilled water and lyophilized.
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HBC solution (3 wt %) was prepared at 4℃. As the main matrix of composite material is hydroxybutyl chitosan, the purpose for adding
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chitosan is to enhance its antibacterial effect and mechanical strength.
The composite material has not enough mechanical strength to main-
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tain certain shape after exudation absorption with too much hydroxy-
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butyl chitosan, and the bacteriostatic effect is also not ideal with low
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content chitosan. In contrast, the composite material has relatively poor
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hydrophilic and water-retention capacity with too much chitosan. So
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the final CS:HBC solution contained chitosan/hydroxybutyl-chitosan at the respective weight ratios of 1:3, 1:2 and 1:1 designated HC-1, HC-
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2, HC-3 respectively. Then, 5 mL solution (3 wt %) was poured into
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each well of polystyrene 6-well plates (Corning, Inc.; CS016-0092; USA), frozen at −20 ℃ in a freezer for 24 h and lyophilized in a freeze-
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dryer (SIM, Inc.; USA) under vacuum for 48 h. 2.3 Physicochemical Characterizations The structure of HBC was analyzed with the KBr pellet method on a NEXUE470 Fourier Transform Infrared Spectrophotometer (Nicolet,
Madison, USA). The powder samples were characterized by FTIR using a spectral width ranging from 4000 to 400 cm−1, with a 4 cm−1 resolution and an accumulation of 20 scans. And the degree of substitution (DS) of HBC was calculated by 1H NMR (Wang et al., 2017) on a
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Bruker ARX 400 MHz spectrometer (Germany). Then the samples were cut into cylindrical shape with a diameter of 13 mm and a thick-
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ness of 10 mm, and the surface morphology were elucidated by scanning electron microscope (SEM, JSM-810, JEOL Ltd, Japan).
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2.4 Swelling and water retention test
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The swelling test was performed by immersing a pre-weighed dry
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sample in SBF solution. The samples were weighed 15 min later as
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initial weight after wiping off excess water with filter paper. The swell-
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ing sponges were then incubated for 8 h at 37℃, 50% with humid atmosphere and the following weight was evaluated with completely
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dried sample. Same experiment was repeated for three times (Liang,
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Lu, Yang, Gao & Chen, 2016). The water uptake rate (𝑊𝑢 %) and water retention rate (𝑊𝑟 %) were
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determined as follows: 𝑊𝑢 % = (𝑊𝑑 − 𝑊𝑠 )⁄𝑊𝑠 × 100 𝑊𝑟 % = (𝑊𝑛 − 𝑊𝑠 )⁄(𝑊𝑑 − 𝑊𝑠 ) × 100 Where 𝑊𝑠 , 𝑊𝑑 and 𝑊𝑛 are the dry weight, the weight of swol-
len sample at equilibrium and the last weight of the sample, respectively. 2.5 Mechanical Testing Sponge samples were sliced into uniform parallel discs of diame-
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ter of 13mm and thickness of 5 mm. The mechanical stability of samples were investigated by applying uniaxial compression using
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Zwick/Roell Z010 machine (Germany). Samples were compressed up
to 80% of their original length at the displacement rate of 1 mm min −1.
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The compressive modulus of the sponge samples was calculated from
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Yang, Yang, Peng & Hu, 2016).
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the slope of the graph obtained by stress (MPa) versus strain (%) (Fan,
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2.6 Porosity of the sponge dressings
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The porosity of the prepared sheets was determined using the reported method (Tran & Mututuvari, 2016). Briefly, the sheets were im-
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mersed into absolute ethanol until it was saturated. The sheets were
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weighed before and after the immersion in alcohol. The porosity was cal-
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culated using the formula: P = (𝑊2 − 𝑊1 )⁄(𝜌𝑉2 − 𝜌𝑉1 ) × 100
In the equation, 𝑊1 and 𝑊2 indicate the weight of samples before
and after immersion in alcohol, respectively. 𝑉1 is the volume of alcohol before immersion, 𝑉2 is the volume of alcohol after immersion, 𝜌 is a constant (the density of alcohol at normal temperature). All samples
were triplicated in the experiment. 2.7 In Vitro Hemolysis Test The hemolysis ratios of samples at different concentration were tested in vitro (Feng et al., 2016). The sample was immersed in normal saline
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and incubated to 37 °C. Then, 20μL erythrocyte stock dispersions were added into sample suspensions (1 mL) and incubated at 37 °C for 1 h.
