Biodegradable and injectable thermoreversible xyloglucan based hydrogel for prevention of postoperative adhesion

Accepted Manuscript Biodegradable and Injectable Thermoreversible Xyloglucan Based Hydrogel for Prevention of Postoperative Adhesion Ershuai Zhang, Junjie Li, Yuhang Zhou, Pengcheng Che, Bohua Ren, Zhihui Qin, Litao Ma, Jing Cui, Hong Sun, Fanglian Yao PII: DOI: Reference:

S1742-7061(17)30229-5 http://dx.doi.org/10.1016/j.actbio.2017.04.003 ACTBIO 4827

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

6 January 2017 21 March 2017 3 April 2017

Please cite this article as: Zhang, E., Li, J., Zhou, Y., Che, P., Ren, B., Qin, Z., Ma, L., Cui, J., Sun, H., Yao, F., Biodegradable and Injectable Thermoreversible Xyloglucan Based Hydrogel for Prevention of Postoperative Adhesion, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio.2017.04.003

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Biodegradable and Injectable Thermoreversible Xyloglucan Based Hydrogel for Prevention of Postoperative Adhesion Ershuai Zhang a, b, Junjie Li c, Yuhang Zhou a, Pengcheng Che b, Bohua Ren a, Zhihui Qin a, Litao Ma b, Jing Cui b, Hong Sun b, a,*, Fanglian Yao a, d,* a

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China

b

Department of Basic Medical Sciences, North China University of Science and Technology,

Tangshan 063000, China c

Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and

Tissue Engineering Research Center, Academy of Military Medical Sciences, Beijing 100850, China d

Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University,

Tianjin 300350, China

Corresponding Authors *E-mail: [email protected] (F. Yao). Tel.: +86-22-27402893. Fax: +86-22-27403389. *E-mail: [email protected] (H. Sun). Tel.: +86-315-3725740. Fax: +86-315-3726552.

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ABSTRACT: Peritoneal adhesion is very common after abdominal and pelvic surgery, which leads to a variety of severe complications. Although numerous pharmacological treatments and barrier-based devices have been investigated to minimize or prevent postoperative adhesion, the clinical efficacy is not very encouraging. In this work, a biodegradable and thermoreversible galactose modified xyloglucan (mXG) hydrogel was developed and the efficacy of mXG hydrogel in preventing postoperative peritoneal adhesion was investigated. The 4% (w/v) mXG solution was a free flowing sol at low temperature, but could rapidly convert into a physical hydrogel at body temperature without any extra additives or chemical reactions. In vitro cell tests showed that mXG hydrogel was non-toxic and could effectively resist the adhesion of fibroblasts. Moreover, in vitro and in vivo degradation experiments exhibited that mXG hydrogel was degradable and biocompatible. Finally, the rat model of sidewall defect-cecum abrasion was employed to evaluate the anti-adhesion efficacy of the mXG hydrogel. The results demonstrated that mXG hydrogel could effectively prevent postoperative peritoneal adhesion without side effects. The combination of suitable gel temperature, appropriate biodegradation period, and excellent postoperative anti-adhesion efficacy make mXG hydrogel a promising candidate for the prevention of postsurgical peritoneal adhesion.

Keywords: Postoperative adhesion, Modified xyloglucan, Thermoreversible hydrogel, Adhesion prevention, Biodegradable

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1. Introduction Peritoneal adhesions are common and severe complications after almost any types of abdominal and pelvic surgery, which can be found in up to 93% of patients [1,2]. The peritoneal adhesions can lead to many adverse consequences, such as chronic pain, female infertility, intestinal obstruction, and even death [3,4]. To prevent or minimize the formation of peritoneal adhesions after surgery, many approaches have been advocated and used in clinic, which can be generally classified as pharmacological treatments and physical barrier devices [4-6]. For the pharmacological treatments, many drugs such as aspirin, dexamethasone, and heparin have been used during the surgery, but the rapid clearance of drugs in abdominal cavity greatly limits their therapeutic effects [7,8]. Recently, some physical barrier systems, including polymer solutions and solid sheets, are regarded as the more effective adjuncts to reduce adhesion formation. For example, some solid anti-adhesion sheets have been commercialized, such as Interceed® (Johnson & Johnson, Cincinnati, USA), Seprafilm® (Genzyme, Cambridge, USA). However, it is difficult to cover and fix them on the injured tissues with irregular shape [9,10]. Moreover, some of them can easily adhere to any moist surface such as the surgeon’s gloves during placement. For the non-degraded solid sheets (e.g., Polytetrafluoroethylene (PTFE)-based membranes), the second surgery is necessary to remove them from the surgical site, which may suffer a risk of readhesion [11]. The polymer solutions can overcome these disadvantages to some extent, but their applications also are limited due to the short residence time [12,13]. The injectable hydrogels may be ideal agents for postoperative adhesion prevention because they are free flowing solutions before administration and can form gels under physiological conditions to separate the wound surface from the surrounding tissue or organs with minimal invasiveness. A series of in situ chemically cross-linked hydrogels derived from natural-occurring materials [14-17] or synthetic macromolecules [18] have been prepared for

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preventing postoperative adhesion. For example, Zhu et al. developed metal and light free PEG hydrogels through the thiol–ene click reaction under physiological conditions for prevention of postoperative peritoneal adhesions [18]. Moreover, many injectable physical hydrogels derived from ionic crosslinking [19] or temperature-response [20-23] have been tried for postoperative adhesion prevention. For example, Ishiyama et al. prepared ion-crosslinked physical hydrogels by mixing two phospholipid polymers in the presence of Fe3+ and successfully prevented peritendinous adhesion in a chicken model [19]. Recently, some enzymatic cross-linking hydrogels also have been developed. For example, Sakai et al. prepared a novel biodegradable hyaluronic acid-based in situ hydrogel through a cascade enzyme reaction initiated by contact with body fluid containing glucose and successfully reduced peritoneal adhesions in a bowel abrasion-abdominal sidewall defects model [24]. Among various injectable hydrogels, the thermoresponsive hydrogels have attracted increasing attention in prevention postsurgical adhesion due to their spontaneous gelling behaviors at the body temperature without any in vivo chemical reactions or extra factors such as ultraviolet light irradiation [20-23]. However, they are still suffering some problems when used for biomedical fields in vivo [25]. For example, thermoresponsive Pluronic hydrogels have the disadvantages of non-biodegradability, low mechanical strength, and relatively rapid erosion at the injection site [26,27]. In particular, Pluronic hydrogels suffer from short persistence time (usually less than 2 days) when used as anti-adhesion agent for prevention of postoperative adhesion in vivo [28]. Therefore, it is still urgent to develop a novel thermosensitive hydrogel with excellent biodegradability and biocompatibility for the adhesion prevention application. Polysaccharides (including cellulose, hyaluronic acid, chitosan, alginate, dextran, and others) are among the most abundant natural polymers on earth, which have been widely applied in biomedical fields by virtue of their excellent biocompatibility and biodegradability. In recent

