Preventing postoperative tissue adhesion using injectable carboxymethyl cellulose-pullulan hydrogels

Accepted Manuscript Title: Preventing Postoperative Tissue Adhesion using Injectable Carboxymethyl Cellulose-Pullulan Hydrogels Authors: Sumi Bang, Young-Gwang Ko, Won Il Kim, Donghwan Cho, Won Ho Park, Oh Hyeong Kwon PII: DOI: Reference:

S0141-8130(17)31292-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.07.103 BIOMAC 7911

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

12-4-2017 10-7-2017 17-7-2017

Please cite this article as: Sumi Bang, Young-Gwang Ko, Won Il Kim, Donghwan Cho, Won Ho Park, Oh Hyeong Kwon, Preventing Postoperative Tissue Adhesion using Injectable Carboxymethyl Cellulose-Pullulan Hydrogels, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.07.103 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.

Preventing Postoperative Tissue Adhesion using Injectable Carboxymethyl Cellulose-Pullulan Hydrogels Running head: Injectable carboxymethyl cellulose-pullulan hydrogels

Sumi Banga, Young-Gwang Koa, Won Il Kimb, Donghwan Choa, Won Ho Parkc, Oh Hyeong Kwona,* a

Department of Polymer Science and Engineering, Kumoh National Institute of Technology,

Gumi, Gyeongbuk 39177, Korea b

c

R&D Center, Wonbiogen Inc., Gumi, Gyeongbuk 39372, Korea

Department of Advanced Organic Materials and Textile System Engineering, Chungnam

National University, Daejeon 34134, Korea *Corresponding author: Professor Oh Hyeong Kwon E-mail: [email protected] Tel: +82-54-478-7690 Fax: +82-54-478-771

Abstract An injectable adhesive hydrogel composed of carboxymethyl cellulose (CMC) and pullulan is developed and evaluated as a postoperative anti-adhesion barrier. CMC was modified with tyramine to introduce crosslinking via an EDC-NHS reaction. The in situ hydrogel was developed by an enzyme-mediated reaction of tyramine-immobilized CMC with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Pullulan was added to the hydrogel solution to improve adhesiveness to the wound area and accelerate biodegradation. The modified CMC was confirmed by ATR-FTIR spectroscopy. The gelation time, storage modulus (G'), and weight loss of the hydrogels were measured as functions of the amounts of HRP and H2O2. The hydrogel group showed negligible cell proliferation and cytotoxicity, compared to that shown by the control group. The in vivo animal test demonstrated that significant

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decrease of postoperative tissue adhesion by applying the hydrogels. The CMC-pullulan hydrogel could be a useful treatment as an injectable in situ anti-adhesive agent. Keywords: carboxymethyl cellulose; pullulan; hydrogels; enzyme reaction; anti-adhesion 1. Introduction Tissue adhesions take place in 68–100% of surgical operations, despite research endeavors to prevent them [1-3]. Adhesion pathogens can occur from increasing the vascular permeability of the submesothelial layer, leaking body fluid, or inflammatory responses. When recovering injured tissue, fibrin adheres to nearby organs owing to the adhesive properties of fibrinogen produced by thrombin [4]. When fibrinolysis does not occur normally, the fibrin matrix makes cellular structures containing blood vessels and nerves, which causes tissue adhesion [5,6]. Tissue adhesions can occur at various biological locations, such as the pelvis, peritoneum, bowel, uterus, and nasal cavity, and can cause intestinal obstruction, chronic abdominal pain, chronic pelvic pain, and infertility [7-10]. Methods of preventing tissue adhesion are classified as either pharmaceutical adhesion based on adhesion pathogenesis or adhesion prevention barriers based on separating tissues. The drugs used in the pharmaceutical adhesion method include steroids, warfarin, urokinase, direct thrombin inhibitors, and heparin [11]. However, this method has not been tested clinically and can lead to side effects including the retardation of wound healing. The products of adhesion prevention barriers can be developed as solutions, gels, or films. Commercial products include Interceed (Ethicon, OH, USA), Gore-Tex (W. L. Gore & Associates Inc., AZ, USA), Seprafilm (Genzyme Biosurgery, MA, USA), and SprayGel (Confluent Surgical Inc., MA, USA). However, these products have drawbacks, including issues with flowability, non-biodegradability, aggregation, and difficult applications, especially when used in the film form [12]. On the basis of this context, we fabricated an anti-adhesive hydrogel barrier that is biocompatible and anti-inflammatory, and has biodegradable properties, using carboxymethyl cellulose (CMC) as a base material. Hydrogels have several advantages compared to films such as being able to diffusing oxygen, nutrients, and waste, protect cells against inflammation, and protect tissues from physical impacts [13-16]. CMC is soluble in water and organic solvents owing to its carboxyl group,

