Fabrication of a photo-crosslinked gelatin hydrogel for preventing abdominal adhesion

Abstract

Natural hydrogels are promising membranes used to prevent intra-abdominal adhesion formation. Currently, natural hydrogels such as chitosan-, gelatin- or hyaluronic acid-based hydrogels are utilized to prevent adhesion. However, their uncontrollable mechanical properties and quick degradation result in an unsatisfying short effect-time. In this study, a photocrosslinkable gelatin (GelMA) prepolymer was synthesized and developed for preventing intra-abdominal adhesion formation. Hydrogel membranes based on GelMA showed easy-handleability, non-toxic degradation and a long-lasting excellent barrier effect for up to 1 month. We have found that a 20% GelMA hydrogel membrane concentration could be employed to meet the requirements of excellent barrier effect, and the implantation of GelMA hydrogel membranes in rat abdominal cavities in experimental groups led to a considerable decrease in adhesion formation in comparison to the control group. The present study established the initial foundation for a novel and practical approach to prevent abdominal adhesion in surgery.

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Introduction

Intra-abdominal adhesion is a common complication that follows abdominal surgery where tissues that are normally separated become adhered together by an insoluble fibrotic tissue.^1,2 The formation of the adhesion between normally separated tissues stems from various events such as burns, infections, trauma, peritoneal dialysis and foreign materials.^3–5 The adhesion may lead to severe abdominal pain, infertility and bowel obstruction.^6,7 In approximately 10% of adhesion cases, a portion of the bowel may become entwined with the fibrotic tissue, resulting in tissue infarction and ischemia, which eventually leads to tissue necrosis.⁸ Current attempts to prevent intra-abdominal adhesion formation include pharmacological approaches which target the biochemical and cellular events, as well as membranes, films and viscous intraperitoneal solutions which act as a physical barrier to limit adhesion formation.^9,10 A physical barrier can prevent peritoneal adhension during the healing process by separating injured regions, and thus becomes an attractive approach.¹¹ However, shortcomings remain with commercially available physical barriers based on both natural and synthetic polymers. Apart from efficacy and practicality questions, natural polymer devices showed short effect-time and synthetic polymer devices could induce the tissue inflammation after implantation and degradation.

Recently, various physical membranes, such as electrospun fibrous membranes, films, gels, sponges and so on, have been used as barriers to minimize extrinsic healing, an important part of adhesion formation.¹² Hydrogel membranes based on crosslinkable polymers draw tremendous attention because of their biologically and physically advantaged properties, such as excellent biocompatibility, in situ crosslinkability and adjustable mechanical, swelling and degrading properties.¹³ Moreover, synthetic polymers, possessing better handleability and mechanical properties and containing fewer bio-contaminants and immunogens compared to natural polymers, are considered more preferable to engineer hydrogel membranes as physical barriers.¹⁴ Yet, there are certain limitations in the application of synthetic hydrogel membranes to prevent intra-abdominal adhesion. Membranes based on nonabsorbable hydrogels stay undegraded at where they are applied until removal operation, while absorbable membranes degrade faster than requirement and often cause serious acute inflammation which brings about tissue adhesion.^15–17 Therefore, the aim of this project is to fabricate natural polymer membranes with good water retention, mechanical and degradation characteristics.

Previously, by incorporating reactive methacryloyl groups to gelatin of natural polymers, we synthesized a photocrosslinkable gelatin (GelMA) prepolymer.¹⁸ Prior studies indicate that GelMA hydrogels are biocompatible materials which can be crosslinked in vitro to form a physical barrier.¹⁹ The present work innovatively applies GelMA hydrogels containing hyaluronic acid in the prevention of intra-abdominal adhesion formation which requires good handleability, non-toxic degradation and excellent and long-lasting barrier effect for up to 1 month. The physical properties of GelMA hydrogels containing hyaluronic acid are investigated to evaluate their clinical operation characteristics. Furthermore, the anti-adhesion efficacy of the GelMA-based membrane was assessed in vivo through a rat cecum abrasion model. The objective of this study is to investigate the efficacy of the GelMA hydrogel barrier in preventing the formation of intra-abdominal adhesive tissues.

Materials and methods

Materials

Gelatin (derived for porcine skin) was purchased from Yuancheng Technology Co. (Wuhan, China) and used without further purification. Methacrylate anhydride, dialysis membranes (10 000 to 14 000 kDa), PBS solution was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). PI (sigma) was obtained from Shanghai Qiangshun Chemical Reagent Co., Ltd (Shanghai, China). The sodium salt of hyaluronic acid powder (HA, M_w = 1.0 MDa) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and used without further purification. All other chemicals and solvents were of reagent grade or better and were purchased from GuoYao Regents Company (Shanghai, China), unless otherwise indicated.