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years, many polysaccharide-based hydrogels have been developed and applied to prevent postoperative adhesions [14-17, 20]. For example, Ito et al. prepared a series of cross-linking hydrogels based on dextran, hyaluronic acid, and cellulose derivatives [15-17], which greatly reduced the formation of adhesions in a rabbit sidewall defect-bowel abrasion model. However, for these chemically cross-linked hydrogels, the application of chemical crosslinkers or any other chemical reactions in vivo may lead to some biocompatibility problems, such as acute inflammation and foreign body reaction. Besides, the relatively long gelation time may limit the clinical application of some chemical hydrogels [29]. Therefore, it should be a preferable choice to developed thermoresponsive physical hydrogels with an appropriate gelation time based on natural polysaccharides for the prevention of postoperative adhesion. Xyloglucan (XG) is the major storage or structural heteropolysaccharides of higher plant cell walls, which has a basic structure of a (1-4)-β -D-glucan backbone chain with (1-6)-α -D-xylose branches partially substituted by (1-2)-β -D-galactoxylose [30,31]. Native XG obtained from the tamarind seed cannot form hydrogel, whereas thermoreversible XG hydrogel can be obtained by removing partial galactose residues with fungal β -galactosidase [31]. Miyazaki et al. found that XG with a galactose removal ratio of 35% could form hydrogel at 37 °C in dilute aqueous solution at concentration of 1-2 wt% [32]. The thermoresponsive XG based hydrogel has been widely investigated as the carriers for drug delivery [31,33,34]. However, no report could be found on its application in postoperative adhesion prevention. In the present study, thermoreversible galactose modified xyloglucan (mXG) hydrogel was developed and evaluated as a physical barrier device for postsurgical adhesion prevention. First, the mXG was prepared from the native XG by partially removing the galactose side-chains. Then, mXG hydrogel were prepared and characterized in terms of thermosensitivity, degradability, cytotoxicity, hemocompatibility, and biocompatibility. Moreover, the anti-adhesion mechanism

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of the developed hydrogel was investigated using L929 cells as the model cells. Finally, the efficacy of mXG hydrogel in preventing postoperative adhesion in a rat sidewall defect-cecum abrasion model in vivo was evaluated. 2. Materials and methods 2.1 Materials, cell lines, and animals Tamarind gum was purchased from Tokyo Chemical Industry Co., Ltd (Japan). Roswell Park Memorial Institute 1640 medium (RPMI-1640), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (USA). β -galactosidase from Aspergillus oryzae, trypsin solution, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), standards of glucose, xylose, and galactose were purchased from Sigma-Aldrich (USA). All other chemicals were analytical grade and used without further purification. L929 fibroblasts were obtained from the American Type Culture Collection (ATCC, USA). L929 fibroblasts labeled with green fluorescent were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Spraguee-Dawley (SD) rats (180 ± 20 g), C57 mice (15 ± 2 g) were purchased from the Experimental Animal Center, Academy of Military Medical Science (Beijing, China). All the animals were fed separately with enough food and water at a temperature of 22 ± 2 °C and a relative humidity of 50 ± 5%. All animals were acclimatized in specific pathogen-free (SPF) conditions for 1 week before experiment. All animal experimental procedures were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the North China University of Science and Technology. 2.2 Extraction and characterization of XG XG was extracted from tamarind gum using an ethanol precipitation method according to previous studies [35,36]. Briefly, 1 wt% tamarind gum aqueous solution was centrifuged at 9000

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rpm for 15 min to remove the water-insoluble impurities and protein fraction. The supernatant was gradually added to twice the volume of ethanol to precipitate the XG. After three cycles of redispersion in water/precipitation in ethanol for purification, the pure XG was obtained by filtrating and freeze-drying. The chemical character of prepared XG was determined by 1H-NMR (Bruker AVANCE Ⅲ spectrometer, 400MHz) using D2O as the solvent. The FTIR spectrum of dried XG was recorded using Bruker TENSOR 27 Infrared Spectrophotometer (Switzerland). Gel permeation chromatography (GPC) equipped with a Viscotek TDA 305 triple detector array (Malvern, UK) was used to characterize the Mw and polydispersity index (Mw/Mn) of XG. Measurement was carried out at 30 °C in sodium nitrate solution (0.1 M) as eluent at a flow rate of 0.5 mL/min. The monodisperse poly(ethylene glycol) with Mw of 99 k was used as standard. 2.3 Enzymatic modification of XG The side-chain galactose of XG was partially removed by the β -galactosidase reaction according to previous contributions [36,37]. Briefly, 1 g of XG was dissolved in 50 mL of sodium acetate buffer (pH 4.5) containing 50 mg β -galactosidase. The mixture was kept at 50 °C under stirring. After 24 h, the reaction was terminated by heating up to 100 °C for 30 min to inactivate the enzyme. Then, the mixture was dialyzed against deionized water for 72 h using a dialysis bag (MWCO 300 kDa). Finally, the prepared galactose modified xyloglucan (mXG) was purified by precipitation in ethanol and dried at 60 °C for 48 h under vacuum. In order to quantify the galactose removal ratio (GRR), XG and mXG were completely hydrolyzed by heating with 2 N sulfuric acid at 100 °C for 3 h [31]. The hydrolyzates were analysed using high performance anion exchange chromatography equipped with a pulsed amperometric detector (HPAEC-PAD, Dionex ICS-5000). Standard solutions of glucose, xylose, and galactose were

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used for calibration. The GRR was determined according the following equation, where N0 and Nr are the amount of galactose of XG and mXG, respectively. GRR % =

