2.1 PCL membrane preparation
PCL (Sigma-Aldrich, USA) membranes were fabricated using solvent-casting method. Briefly, 20% PCL solution dissolved in acetic acid was cast on glass plate at room temperature, dried in the oven at 60°C overnight, immersed in the water, and neutralized by 0.5 N NaOH aqueous solution to obtain the dense membrane with a thickness of 200±50 µm. The porous PCL membrane was prepared by adding 8.8%
sodium chloride to the PCL solution. In this study, sodium chloride was used as the pore former to produce porous morphology with a thickness of 250±50 µm. Detailed membrane structure was examined using a scanning electron microscope (SEM) (Hitachi S-4800).
2.2 Cell culture
Human fibroblast Hs68 cells were purchased from Bioresource Collection and Research Center (BCRC, Taiwan). Cells were expanded in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Biological industries) and antibiotic/antimycotic (penicillin G sodium 100 U/ml, streptomycin 100 m g/ml, amphotericin B 0.25 m g/ml, Gibco-BRL Life Technologies, UK) at 37°C
in a humidified atmosphere containing 95% air and 5% CO2. Once 90% confluent, cells were trypsinized using 0.2% trypsin with 0.1% ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, USA) and re-suspended in the same medium until ready for experiments.
2.3 Hs68 cell proliferation on PCL membranes
The alamar blue assay was employed for proliferation estimation of Hs68 cells cultured on dense and porous PCL membranes. Circular samples (1.5cm in diameter) were cut from the PCL membranes, rinsed extensively with phosphate-buffered saline (PBS) and then placed in the 24-well tissue culture plate (Corning, USA). After ultraviolet light sterilization overnight, 2 x 104 cells were added to the well. On days 1, 4, and 7, alamar blue solution (AbD Serotec, UK) was added into culture well and incubated at 37ºC for 2 hours. The fluorescent intensity, correlated with the number of viable cells, was monitored using a spectrophotometer (Tecan, Taiwan) at the wavelengths of excitation and emission of 570 and 600 nm, respectively.
2.4 Immunocytochemistry
After 4 days of incubation, cells were washed with PBS, fixed in 4%
paraformaldehyde for 20 min and permeabilized with 0.1% triton X-100 for 10 min.
The cells were blocked in 2% bovine serum albumin overnight and stained by incubation with anti-fibronectin antibody (MAB1937, EMD Millipore, USA).
Fibronectin was then visualized using fluorescence-conjugated secondary anti-mouse IgG (Santa Cruz, USA). DAPI (Invitrogen) was used as the nuclear marker. Images were taken with a fluorescent microscope (Leica DMI 6000).
2.5 Western blot analysis (in vitro)
After 7 days of incubation, cells were lysed with a lysis buffer containing Complete Protease Inhibitor Cocktail Tablets (Roche Diagnostics GmbH, Germany) for 30 min on ice and then proteins were collected followed by centrifugation. The protein concentration in the supernatants was measured by a Bicinchoninic acid (BCA) protein quantification kit (Pierce Biotechnology, USA). Proteins were separated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were immersed in the primary antibodies, anti-fibronectin & anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), overnight at 4°C. After washing several times, the membranes were immersed in the horseradish peroxidase-conjugated secondary antibodies for 2 h and visualized with an enhanced chemiluminescence detection system (Millipore, USA).
2.6 Western blot analysis (in vivo)
Pleurodesis induced by PCL mesh in pleural cavity of rabbit were harvested.
Protein from pleurodesis was extracted by Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA). Equal amounts of protein were separated in SDS-PAGE gels and transferred to PVDF membrane. Then the membrane was incubated with primary antibodies against FNDC4 (fibronectin type III domain containing 4; Novus Biologicals, NBP1-59690) and β-Actin ( Novus Biologicals, NB600-501) overnight at 4 °C. After washing with TBST three times, the specific bands were detected by goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horse radish peroxidase. The immune complexes were visualized with an enhanced chemiluminescent system (Western Lightning® Plus-ECL; PerkinElmer Inc.).
2.7 Animal Subjects
The protocol was approved by the Institution’s Committee on Investigations Involving Animal Subjects at National Taiwan University. All animals were housed in, and the procedures were performed at the facilities of National Taiwan University. The methods were carried out in accordance with the relevant guidelines, including any relevant details.
2.8 Pilot abdominal adhesion study in Rat
We use C57BL/6 rats for pilot study. The abdominal incision was made at midline.
The PCL membrane, 2 X 2 cm2, was inserted through the wound. The PCL membrane was well covering the intestine. We evaluate the adhesion degree ten days after the operation.