Gelatin methacrylate synthesis

GelMA was synthesized using methods previously described.²⁰ Type A gelatin derived from porcine skin was mixed at a 10% (w/v) in PBS and stirred at 60 °C until the gelatin was fully dissolved. Methacrylate (MA) was added dropwise to the gelatin solution over a period of one hour. Following the addition of MA, the reaction system was covered with foil and left to react in the dark for 3 hours. The solution was diluted by 5 times with 40 °C PBS in order to stop the reaction. Unreacted MA and other salts were removed from the solution via 1 week of dialysis using 10–14 kDa cut-off membranes. After the solution was dialyzed for one week, the solution was vacuum filtered via a membrane with a pore size of 0.22 μm. The filtered solution was then lyophilized to obtain a white, spongy foam. The foam was stored in a desiccator until needed.

Preparation of GelMA hydrogel membranes

The GelMA foam was dissolved in PBS to achieve GelMA solutions with concentrations of 10% and 20% (w/v), and 1.5% HA (w/w) was also dissolved into 10% and 20% GelMA solution. Following the addition of GelMA, 1% (w/v) of 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone was added to the solution as a photoinitiator. The prepolymer solution was then pipetted into a culture dish (Φ = 8 cm, made from polypropylene) and exposed to UV light (360–480 nm, 1.5 W, 85 mm working distance, 6.9 mW cm⁻²) for 2 minutes to form a 0.4 cm thick membrane. The GelMA samples were freeze dried and used for scanning electron microscope (SEM, QUANTA 250, FEI, US).

Swelling and degradation measurements

GelMA samples were lyophilized and weighed to find their initial mass. The lyophilized samples were then immersed in PBS for 24 hours. Samples were blotted dry using a KimWipes and weighed (W_s). Swelling percentages were calculated.²¹

The degradation study of the membranes was carried out in vitro by placing the samples in PBS solution at pH 7.4 and 37 °C at different time points. At each measurement, the samples were then removed from the recipient and weighed after drying at 40 °C. The weight loss was calculated using the before–after weight changes equation. Each degradation experiment was repeated three times and the average value was considered.

Rat cecum abrasion surgical model

The surgical procedures were performed under the institutional review committee of Shanghai Jiao Tong University, School of Medicine and the National Institutes of Health. Sprague-Dawley rats weighted 350–450 g were randomized into three groups as follow: 10% GelMA membranes group, 20% GelMA membranes group and control group. After anesthesia with ketamine (20 mg kg⁻¹) by intramuscular injection, all the rats underwent a mid-line laparotomy incision to remove the peritoneum so that a 1 cm × 1 cm defect was created between the abdominal wall and the cecum.²² For 10% and 20% GelMA membranes group, 2 cm × 2 cm 10% or 20% GelMA membranes were applied to each wound. No treatment was given to the control group. 4–0 silk sutures were applied to close the mid-line incision layer by layer.

Macroscopic evaluation

Visual check was conducted to examine the inflammation or ulcer signs on the skin incision. Fourteen days after the operation, the rats were sacrificed. A semiquantitative grading scale (Table 1) was designed to classify the extent of adhesion based on the macro view.^23–25

Table 1 Semiquantitative grading scale

Grade	Macro view
0	No adhesions
1	Thin and film adhesions easily separable with blunt dissection
2	Moderate adhesions with freely dissection plane
3	Severe adhesions with fibrosis difficult dissection plane

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The macroscopic evaluation was conducted by two independent investigators who were blind to this study.

Statistical analysis

Ten animals in each sample were studied to determine the abdominal adhesion. The results were presented as mean ± standard deviation and analyzed by one way ANOVA (analysis of variance) in SPSS 10.0 (p < 0.05).

Results

Morphology of hydrogel membrane

Synthesis of GelMA was described previously. A water-soluble initiator was applied to conduct the photocrosslinkage of GelMA under exposure to UV (Fig. 1a). It was found that the hydrogel membrane formed after UV polymerization which showed good handleability and can be easily cut for animal experiment and clinical applications (Fig. 1b). The internal microstructure of the GelMA is revealed by aid of SEM, illustrating a loose, scaly cross-section with pores averagely sized 238.5 ± 89.7 μm.

Fig. 1 Chemical structure of GelMA hydrogel network (a), GelMA hydrogel membrane in the hand (b) and microstructure of GelMA membranes after freezing dry (c).

Characterization of hydrogel membrane

The GelMA hydrogels with different concentrations were weighed before and after incubation in DPBS at 37 °C (±0.5 °C) for 24 h until equilibrium was established.²⁶ Swelling ratio (Fig. 2) of the GelMA hydrogels is analyzed from the weight data and utilized to study their swelling behaviors such as water absorption capacity from which the degradation ratio could be inferred. As shown in Fig. 2, the swelling ratio of GelMA declines from 720% to 510% as the concentrations rise from 10% to 20% (p < 0.05), which is believed to result from the significant increase in crosslinking densities brought about by the increasing GelMA concentration,²⁷ a property that sets a limitation of both water penetration capacity and degradation rate for GelMA hydrogel.