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while cellulose is insoluble in water and most organic solvents [17]. CMC is widely used as an ecofriendly, biocompatible material in drug delivery, dentistry, and tissue engineering [18]. Moreover, CMC is biodegradable in rats and does not require a secondary surgery to remove the barrier [15,17,18]. Therefore, CMC is a suitable hydrogel material as an injectable anti-adhesive barrier. Pullulan was added to the CMC solution to improve its adhesive properties on wound tissues [19]. Pullulan is a glucan produced from starch by a fungus of the genus Aureobasidium. Pullulan dissolves in water to form a stable, adhesive solution and degraded by many microorganisms. For these reason, pullulan is used in various ways as a thickner, adhesive enhancer of foods, packaging materials, edible coating, and elastic binder agents. Because the adhesive nature of pullulan, we can adopt a pullulan hydrogel as a biodegradable wound coverage agent. In this study, CMC was modified with tyramine to include a phenol group via the 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling reaction as a crosslinkable hydrogel by in situ injection. The horseradish peroxidase (HRP) mediated crosslinking system was established by M. Kurisawa et al [20-24]. The CMC-pullulan hydrogel was formed with HRP and hydrogen peroxide (H2O2) via an enzyme reaction [25]. The enzyme reactants initiate phenol radicals of tyramine to crosslink CMC chains. The CMC-pullulan hydrogel was characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and a rheometer. We also investigated the gelation time, biodegradation, cell proliferation, and cytotoxicity of the CMC-pullulan hydrogel. Its ability to prevent tissue adhesion was evaluated through in vivo animal tests of defected abdominal wall and cecum. 2. Materials and methods 2.1. Materials CMC (Mw 250,000 g/mol) and 4-(2-Aminoethyl)phenol (tyramine) were purchased from Sigma-Aldrich (USA). EDC was purchased from Tokyo Chemical Industry (Japan). NHS was obtained from Wako (Japan). Ethanol (99%) and H2O2 (30%) were obtained from Daejung Chemicals & Metals (Korea). HRP (specific activity 223.0 unit/mg) was purchased from Amresco (USA). Purified water was obtained by using a water purification system (Pure power I+, Human Corporation, Korea). 2.2. Reaction

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To introduce tyramine to the side chain of CMC, EDC and NHS were incorporated by a coupling reaction [26,27]. The phenol group of tyramine formed a crosslink site on the CMC backbone (Fig. 1). CMC was dissolved in deionized water at 0.01% (w/v) for 6 h. Then, EDC, NHS, and tyramine were added to the CMC solution at 0.000383%, 0.000230%, and 0.000348% (w/v), respectively. The solution was stirred for 24 h. The reactant was dialyzed as follows to remove the unreacted materials: 100 mM of NaCl solution for 2 days, 25% (v/v) ethanol solution for 1 day, and deionized water for 2 days. The dialyzed polymer solution was poured into an ice tray and frozen at -80oC for a day. The frozen cubes were lyophilized using a freeze dryer (FDU-1200, Tokyo Rikakikai Co., Ltd., Japan) for 3 days. The modified CMC sponges were stored in a freezer (-20oC) until required. Figure 1 2.3. Fabrication of CMC-pullulan hydrogel Through the EDC-NHS coupling reaction, a phenol group was added to the lyophilized CMC. The lyophilized CMC was dissolved in a phosphate-buffered saline (PBS) solution at 3% (w/w). Then, pullulan (3% w/w) was added to the polymer solution to improve the adhesive property of the hydrogel. HRP and H2O2 were added into the polymer solutions. The weights of modified CMC, PBS, and pullulan were fixed at 0.3, 9.7, and 0.3 g, respectively. The CMC-pullulan hydrogel, with different amounts of HRP and H2O2, was characterized by using a rheometer (at 44.6-1115 unit/10 g and 2-100 μL/10 g) as well as cell studies (at 44.6-1115 unit/10 g and 2-10 μL/10 g) and animal tests (at 44.6-223.0 unit/10 g and 2-10 μL/10 g). 2.4. Characterization of CMC-pullulan hydrogels The chemical structure of modified CMC with tyramine was confirmed by an ATR-FTIR spectrometer (VERTEX 80V, BRUKER, Germany) with Ge ATR crystal. The samples were lyophilized using a freeze dryer, reconstituted in deuterium oxide, and analyzed by 1H NMR spectrometer (BIOSPIN/ADVANCE III, 400 MHz FT-NMR, BRUKER, Germany). Gelation time was measured by the tube inversion method, with reference to the amount of HRP and H2O2. Hydrogel samples were prepared at room temperature (25oC) and transferred to a humidified incubator (37°C), then monitored at by inversing test tubes every 5 s. The gelation time was determined when the hydrogel stopped flowing (peak yield stress) [28].

