Article pubs.acs.org/Biomac
A Biodegradable Trilayered Barrier Membrane Composed of Sponge and Electrospun Layers: Hemostasis and Antiadhesion Qinghua Xia,†,‡,∥ Ziwen Liu,§,∥ Chenhong Wang,†,‡ Zixin Zhang,†,‡ Shanshan Xu,† and Charles C. Han*,† †
State Key Laboratory of Polymer Physics and Chemistry, Joint Laboratory of Polymer Science and Materials, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China § Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, P. R. China ABSTRACT: Placing a physical barrier between the injured site and the adjacent tissues is a very common and highly effective approach to prevent abdominal adhesions in these days. A biodegradable trilayered barrier was fabricated to prevent formation of abdominal adhesions, in which a poly(lactide-co-glycolide)/ poly(lactide)-b-poly(ethylene glycol) (PLGA/PLA-b-PEG) electrospun layer was sandwiched between layers of carboxymethyl chitosan (CMCS) sponge. The hydrophilic CMCS sponge layers with glycerin (GL) could adhere to the surface of wound easily, and present great hemostatic capability. The mechanism of the formation of adhesion related to blood clots acting with fibroblast cells was evaluated in detail. The blood clot acted as a “medium” inducing the fibroblast cells growth and proliferation, but had no special attraction on epithelial cells. CMCS sponge layer took away the blood clots during the swelling and dissolution stages. The electrospun layer promoted the growth of epithelial cells, but exhibited inhibition on the adhesion and spread of fibroblast cells, which ensured excellent effect of adhesion prevention. Evaluated by a rat model of sidewall defect-bowel abrasion, significant reductions of postoperative adhesion in its level and occurrence were observed in animals treated by the trilayered barrier. effective in all situations.1,2,4 Thus, there remains an urgent need for an improved product, which is highly effective and suitable for various surgical conditions. Hemorrhage is common in surgery and hard to stop.8 The role of blood in the formation of adhesions is still controversial.9 Some works focus on the collagen membrane10 or carboxymethyl chitosan products11 for antiadhesions in cardiac surgery, however, hemostasis still has not been paid enough attention in investigations of abdominal antiadhesive materials so far. Previous works12,13 of our research group has shown that PLGA/PLA-b-PEG (85/15, w/w) electrospun membrane (e-membrane), which has appropriate degradation behavior in vitro and in vivo, is suitable as a drug carrier for hemostasis and anti-infection. However, the metabolism of drug is complicated in general, and the usage of drugs may cause immunosuppression and delay of wound healing.14 In this study, bioabsorbable materials with hemostatic properties instead of the drugs were used to combine with the emembrane in order to provide reasonable assurance of safety and effectiveness. Carboxymethyl chitosan (CMCS) is the choice for hemostasis in this work. As a water-soluble chitosan
1. INTRODUCTION Postoperative abdominal adhesions, which are the most common adverse effects after surgical operation, affect millions of individuals around the world and cause billions of dollars in the health-care costs.1 One common and highly effective approach to prevent adhesion is to place a physical barrier between injured site and the adjacent tissues.1,2 The need to reduce adhesions and the other associated postoperative complications has driven the development of a number of biodegradable antiadhesion barriers.3 Some have been used commercially and clinically.4 Seprafilm, which is composed of sodium hyaluronate and carboxymethyl cellulose, could adhere to a wound surface and mechanically separate the tissues during the postoperative healing process.5 Nevertheless, the film is hard to handle because of its brittleness and would strongly adhere to objects with any moisture on the surface even on the surgeon’s gloves during placement.6 Interceed, which is composed of oxidized regenerated cellulose, is reported to significantly reduce adhesions in some animal models and clinical studies.1,7 However, this is true only when the entire area is completely hemostatic. Normally, it is difficult to ensure that all blood is cleared from the surgical field.8 Although many newer products for practical application have been attempted to fulfill the criteria for adhesion prevention, none of them is © 2015 American Chemical Society
Received: August 14, 2015 Published: August 25, 2015 3083
DOI: 10.1021/acs.biomac.5b01099 Biomacromolecules 2015, 16, 3083−3092
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Biomacromolecules