Fig. 2 Equilibrium swelling properties of hydrogel with various GelMA concentrations.

GelMA hydrogel is able to simulate microenvironment of flexible, hydrated natural soft tissues and is enzyme-degradable because of their natural amino acid constituents. Degradation by protease enzyme has been found to be important for regulating tissue functions. The feature of hydrogel degradation is further studied in this research. As shown in Fig. 3, the residual percentage of 10% GelMA dropped sharply while 20% GelMA degraded steadily. 10% GelMA hydrogels membrane disappeared within 15 days. However, the 20% GelMA hydrogel membranes kept a residual percentage of 30% even after 5 weeks of incubation. This discovery makes 20% GelMA more preferable than 10% GelMA to be applied in long-term prevention of abdominal adhesion.

Fig. 3 Mass retention during degradation of GelMA hydrogel of 10% and 20% concentrations.

Gross observation

Tissue adhesion extent was evaluated by macroscopic examination. No signs of inflammation were exhibited around the skin wound in all groups. Both hydrogel membrane groups experienced an amazing reduction in adhesion formation compared to the control group, and 20% GelMA hydrogel membranes showed superior anti-adhesion effects than 10% GelMA hydrogel membranes did (Fig. 4).

Fig. 4 Gross evaluation of a rat cecum abrasion model after 14 days.

Large area of severe adhesion happened to all the animals in the control group that received no treatment for peritoneal defect, resulting in high average scores in the semiquantitative grading of the adhesion extents, while the average scores of both GelMA hydrogel membranes groups were substantially lower. Furthermore, the 20% GelMA hydrogel membranes group achieved an even lower average score than the 10% group. Consequently, notable potential is seen in 20% GelMA hydrogel membranes to prevent intra-abdominal adhesion (Fig. 5).

Fig. 5 The average scores of intra-abdominal adhesion in three group (n = 5). (*p < 0.05).

Discussion

This investigation focuses on the applicability of natural polymers membranes to the prevention of intra-abdominal adhesion formation. GelMA hydrogel was chosen because it was previously found to remain for at least 1 month in PBS and prevent adhesion in animal models, as well as showing high degree of biocompatibility, easy-handling properties, non-toxic degradaion and excellent barrier effect. The main consideration of the design was presented in the first part of the paper. By means of in vivo animal study, prevention of tissue adhesion in abdominal cavity by GelMA hydrogel membranes was observed, and better performance of anti-adhesion in rat abdominal cavity was found in 20% GelMA than in 10% GelMA.

The GelMA hydrogel membrane is an ameliorated natural material based barrier system without cytotoxic crosslinking agent, which other systems such as gelatin hydrogel inevitably contain.^28,29 As clinical proved degradable barriers, chitosan and gelatin inhibit adhesion formation by separating injured tissue while promoting healing process.^29,30 However, their physical fragility and tendency to adsorb moisture add difficulty to the placement procedure and thus limits their application in open surgical procedures. Oppositely, GelMA hydrogel shows improved tensile strength, making itself more suitable as an anti-adhesion physical barrier.

As the in vivo anti-adhesion result demonstrates, 20% GelMA hydrogel membranes showed superior anti-adhesion effects in rat abdominal cavity than 10% GelMA hydrogel membranes did possibly owing to the synergistic activity of natural materials as well as the barrier effect of the hydrogel film. In conclusion, hydrogel membrane is potentially an effective approach to prevent adhesion formation. In our research, the implantation of GelMA hydrogel membranes in rat abdominal cavity in experimental groups led to a considerable decrease in adhesion formation in comparison to the control group. The present study established the initial foundation for a novel and practical approach to prevent abdominal adhesion in surgery.

Conclusions

In this study, GelMA hydrogels were developed for preventing intra-abdominal adhesion formation for their easy-handle properties, non-toxic degradation as well as excellent and long-lasting barrier effect for up to 1 month. We have found that the 20% GelMA hydrogels membranes could be employed to meet the requirements of excellent barrier effect. The implantation of GelMA hydrogel membranes in rat abdominal cavity in experimental groups led to a considerable decrease in adhesion formation in comparison to the control group. The present study established the initial foundation for a novel and practical approach to prevent abdominal adhesion in surgery.

Acknowledgements

This work was supported in part by Social development project of Yangzhou Key Research Program (YZ2015070). We would like to thank Dr Shen Liu of Shanghai Jiao Tong University for help of their animal experiments.

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Footnote

† These authors contributed equally to this work.

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