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After then, the mixtures were centrifuged at 2000 rpm/min for 5 min. The absorbance of the supernatant was determined at 545 nm by UV−vis
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spectrophotometer (UV-1200 MAPADA, China). In order to eliminate
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the influence of the absorbance of the samples, distilled water mixed with
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samples and normal saline mixed with samples were set as positive con-
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trol and negative control respectively. Same experiment was repeated for
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three times. The hemolysis rate (HR, %) was calculated using the formula： HR% = (𝐷𝑠 − 𝐷𝑛 )⁄(𝐷𝑝 − 𝐷𝑛 ) × 100
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where 𝐷𝑠 , 𝐷𝑛 , and 𝐷𝑝 are the absorbance of the sample, normal
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saline mixed with samples, and the distilled water mixed with samples respectively.
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2.8 Whole blood clotting and blood cell adhesion tests The blood clotting studies were done based on reported literature
(Lan et al., 2015; Ong, Wu, Moochhala, Tan & Lu, 2008). Blood was drawn from rabbit heart and mixed with anticoagulant agent acid citrate dextrose at a ratio of 85%:15%. Triplicate samples were used for this
study and blood without sample was used as negative control. Blood was added to each sample and placed in a 25-mL plastic Petri dish, which was followed by the addition of 10 μ L of 0.2 M CaCl2 solutions to initiate blood clotting. These samples then were incubated at 37 °C
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for 10 min. Fifteen milliliters (15 mL) of distilled water was then added dropwise without disturbing the clot. The sample sheets were then
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treated according to the above-mentioned method prior to SEM obser-
vation. Subsequently, 10 mL of solution was taken from the dishes and
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was centrifuged at 1000 rpm for 1 min. The supernatant was collected
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for each sample and kept at 37 °C for 1 h. Two hundred microliters
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(200 μ L) of this solution was transferred to a 96-well plate. The optical
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density was measured at 540 nm using a plate reader (BioTek
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PowerWave XS). The absorbance of citrated whole blood in deionized water was used as the reference (Abs of blank). Finally, the blood-clot-
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ting index (BCI) of the CG biomaterial was calculated from the follow-
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ing equation:
BCI = 𝐴𝑠 ⁄𝐴𝑏 × 100
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Where 𝐴𝑠 is the Abs of sample and 𝐴𝑏 is the Abs of blank. 2.9 In Vitro Cytotoxicity Test Cell viability was estimated by Cell Counting Kit-8 (CCK-8) based
on WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium), and assessed by contacting extracts of
the samples (Quan, Li, Luan, Yuan, Tao & Wang, 2015; Zhou, Yang, Liu, Mao, Gu & Xu, 2013). Briefly, L929 mouse fibroblast cells and HUVEC cells were seeded in 96-well plates at 1−3 × 105 cells/well and allowed to attach for 24 h, respectively. Then, the culture media was
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respectively replaced with media containing extracts of the samples at different concentration (0.3125, 0.625, 1.25, 2.5 and 5 mg/mL). The
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blank control without sample was also carried out under identical con-
ditions. After 24, 48, and 72 h incubation, the cell viability was deter-
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mined by measuring the absorbance at 450 nm on a microplate reader
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(Multiskan MK3). Measurements at each time-point were replicated
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five times. Finally, the toxicity grade was assessed based on the relative
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lowing formula:
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growth rate (RGR) of the cells which calculated according to the fol-
RGR = 𝐴𝑡 ⁄𝐴𝑐 × 100
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Where 𝐴𝑡 is the Abs of treatment group and 𝐴𝑐 is the Abs of con-
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trol group.