used to observe the surface and cross-section morphology of all prepared materials after being sputter-coated with platinum. 2.3.2. Tensile Test. The tensile test was performed using a universal electromechanical tester (Instron 3365, USA) at a speed of 50 mm/ min. E-membrane and trilayered barrier were both cut into 5 cm × 1 cm rectangular specimens, respectively, and six repetitions for each sample were carried out in the test. 2.3.3. FT-IR Analysis. Fourier transform infrared (FT-IR) spectra were obtained from a TENSOR 27 (Bruker, Germany) with resolution of 4.0 cm−1 and 32 scans in the range of 4000−400 cm−1. Surface chemical structure of trilayered barrier before and after incubated in phosphate buffer solution (PBS, pH = 7.4, 37 °C) for 6 h was evaluated, compared with that of e-membrane. 2.4. In Vitro Blood Coagulation Test of CMCS Sponge. The CMCS sponge was cut into about 1 × 1 cm2 squares and placed in culture dish. 100 μL of blood (containing the anticoagulant sodium citrate at a ratio of 1:9) was dispensed onto the surface of each sample. The same amount of blood was dropped onto the culture dish directly in control group. All dishes were incubated at 37 °C for 5 min. Then, 50 mL of distilled water was carefully added into the culture dish. Red blood cells, which were not entrapped in the clots, would hemolyze in water. Subsequently, a hemoglobin assay kit (DIHB-250, BioAssay Systems, USA) was used for quantitative determination of blood clots on the different samples. Blood clots on sponge were gathered and dissolved in the reagent. After being diluted to the concentration range of the calibration curve, the specimens were measured by UV spectrophotometry (Beijing Purkinje General Instrument Co. Ltd., China) at 400 nm wavelength. Five samples were averaged for each group in this test. 2.5. In Vitro Cytocompatibility and Cell Adhesive Behavior. 2.5.1. Cell Culture. Fibroblasts cells (L929, Cell Resource Center, IBMS, AMS/PUMC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Hyclone), 1% Pen Strep (10,000 IU/mL penicillin and 10 000 μg/mL streptomycin solution, Invitrogen). Epithelial cells (IEC-6, Cell Resource Center, IBMS, AMS/PUMC) were cultured in DMEM containing 5% FBS, and 0.01 mg/mL insulin. All the cells were cultured at 37 °C in a 5% CO2 atmosphere incubator. The medium was changed every other day. 2.5.2. Cytocompatibility Assay. In vitro cell viability in the presence of CMCS sponge, PLGA/PLA-b-PEG e-membrane, and trilayered barrier was investigated by the cell counting kit-8 (CCK-8, Dojindo, Japan) testing. All the materials were decontaminated under UV light for 2 h. One ×104 cells per well were transferred into 96-well plates to incubate 24 h before the materials were added. The group without any material was set as control. At given time intervals, all materials were taken out and the cell-seeded wells were washed twice with PBS to remove unattached cells. Then, fresh medium with 10% CCK-8 was added and cells were further incubated for 2 h. The optical density (OD) value at 450 nm was measured by a microplate reader (Multiskan GO, Thermo Scientific, USA). Seven wells were averaged for each group in this test. 2.5.3. Cell Adhesive Behavior. In this test, the materials included PLGA/PLA-b-PEG e-membranes covered with and without clots, as well as trilayered barrier covered with clots. Before cell seeding, all samples were placed in 96-well tissue culture plates (TCP) and decontaminated under UV light for 2 h, then the cells were transferred at a density of 1 × 104 cells per well, respectively. The cells were tested after given time intervals. For CCK-8 testing, all cell-seeded materials were washed twice with PBS to remove unattached cells. Fresh medium with 10% CCK-8 was added and cells were further incubated for 2 h. Then, 100 μL of the indicator solution was removed from each well into another plate for absorbance measurement. Seven wells were averaged for each group in this test. For confocal laser scanning microscope (CLSM, Olympus, Japan) observation, the specimens were taken out and washed twice with PBS to remove unattached cells and fixed in 4% (v/v) formaldehyde solution for 30 min. Then, the cell seeded samples were incubated
derivative and overall negatively charged after the introduction of carboxymethyl groups on chitosan chains,15 CMCS has promising potential in many biomedical applications for great properties, such as biocompatibility, hemostasis and antibacterial activity.15,16 It has been reported that CMCS-based freezedrying superabsorbent was applied for its high water binding capacity in water and saline solution.17 Also, calcium could chelate with chitosan and its derivatives, improving intermolecular cross-linking reaction gradually.18 Thus, a certain amount of CaCl2 (about 6% to mass of CMCS), which is determined by clinical dosage, is added into CMCS solution to transform linear structure into a grid structure and improve the hemostatic effect in this study.19 Since the CMCS sponge can easily be broken for its brittleness, glycerine (GL) is added as a softener and humectant20 to keep the membrane soft and flexible. The aim of this study is achieving a safe and effective material to prevent the formation of postoperative intestinal adhesion. The PLGA/PLA-b-PEG e-membrane was employed as the main antiadhesion material, while the CMCS layer was designed to stop bleeding fast and take the clots away. The cell adhesive tests were performed to find out the effects of the material and blood clots on cells growth, which had close relation with the formation of adhesion. It is expected that this study could help in understanding the antiadhesion mechanism and contribute to the design of an effective antiadhesion barrier material for the application.