N − N × 100 1 N

2.4 Observations of sol-gel transition The sol-gel phase transition of mXG was roughly estimated by vial-inverting approach. Briefly, 0.5 mL of 4% (w/v) mXG solution was added into a 2 mL tightly screw-capped vial and stored at 4 °C for 1 h, and then the vial was incubated at 37 °C for a certain time. The sample was regarded as a “gel” when the visual flow could not be observed within 30 seconds by inverting the vial. 2.5 Rheological measurements The thermosensitivity of mXG was further evaluated using a rheometer (Ar1000, TA Instruments, USA) equipped with a cone-plate geometry (20 mm diameter, 2° angle, and gap size of 500 µm). m). A strain amplitude of 1% was used to ensure all measurements were conducted within the linear viscoelastic region, where the dynamic storage modulus (G•)) and loss modulus (G•)) are independent of the stress amplitude. Then rheological behavior of the sol-gel sol transition was measured by performing a temperature sweep. 200 µL L cold 4% (w/v) mXG solution was loaded onto the plate (5 °C) of the rheometer and allowed to equilibrate for 5 min. Temperature dependence of G• and G• were recorded by heating the systems from 5 to 50 °C at a rate of 1 °C/min. In addition, the time dependence of G• and G• of 4% (w/v) mXG solution was investigated at 37 °C ranges from 0 s to 1200 s, and the gelation time was determined as the time when G• became higher than G•. 2.6 In vitro cell viability assay

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Cytotoxicity of mXG and hydrogel extract were assessed by MTT assay using L929 cells. The L929 cells were seeded into 96-well plates (1 × 104 cells/well) and incubated at 37 °C in 5% CO2 atmosphere for 24 h. Then the RPMI-1640 medium was replaced with fresh medium with a series concentration (0, 1, 10, or 100 µg/mL) of mXG for another 24 h or 48 h incubation, respectively. For the cytotoxicity of hydrogel extract, the extraction media were prepared by incubating 4% mXG hydrogel in RPMI-1640 with 10% FBS at a 1:10 volume ratio (hydrogel volume to medium volume) for 48 h at 37 °C. Then, the resulting extraction media were harvested as stock solution and sequential dilutions were carried out to obtain the leachates with different concentrations (25%, 50%, 75%, and 100%). Cells were cultured with 200 µL L of the leachates with different concentrations at 37 °C in 5% CO2 atmosphere. The untreated cells incubated with plain culture medium were set as control. After incubation for 24 h or 48 h, the medium was replaced with 200 µL L of fresh RPMI-1640 medium containing 20 µL L of MTT (5 mg/mL), followed by a further incubation at 37 °C for 4 h. The supernatant was carefully removed and 200 µL L dimethyl sulfoxide (DMSO) was added to each well. The absorbance (A) ( was measured at the wavelength of 492 nm using a microplate reader (Bio-Rad, Berkeley, CA). The relative cell viability was determined using the following equation: Relative cell viability % =

A − A ! × 100 2 A" #  − A !

2.7 In vitro cell adhesion assay The cell adhesion on the surface of mXG hydrogel was assessed using L929 fibroblasts labeled with green fluorescent. Briefly, 0.2 mL of 4% (w/v) mXG solution was added into the 48-well plate and then incubated at 37 °C for 1 h to sufficiently congeal. Subsequently, 1 mL of RPMI-1640/10% FBS solution containing 1 × 104 cells was added into each well, followed by incubation at 37 °C in a 5% CO2 atmosphere for 48 h. After removing the medium, the hydrogel was gently rinsed with sterile PBS to remove the unattached cells. The morphology of L929 cells 9

attached on the surface of mXG hydrogel was observed by a fluorescence microscope (IX-51, Olympus, Japan). 2.8 In vitro hemolysis assay The hemocompatibility of mXG solutions and hydrogel extract was evaluated by hemolytic tests in vitro. The hydrogel extract was prepared by immersing 1 mL 4% mXG hydrogel in 10 mL of sterile normal saline (NS) for 48 h at 37 °C. 0.2 mL of the whole blood of rabbits was added to 10 mL of mXG hydrogel extract or mXG solutions with different concentrations (0.1 mg/mL or 1 mg/mL), respectively. Double distilled water and NS were employed as the positive and negative control, respectively. After incubating at 37 °C for 1 h, the samples were centrifuged at 2000 rpm for 5 min, and the absorbance (A) of the supernatant was detected by a UV/Vis spectrophotometer (Perkin Elmer, USA) at 540 nm. The hemocompatibility was characterized quantitatively by hemolytic ratio, which was calculated using Equation (3). Hemolytic ratio % =

A − A )#*+ × 100 3 A *#*+ − A )#*+

2.9 In vitro degradation of mXG hydrogel In vitro degradation of mXG hydrogel was monitored as a function of weight loss over time under simulated physiological conditions. 0.5 mL of 4% (w/v) mXG solution was injected into a test tube and incubated at 37 °C for 1 h to complete gelation. Then 9.5 mL of phosphate buffered saline (PBS) (10 mM, pH 7.4) was added and refreshed weekly. The pH change was measured with a pH meter before the medium was replaced by a fresh one. At predetermined time points, the gels were taken out, lyophilized, and weighed (Wt). The weight loss percentage (∆ W %) at each time interval was calculated using Equation (4), where W0 is the initial weight of each sample.

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∆W ( %) =

W0 − Wt × 100 W0

(4)

2.10 In vivo degradation and biocompatibility of mXG hydrogel

To investigate the in vivo degradation behaviors and biocompatibility of mXG hydrogel, 0.2 mL of 4% (w/v) mXG solution was subcutaneously injected into the back of C57 mice. 1 h later, one animal was sacrificed to check initial size of the formed mXG hydrogel. Thereafter, three of the mice were sacrificed to evaluate the degradation and biocompatibility of mXG hydrogel at 1 d, 1 w, 2 w, 4 w, 6 w, and 8 w, respectively. The remaining gels in the animals were photographed to observe the degradation of hydrogel. The tissue surrounding the implants was removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for further histological examination. 2.11 In vivo anti-adhesion evaluation of mXG hydrogel

A rat model of sidewall defect-cecum abrasion was used to evaluate the anti-adhesion efficacy of the mXG hydrogel. Forty-eight SD rats were randomly divided into three groups, the sidewall defect-cecum abrasion model was performed as previously described with some modifications [14,15]. Briefly, SD rats were intraperitoneally anesthetized with 10% chloral hydrate, the peritoneum was opened by a 5 cm long incision along the linea alba on the abdominal wall. The cecum defect was prepared by abrading with sterile surgical gauze until the serosa was damaged and hemorrhaging but not perforated. A corresponding 1 × 2 cm2 peritoneal defect on the right lateral abdominal wall was created using scalpel. Then the damaged cecum and injured abdominal wall were approximated with 3-0 silk suture. For the hydrogel group, 1 mL of 4% (w/v) mXG solution was injected on the injured sites, and gelation occurred in situ within 3 min. For the film group, the damaged abdominal wall was covered by a 2 × 3 cm2 commercialized anti-adhesion chitosan film (Yantai Wanli Medical Equipment Co., Ltd., China) without suturing.