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The effects of the amount of HRP and H2O2 on the storage modulus (G') were investigated by using a rheometer (ARES, TA instruments, USA; 25oC, gap: 1 mm, plate-plate diameter = 10 mm). The anti-adhesive barrier does not require a second surgery to remove the non-biodegraded materials. We performed experiments to evaluate the biodegradability of the CMC-pullulan hydrogel. The hydrogel disc (diameter 1.0 cm, thickness 0.5 cm) was immersed in PBS solution and shaken at 80 ppm at 37oC. Then, we observed the biodegradability of CMC-pullulan hydrogel by evaluating the weight loss and using scanning electron microscopy (SEM) (JSM-6380, JEOL Ltd., Japan) of the fracture surface on days 0, 2, 4, 7, 14, 21, and 28. Weight loss was calculated using the following the equation: Weight loss (%) = 1 - [Wa/Wi] × 100

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where Wi is the initial average hydrogel mass and Wa is the hydrogel mass at the day of evaluation. 2.5. Cell study Embryonic fibroblasts (NIH3T3, mouse) from the Korean Cell Line Bank (Korea) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin G-streptomycin, and incubated at 37°C under 5% CO2. All reagents used in the cell study were obtained from WELGENE Inc. (Korea). The hydrogel samples were sterilized by ultraviolet rays. Cells were seeded on 1 mL of CMC-pullulan hydrogel in 12-well plates at 3.0 × 104 cells per well. Analysis of the proliferation of cells on the hydrogel was conducted by a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay on day 1, 3, 5, and 7. MTT solution consisting of a 1:9 volume ratio of MTT and DMEM was added to the cells seeded on the hydrogel after removal of the culture media. Then, the cells seeded on the hydrogel were incubated for 2 h at 37°C. After 2 h, the MTT solution was removed and MTT-formazan was dissolved by the addition of 1 mL of dimethyl sulfoxide. The MTT-formazan solution (200 μL) was added to the 96-well plates (n = 4). The optical density of the MTT-formazan was measured at 540 nm using a microplate reader (PHOmo, Autobio Labtec Instruments Co., Ltd., China). Adhesion prevention barrier is sorted as medical device by regulation and cytotoxicity test is an essential requirement for biomedical applications. The in vitro cytotoxicity of hydrogels was evaluated by extraction liquid method (ISO 10993-5). In brief, the extracted solution was prepared by 4 g of the