2. MATERIALS AND METHODS 2.1. Materials. Carboxymethyl chitosan (the degrees of substitution of carboxymethyl groups on chitosan backbone were approximately 95%) were purchased from Hailian Co. Ltd. (China). Two copolymers PLGA (Mw = 60k, LA/GA = 75/25) and PLA-b-PEG (Mw = 10k, LA/EG = 50/50) were purchased from Jinan Daigang Enterprise Biological Technology Co. Ltd. All other materials and reagents were of analytical grade. 2.2. Fabrication of Trilayered Barrier. In this study, the trilayered barrier was made, which was composed of CMCS sponge layers and PLGA/PLA-b-PEG electrospun layer. Procedures for the preparation of PLGA/PLA-b-PEG e-membranes were the same as those described previously.12,13 In brief, N,N-dimethylformamide (DMF) and acetone (VDMF/VAcetone = 5/5) mixed solvent was employed, and the total concentration of PLGA/PLA-b-PEG (85/15) was 50 w/v%. The fibrous membranes were fabricated by a ten-jet electrospinning device at 20 kV and a steady flow rate of 20 μL/min. The thickness of membrane was 110 ± 10 μm each. The resulting emembranes were dried under vacuum at room temperature for 72 h prior to use. 3% CMCS (w/v) were dissolved in distilled water with stirring for 12 h first, and CaCl2 aqueous solution were added very slowly to avoid precipitation action. The concentration of CaCl2 was about 6% of the total polymer content. Subsequently, different amounts of glycerin (0%, 1%, 2% to the solutions, v/v) was added respectively to the solution as a humectant. Half of the mixture was poured into the culture dish before already prepared PLGA/PLA-b-PEG e-membrane was dropped in. The electrospun layer was then trapped in the middle of CMCS solution. The culture dish with its content was immediately frozen at −20 °C for 24 h and then lyophilized in a freeze-dryer (Beijing Detianyou Technology Co. Ltd., China). Finally, the trilayered barriers were stored in a vacuum drying oven at room temperature. Additionally, CMCS sponge was also fabricated alone by pouring CMCS solution into the culture dish and was frozen at −20 °C immediately. The rest of the preparation steps were performed with the same procedures and conditions as those mentioned above. 2.3. Characterizations. 2.3.1. Scanning Electron Microscopy. Scanning electron microscope (SEM, JEOL JSM-6700F, Japan) was 3084
DOI: 10.1021/acs.biomac.5b01099 Biomacromolecules 2015, 16, 3083−3092
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sliced structure or in a wide area; 3 = besides adhesions in a wide area, the visceral organs adhered to the abdominal wall and adjacent tissues. The scorers were blinded to the groups. Additionally, specimens were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (HE) staining for histological examinations. 2.7. Statistical Analysis. The scores of adhesion level were compared between treated and control groups using the Fisher exact test. A p value less than 0.05 was considered significant, and less than 0.01 was considered extremely significant. Other results were presented as mean ± standard deviation (SD). Differences between groups were determined by Student’s t-test, with p < 0.05 considered to be significantly different, and p < 0.01 meant the difference was extremely significant.
with 0.1% (v/v) Triton X-100 for 5 min, Phalloidin−FITC (Sigma, USA) for 40 min and 4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 10 min under dark condition. 2.6. In Vivo Evaluation. 2.6.1. Animals. Adult male Sprague− Dawley rats (Vital River Laboratory, China) weighing 320−350 g were used in this study. All rats were kept at least 1 week before operation to become adjusted to the laboratory environment. All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All animals were treated humanely throughout the experimental procedure. The procedures and handling of the animals were carried out under aseptic conditions in animal research laboratory. In both the hemostasis and antiadhesion evaluation, the rats were anesthetized by intraperitoneal administration of 10 wt % chloral hydrate solution after inhalation of diethyl ether. 2.6.2. In Vivo Hemostatic Capability. The hemostatic effect of the trilayered barrier was compared to that of Surgicel and PLGA/PLA-bPEG e-membrane by covering the materials on the abraded cecum of rats. Surgicel, a sterile absorbable knitted fabric prepared by oxidized regenerated cellulose, has been reported to effectively assist in the control of capillary, venous, and small arterial hemorrhage.21 After anesthesia, the animals underwent a 4 cm midline incision on the abdominal wall and the cecum was abraded until a bleeding surface was obtained. Then, different materials were put on the abraded cecum. Animals were randomized into four groups, consisting of 10 rats each: (i) control group without treatment; (ii) put a 3 × 3 cm2 piece of PLGA/PLA-b-PEG e-membrane on the abraded cecum; (iii) put a 3 × 3 cm2 piece of Surgicel on the abraded cecum; (iv) put a 3 × 3 cm2 piece of trilayered barrier on the abraded cecum. Then, all the animals were blanketed with saline soaked gauze to simulate a normal abdominal environment. The gauze was removed gently in 5 min, and the hemostatic effects of the materials were evaluated through direct observation. The amount of bleeding in the first 30 min was also observed using a hemoglobin assay kit. The