2.10 In Vitro Antibacterial Studies
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To evaluate the antimicrobial activity of the samples, Gram-positive
bacteria S. aureus and Gram-negative bacteria E. coli were used as the model microorganism. The samples were packed (0.3 g each) and placed under the UV light 48h for irradiation sterilization. S. aureus and E. coli were inoculated in sterilized LB broth overnight at 37 °C in
a shaking incubator, respectively. The concentration of bacteria was 106 colony-forming units per milliliter (CFU/mL). Each sample was mixed with 1 mL bacterial suspension containing ∼106 CFU/mL in conical flasks and added 9 mL PBS solutions(pH = 6.2). The blank without
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sample was as the control group. The test conical flasks were incubated at 37 ℃ in a shaking incubator operating at 130 rpm for 24 h.
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After the incubation period, the quantification of viable bacteria was done by serial dilution of the bacteria culture in PBS solutions followed
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by plating on LB agar plate. The colonies were calculated by the plate
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Lee, 2015; Xia et al., 2016).
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count method. Three parallels were done in the test (Kim, Kim, Ryu &
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2.11 Animal experiment for wound healing
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In vivo wound healing studies were performed on male Sprague−Dawley (SD) rats, weighing 200−250 g and 4−6 weeks of age. The
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twenty rats were divided into five groups and allowed to take normal rat
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feed and water without restriction (Loo et al., 2014). After hair removal and anesthesia, two full-thickness wounds of 1 cm × 1.5 cm were created
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on the dorsum of each rat. The right side was the test group fixed with radiation-sterilized samples (CS, HBC, HC-1 and HC-2), and the other side was the control group covered with medical gauze. Photographs were taken and the wound area was measured every three days. The skin wound tissue of the rat was excised, fixed with 10% formalin, and stained
with a hematoxylin−eosin (H&E) reagent for histological observations at days 3, 6, 9 and 12 (Singh & Dhiman, 2016). All above mentioned experimental rabbits were treated with the National Research Council’s Guide for the care and use of laboratory ani-
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mals. 2.12 Statistical analysis
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All the data were expressed as the mean ± standard deviation (SD),
and assessed by a one-way analysis of ANOVA to demonstrate differ-
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ences between groups. P values <0.05 were considered to be statistically
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significant.
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3. Results and Discussion
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3.1 Material Characterization
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In present research, hydroxybutyl chitosan was synthesized by introducing hydroxybutyl groups into the C-6 hydroxyl groups of chitosan
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in alkaline conditions. The FTIR spectrum of chitosan and HBC
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showed peaks assigned to the C–H stretching and bending of the CH3 group at 2965.48 cm-1 and 1460.15 cm-1 in Fig. 1A. These peaks indi-
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cated that -CH3 group was introduced into the chain of chitosan after reaction with 1, 2 -epoxy butane (Wang et al., 2013). In addition, the characteristic band of C-6 hydroxyl in chitosan at 1154.88 cm-1 became weaker and almost disappeared in HBC, suggested that the hydroxy-
butyl substitution mainly occurred at C-6 hydroxyl groups. Overall, hydroxybutyl was successfully conjugated onto the hydroxyl groups of chitosan. The 1H-NMR spectra of the HBC was shown in Fig. 1B. The peak-1 in the1H NMR spectra of HBC was the C-1 hydrogen of HBC,
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and the peak-2 was attributed to the appearance of -CH3 on the hy-
droxybutyl group (Wang et al., 2016). The degree of substitution of
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HBC was determined to be about 129% based on the integrated area
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rate of peak-2 and peak-1.
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Figure 1. A) FT-IR spectra of hydroxybutyl chitosan (spectrum pink) and chitosan control (spectrum red). B) 1H NMR spectra of HBC.