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hydrogel place in 20 mL of media. Then, the extracted solution was applied to 2.0 × 104 cells/mL incubated for 24 hours. The cells viability was measured by MTT assay after 48 hours of incubation. 2.6. Animal tests Outbred male Sprague-Dawley rats (230-280 g, 8 weeks old) were used as the experimental model to evaluate the ability of the CMC-pullulan hydrogel to prevent tissue adhesion. The animal experiments were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the Kyungpook National University School of Medicine. The procedure was approved by the Animal Care Committee and was performed as follows: rats were anesthetized by a 4:1 volume ratio of Zoletil (Virbac, France): Rompun (Bayer, Germany). After splitting the abdomen, the cecal serosa was abraded using sandpapers and a wound was induced on the abdominal wall with a no. 10 blade to induce tissue adhesion. The control group was sutured but received no treatments. The five experimental groups were sutured after applying CMC-pullulan hydrogel using a dual syringe (7 x 7 mL, Plas-Pak Industries Inc., USA) equipped with a static mixing nozzle as an anti-adhesive barrier. After a 4-week breeding period, the rats were euthanized with potassium chloride. The abdomen was reopened and the adhesion score was rated according to the method by Vlahos et al [29]. 3. Results and discussion 3.1. Crosslinking of modified CMC and its chemical structure The main chain of CMC is crosslinked to maintain its physical structure during the in vivo experiments. To crosslink the CMC chains, the backbone was modified with tyramine by an EDC-NHS reaction (Fig. 1) [20-25]. Through this modification, a phenol group was introduced on to the carboxyl group of CMC. HRP and H2O2 form phenol radicals, which cause the CMC chain to crosslink. The crosslinking reaction occurred between either an oxygen radical and an ortho-carbon radical or two ortho-carbon radicals. The characteristic groups of CMC modified with tyramine was analyzed by ATR-FTIR spectroscopy and compared with that of unmodified CMC and tyramine (Fig. 2). CMC showed a broad peak of O-H stretching at 3,400 cm-1, a C-H stretching peak at 2,900 cm-1, a C=O peak at 1,680 cm-1, and a C-O peak at 1,100 cm-1. Tyramine showed a strong peak of N-H stretching at 3,100 cm-1, an aromatic C=C group at around 1,615 and 1,590 cm-1, a C-O peak at 1,210 cm-1, and a C-N peak at 1,115

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cm-1. CMC modified with tyramine showed a strong and broad peak of O-H and N-H stretching at 3,500– 3,000 cm-1, a C-H stretching peak at 2,900 cm-1, a strong C=O peak overlapped with a C=C peak at around 1,600 cm-1, and a strong C-O peak overlapped with a C-N peak at 1,115 cm-1. Modified CMC with tyramine showed increased intensity at around overlapped peaks, while no significant differences can be observed from the recorded spectra. Thus, the ATR-FTIR spectroscopy does not demonstrated whether tyramine was successfully immobilized onto the carboxyl group of CMC. For this reason, we carried out 1H NMR spectroscopy to characterize the peaks of CMC, tyramine and tyramine immobilized CMC. The 1H NMR showed that benzene peaks of tyramine appeared in the modified CMC spectrum at 6.9 and 6.7 ppm (Fig. 3c). This means that CMC had a phenol group to ensure crosslinking in the backbone. The amount of tyramine substitution on CMC was about 3.5%, quantitatively. The quantitative value of substitution degree of tyramine was calculated by an integral proton ratio of tyramine from methine of benzene at 6.9 and 6.7 ppm to that CMC from methine of tetrahydropyran at 3.7 ppm. 3.2. Formulation of CMC and pullulan mixtures The optimization of the CMC and pullulan formulation is very critical. Because, we added the pullulan in the CMC hydrogels to improve the attachment and wound coverage. In the preliminary experiment, the pullulan was added to 3 wt% CMC solutions as pullulan:CMC = 0:3, 1:3, 1:2, 1:1, 2:1, 3:1, respectively. We have measured the adhesive strength of CMC-pullulan hydrogels as a function of ratio using a Universal Testing Machine (AG-50kNX, Shimadzu Corporation, Japan) by the modified ASTM method F2255-05 [30,31]. The adhesive strength was increased as a function of pullulan amount from 7 to 36 kPa. The adhesive strength of CMC and pullulan-CMC (1:3) hydrogel was 7 and 13 kPa, respectively. This result means that small amount of pullulan improves the adhesive strength of the hydrogels, significantly. We selected the pullulan:CMC = 1:1 ratio. Because, the stirring of pullulan solution, injectability of CMC-tyramine and adhesive force were adequate as an in situ injectable hydrogel. The final mixture formulation of each material was set as CMC-tyramine 0.3 g, pullulan 0.3 g and PBS 9.7 g, respectively for other experiments. Figure 2