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For the control group, the defects were washed with 1 mL of sterile normal saline (NS). Finally, the peritoneum was closed with interrupted 3-0 silk sutures, and the skin was closed with 4-0 silk sutures, respectively. At determined time points after the surgery (1 or 2 weeks), eight rats for each group were euthanized with an overdose of sodium pentobarbital via intravenous injection. Subsequently, the peritoneum was opened and the extent of adhesion was checked and scored in a double-blind manner according to the standard adhesion scoring system as follows: score 0, no adhesion; score 1, one thin filmy adhesion; score 2, more than one thin adhesion; score 3, thick adhesion with focal point; score 4, thick adhesion with plantar attachment or more than one thick adhesion with focal point; score 5, very thick vascularized adhesion or more than one plantar adhesion [14,22]. Specimens were taken from the damaged abdominal wall, injured cecum, and adhesion-associated tissues containing cecum and abdominal wall for histopathological examination. The obtained tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE, Masson trichrome, and Van Gieson technique, respectively. All tissue slides were analyzed using an optical microscope (BX-53, Olympus, Japan) by two pathologists in a blinded manner. 2.12 In vivo toxicity assessment

After surgery, all rats were observed to evaluate possible side effects caused by mXG hydrogel. The observation included their general conditions (activity, energy, hair, feces, and behavioral pattern), body weight, mortality, food intake, and water intake. Major organs, including heart, liver, spleen, lung, and kidney, were harvested at day 7, 14, 28, and 56 to stained with HE for histological analysis. 2.13 Statistical analysis

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Statistical analysis was carried out using SPSS 15.0 software. Statistical analysis for cell viability was performed using one-way analysis of variance (ANOVA). Adhesion scores did not always follow a normal distribution, so statistical analyses were performed using Manne-Whitney U-tests, or Fisher’s exact test. Differences were considered statistically significant if p < 0.05 on a 2-tailed test. 3. Results 3.1 Preparation and characterization of XG and mXG

XG was obtained from tamarind gum with the method of water abstraction/ethanol precipitation. The chemical structure of XG was displayed in Fig. 1A and was confirmed by 1

H-NMR and FTIR. 1H-NMR spectrum (Fig. 1B) of XG showed the characteristic peaks for

anomeric hydrogens of three different residues, i.e. galactose, xylose, and glucose. The peak at 5.1 ppm was attributed to the galactose, and the chemical shift at 4.8 and 4.4 ppm were attributed to the xylose and glucose, respectively. According to the FTIR spectrum (Fig. 1C), the wide absorption band observed at 3400 cm-1 can be attributed to the O-H stretching. The bands at 2895 cm-1 and 1373 cm-1 were attributed to the asymmetric stretching and angular deformation of C-H, respectively, while the band at 1030 cm-1 was ascribed to C-O stretching. According to the GPC trace (Fig. 1D), the Mw and polydispersity index (Mw/Mn) of the obtained XG were 1.1 × 106 and 1.4, respectively. The molecular weight of the xyloglucan may have been overestimated when Polyethylene glycol was used as standard because Polyethylene glycol has linear backbone and is more flexible than xyloglucan, which has the highly branched chain (Fig. 1A). The mXG was prepared by partially removing the galactose side-chains from the native XG with β -galactosidase reaction. In order to quantify the galactose removal ratio (GRR), XG and mXG were heated with 2 N sulfuric acid at 100 °C for 3 h, which could ensure the polysaccharides were completely hydrolyzed while the resulting monosaccharides could not be 13

degraded. High performance anion exchange chromatography equipped with a pulsed amperometric detector (HPAEC-PAD) was used to determine the monosaccharide composition of XG and mXG. The amount of monosaccharide was calculated from the area of the corresponding peak (Fig. 1E). The monosaccharide composition/100 mg of the sample was calculated and the results were presented in Table 1. In native XG, the molar ratio of galactose, xylose, and glucose was 1.0: 2.8: 3.7, whereas that for mXG was 1.0: 5.5: 6.1. Therefore, the corresponding galactose removal ratio for mXG was 41%. 3.2 Thermosensitivity of mXG hydrogel

As is showed in Fig. 2A, 4% (w/v) mXG solution exhibited a temperature-dependent sol-gel phase transition behavior with the visual observation. The polymeric solution was a free flowing liquid at low temperature (4 °C), but it rapidly converted into a hydrogel at 37 °C without any chemical reactions or crosslinkers, indicating that mXG hydrogel has excellent thermosensitivity. Moreover, the heat-induced gelation was thermoreversible and the mXG hydrogel can revert to liquid when the temperature decreases to 4 °C. It is easy to realize the sol-gel transition via adjusting the temperature, which is convenient for its storage and application. The thermosensitive behaviors of XG and mXG were further evaluated by rheological analysis. For XG, as shown in Fig. 2B, both storage modulus (G•)) and loss modulus (G•)) were very low and the G• was always smaller than G• in all the temperature range (5 to 50 °C), indicating the sol nature of XG. For the mXG (Fig. 2C), the G′ was inferior to G″ when the temperature was less than 20 °C. However, both G• and G• increased with the temperature and achieved the same value when the temperature increased to 23 °C, suggesting that the transition occurred from the initial dominant viscous liquid to elastic solid. The corresponding temperature was defined as gel temperature (Tgel) [35]. Following the cross-over point, there was a relatively rapid increase in G• due to the formation of the gel. When temperatures were above 40 °C, the maximum elastic