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Though dressing samples made from the pure chitosan sponge, the pure hydroxybutyl chitosan and the composite sponge were porous and
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no obvious difference on the macroscopic observation as shown in Fig. 2 (A1-E1). The composite sponges were named HC-1, HC-2, HC-3 ac-
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cording the weight of hydroxybutyl chitosan/chitosan ratios: 1/1, 2/1 and 3/1, respectively. The microscopic difference of the sample sheets illustrated by SEM shown as the Fig. 2 (A2-E2). Hydroxybutyl chitosan (named as HBC) sponge had the smallest pore size compared with chitosan (named as CS) sponge, which had the biggest pore size
(100±10μm vs 250±20μm). The pore size of the composite sponge (HC-1, HC-2 and HC-3) were about 200μm, and the pore wall was filled with a particular microporous (40μm) structure. The composite sponge blended with CS converted the pore size and structure, enlarged
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the specific surface area and permeability.
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Figure 2. A1-E1 were the photographs of the sponge samples for CS, HC-1, HC-2, HC-3 and HBC (the size of sponge: 13 mm diameter, 10 mm thickness), and A2-E2 were the SEM images of the interior porous structure of the CS, HC1, HC-2, HC-3 and HBC respectively.
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3.2 Swelling and water retention of the dressings
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Water absorption and retention capability of wound dressing is an important property to evaluate the efficacy of clearing the wound exu-
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date and keeping moist. The excellent swelling properties of the
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sponges are conduce to stop bleeding, and a moist environment can promote the healing of the wounds. The maintenance of a moist wound
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bed has been widely accepted as the most ideal environment for effective wound healing (Fan, Yang, Yang, Peng & Hu, 2016). CS sponge and composite sponge absorbed large amounts of SBF solution, and the order of swelling ratios for samples (CS＞HC-1＞HC-2＞HC-3＞ HBC) as shown in Fig. 3 (A). The swelling property decreased with the
increase of the weight ratio of HBC. It is maybe because HBC porous sponge is too dense to reach the swelling equilibrium within 15 minutes, and the composite sponge containing hydrophilic HBC delay further water absorption due to the formation of sticky spots after absorbing
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water. Despite CS sponge had the highest water absorption rate (Fig. 3 A),
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the capacity of water retention was not optimistic compared to compo-
site sponge as shown in Fig. 3 B, and the water content dropped to 20%
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within 2 hours. All the composite sponge (HC-1, HC-2 and HC-3
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sponge) had the better water retention compared to CS and HBC
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sponge, and HC-2 sponge had the best water retention. Therefore, the
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addition of a certain of HBC into chitosan sponge could significantly
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improve water retention, and the excessive HBC also reduced its water retention capacity. The main reason is that the water in pure chitosan
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sponge can evaporated easily as the form of free water. However, the
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most water in HC sponge groups can maintain effectively for a long time because the addition of HBC transfer free water into bound water
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by hydrophilicity. Anyway, chitosan also plays an important role of support when HBC is in contact with water to form a sticky colloid in the process of evaporation of water. In a word, HC-2 composite sponge have favorable water absorption and retention capability.
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Figure 3. Swelling ratios of various sponge samples including CS, HC-1, HC2, HC-3 and HBC in SBF solution after incubation at room temperature for 15 minutes (A) and the changes of water retention rate with time at 37 °C and humid atmosphere of 50% (B).
3.3 Porosity of the sponge dressings
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Porosity of samples was evaluated using an alcohol displacement
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method. It was found that the porosity of the CS sponge was the mini-
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mal (73 %), the HBC sponge showed the highest porosity (90 %), the
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porosity of HC-1, HC-2 and HC-3 composite sponge were about 85 %
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as shown in Fig. 4. The porosity of composite sponge decreased after mixing with CS, and there was little difference in porosity between the
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composite sponges. Because the pores of CS sponge is macroporous
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but less, however the pore of HBC sponge is microporous but more
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according to the previous image of the electron microscope (Fig. 2).
Figure 4. Porosity evaluation of CS, HC-1, HC-2, HC-3 and HBC sponge by
an alcohol displacement method.