blood and blood clots on the materials and cecum were all gathered to dissolve in the reagent and measured by UV spectrophotometry at 400 nm. 2.6.3. In Vivo Antiadhesion Evaluation. In this test, peritoneal adhesions were induced by making a 2 × 2 cm2 defect on the right lateral abdominal wall and abrading the cecum until a bleeding surface was obtained. Animals were also randomized into six groups: (i) Normal group without operation or any other treatment (10 rats); (ii) Control group, dropped approximately 1.0 mL of normal saline into the abdominal cavity (15 rats); (iii) Surgicel group, put a 3 × 3 cm2 piece of Surgicel material between the abdominal wall defect and abraded cecum (15 rats); (iv) CMCS group, put a 3 × 3 cm2 piece of CMCS sponge between the abdominal wall defect and abraded cecum (15 rats); (v) PLGA/PLA-b-PEG group, put a 3 × 3 cm2 piece of PLGA/ PLA-b-PEG e-membrane between the abdominal wall defect and abraded cecum (30 rats); (vi) Trilayered group, put a 3 × 3 cm2 piece of trilayered barrier between the abdominal wall defect and abraded cecum (30 rats). After surgery for 10 days, rats were sacrificed to examine postoperative adhesion, and the tissue regeneration on defect was checked. Adhesion level and occurrence were recorded. The normalized mean score of adhesion level was calculated via the following equation: 3
S̅ =
3. RESULTS AND DISCUSSION 3.1. Fabrication of Trilayered Barrier. In the trilayered barrier, the PLGA/PLA-b-PEG electrospun layer was sandwiched between layers of CMCS sponge. The cross-section morphology of the trilayered barrier (Figure 1A(i)) showed that the electrospun layer stuck together with the CMCS sponge layers tightly, which indicated that the CMCS sponges may surround the electrospun layer until they were dissolved completely. The surface morphology of PLGA/PLA-b-PEG emembrane before and after freeze-drying process was also observed (Figure 1A(ii),(iii)). The diameters of fibers had little change, and the membrane still presented dimensional flexibility after the freeze-drying process. All fibers were smooth and uniform, which would supply large surface area, easy handling ability and safe texture for the application in vitro and in vivo. In this study, glycerin (GL) was added into the CMCS sponge to get over the property of brittleness first. The crosssection and surface micrographs, as well as photo images of a CMCS sponge with different ratios of GL were shown in Figure 1B. Obviously, there were some changes in morphology when the amount of GL increased from 0% to 2%. For the same surface area, the number of pores decreased as the GL added, which meant that the average pore size increased. That may be due to the faster phase separation of water from the CMCS matrix during freezing process with the addition of GL. Moreover, the wall of pore thickened slightly, while the lamella structure on cross-section also became irregular little by little as the amount of GL increased. The lamella boundary on a crosssection of 0% GL samples were apparent and clear, while there were some fibers turned up in 1% GL sample. Also, the lamella boundary for the CMCS sponge with 2% GL was very fuzzy. The photo images for 10 cm culture dishes revealed that the CMCS sponge without GL was easy to break, while the samples containing GL were soft and flexible. However, the CMCS sponge containing 2% GL soaked up water molecules in air and shrank seriously, which may limit the operability of the material. The tensile properties of trilayered barrier and PLGA/PLAb-PEG e-membrane were shown in Figure 1C(i). It was found that there was no obvious difference between the two samples, which indicated that the tensile property of trilayered barrier was mostly controlled by the electrospun layer. The surface FTIR was used to analyze the chemical structure of the trilayered barrier before and after being incubated in PBS for 6 h, compared with that of PLGA/PLA-b-PEG e-membrane (Figure 1C(ii)). The surface structure of trilayered barrier after incubated for 6 h was similar to that of PLGA/PLA-b-PEG emembrane, and had an obvious difference with that of trilayered barrier before incubation. Their major difference of
3
∑ (s × as)/∑ as s=0
s=0
where S̅ was normalized mean score of adhesion level, s was score of adhesion level, as was the number of rats suffered in the s score of adhesion. Levels of abdominal adhesions were determined using the grading system synthesized from the previous research:22,23 0 = no adhesion; 1 = adhesions with filamentous structure; 2 = adhesions with 3085
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⎛W ⎞ T × A − ⎜ ρm ⎟ ⎡ Vm − Vp ⎤ ⎝ p⎠ P(%) = ⎢ × 100% ⎥ × 100% = T×A ⎣ Vm ⎦
in which Vm was the volume of the CMCS sponge. Vp was volume occupied by polymer, which was obtained by compacting the same amount of CMCS powder into a graduated cylinder. ρp was the density of CMCS powder and had a value of 0.312 g/cm3. After measuring the mass Wm, the area A and the thickness T of CMCS sponge, the overall porosity could be estimated (Table 1). The results showed that Table 1. Thickness and Porosity of the CMCS Sponge with Various Ratios of Glycerina
a
CMCS sponge
thickness (μm)
porosity (%)
0% GL 1% GL 2% GL
513 ± 22 452 ± 13 187 ± 17
92.1 ± 2.1 89.2 ± 1.7 68.4 ± 2.6
Data are average ± standard deviations (n = 5).