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Figure 3 3.3. Gelation behavior of CMC-pullulan hydrogel Hydrogels are convenient materials as anti-adhesive barriers because they are minimally invasive during surgery. The CMC-pullulan hydrogels are prepared for the in situ injectable and crosslinkable purpose in the body. At the beginning, to observe the physical state of the hydrogel, the gelation time was measured using the tube inversion method. We measured the effect of concentrations of HRP (223.0-1115 unit/10 g) and H2O2 (10-50 µL/10 g) on the gelation time of the CMC-pullulan (Fig. 4). When H2O2 was fixed at 10 μL/10 g, the gelation time decreased as the amount of HRP increased. As the amount of HRP increases, it produces more oxygen radicals, which leads to quicker crosslinking. When HRP was fixed at 223.0 unit/10 g, the gelation time remained around 30-35 s regardless of the amount of H2O2. This is because the formation of oxygen radicals is limited by the amount of HRP (223.0 unit/10 g). Therefore, the decrease in gelation time with H2O2 concentration was minimal. The H2O2 amount should be adjusted less than 10 µL/10g. As shown in graphs, the gelation time correlated with the amount of HRP, if the amount of H2O2 was sufficient to activate the HRP. The major advantage of hydrogel type anti-adhesive barriers is convenient handling on laparoscopic surgery. In situ hydrogel should be injected onto a wound area by dual syringe with a static mixing nozzle. Thus, we have considered the approximately 35 s of gelation time is better than quick gelation time to prevent clogging a nozzle during surgical operation. Figure 4 3.4. In vitro biodegradation of CMC-pullulan hydrogels The CMC-pullulan hydrogel is a natural biodegradable polymer and an adequate anti-adhesive barrier. To examine the biodegradability of the CMC-pullulan hydrogel, we performed an in vitro biodegradation test. The rate of biodegradation depends on the dominant material, crosslinking density, and physical, chemical, and biological conditions [32]. The in vitro biodegradation data over the course of 4 weeks are shown in Fig. 5a (relevant to the HRP concentration) and Fig. 5b (relevant to the H2O2 concentration). All of hydrogels showed 30–50% weight loss after 1 week. The hydrogel prepared with the HRP 223.0 unit/10 g and H2O2 10 μL/10 g, showed a 70% weight loss. Higher concentrations of HRP increase the rigidity of the hydrogel because of an increase in the crosslinking density. Therefore,

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CMC-pullulan hydrogels with higher concentrations of HRP degraded slower. Weight loss was about 60%, 55%, and 50% when H2O2 was 3 μL/10 g, 5 μL/10 g, and 7 μL/10 g, respectively. However, when H2O2 was 10 μL/10 g, the weight loss was 70%. This is most likely because higher concentrations of H2O2 cause the deactivation of HRP. As the amount of H2O2 increases, weight loss increases. Fractures of the freeze-dried hydrogels were analyzed by SEM (Fig. 6). The frequency of new pores and collapsed hydrogels increased with time, however, the amount of HRP did not affect the biodegradation of the hydrogels. On 7 day, with 10 μL/10 g of H2O2, there was a marked change in their structure. In addition, pores and the collapse of structures were observed after 28 days. Figure 5, 6 3.5. Rheology We investigated the viscoelastic behavior of the CMC-pullulan hydrogel by measuring the storage modulus (G') with a rheometer. Samples were prepared with different H 2O2 and HRP concentrations. We fixed the amount of H2O2 at 10 μL/10 g and measured the change in G'. The G' was 40 kPa at 223 unit/10 g of HRP, 50 kPa at 669 unit/ 10 g, and 70 kPa at 1115 unit/10 g (Fig. 7). G' increased gradually with the concentration of HRP, to a plateau. Increasing the concentration of HRP reduces the gelation time and increases rigidity. We concluded that hydrogels with higher concentrations of HRP are created faster and are more rigid. When the amount of HRP was fixed at 223 unit/10 g, the G' was 260 kPa at 10 μL/10 g of H2O2, 225 kPa at 50 μL/10 g, and 150 kPa at 100 μL/10 g (Fig. 7). HRP is deactivated when an excess of amount H2O2 is used to create the hydrogel [28, 33]. The deactivation of HRP causes the storage modulus of the CMC-pullulan hydrogel to decrease. As shown in Fig. 8, the G' of pullulan, CMC, and CMC + pullulan was 22, 30, and 60 kPa, respectively. When HRP and H2O2 are increased at the same time, the G' of the CMC-pullulan hydrogel also increases because the crosslinking density is subsequently increased. The structure of CMC, a cellulose-derived polymer, is poly(β-D-1,4-glucose), while the structure of starch is poly(α-D-1,4-glucose). Owing to its structure, the CMC-pullulan hydrogel has a greater G' compared with that of general hydrogels. Furthermore, we hypothesized that the flow ability of this hydrogel is not affected by gravity in the body. Figure 7, 8 3.6. Cell study