3.4 Mechanical Testing As a wound dressing, which requires it to be in close contact with the skin surface, and its mechanical strength needed to be tested to de-
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termine the comfort of the materials (Singh & Dhiman, 2016). When rubbing against, it should not be easy to be torn out. Therefore, the
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compressive strength of sponges were tested by compressive testing
(Kumar et al., 2012). The compressive strength of CS, HBC, HC-1, HC-2 and HC-3 sponge appeared in Fig. 5. It could be seen that the
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curve of pure HBC sponge was almost a straight line indicating that
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HBC sponge alone was poor soft and fragile compared to the composite
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sponge. The HC-1 sponge had the best comfortable performance because of its lowest deformation pressure with the same strain compared
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to other samples at 40% & 80% as shown in Table 1. Furthermore, the
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curve of HBC sponge showed fit elastic curve and could be compressed
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repeatedly after the incorporation of CS.
Figure 5. Compressive strength (MPa) versus strain (%) of the sponge (CS, HBC,
HC-1. HC-2 and HC-3) at the displacement rate of 1 mm min−1. Table 1. The compression stress values (kPa) at 40% & 80% strain in compressive testing. CS
HBC
HC-1
HC-2
HC-3
40 % strain
53.60 ± 0.83
72.63 ± 1.21
44.47 ± 1.32
52.48 ± 1.09
54.16 ± 0.99
80 % strain
268.88 ± 1.24
137.89 ± 3.78
98.31 ± 2.25
145.34 ± 3.44
226.64 ± 2.11
3.5 In Vitro Hemolysis Test
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Hemolysis is a very simple and reliable way to evaluate the blood compatibility of wound dressings. Generally, the lower the hemolysis
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rate of wound dressings, the better the blood compatibility was (Feng et al., 2016). According to the national assessment criteria, the wound
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dressing could be safe which can be applied for clinic biomedical ma-
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terials in the condition of hemolysis rate lower than 5% (Archana,
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Singh, Dutta & Dutta, 2015). All the samples could absorb some blood
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cells, and the supernatant was clear and transparent as shown in the Fig.
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6 (A). The hemolysis rate of CS, HBC, HC-2, HC-2 and HC-3 sponges were all lower than 5% and hardly cause the hemolysis shown as Fig.
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6 (B). The evidence suggests that the composite sponge is a material
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with good blood compatibility.
Figure 6. (A) Photographs of RBCs treated with CS, HBC, HC-1, HC-2 and HC-3 sponges. P-C group and N-C group were the positive control and negative control respectively. (B) Hemolysis ratio of CS, HBC, HC-1, HC-2 and HC-3 sponges. Data represents the mean ± SD (n = 5).
3.6 Whole blood clotting and blood cells adhesion tests
The study on blood clotting mainly evaluates the performance of the hemostatic material to induce thrombosis in blood using a performance evaluation parameter called blood-clotting index (BCI). The smaller the BCI is, the stronger the hemostatic potential of the material is (Gu,
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Park, Kim, Kang, Kim & Kim, 2013; Gu, Park, Kim, Lee, Kim & Kim, 2016). The BCI among the different materials showed that the compo-
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site sponges had smaller blood clotting index than the single CS sponge or HBC sponge (Table 2). The BCI of composite sponge varied with
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the content of CS, and the best hemostatic activity was observed in HC-
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2 sponge. The composite sponge has a greater porosity and higher me-
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chanical strength being favor to maintain the three-dimensional porous
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structure compared to HBC sponge. The result was consist with hemad-
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sorption analysis by SEM, which composite sponge could absorb more red cells permeated all over the porous structure (Figure 7B-F). Mean-
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while, the hydrophilic component of composite sponge could make the
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blood form viscous gel conducive to promote blood coagulation. The reason was also due to the interaction between cationic CS and nega-
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tively charged blood cells (Tseng, Tsou, Wang & Hsu, 2013). Table 2. The effects of different samples on blood clotting index (BCI), Control—Negative control without hemostatic material. BCI (%)
HBC
CS
HC-1
HC-2
HC-3
96.8 ± 1.3
56.1 ± 0.4
16.1 ± 0.5
14.4 ± 1.2
32.8 ± 1.4
s
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Figure 7. A was the photo of the composite sponge with blood; B-F were the SEM images of blood cells adhesion of the CS, HBC, HC-1, HC-2 and HC-3 respectively.