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modulus was achieved. The tan δ (G•/G•) of mXG hhydrogel was smaller than 0.1, indicating that it was a conventional elastic gel [38,39]. As displayed in Fig. 2D and E, the gelation of 4% (w/v) mXG solution occurred at 20 s and almost completed within 3 min at 37 °C, which was a suitable time when used for the application in adhesion prevention. 3.3 In vitro cytotoxicity of mXG hydrogel

The cytotoxicity of mXG was evaluated using L929 cells by MTT assay because fibroblasts played very important roles in the postoperative adhesion formation and wound repair. Due to 4% mXG solution formed a gel at 37 °C, mXG hydrogel extract with different concentrations was evaluated. As shown in Fig. 3A, the cell viabilities of L929 cells cultured with different concentrations of hydrogel extract were approximately 100% compared with the control after 24 h or 48 h culture (p < 0.01). Moreover, L929 cells that exposed to mXG solution with the concentration from 1 µg/ g/mL to 100 µg/ g/mL showed high cell viability after 24 h or 48 h incubation (Fig. 3B). Even at a higher concentration of 100 µg/mL, the cell viability was higher than 80% compared with the control after 48 h culture (p < 0.01). These results suggested that the mXG had almost no adverse impact on cell viability and could be a safe anti-adhesion barrier agent. 3.4 In vitro anti-adhesion to fibroblasts of mXG hydrogel

The fibroblasts adhesion plays an important role in the formation of postsurgical peritoneal adhesion [1,3,6]. Therefore, the ideal barrier agent for adhesion prevention should efficiently prevent the fibroblasts adhesion. Fig. 3D shows the adhesion of L929 cells on the surface of mXG hydrogel after cultured for 48 h. There were very few cells could be observed on the surface of the mXG hydrogel, while many cells adhered on TCPS and adopted elongated shapes (Fig. 3C). Moreover, the attached cells on the surface of the mXG hydrogel were spherical and emitted relatively weak fluorescence. It indicated that the mXG hydrogel had a good

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performance on prevention of fibroblasts adhesion, which plays an important role to minimize or prevent the formation of postoperative adhesion. 3.5 In vitro hemocompatibility of mXG

The hemocompatibilities of mXG solutions and hydrogel extract were evaluated by in vitro hemolytic tests. As shown in Fig. 4C, none of mXG solutions or hydrogel extract cause hemolysis, which were similar to that in the negative control (the normal saline solution). However, the obvious hemolysis was observed in the positive control (distilled water). Compared with the positive control (100% hemolytic ratio), all of mXG solutions and hydrogel extract exhibited less than 1% hemolytic ratio (Fig. 4D), indicating that mXG has excellent hemocompatibility and may be suitable for biomedical applications. 3.6 In vitro degradation of mXG hydrogel

In vitro degradation of mXG hydrogel was monitored as a function of weight loss over time in PBS (10 mM, pH 7.4) at 37 °C. The weight loss of mXG hydrogel at specific time point was presented in Fig. 4A. It is clearly observed that the weight loss of hydrogel samples gradually increased over time, which was about 30% at 10 weeks. However, the hydrogel samples had no significant change in shape during the in vitro degradation tests (data not shown). In addition, the degradation of mXG hydrogel nearly had no impact on pH of the degradation medium, which stayed approximately 7.4 during the degradation tests (Fig. 4B). 3.7 In vivo degradation and biocompatibility of mXG hydrogel

In vivo degradation of mXG hydrogel was investigated by subcutaneous injection of mXG solution into the backs of C57 mice. As presented in Fig. 5A, mXG hydrogel was formed in situ at the injection site. On gross observation, the size of mXG hydrogel was decreased gradually over time, indicating that the hydrogel degraded continuously. At 2 weeks after injection, there was a significant decrease in the amount of mXG hydrogel. The hydrogel completely

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disappeared after 6 weeks. In contrast with the degradation in vitro, mXG hydrogel displayed a faster degradation rate in vivo. In vivo biocompatibility of mXG hydrogel was evaluated by histological analysis of the tissues surrounding the injection site. As shown in Fig. 5B, a few of neutrophils were observed at the interface between surrounding tissues and the hydrogel at 1 day post-injection. A mild inflammation with enhanced neutrophils and macrophages in the tissues were observed at 2 weeks. At 4 weeks, there were only few inflammatory cells in the tissues as the hydrogel degraded gradually. After injection for 6 weeks, the hydrogel completely disappeared and the surrounding tissue was similar to normal tissue. Additionally, few hemorrhaging, hyperemia, necrosis, or edema could be observed around the injection site during the whole experiment. These results suggested that the inflammatory response of mXG hydrogel was acceptable and the mXG hydrogel was biocompatible for the further application in vivo. 3.8 In vivo evaluation of anti-adhesion efficacy

The in vivo anti-adhesion efficacy of mXG hydrogel was evaluated in a rat model of sidewall defect-cecum abrasion. As shown in Fig. 6B, the defects were created by abrasion of the cecum and excision of a part of adjacent abdominal wall. For the film group, the injured abdominal wall was covered by a commercialized chitosan film (Fig. 6C). For the hydrogel group, the 4% (w/v) mXG solution was injected onto the defects (Fig. 6D), and then gelated in situ within 3 min (Fig. 6E). For the control group, the defects were only washed with sterile normal saline. On day 7 after surgery, the incision was reopened to check the peritoneal adhesion (Fig. 7 and Fig. S1), and the adhesion scores were displayed in Table 2. All of the 8 rats in the control group suffered from severe abdominal adhesions (Fig. 7A), two developed score 5 adhesions, the remaining developed score 4 adhesions (p < 0.001, Fisher’s exact test). Besides, a large amount of ascites was observed in the abdominal cavity. For the animals treated with commercialized