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Adhesive barriers have poor cell proliferation on their surface to prevent the formation new tissues. To test this property, cells were seeded on the CMC-pullulan hydrogel and cell viability was evaluated by an MTT assay on days 1, 3, 5, and 7 (Fig. 9). The optical density of the control group was 0.2 on day 1 and 1.15 on day 7. Cell growth of control group gradually increased during the incubation period. On the other hand, the optical density of hydrogel group was around 0.1–0.2. The hydrogel showed lower cell proliferation compared with the commercial cell culture dish, owing to its poorer protein adsorption onto hydrophilic materials [34-37]. To test biocompatibility (cytotoxicity), we compared the cell viability of the control and hydrogel groups using fibroblasts (Fig. 10). The cell viability of the control group was 100%, while that of the hydrogel group was around 80–82%, regardless of the HRP or H2O2 concentration. Thus, the CMCpullulan hydrogel demonstrated high biocompatibility in these experiments. Figure 9, 10 3.7. Animal tests To investigate the anti-adhesive effect of the CMC-pullulan hydrogel, we performed two animal tests. The hydrogel was applied to abdominal walls and compared with controls. The adhesion score was evaluated using Vlahos method (Fig. 11a-b, Table 1) [29]. The first animal test contained the hydrogel at one concentration, in order to investigate its anti-adhesive effect. HRP and H2O2 were fixed at 223 unit/10 g and 10 μL/10 g, respectively. The control group had an average adhesion score of 3.7 ± 0.2 and 100% adhesion in 15 rats. The hydrogel group had an average adhesion score of 0.7 ± 0.3 and 27% adhesion in 15 rats. The degree and incidence rate was considerably decreased in the group treated with the hydrogel barrier by physical separation between an wounded abdominal wall and cecum. However, 27% of abdominal adhesion was occurred between cecum and abdominal walls on the CMCpullulan hydrogel group. To investigate the optimum concentration of HRP and H2O2 of injectable CMC-pullulan hydrogels as an anti-adhesion barrier, the second animal test was conducted with five hydrogel groups using different concentrations of HRP and H2O2. As shown in Fig. 8, low amount of HRP diminish the storage modulus of hydrogels. And high amount of H2O2 decreases the storage modulus of hydrogels by deactivation of HRP. The control group had an average adhesion score of 3.8 ± 0.2 and 100%

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adhesion, similar to the first animal test (Table 1, Fig. 11a). As the concentration of HRP and H2O2 decreases, the hydrogel becomes softer. The soft hydrogel could not secure the coverage of wound area as a physical barrier. Softer hydrogels (group A) have higher adhesion scores and incidence rates owing to the effect of gravity and movement of organs. The more rigid hydrogels acted as better anti-adhesive barriers, except the group D at a concentration of 178.6 unit/10 g and 8 μL/10 g of HRP and H 2O2, respectively. The hydrogel B, C, E groups had an average adhesion score of 0 and no adhesion. Furthermore, after 4 weeks, there was no evidence of any hydrogel residues in the rats. Thus, the CMCpullulan hydrogels had biodegraded during the 4-week wound-healing period. This indicates that the hydrogel perfectly covered and isolated the injured abdominal wall from the abraded cecal serosa. Figure 11 Table 1 3.8. Histology In the first animal test, the cytoplasm of new tissues in the control group were dyed light pink between the abdominal wall and cecum mucosa of the rat (Fig. 11a, c). The control group showed firm adhesion band between between the cecum and abdominal wall after 8 weeks of surgery. On the other hand, the injured abdominal recovered perfectly in hydrogel group (Fig. 11b, d). This indicates that the CMC-pullulan hydrogel blocked tissue adhesion and improved wound recovery. 4. Conclusion