3.7 In Vitro Cytotoxicity Test
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The cytotoxicity of HBC, HC-1, HC-2 and HC-3 sponges were eval-
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uated by CCK-8. The cytocompatibility results indicated that all sam-
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ples were no toxic with up to 5 mg/mL extraction concentrations as
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shown in Fig. S1. The relative growth rate of HUVEC cells and the
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L929 cells reached 110% -120% after composite sponge group incubation within 48 hours as depicted in Table 3 and Table 4. The results
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demonstrated the composite sponge don’t only have no cytotoxicity but
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also promoted the growth of L929 cells and HUVEC to a great extent, which indicated that composite sponge was a good candidate to be used as wound dressing.
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Table 3. The relative growth rate (RGR) of different concentration samples in L929 fibroblasts cells after incubation for 24h, 48h and 72h. 24 h
48 h
72 h
0.312
0.625
1.25
2.5
5
0.312
0.625
1.25
2.5
5
0.312
0.625
1.25
2.5
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
119.2 ±
115.8 ±
117.3 ±
118.7 ±
106.5 ±
101.1 ±
106.5 ±
121.1 ±
97.8 ±
102.3 ±
107.1 ±
113.5 ±
110.1 ±
101.3 ±
1.0
7.9
6.6
4.3
2.9
3.9
5.4
2.0
4.8
3.8
4.3
4.2
3.6
2.2
%)
%)
%)
)
110.6 ±
108.8 ±
101.3 ±
100.2 ±
106.6 ±
114.2 ±
109.5 ±
112.4 ±
104.6 ±
3.0
4.4
4.3
3.1
1.3
6.4
51.9
4.9
3.9
5.5
2.2
2.5
2.5
3.3
111.3 ±
111.6 ±
108.0 ±
102.0 ±
100.0 ±
104.3 ±
111.2 ±
99.1 ±
104.1 ±
108.8 ±
122.7 ±
121.4 ±
112.1 ±
108.1 ±
5.3
7.6
6.2
5.0
3.2
3.1
1.7
5.0
2.5
6.9
3.8
3.8
5.1
4.4
103.1 ±
119.6 ±
120.9 ±
102.1 ±
96.9 ±
113.1 ±
102.1 ±
117.8 ±
102.9 ±
109.3 ±
116.2 ±
113.2 ±
101.2 ±
101.3 ±
3.9
3.2
5.5
5.3
4.4
5.0
1.8
3.0
4.3
2.3
4.7
2.6
5.3
4.3
114.1 ±
115.9 ±
107.2 ±
105.6 ±
103.7 ±
102.3 ±
102.4 ±
107.7 ±
101.9 ±
111.8 ±
105.9 ±
114.0 ±
105.0 ±
103.4 ±
6.5
3.8
5.7
5.3
5.6
3.7
2.8
3.7
7.4
7.2
4.0
1.8
4.0
5.7
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103.4 ±
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Table 4. The relative growth rate (RGR) of different concentration samples in HUVEC cells after incubation for 24h, 48h and 72h. 24 h
48 h
0.312
0.625
1.25
2.5
5
0.312
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
111.2 ±
109.8 ±
117.6 ±
117.0 ±
4.1
5.3
2.9
109.8 ±
100.0 ±
6.3
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s
102.7 ±
72 h
0.625
1.25
2.5
5
0.312
0.625
1.25
2.5
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
102.1 ±
111.5 ±
104.7 ±
105.0 ±
106.0 ±
105.9 ±
114.9 ±
113.2 ±
116.2 ±
115.0 ±
6.7
2.2
5.7
2.9
5.7
3.7
3.4
2.1
3.6
3.0
2.6
105.3 ±
108.3 ±
103.5 ±
110.2 ±
106.9 ±
104.7 ±
108.7 ±
111.3 ±
118.0 ±
115.7 ±
114.2 ±
121.7 ±
5.3
1.8
2.3
1.5
3.9
6.2
3.1
6.0
4.7
2.6
6.4
6.4
6.4
99.8 ±
103.1 ±
103.0 ±
111.3 ±
100.6 ±
113.5 ±
107.5 ±
106.8 ±
111.0 ±
105.9 ±
119.4 ±
123.0 ±
117.4 ±
116.7 ±
3.3
0.4
6.0
6.7
4.1
1.6
6.1
1.7
5.4
5.1
2.4
3.9
3.2
0.9
102.2 ±
103.8 ±
105.5 ±
107.6 ±
104.5 ±
115.2 ±
118.3 ±
111.3 ±
112.4 ±
110.3 ±
120.3 ±
113.0 ±
115.7 ±
114.6 ±
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)
102.1 ±
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%)
107.1 ±
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%)
101.4 ±
1.9
5.5
5.7
3.1
6.2
5.4
3.9
3.4
4.8
2.1
2.5
1.5
4.7
2.4
112.7 ±
109.6 ±
109.3 ±
104.6 ±
103.6 ±