2.9 General Design
Thirty-six New Zealand White rabbits weighing 2.5-4.0 kg were randomized into three groups to undergo thoracoscopic procedures in the hemithorax as follows: (a) dense PCL membrane pleurodesis [dense PCL group (n=12)]; (b) porous membrane pleurodesis [porous PCL group (n=12)]; (c) control [control group (n=12)].
2.10 Surgical Techniques
Light general anesthesia was induced with intramuscular ketamine hydrochloride (Ketalar, 35 mg/kg; Pfizer, Shinchu, Taiwan) plus xylazine hydrochloride (Rompun®, 5 mg/kg, Bayer, Leverkusen, Germany). Arterial blood pressure and transcutaneous oxygen saturation were continuously monitored via percutaneous ear arterial catheter and pulse oximeter, respectively. ECG monitoring using standard limb leads was continuous. Penicillin G benzathine (40,000 U/kg IM) was administered
prophylactically. An antimicrobial skin preparation (povidone-iodine) was used prior to all invasive procedures, all performed with aseptic surgical techniques. A tracheostomy using a 3-mm endotracheal tube was made through a transverse incision between the third and fourth tracheal ring, with the tube advanced into the right main bronchus. The animals were ventilated (Servo 900 C, Siemens-Elema AB, Solna, Sweden) based on body weight and oxygen saturation, with a respiratory rate of 35–45 breaths/min, I:E ratio of 1:2, and inspiratory and expiratory pressures of 8–10 and 2 cm H2O, respectively.
Three 5-mm incisions were made in the chest overlying the sixth and eighth intercostal spaces for insertion of the telescope and instruments. Visual examination of the hemithorax was performed using a 5-mm, 30° rigid telescope (Karl Storz, Tuttlingen, Germany). After instillation of 2 mL 2% lidocaine hydrochloride (40 mg) into the pleural cavity, three types of thoracoscopic procedures were performed. Rabbits in the film group underwent thoracoscopic film placement at the parietal pleura. The upper half of the parietal surface, including areas above the 5th rib anteriorly and 7th rib posteriorly, was covered by the film. The incision was then sutured in layers. Animals in the control group underwent thoracoscopic examination of the pleural cavity only. At the conclusion of each thoracoscopic procedure, a chest tube made from sterile suction catheter (14 Fr; Symphon Chemical Corporation, Taipei, Taiwan) was inserted into the pleural space for aspiration of the retained air and fluid. After resumption of
spontaneous breathing, the endotracheal tube was removed and the tracheostomy incision closed using 6-O Prolene. The rabbits were then sent to the animal center for post-operative care.
2.11 Macroscopic Evaluation
The rabbits were euthanatized 30 days after the operation by injection of pentobarbital sodium (Nembutal; 120 mg/kg) into an ear vein. The thorax was removed from the remainder of the animal en bloc. Small incisions were made in the diaphragm to allow better access of the fixative (10% formalin) into the pleural cavities. Attempts were made to expand the lungs by injection of the fixative into a plastic catheter (6-mm-diameter) inserted into the exposed trachea. The entire thorax was then submerged in 10% formalin solution for 72 h. Necropsy and macroscopic evaluation were
performed blinded with respect to method. The degree of pleurodesis observed grossly was graded according to the following scheme: 0, normal pleural space; 1, no adhesions but pleural space inflamed as evidenced by redness and fibrin deposition; 2, a few scattered adhesions (25%); 3, generalized scattered adhesions (25-75%); and 4, complete obliteration of the pleural space by adhesions (75%) [21, 22].
2.12 Microscopic Evaluation
During the gross assessment of the thorax, samples of the parietal pleura, contiguous visceral pleura, and lung from each hemithorax were obtained from the areas of greatest adhesion and placed in neutral-buffered 10% formalin. If there were no adhesions, samples of the parietal pleura, contiguous visceral pleura, and lung were taken from the middle portion of the fourth intercostal space. These tissue samples were sent for routine histological examination and stained with hematoxylin-eosin.
The microscopic slices were evaluated blindly by two of the investigators (M.-S.H., K.-C.C.) for the presence of inflammation and fibrosis and graded on a five-point scale (0-4: absent, equivocal, mild, moderate, or marked, respectively) [21, 22].
2.13 Statistical Analyses
Categorical variables were compared using χ2 and Fisher’s exact tests and continuous variables, with Student’s t test. P values of less than 0.05 were regarded as significant.
Statistical analyses were performed using SPSS release 18 (SPSS Inc., Chicago, Ill), and all statistical tests were two-sided.