CMC was modified with tyramine via the EDC and NHS coupling reaction and tyramine was introduced into the carboxyl group. The chemical structure of the modified CMC was confirmed by ATR-FTIR and 1H NMR spectroscopy, and constructed with HRP and H2O2. The gelation time was around 30–40 s, relevant to the concentration of HRP and H2O2. When the rheometric behavior of the CMC-pullulan hydrogel was measured, the G' increased with the amount of HRP. G′ also increased with the amount of H2O2 if an excess amount of H2O2 was not used to create the hydrogel. Biodegradation of the hydrogel was analyzed by observing weight loss and SEM images. The CMC-pullulan hydrogel demonstrated significantly low cell proliferation without cytotoxicity. In the animal tests, prevention of tissue adhesion was significantly better in the hydrogel group compared with that in the control group.

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We expect that the CMC-pullulan hydrogel will be useful as anti-adhesive barrier for laparoscopy and normal surgeries.

Acknowledgements

This research was supported by the Human Resource Training Program for Regional Innovation and Creativity funded by the Ministry of Education and National Research Foundation (NRF2014H1C1A1066917); the Basic Science Research Program (2015M2A2A6A03044942); and Research Program to Solve Social Issues (2015M3A9D7067457) of NRF funded by the Ministry of Science, ICT & Future Planning.

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[25] S. Bang, E. Lee, Y.-G. Ko, W.I. Kim, O.H. Kwon, Int. J. Biol. Macromolec. 87 (2016), 155-162. [26] Y.D. Park, Nicola Tirelli, Jeffrey A. Hubbell, Biomaterials 24 (2003) 893-900. [27] D. Pasqui, M. De Cagna, R. Barbucci, Polymers 4 (2012) 1517-1534. [28] S.P. Zustiak, J.B. Leach, Biomacromolecules 11 (2010) 1348-1357. [29] A. Vlahos, P. Yu, C.E. Lucas, A.M. Ledgerwood, Am. Srug. 67 (2001) 15-21. [30] P.L. Thi, Y. Lee, D.H. Nguyen, K.D. Park, J. Mater. Chem. B. 5 (2017) 757-764. [31] Y. Lee, J.W. Bae, D.H. Oh, K.M. Park, Y.W. Chun, H.-J. Sung, K.D. Park, J. Mater. Chem. B. 18 (2013) 2407-2414. [32] A. Al-Abboodi, J. Fu, P. M. Doran, T.T.Y. Tan, P.P.Y. Chan, Adv. Health. Mater. 3 (2014) 725-736. [33] A. Schmidt, J.T. Schumacher, J. Reichelt, H.-J. Hecht, U. Bilitewski, Anal. Chem. 74 (2002) 30373045. [34] J. Carvalho, S. Moreira, J. Maia, F.M. Gama, J. Biomed. Mater. Res. Part A 93A (2010) 389-399. [35] E. Österberg, K. Bergström, K. Holmberg, T.P. Schuman, J.A. Riggs, N.L. Burns, J.M. Van Alstine, J.M. Harris, J. Biomed. Mater. Res. 29 (1995) 741-747. [36] R.A. Frazier, G. Matthijs, M.C. Davies, C.J. Roberts, E. Schacht, S.J.B. Tendler, J. Biomed. Mater. Res. 21 (2000) 957-966. [37] C.J. De Groot, M.J.A. Van Luyn , W.N.E. Van Dijk-Wolthuis, J.A. Cadée, J.A. Plantinga , W. Den Otter , W.E. Hennink, Biomaterials, 22 (2001) 1197-1203.

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Figure captions

Fig. 1. Schematic procedure of modified carboxymethyl cellulose (CMC) with 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS), and tyramine. And subsequent crosslinking reaction of modified CMC-pullulan mixture by enzymatic reaction.

Fig. 2. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectra of carboxymethyl cellulose (CMC) (a), tyramine (b), and modified CMC with tyramine (c).

Fig. 3. 1H NMR spectra of carboxymethyl cellulose (CMC) (a), tyramine (b) and modified CMC with tyramine (c).