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films, both the adhesion scores and adhesion area were reduced significantly compared with the control group (p < 0.01, Mann-Whitney U test), but most of them still suffered from intestinal adhesions (Fig. 7B). For the hydrogel group, most of the rats did not suffer from adhesions and the damaged cecum and injured abdominal wall were partly recovered (Fig. 7C, p < 0.001, Fisher’s exact test), which showed significant differences compared to the NS group (p < 0.001, Mann-Whitney U test). On the 14th day, the more serious adhesions between the injured cecum and abdominal wall were observed in the control group (Fig. 7D). The rats in the film group also developed compact adhesions (Fig. 7E). In contrast, few adhesion was observed in the hydrogel-treated group (Fig. 7F) compared to the NS group (p < 0.001, Mann-Whitney U test), indicating that mXG hydrogel could effectively prevent postsurgical peritoneal adhesions. Furthermore, the defects were completely recovered after treated with mXG hydrogel and the hydrogel was completely disappeared from the abdominal cavity on day 14. As shown in Fig. 8A, histological observations of the adhesion sites were performed by HE, Masson trichrome, and Van Gieson staining, respectively. On day 7, the tissues taken from the adhesion site in the control and film group showed that the injured cecum was fused to the muscles of damaged abdominal wall, and the resulting adhesion contained a great deal of collagen fibers, fibroblasts, and inflammatory cells such as foamy macrophages, macrophages (Fig. S2). It is well known that collagen can be stained in blue while skeletal muscle show red in Masson trichrome staining. Therefore, it can be clearly observed that the deposited collagen in the adhesion site, which displayed intense blue. The deposited collagen in the adhesion site was further illustrated by Van Gieson staining, which was stained in red. Compared with the control group, there was less collagen deposition in the adhesion site in the film group and the adhesion region was much looser. For the rats in hydrogel-treated group, the histological observations of the injured abdominal wall and cecum were performed separately due to few adhesion was

18

observed. The injured abdominal wall and cecum were recovered with new peritoneal mesothelium without postoperative adhesion within 7 days, although there were still some fibroblasts, inflammatory cells, and fibrosis appeared at the damaged sites. After 2 weeks, dense adhesion with much collagen deposition was observed between the injured cecum and damaged abdominal wall, and the thickness of adhesion region was increased in rats of both the control and film group (Fig. 8B). Besides, some new blood vessels could be found in the adhesion site. However, for the rats treated with mXG hydrogel, the damaged abdominal wall has been completely remesothelialized (Fig. 8B), and the histological structure was similar to that in the normal tissue (Fig. S3). The similar result was also observed in the injured cecum. All these results indicated that the developed mXG hydrogel could serve as an effective barrier to prevent the postoperative adhesion. In addition, these results also illustrated that mXG hydrogel and its degradation product did not affect the wound healing. 3.9 In vivo toxicity observation

In order to evaluate the potential toxicity of mXG hrdrogel in vivo, all of the rats treated with mXG hydrogel were observed and recorded after surgery. All the animals displayed free movement and normal behavior without any adverse reactions throughout the experiments. Moreover, few side effects such as slow movement, eye secretion, or running nose were observed. The major organs were harvested on day 7, 14, 28, and 56 to stain with HE for further histopathologic examination. As shown in Fig. 9, no significant differences were observed in the organs of the hydrogel-treated rats when compared with that in the normal tissue. For example, the cardiac myocytes from rats treated with mXG hydrogel was similar to that in the normal tissue, which were clear and arranging in good order, and no hemorrhage or necrosis was observed. It indicated that the mXG hydrogel was non-toxic when used for adhesion prevention in vivo.

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4. Disscussion

Peritoneal adhesion is a common complication after abdominal surgery. Although numerous drugs or barrier-based devices have been developed and used to prevent postsurgical adhesion, few of these measures have proven to be uniformly effective in the subsequent clinical trials. An ideal barrier system for adhesion prevention not only should be convenient to handle and apply, but also can provide unrestricted coverage on the surface of the defects with irregular shape [6,40,41]. Additionally, the anti-adhesion material should have excellent biocompatibility and an appropriate degradation rate [41,42]. In the present work, we successfully developed in situ injectable mXG hydrogel for adhesion prevention. XG is a natural polysaccharide and was extracted from tamarind gum with water and ethanol. The mXG was prepared by β -galactosidase reaction, which was a green chemical reaction without using any organic reagents. All these processes can ensure the resulting mXG is biocompatible. 4% (w/v) mXG solution exhibited reversible sol-gel transition with the change of temperature (Fig. 2A) and the sol-gel transition occurred at 23 °C (Fig. 2C). The appropriate Tgel enables its potential as injectable material for biomedical applications. Moreover, it should be noted that mXG solution can form hydrogel at a lower concentration (less than 4 wt%) than that for

the

thermosensitive

synthetic

polyester-polyether

block

copolymer,

including

PLGA-PEG-PLGA [21], PCGA-PEG-PCGA [23], PEG-PCL-PEG [22], PCL-PEG-PCL [42], and PECT [43], etc, which always need a high concentration above 20 wt%. Besides, compared with the in situ chemically cross-linked hydrogel, 4% mXG solution could spontaneously form hydrogel without any extra additives or chemical reaction. However, for a chemical hydrogel, the application of chemical initiators, cumbersome ultraviolet (UV) illumination or any other treatments for triggering chemical reactions in vivo may bring with biocompatibility problems to a certain extent, and also restrict its practical clinical applications.

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The gelation time has an important influence on the application of the injectable hydrogel for adhesion prevention. If the gelation rate is too fast, the injured surfaces cannot be completely covered before gelation, whereas a long gelation time may prolong operation time and increase the risk of infection. For example, Liu et al. prepared chemically crosslinked hyaluronan hydrogels to reduce postoperative abdominal adhesions, while the relatively long gelation time (10–45 minutes) greatly hampered its application in clinical practice [29]. In the present work, the gelation of 4% (w/v) mXG solution occurred at 20 s and almost completed within 3 min at physiological temperature (Fig. 2D and E). As a result, the viscous solution could maintain on the damaged tissue with irregular shape after injection and rapidly turned into a non-flowing hydrogel. Therefore, it was an appropriate time for the application in adhesion prevention. As mentioned above, the physical mXG hydrogel has excellent thermosensitivity, injectability, and appropriate gelation time and temperature, which make it easy to handle and cover the affected tissues unrestrictedly as compared with the solid anti-adhesion sheets, particularly in minimally invasive laparoscopic surgery. Besides, the final elastic moduli of the 4% mXG hydrogel at 37 °C were approximately 1000 Pa (Fig. 2D), which is much higher than that of many synthetic physical hydrogels [21,22,23,43]. As a result, the mXG hydrogel could present as a durable physical membrane after being injected onto the defects and maintain its integrity, even in the environment of abdominal cavity with gastrointestinal peristalsis. Both in vitro MTT assay indicated the mXG hydrogel was non-toxic (Fig. 3A and B). The good hemocompatibility of mXG hydrogel was confirmed by hemolysis test (Fig. 4C and D). These results suggested mXG hydrogel might be suitable for biomedical applications. In vitro degradation experiment showed the weight loss of mXG hydrogel gradually increased over time (Fig. 4A) and the degradation products had no impact on the pH of the medium (Fig. 4B). In vivo degradation experiment suggested that mXG hydrogel was biocompatible (Fig. 5B) and could be