121.7 ±
111.3 ±
113.8 ±
112.5 ±
109.9 ±
113.2 ±
113.9 ±
110.1 ±
113.7 ±
5.4
2.1
2.3
6.2
3.3
3.2
5.1
1.5
4.9
3.4
1.4
3.0
3.6
3.0
3.8 In Vitro Antibacterial Studies
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The antibacterial activity of samples against S. aureus and E. coli were tested as shown in Fig. 8. The CS, HC-1, HC-2 and HC-3 sponges had significant antibacterial activity (>99.9% reduction), compared with the control group. HC-1 sponge had the better antibacterial activity among
composite sponge, and the poor bacteriostatic activity showed in HBC sponge (Fig. 8). Meanwhile, the pH of the test medium maintained 6.206.89 after co-cultivation of bacteria and samples (Table 5), it eliminated the impact of low pH on bacterial growth. The results showed that the
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sponge mixed with CS could significantly improve the antibacterial ef-
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fect, and the higher the CS content, the better the antibacterial effect was.
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Figure 8. The antibacterial activity of HBC, CS, HC-1, HC-2, and HC-3 sponges against S. aureus and E. coli. (Star symbols (*) represents the p < 0.05 level, indicating that the means are significantly different compared with the control.)
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The reason was supposed that the amino group reduced due to it partially substituted by hydroxybutyl group in HBC sponge, partially, and
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composite sponge made up for this drawback. It has been reported earlier that chitosan does not show antibacterial activity at neutral pH, because
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the amino group will not dissociate the positive charge (Jayakumar, Prabaharan, Kumar, Nair & Tamura, 2011). However, the CS sponge had a good antibacterial activity, which indicating that the synergistic effect of chitosan and weak acidity (pH=6.3) could enhance the bacteriostatic
effect, albeit in small amounts. The composite sponge showed higher activity against E. coli, compared to S. aureus, which can be attributed to the presence of a thick layer of peptide glycans in the cell wall of S. aureus, compared to E. coli.(Kumar et al., 2012) Table 5. The pH of the test medium after co-cultivation of bacteria and samples. HBC
HC-1
HC-2
E. coli
6.51 ± 0.12
6.21 ± 0.09
6.66 ± 0.15
6.20 ± 0.08
6.34 ± 0.12
S. aureus
6.45 ± 0.09
6.37 ± 0.10
6.89 ± 0.12
6.59 ± 0.13
6.66 ± 0.14
HC-3
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CS
6.47 ± 0.11
6.78 ± 0.18
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Control
3.9 Wound Healing Efficiency and Histological Observation In vivo wound healing study conducted in Sprague−Dawley (SD)
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rats was to evaluate the effect of the skin tissue reconstruction. HBC,
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CS sponge as well as composite sponge showed faster healing capacity
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after 6 days compared to that of a commercial dressing of gauze, HC1 and HC-2 sponges had the faster wound healing rate visually as
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shown in Fig. 9A. In the wound healing process, the surface of wound
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treated with CS sponge and gauze formed a scab over the wound, while the composite sponge group had smooth wound surface with no scab.