Fig. 4. Gelation time as a function of the horseradish peroxidase (HRP) concentration (223, 446, 669, 892, and 1115 unit/10 g) and H2O2 concentration (10, 20, 30, 40, 50 μL/10 g) (mean ± SD, n = 4).

Fig. 5. In vitro biodegradation of hydrogels at 2, 4, 7, 14, 21, and 28 days in phosphate-buffered saline (PBS, 37oC). The weight loss of hydrogels according to the amount of horseradish peroxidase (HRP) (a) and H2O2 (b) (mean ± SD, n = 6).

Fig. 6. Scanning electron microscopy (SEM) images of lyophilized hydrogels according to the biodegradation time. Each hydrogel sample was prepared by HRP 66.9 (a), HRP 111.5 (b), HRP 156.1 unit/10 g (c) with H2O2 10 μL/10 g, and H2O2 3 μL (d), H2O2 5 μL (e), H2O2 7 μL (f), H2O2 10 μL (g) with HRP 223 unit/10 g.

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Fig. 7. Effect of the horseradish peroxidase (HRP) and H2O2 concentration on storage modulus (G') of the hydrogels; carboxymethyl cellulose (CMC)-tyramine 0.3 g, phosphate-buffered saline (PBS) 9.7 g.

Fig. 8. Storage modulus (G') of carboxymethyl cellulose (CMC), pullulan, mixture of CMC with pullulan, and CMC-tyramine hydrogels crosslinked with five composition of horseradish peroxidase (HRP) and H2O2.

Fig. 9. Cell proliferation on tissue culture polystyrene (TCPS) and carboxymethyl cellulose (CMC)tyramine hydrogels crosslinked as a function of HRP and H2O2 composition (mean ± SD, n = 4, *** p<0.001 as compared with the control).

Fig. 10. Cell viability on tissue culture polystyrene (TCPS) and carboxymethyl cellulose (CMC)tyramine hydrogels crosslinked as a function of horseradish peroxidase (HRP) and H2O2 composition (mean ± SD, n = 4, ***p < 0.001 as compared with the control).

Fig. 11. Photographs of the tissue adhesion between the cecum and abdominal wall after 8 weeks of surgery. The experimental groups are control (a) and carboxymethyl cellulose (CMC)-pullulan hydrogels with 223 units of horseradish peroxidase (HRP) and 10 μL of H2O2 (b). Photomicrographs of cross-sectional cecum-abdominal wall construct with hematoxylin and eosin (HE) staining after 8 weeks of surgery. Tissue adhesion between cecum and abdominal wall of the control group (c); healed abdominal wall of hydrogel group (d). The dotted square and arrows indicates wound area and adhesion bands, respectively. Abdominal wall (AW), cecum mucosa (CM), muscularis and serosa (MS) (scale bars = 10 μm).

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 1

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 2

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 3

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 4

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 5

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 6

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 7

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 8

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 9

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 10

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International Journal of Biological Macromolecules, Sumi Bang, Fig. 11

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Tables Table 1.: Adhesion score of the hydrogels (horseradish peroxidase (HRP) 223 unit and H2O2 10 μL in carboxymethyl cellulose (CMC)-tyramine hydrogel 10 g) on the first (n = 15) and second (n = 5) animal test (**p < 0.01, ***p < 0.001 compared with the control) st

1 animal test

nd

2 animal test (n=5)

(n=15)

Score

CMC-

Control

pullulan

CMC-pullulan Control A

B

C

D

E

0

0

11

0

3

5

5

4

5

1

0

1

0

0

0

0

0

0

2

1

1

0

1

0

0

0

0

3

3

1

1

1

0

0

0

0

4

11

1

4

0

0

0

1

0

Mean±SE

3.7±0.2

Adhesion (%)

0.7±0.3

100

27

***

3.8±0.2

100

1.0±0.6

40

A) HRP 44.6 unit, H2O2 2 μL B) HRP 89.2 unit, H2O2 4 μL C) HRP 133.8 unit, H2O2 6 μL D) HRP 178.6 unit, H2O2 8 μL E) HRP 223.0 unit, H2O2 10 μL

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**

0

***

0

0

***

0

0.8±0.7

20

***

0

***

0