the thickness and porosity of the CMCS sponge were decreased dramatically with the increase of GL ratio. That may be because the GL prevented the loss of water during the preparation, resulting in contraction of thickness and area in 2% GL sponge, as well as the fuzzy pore boundary on cross-section in 2% GL sample compared to 0% and 1% samples (shown in Figure 1B). 3.2. In Vitro Blood Coagulation Test of CMCS Sponge. The hemostasis of the CMCS sponge significantly correlated with the blood coagulation.15 In order to find out the influence of glycerin on blood-clotting in vitro, CMCS sponges with various ratios of GL were tested by incubating 100 μL of the blood on the materials for 5 min before rinsing with distilled water. The blood was added onto the culture dish directly in the control group. Photo images of the sample (Figure 2A) showed that little clotted blood was left in the control group and the rinsing water turned to red finally, which meant the blood could not be automatically coagulated on culture dish in 5 min. However, blood was mostly clotted on all sponge samples, and the color of rinsing water showed little change. A lighter color of rinsing water meant a quicker rate of blood coagulation. Although all groups of CMCS sponges showed excellent blood coagulation effects compared with the control group, the color of rinsing water was found to vary with the amount of GL added. Compared to the samples with GL, the sponge without GL produced redder rinsing water. It meant that the blood could not be coagulated well on the sponge without GL in 5 min, resulting in some dissolution of blood when rinsed with distilled water. The concentration of the hemoglobin in blood clots on the sponge was shown in Figure 2B, in which the concentration of the hemoglobin in 100 μL blood was set as a reference value. A higher concentration of hemoglobin indicated the blood was clotted more completely. The concentration of hemoglobin in the control group was close to 0, which confirmed that the blood clots could not be formed on culture dish in 5 min. All of the CMCS samples showed good clotting ability compared with control group (p < 0.01). It was also found that the concentrations of hemoglobin in blood clots on CMCS sponges with 1% and 2% GL were nearly equal to 100 μL blood, while that for the sample without GL was a little lower (p < 0.05). Thus, this proved numerically that the addition of
Figure 1. (A) (i) Cross-section micrograph of trilayered barrier, and surface micrographs of e-membrane (ii) before and (iii) after the freeze-drying process. (B) (a1−c1) Cross-section micrographs, (a2− c2) surface micrographs and (a3−c3) photo images of CMCS sponge with 0%, 1%, and 2% GL. (C) (i) Tensile properties of PLGA/PLA-bPEG e-membrane and trilayered barrier; (ii) FT-IR of the trilayered barrier before and after being incubated in PBS for 6 h, compared with PLGA/PLA-b-PEG e-membrane. The trilayered barrier tested in this study was composed by a PLGA/PLA-b-PEG electrospun layer and a CMCS layer with 1% GL.
peaks was at 1317 cm−1, 1408 cm−1, and 1586 cm−1. In this case, 1408 cm−1 was characteristic peak of the stretching vibration of C−N, while 1586 and 1317 cm−1 were the coupling peaks of deformation vibration of N−H and stretching vibration of C−N. The original trilayered barrier was covered with CMCS sponge, in other words, the surface FT-IR of trilayered barrier before incubation showed the chemical structure of CMCS sponge. There only remained a weak peak at 1586 cm−1 for trilayered barrier after incubated in PBS (pH = 7.4, 37 °C) for 6 h, which suggested that most of the CMCS sponge layer was dissolved. Porous structure, which would enlarge the surface area of materials, was important for a hemostatic agent. The porosity (P) of CMCS sponge can be approximately calculated as follows: 3086
DOI: 10.1021/acs.biomac.5b01099 Biomacromolecules 2015, 16, 3083−3092
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Figure 2. Results of the blood-clotting effects of a CMCS sponge with different ratios of GL: (A) the photo images during the blood-clotting process and (B) the concentration of the hemoglobin in blood clots on the materials. Data are average ± standard deviations (n = 5). (# indicates extremely significant difference from the control group, p < 0.01, and * indicates significant difference from the 100 μL group, p < 0.05).
Figure 3. Process and results of the hemostasis with different materials. (A) The photo images of rats’ cecum taken during the process of hemostasis: the abraded cecum was covered by the salinesoaked gauze alone (control), PLGA/PLA-b-PEG fibrous membrane, Surgicel and trilayered barrier for 5 min. The left insets are the cecum images taken right after being abraded. The right insets are the cecum images taken right after being covered by materials. (B) The concentration of the hemoglobin in blood and blood clots gathered from the abraded cecum and materials after 30 min. Data are average ± standard deviations (n = 10). (* indicates significant difference from the control group, p < 0.05).