21

degraded completely within 6 weeks (Fig. 5A), which was a satisfactory persistence period for the adhesion prevention application. In addition, the biodegradability and bioabsorbability of the mXG hydrogel could avoid the second surgery and enhance patient compliance. Generally, peritoneal adhesions usually form within 7-10 days after surgery and result from a complex cascade regulated by different cellular and humoral factors [1,2]. On day 4 or 5 after trauma, fibroblasts get to invade the fibrinous adhesion, and the initial fibrin-composed matrix is gradually replaced by deposited collagen, leading to the formation of permanent fibrous adhesion. According to the process of adhesion formation, it is very important to block the invasion of fibroblasts to prevent peritoneal adhesion [1,3,6]. The mXG hydrogel displayed a good performance on prevention of fibroblasts adhesion (Fig. 3D), which might be explained by the hydrophilicity of mXG hydrogel surface. According to the gelation mechanism of mXG, hydrophobic domains (main chain) of mXG aggregate to minimize the hydrophobic surface area contacting the bulk water with increasing temperature, thus exposing the hydrophilic segments (galactose moieties) to the surrounding water and increasing the hydrophilicity of the surface of resulting hydrogel [38]. As a result, the excessively hydrophilic surface of mXG hydrogel could effectively resist the adhesion of fibroblasts. It may be the potential anti-adhesion mechanism of mXG hydrogel. These results suggested that the mXG hydrogel with good resistance to fibroblasts adhesion could be a potential candidate for adhesion prevention. Finally, the rat model of sidewall defect-cecum abrasion was employed to evaluate the anti-adhesion efficacy of the mXG hydrogel, while the commercialized chitosan film was used for comparison. Compared with the normal saline group, the adhesion score and adhesion area of the film group was obviously reduced. However, the results revealed that mXG hydrogel could more effectively prevent postoperative peritoneal adhesions without side effects and promote the remesothelialization of injured cecum and abdominal wall (Fig. 7C and F) as compared with

22

commercialized chitosan film group and normal saline group. After being injected onto the defects, the in situ formed mXG hydrogel was able to firmly adhere to the peritoneal wounds and could serve as a physical barrier to prevent the coalescence of the damaged cecum and injured abdominal wall (Fig. 6D and E). Then, the hydrophilic surface of covered Mxg hydrogel could prevent the invasion of fibroblasts, and also prevent the adhesion and deposition of the fibrous proteins, which enhanced the anti-adhesion efficacy and created favorable conditions for mesothelial regeneration during the critical period after surgery. Therefore, the excellent anti-adhesion effects may attribute to the combination of barrier function and fibroblast-adhesion resistance property of the mXG hydrogel. 5. Conclusion

In this work, the injectable thermoreversible mXG hydrogel was successfully prepared and characterized, which was a free flowing sol at low temperature and could rapidly form a gel spontaneously at the body temperature. The developed physical hydrogel showed excellent biodegradability and biocompatibility both in vitro and in vivo. In a rat model of sidewall defect-bowel abrasion, the mXG hydrogel was easy to handle and showed significant efficacy in preventing the formation of postoperative peritoneal adhesion. These results suggested that the biodegradable and thermosensitive mXG hydrogel could serve as a safe and effective anti-adhesion material and may have potential applications in clinical practice. Disclosure

The authors report no conflicts of interest in this work. Acknowledgments

This work is supported by National Natural Science Foundation of China (Grant No. 31271016 and 51573127).

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Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version. References

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[23] L. Yu, H. Hu, L. Chen, X. Bao, Y. Li, L. Chen, G. Xu, X. Ye, J. Ding, Comparative studies of thermogels in preventing post-operative adhesions and corresponding mechanisms, Biomater. Sci. 2 (2014) 1100-1109. [24] S. Sakai, K. Ueda, M. Taya, Peritoneal adhesion prevention by a biodegradable hyaluronic acid-based hydrogel formed in situ through a cascade enzyme reaction initiated by contact with body fluid on tissue surfaces, Acta Biomater. 24 (2015) 152-158. [25] J. Yang, J. Yeom, B. Hwang, A. Hoffman, S. Hahn, In situ-forming injectable hydrogels for regenerative medicine, Prog. Polym. Sci. 39 (2014) 1973-1986. [26] U.P. Shinde, B. Yeon, B. Jeong, Recent progress of in situ formed gels for biomedical applications, Prog. Polym. Sci. 38 (2013) 672-701. [27] S.H. Oh, J.K. Kim, K.S. Song, S.M. Noh, S.H. Ghil, S.H. Yuk, J.H. Lee, Prevention of postsurgical tissue adhesion by anti‐inflammatory drug‐loaded pluronic mixtures with sol–gel transition behavior, J. Biomed. Mater. Res. A. 72 (2005) 306-316. [28] J.L. West, J.A. Hubbell, Comparison of covalently and physically cross-linked polyethylene glycol-based hydrogels for the prevention of postoperative adhesions in a rat model, Biomaterials 16 (1995) 1153-1156. [29] Y. Liu, H. Li, X.Z. Shu, S.D. Gray, G.D. Prestwich, Crosslinked hyaluronan hydrogels containing mitomycin C reduce postoperative abdominal adhesions, Fertil. Steril. 83 (2005) 1275-1283. [30] A. Mishra, A.V. Malhotra, Tamarind xyloglucan: a polysaccharide with versatile application potential, J. Mater. Chem. 19 (2009) 8528-8536. [31] S. Miyazaki, F. Suisha, N. Kawasaki, M. Shirakawa, K. Yamatoya, D. Attwood, Thermally reversible xyloglucan gels as vehicles for rectal drug delivery, J. Control. Release 56 (1998) 75-83. [32] M. Shirakawa, K. Yamatoya, K. Nishinari, Tailoring of xyloglucan properties using an enzyme, Food hydrocolloids 12 (1998) 25-28. [33] S. Miyazaki, S. Suzuki, N. Kawasaki, K. Endo, A. Takahashi, D. Attwood, In situ gelling xyloglucan formulations for sustained release ocular delivery of pilocarpine hydrochloride, Int. J. Pharm. 229 (2001) 29-36. [34] D.R. Nisbet, A.E. Rodda, M.K. Horne, J.S. Forsythe, D.I. Finkelstein, Implantation of functionalized thermally gelling xyloglucan hydrogel within the brain: associated neurite infiltration and inflammatory response, Tissue Eng. A 16 (2010) 2833-2842. [35] A.P. Busato, F. Reicher, R. Domingues, J.L.M. Silveira, Rheological properties of thermally xyloglucan gel from the seeds of Hymenaea courbaril, Mater. Sci. Eng. C 29 (2009) 410-414.