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The extent of wound closure was evaluated macroscopically as shown in Fig. 9B. After 12 days, the wounds treated with the composite
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sponge achieved significant closure to ∼90%, compared to the gauze, which showed ∼80% wound closure. It was also noted that the composite sponge (HC-1 and HC-2) dressing attached to the wounds with infiltration growth of epithelial cell (Fig. 9C). This was due to the higher water absorption and stability of the three-dimensional porous structure
are conducive to preserve nutrients and keep the wound moist, which could promote epithelial cells crawling growth in the composite sponge,
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and accelerate wound healing (Xu et al., 2015).
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Figure 9. In vivo study of rats healed wounds with the sponge samples. (A) Representative wound images of visual appearance; (B) wound size (%), at days 3, 6, 9 and 12 for each treatment regimen, (C) tissue adhesion of composite sponge. The wound of control group is treated with gauze.
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Results of histological analysis are depicted in Fig. 10. Several his-
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tological indicators can be used to evaluate the efficiency of wound dressings, including the angiogenesis, the presence of fibroblasts, se-
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baceous gland and the hair follicle development (Lih, Lee, Park & Park,
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2012). HBC sponge and composite sponge absorbed wound exudate to form a layer of gel on the surface of wound to lock the nutrients and
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kept the wound moist before 3 days. After 6 days, wounds covered with composite sponge presented significant development of sebaceous glands and fibroblasts with a layer of jelly, on the other hand, wounds covered with gauze were still in the early stage of healing without any glands, drying wound seemed to delay the wound recovery (Farrugia
et al., 2014). Therefore, granulation cells had infiltrated into the interior of the gel indicating the three-dimensional pore structure of the composite sponge dressing was suitable for supporting epithelial cell infiltration growth (Chang et al., 2010). After 12 days of contact with com-
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posite sponge, thickness of the granulation layer was similar to that of the unwounded skin, indicating optimal healing. The granulation layer
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was neither thinner nor thicker, suggesting that the healing was almost complete with no visible differences between new and old tissues (thus
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no scar). HC-1 sponge dressings exhibited either complete formation
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of the granulation layer or presence of numerous blood capillaries, in-
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dicating that the healing process was done (Gong et al., 2013). How-
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ever, fewer blood vessels or glands and the thinner granulation layer
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were observed in the dermis of the wound and deeper layers of epidermis covered with chitosan or gauze dressing. Given all that, composite
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sponge especially HC-1 sponge present an excellent ability to promote
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wound healing.
Figure 10.The H&E-stained images of rat epithelial wound tissue with control (gauze), CS, HBC, HC-1 and HC-2 sponges at 3 day, 6 day and 12 day respectively.
4. Conclusions A composite sponge was produced by physically mixing hydroxy-
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butyl chitosan with chitosan to form a porous spongy material through vacuum freeze-drying. The prepared composite sponge that have inter-
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connected pores showed ∼85% porosity of the total volume and were helpful with regard to absorbing large volumes of blood cells and
wound exudate (15-20 times). In vitro Cytotoxicity studies revealed
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that the sponge showed enhanced cell viability and proliferation. Initial
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investigations showed that HC-2 composite sponge performed better in
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water retention and erythrocyte aggregation than chitosan sponge and hydroxybutyl chitosan sponge separately, which is likely due to the sta-
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ble three-dimensional-reticulated porous structure of the composite
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sponge. Cytotoxicity tests had shown that the composite sponge induced higher cell proliferation with high cell viability and no obvious
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cytotoxicity in the mouse fiber cell line L929. Excellent antibacterial activity of composite sponge was also proved. Rats wound healing ex-
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periments showed that the composite sponge could support the creeping growth of epithelial cells to promote wound healing compared to chitosan sponge and hydroxybutyl chitosan sponge alone, and HC-1 sponge had the best ability to promote wound healing. All these studies indicated that the composite sponge especially HC-1 sponge had great
potential to be applied for a wound dressing. Acknowledgments This work was supported by National Natural Science Foundation of China (81671828), Applied Basic Research Plan of Qingdao (no.16-5-
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1-70-jch), and the Taishan Scholar Program, China. References
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92.