GL increased the blood coagulation effects of the CMCS sponge. It had already been found that the porosity of the sponge, which would enlarge the contact area for the blood and sponge, was reduced with the addition of GL (shown in Table 1). Thus, in this case, the water binding capacity of GL was more important for blood coagulation than high porosity for the CMCS sponge with GL. What’s more, considering that the addition of GL would overcome the brittleness, increase the shrinkage, and enhance the hemostatic effects, the ratio of GL in CMCS sponge should be controlled to meet the functional needs of the trilayered barrier. 3.3. In Vivo Hemostatic Capability. On the basis of the preliminary study on the characteristics and blood coagulation of CMCS sponges, the sample with 1% GL was chosen to fabricate a trilayered barrier with a fibrous membrane. The in vivo hemostatic capability of the trilayered barrier was evaluated using rat models and compared with that of PLGA/PLA-b-PEG fibrous membrane and Surgicel (Figure 3). The animals treated with only saline soaked gauze were set as a control group. The photo images (Figure 3A) showed the process of the in vivo hemostasis of various materials. The hemorrhage was still active when only saline-soaked gauze was used to cover the abraded cecum (control) for 5 min. The defects treated with trilayered barrier and Surgicel stopped bleeding in 5 min, while there were still some bleeding points in PLGA/PLA-b-PEG emembrane group. Thus, this indicated that the CMCS layer was highly effective in hemostasis in vivo. Moreover, both the trilayered barrier and Surgicel could be swelled with blood and fixed well in the wound position, which aided in the clotting of blood. To compare the hemostatic effects of different materials more quantitatively, the concentration of hemoglobin in blood and blood clots gathered from the abraded cecum and material was measured (Figure 3B). The hemoglobin concentration was proportional to the amount of bleeding. The trilayered and Surgicel groups had obviously smaller amounts of bleeding compared with the control group (p < 0.05), while a relatively
greater amount of bleeding existed in animals treated with PLGA/PLA-b-PEG fibrous membrane. Thus, the trilayered barrier was very suitable for hemostasis in vivo. During an operation, the damaged blood vessel will activate clotting factors, leading to the formation of long and sticky threads of fibrin, or even adhesion.1 The coagulation of the CMCS sponge may be related not only to the physical porous structure but also to the chemical structure, such as the presence of an amine group and a carboxyl group.15,24 CMCS also has the capability to bind with Ca2+,15 which would activate coagulation by stimulating platelets and the clotting factors VII, IX and X in the process of blood coagulation.25 In this work, when using a trilayered barrier on the wound surface, the CMCS sponge containing Ca2+ and GL would absorb water from the blood and concentrate the clotting factors to facilitate hemostasis, resulting in a certain amount of clots on the surface of the CMCS sponge and cecum. In this way, a quicker coagulation rate would lead to a smaller amount of blood loss and clots formation. Since a CMCS sponge would swell and dissolve gradually in PBS (pH = 7.4, 37 °C) in about 6 h, most of the blood clots in vivo would be picked up by CMCS gel and then dispersed into the peritoneal fluid. 3.4. In Vitro Cytocompatibility and Cell Adhesive Behavior. Wound healing is a complicated process, during which the activity of mesothelial cells and fibroblast cells are very important.9,26 As we all know, during surgical procedures, the mesothelium is damaged.1 Under normal conditions, the healing appears complete if a continuous sheet of mesothelial 3087
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Figure 4. (A-B) Cytocompatibility of materials on (A) L929 and (B) IEC-6 cells for 1 day and 4 days. (C−D) Cell counting results of (C) L929 and (D) IEC-6 cells adhered on different materials for 6 days. Data are average ± standard deviations (n = 7). (* indicates a significant difference from the e-membrane with clots group, p < 0.05).
cells covers the surface of the wound, during which fibroblast cells contribute collagen to promote healing of the deeper wound.9,26 However, under the ischemic conditions, once the fibrin bands are infiltrated with fibroblasts, they would be organized into adhesions.9 Thus, a good antiadhesion barrier should be able to inhibit the proliferation of fibroblast cells and promote the growth of mesothelial cells on them. Moreover, it has been reported that the role of blood in the peritoneal cavity in the formation of adhesions is controversial,9 while the blood in the surgical field is hard to clean up completely.8 Herein, in order to find out the effect of barrier and blood clots at the cellular level, the adhesion behavior of L929 and IEC-6 cells on different materials with and without clots were investigated, respectively. First of all, it was important to ensure the cytocompatibility of the CMCS sponge, PLGA/PLA-b-PEG membrane, and trilayered barrier, which was assayed using the CCK-8 method for 4 days (Figure 4A−B). The cell viability on the culture plate (control) was set as 100%. As presented, the two kinds of cells treated with various materials showed similar trends in proliferation. The cell viabilities were at 85−95% after 1 day culture, while those were all close to or even more than 100% on the fourth day. The differences among each group were not significant (p > 0.05), which meant that all the materials displayed no toxicity and were suitable for the following study. L929 and IEC-6 cells adhered on different materials for 6 days were assessed by CCK-8 testing (Figure 4C-D). Higher OD value at 450 nm meant more cells adhering on the sample, thus the number of cells for the same group dramatically increased as the culture time went on. As Figure 4C shows, a smaller number of L929 cells adhered on the PLGA/PLA-bPEG membrane without clots than those on an e-membrane with clots and a culture plate (control, p < 0.05), which indicated that the proliferation of the cells was inhibited on
PLGA/PLA-b-PEG e-membrane, but promoted by the blood clots. IEC-6 cells got a similar number on the surface of three kinds of samples, and there were only little difference between experimental groups and the control group (Figure 4D, p > 0.05). Thus, epithelial cells could grow and proliferate well on all materials, and the blood clots had no special influence on them. Moreover, the growth of the two cells on the trilayered barrier with clots was always similar to that on the e-membrane without clots. When the cell-seeded trilayered barrier was taken out, it was found that the CMCS layer was mostly dissolved. Since the CMCS layers would be dissolved gradually in 6 h in PBS, it could be inferred that the blood clots had also been dissolved away during the swelling and dissolution of CMCS sponge. Thus, the cells in the group of trilayered barrier may actually adhere on the electrospun layer without clots. The adhesive morphologies of the two cells in 6 days were also investigated in detail by CLSM imaging using an objective lens at 10× magnification (Figure 5 and Figure 6), which were found to be consistent with the cell counting results. As presented in Figure 5, the morphologies and number of L929 cells among the experimental groups, which showed little difference at 12 h, became more and more different throughout the cell culture. The L929 cells presented fairly homogeneous on the culture plate (control) and the e-membrane with clots, while those on the e-membrane without clots and the trilayered barrier with clots only grow within isolated clusters. It confirmed that the cells in the trilayered barrier group were actually growing on the electrospun layer and were difficult to spread uniformly within clusters. After 4 days, the L929 cells on culture plate and e-membrane with clots had high density, so the proliferating rate of cells markedly reduced. However, L929 cells on the e-membrane without clots and the trilayered barrier with clots failed to cover the membrane all over at the fourth day, and the number of cells was still increasing. Meanwhile, 3088
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The images with objective lens at 10× magnification showed that the number of cells increased dramatically as the time increased, but the morphologies of cells had no obvious change. In order to investigate the influence of materials on the shape of cell, the zoomed pictures of cells for 1 day and 4 day cultures were taken for further observation using an objective lens at 100× magnification (Figure 7). As the culture time went on,
Figure 5. The morphology of L929 cells grown on different materials for 6 days culture. The objective lens was at 10× magnification.