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Table 1. Monosaccharide composition of XG and mXG.

a

Sample

Galactose a

Xylose a

Glucose a

Galactose removal ratio (%)

XG

0.080

0.215

0.293

0

mXG

0.047

0.261

0.291

41

Data are expressed as mmol of monosaccharide per 100 mg of the sample.

Table 2. Distribution of adhesion scores in the control, film, and hydrogel group on day 7 and 14 after surgery. 1 week

2 weeks

Adhesion score Control group (n=8)

Film group (n=8)

Hydrogel group (n=8)

Control group (n=8)

Film group (n=8)

Hydrogel group (n=8)

Score 5

2 (25%)

1(12.5%)

0

7 (87.5%)

2 (25%)

0

Score 4

6 (75%)

3(37.5%)

0

1 (12.5%)

4 (50%)

0

Score 3

0

3(37.5%)

0

0

2 (25%)

0

Score 2

0

1(12.5%)

0

0

0

0

Score 1

0

0

1 (12.5%)

0

0

2 (25%)

Score 0

0

0

7 (87.5%)

0

0

6 (75%)

28

Figure Captions Figure 1. Characterization of the XG and mXG. (A) The structure of the repeating units, (B) 1

H-NMR spectrum, (C) FTIR spectrum, and (D) GPC trace of XG. (E) HPAEC-PAD traces of

monosaccharides in XG and mXG. Figure 2. Thermosensitive behaviors of XG and mXG solution at the concentration of 4% (w/v).

(A) Visual observation of mXG solution at 4 °C (sol) and at 37 °C (gel). Temperature dependence of the storage modulus (G•)) and loss modulus (G'') for (B) XG and (C) mXG solution, respectively. (D and E) Time dependence of G• and G• for mXG solution at 37 °C, the region of dashed rectangle in Figure D was clearly displayed in Figure E. Figure 3. Cytotoxicity and cell adhesion evaluation of the mXG hydrogel. Viability of L929

cells cultured with different concentrations of hydrogel extract (A) and mXG solutions (B) for 24 h and 48 h, respectively. The untreated cells incubated with regular culture medium were set as control. Fluorescence images of L929 cells cultured on TCPS (C) and on the surface of mXG hydrogel (D) for 48 h. Figure 4. In vitro degradation and hemolysis studies of mXG hydrogel. (A) Weight loss of mXG

hydrogel in vitro degradation in PBS at 37 °C. (B) pH change of the medium along the degradation of mXG hydrogel. (C and D) Hemocompatibility of mXG hydrogel extract, mXG solutions of 0.1 mg/mL and 1 mg/mL, with normal saline and distilled water as negative and positive control, respectively. Figure 5. In vivo degradation and biocompatibility of mXG hydrogel after subcutaneous

injection. (A) Gross observations of mXG hydrogel formation and in vivo degradation at different time points. (B) Histological observations of HE-stained slices of tissues surrounding

29

the injection site at different time points. M: muscle tissue; S: subcutaneous tissue; the region of dashed circle indicate inflammatory cells. Figure 6. The establishment of a rat model of sidewall defect-cecum abrasion and the

application of film and mXG hydrogel onto the defects. (A) The normal abdominal sidewall and cecum. (B) The establishment of abdominal sidewall defect and cecum abrasion, the arrows were used to indicate the defect created on the abdominal wall. (C) The film was applied on the damaged abdominal wall. (D and E) mXG hydrogel was applied on the damaged cecum and injured abdominal wall. Figure 7. Prevention of postoperative peritoneal adhesions in a rat sidewall defect-cecum

abrasion model. (A and B) Adhesions were observed in the control and film group on day 7 after surgery. (C) No adhesion between the defected wall and abrased cecum was observed in rats treated with mXG hydrogel on day 7. (D) Serve adhesions were observed in the control group on day 14. (E) Film group clearly presented compact adhesion on day 14. (F) No adhesion was observed in rats treated with mXG hydrogel on day 14 after surgery. Figure 8. Histological examination of tissues from rats treated with normal saline (NS), film,

and mXG hydrogel on day 7 (A) and 14 (B) after surgery, respectively. CE: cecal mucosa; AW: abdominal wall; Me: mesothelial layer; SK: skeletal muscle. For Masson trichrome staining, the deposited collagen in the adhesion site were stained in blue while muscle showed red. For Van Gieson staining, the deposited collagen were stained in red while muscle showed yellow. Figure 9. Histological examinations of major organs (heart, liver, spleen, lung, and kidney) from

normal rat and the rats treated with mXG hydrogel on day 7, 14, 28 and 56 after surgery.

30

31

32

33

34

35

36

37

38

39

Statement of Significance Despite numerous drugs or barrier-based devices have been developed to prevent postoperative adhesion, few solutions have proven to be uniformly effective in subsequent clinical trials. In the present study, we developed a biodegradable and thermoreversible galactose modified xyloglucan (mXG) hydrogel by green enzymatic reaction without using any organic reagents. The developed physical mXG hydrogel not only showed excellent injectability, appropriate gelation time and temperature, but also exhibited excellent biocompatibility and biodegradability both in vitro and in vivo. In addition, mXG hydrogel was easy to handle and could effectively prevent postoperative adhesion without side effects in a rat model of sidewall defect-bowel abrasion. Our study provide a safe and effective postoperative anti-adhesion material which may have potential applications in clinical practice.

40

Graphical Abstract

41