Figure 7. Morphology of L929 and IEC-6 cells grown on various materials for 1 day and 4 days of culture. The objective lens was at 100× magnification.
the number of cells increased, the area of a cell occupied reduced, but the shape of cells almost had no change. For L929 cells, it was found that the cells presented regular spindle shape in control group. However, their shapes changed to spherical on the surface of the e-membrane without clots, as well as the trilayered barrier with clots. That meant that the spread of fibroblast cells was inhibited on PLGA/PLA-b-PEG emembrane, resulting in adhering loosely and forming cell clusters. It may be because cells would prefer to spread and grow on the surface with moderate hydrophilicity, while a very hydrophobic or hydrophilic surface was not suitable for the cells.27 The e-membrane composed of PLGA and PLA-b-PEG may be inappropriate for the adherence and spread of fibroblast cells. Moreover, L929 cells on the surface of the e-membrane with clots spread as an irregular spindle shape or a polygonal shape, which meant the spread and growth of the cells were not inhibited. That may be due to the fibrinous network constituted by clotted blood was suitable for the growth of fibroblast cells. In addition, the morphology of IEC-6 cells in experimental groups and the control group all presented polygonal shape and showed little obvious difference. E-membrane without clots did not show inhibition on the spread and growth of the cells, which indicated that the hydrophilicity of PLGA/PLA-b-PEG was suitable for epithelial cells. It was worth mentioning that the cells cultured on e-membrane with clots showed no special change in number or shape compared to the other two experimental groups. Thus, we can conclude that the fibrinous
Figure 6. Morphology of IEC-6 cells grown on different materials for 6 days of culture. The objective lens was at 10× magnification.
IEC-6 cells spread on various materials uniformly and the morphologies had little difference among experimental groups during the culture (Figure 6). For all the groups, the quantity of cells increased with the culture time, while the proliferating rate slowed down after 4 days due to the cells density had become very high. 3089
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Biomacromolecules Table 2. Adhesion Level and Occurrence in Rats for 10 Days group adhesion level
0 1 2 3 mean of score total (percentage) with adhesions a
normal (n = 10)
control (n = 15)
Surgicel (n = 15)
CMCS (n = 15)
PLGA/PLA-b-PEG (n = 30)
trilayered (n = 30)
10 0 0 0 0 0 (0%)
1 1 4 9 2.4 14 (93%)
0 2 3 10 2.53 15 (100%)
2 3 3 7 2.0 13 (87%)
17 5 4 4 0.83a 13 (43%)a
25 3 2 0 0.23a 5 (17%)a
Indicates extremely significant difference from the control group, p < 0.01; PLGA/PLA-b-PEG group vs Trilayered group: p = 0.012.
Figure 8. Results of the adhesion formation in rats treated with various materials after surgery for 10 days: (a1−f1) photo images of injured cecum, in which the adhesions were pointed out with arrows; (a2−f2, a3−f3) histological examination of injured cecum. SM: Smooth muscle, AW: abdominal wall, SL: Serous layer, GT: granulation tissue, ST: scar tissue.
network formed by clotted blood also had no special promoted effect on the growth of epithelial cells. The results showed that the e-membrane could promote the growth of epithelial cells, but exhibit inhibition on the spread of fibroblast cells, resulting in adhering loosely and difficulty to form adhesion tissues. Meanwhile, the blood clot acted as a “medium”, inducing the adhesion and proliferation of fibroblast cells, but showed no special attraction on epithelial cells, which indicated that the hemostasis was vitally important during the prevention of adhesion. It indicated that the process of the CMCS sponge taking away the clots during the swelling and dissolution would prevent the fibrin band from infiltrating with fibroblast cells. 3.5. In Vivo Prevention of Adhesion. In this study, adhesions were induced in rats through abrasion of the cecum and excision of adjacent abdominal wall, because this peritoneum is one of the most frequently occurring tissue adhesion sites after surgical treatment.1,3 The in vivo antiadhesion of the trilayered barrier was compared with that of Surgicel, CMCS sponge, and PLGA/PLA-b-PEG fibrous membrane. The animals treated only with normal saline were set as the control, while the rats without any operation (normal) were set as the reference. The intestinal adhesion of the animals after surgery for 10 days was examined by direct observation, and the statistical results of the level of adhesion were taken into account in the evaluation of the materials (Table 2). The results showed that all the rats in normal groups grew in good condition. There were 93% of rats in control group suffered from postoperative adhesions, and the mean score of adhesion level was about 2.4. The rats treated with Surgicel had a little higher adhesion level (2.53) and occurrence (100%) than control group, which maybe because the material and structure would promote the formation of adhesion little despite the good hemostatic effect.
The animals treated with the CMCS sponge also had a high adhesion level (2.0) and occurrence (87%), which indicated that the sponge had no special effect on reducing adhesion when used alone. That was mainly because the CMCS sponge would be dissolved completely in less than 12 h in peritoneal fluid; however, the adhesion commonly started to develop on the third to fifth day. There was no significant difference in adhesion level or occurrence among these three groups (p > 0.05), while the results of PLGA/PLA-b-PEG membrane and trilayered barrier groups were significantly different from the control group (p < 0.01). Moreover, the trilayered barrier had a better effect in prevention of adhesion than PLGA/PLA-b-PEG membrane (p = 0.012), especially on the reduction of score of adhesion level. This indicated that the introduction of hemostatic CMCS sponge significantly improved the effect of antiadhesion during the implantation. The photo images and histological analysis of the injured cecum after surgery for 10 days was recorded in detail to assess the antiadhesion effects of materials. The images showed in Figure 8 were at the mean adhesion level for corresponding groups. Obviously, histological images coincided with the results from the direct observation. The normal cecum kept in good condition and was set as a reference group. Most of the rats in the control group, Surgicel group, and the CMCS group suffered from dense adhesions. The original structure of cecum was damaged seriously, and the smooth muscles of the damaged cecum were almost fused to the muscles of the abdominal wall. Mild adhesions appeared between the surface of cecum and mesentery in animals treated with PLGA/PLA-bPEG membrane. The intestinal epithelial cells of cecum were kept intergrity and columnar arranged. Infiltration of the inflammatory cells and edema turned up at submucosa. For the trilayered barrier group, most of animals did not suffer from adhesion, and only the electrospun layer remained in situ at the 3090
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Scheme 1. Schematic Illustration of the Process and Mechanism for Preventing Postoperative Bowel Adhesions of Tri-Layered Barrier, Compared with e-Membrane and Blank Groups
exhibited inhibition for the spread of fibroblast cells, resulting in adhering loosely and difficulty in forming adhesion tissues. Finally, in vivo applications in rats indicated that the trilayered barrier was highly effective in reducing the level and occurrence of intraperitoneal postoperative intestinal adhesion.
10th day after operation. The intestinal epithelial cells also presented as the normal condition, and there was mostly no inflammatory reaction. Most of the granulation tissues close to the electrospun layer were developing into scar tissues, which revealed that the wound was almost healed. The process and mechanism for preventing postoperative bowel adhesions of the trilayered barrier were illustrated in Scheme 1, compared with e-membrane and blank groups. After surgery in the peritoneum, bleeding could not be cleaned up, and lots of clots appeared between the injured sites in the blank group, which induced the fibroblast cells adhesion and proliferation, and thus led to dense adhesion between wound surfaces. When PLGA/PLA-b-PEG e-membrane was used alone, if the bleeding could not be controlled sufficiently, the blood clots would still induce fibroblast cell growth and proliferation. The surfaces of sidewall defect and bowel abrasion would be separated by the membrane; however, the contact between the fibrin deposition and nearby mesentery could not be controlled, which resulted in mild adhesion. In animals treated with the trilayered barrier, the CMCS sponge layers could stop bleeding first and would be dissolved gradually in less than 12 h taking the blood clots away, which ensured the adhesive “medium” between injured sites and the nearby mesentery was removed. Since the PLGA/PLA-b-PEG electrospun layer still remained in situ and the fibroblast cells could not adhere well on the membrane, the formation of adhesion between the wound surfaces was prevented. In this case, the epithelial cells proliferated and grew actively on the emembrane to form a continuous sheet of mesothelium covering the surface of the wound, resulting in wound healing.
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AUTHOR INFORMATION
Corresponding Author
*Address: State Key Laboratory of Polymer Physics and Chemistry, Joint Laboratory of Polymer Science and Materials, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun, Haidian, Beijing 100190, P. R. China. Tel: +86-10-8261-8089. Fax: +86-10-6252-1519. E-mail: c.c.han@ iccas.ac.cn. Author Contributions ∥
Authors Qinghua Xia and Ziwen Liu contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Nature Science Foundation of China (51003110) and the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant KJCX2-YW-H19).
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REFERENCES
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4. CONCLUSIONS In this study, a trilayered barrier composed of a PLGA/PLA-bPEG electrospun layer and CMCS sponge layers was successfully prepared. In vitro and in vivo tests indicated that the CMCS sponge had great hemostatic capability. At the cellular level, the blood clot, which would be taken away during the swelling and dissolution of CMCS sponge layer, acted as a “medium” inducing the fibroblast cells adhesion and growth, but had no special attraction on epithelial cells. The remaining electrospun layer promoted the growth of epithelial cells, but 3091
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