5.1. Obtaining Placenta Tissue and Preparing Amniochorionic Membrane
Placenta tissues (n = 10) were collected from an elective cesarean section with the parents’ written informed consent. All of the procedures in this study were performed in accordance with the declaration of Helsinki and under the supervision and approval of Shahid Beheshti University of Medical Sciences (SBMU) Ethics committee and according to SBMU policies on medical and research ethics (Code: IR.SBMU.MSP.REC.1399.466). All mothers who contributed to this study were tested negative for human hepatitis virus types B and C, human immunodeficiency virus types 1 and 2, cytomegalovirus, syphilis, gonorrhea, and toxoplasmosis. Moreover, there were no signs of premature rupture of the membrane or history of prenatal infection. All mothers were between 39–41 weeks of pregnancy, and pre-term or post-term deliveries were excluded. After delivery, the placental tissues were maintained in normal saline serum at 4–8°C under the sterile condition and immediately transferred to the laboratory. All the procedures were performed in a class 2 laminar flow under sterile conditions, within an hour after surgery. A 10⋅15 cm segment of the amniochorionic membrane was dissected from the placenta with at least 3 cm margin from the placental disc and washed several times in phosphate buffer saline (PBS) (pH 7.4) to remove visible blood clots.
5.2. Decellularization of Amniochorionic Membrane
The ACM was flattened in trypsin-ethylenediaminetetraacetic acid (EDTA) diluted with PBS (trypsin-EDTA, 0.1% w/v, Sigma-Aldrich, USA) for 20–30 min at 37°C. After washing the membrane in Dulbecco’s modified Eagle’s medium (DMEM) culture medium (pH 7.4), the membrane was incubated in Streptokinase (0.02% w/v, Sigma-Aldrich, USA) for 8–10 min at 37°C and subsequently, the membrane was washed with sterile DMEM culture medium (pH 7.4) once and lastly with PBS three times.
Following these treats, trophoblastic cells of the chorionic side of the membrane were gently scraped out in PBS by a plastic scraper without detaching the amniotic membrane from the chorionic membrane or rupture of the membrane for 10–15 min at 25°C. Epithelial cells of the amniotic side of the membrane were scraped out gently by scraping with the same strength in PBS for 10–15 minutes. Finally, the scaffold was washed in PBS three times.
5.3. Cross-linking of Decellularized Amniochorionic Membrane
In this study, we selected glutaraldehyde which has been frequently used for cross-linking of amniotic membrane. 43 After decellularizing the amniochorionic membrane, the scaffold was cross-linked using 0.1% glutaraldehyde (10 mM) for 30 min at room temperature.
5.4. Biomechanical Analysis
The biomechanical properties of the decellularized bi-sided amniochorionic membrane (dbACM) were evaluated and also compared with the natural fresh amniochorionic membrane (fACM) and cross-linked scaffold (cross-linked dbACM). The biomechanical analysis was conducted in Polymer and Petrochemical Institute (IPPI). The average thickness of the samples was measured by a caliper (Absolute AOS Digimatic Caliper, Mitutoyo Europe GmbH, Germany). The maximum load value, maximum elongation on the breaking point, and suture retention strength were measured using a uniaxial universal test machine (STM-20, Santam), with an elongation speed of 10 mm min− 1 with a samples size of 20⋅40 mm. For evaluating the suture retention strength, one side of the samples was sutured (10 mm from the edge) with nylon 5 − 0 round suture, while the opposite edge of the samples was tightly held in clamps of the testing machine. Since the thickness of the spongy layer can significantly decrease after dehydration, which would interfere with biomechanical results during the biomechanical analysis, we used PBS to hydrate the samples.
5.5. In vitro Biodegradation
The biodegradation properties of the scaffold were investigated and compared with the natural fACM and cross-linked scaffold, using in-vitro enzymatic digestion. The samples were cut into 10⋅10 mm pieces and were immersed in 1 cc of the biodegradation solution of collagenase H (Roche, Germany) diluted with PBS (0.01% w/v) (pH 7.4) in 24-well plates and stored at 37°C. The samples were followed on days 1, 3, 7, 14, 18, 21, and 28. On each day, samples were removed from the solution, and subsequently weighed.
5.6. Histological Analysis
The scaffold was stored in 10% formalin for 24 h and fixed using DID SABZ Co. DS 2080/H tissue processor. Hematoxylin and Eosin staining (H&E) technique was used for evaluating histological properties of the scaffold. For further evaluation of ECM content, the scaffold was stained with Mason’s Trichrome staining technique.
5.7. Cytotoxicity, Cell Viability, and Cell Proliferation
Human umbilical vein endothelial cells (HUVECs) line was purchased from Stem Cell Technology Research Center (STRC) and cultured in a medium suggested by the provider consisting of Dulbecco’s modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) + 10% FBS + 90 U/ml heparin and 1% penicillin-streptomycin solution. The HUVECs were cultured on both the chorionic side and amniotic side of the scaffold in a 24-well plate with a density of 40000 cells/well, in the mentioned media and incubated for 24 h, 48 h, and 7 days (d) under 95% air and 5% CO2 at 37°C. HUVECs cultured on standard wells were used as the positive control. To investigate cytotoxicity, cell viability, and cell proliferation rate of the scaffold, the MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide) assay was used. Briefly, 2.5 cc of MTT solution was added to the plates and placed on an incubator for 3–4 hours. The formed formazan crystals were dissolved by adding 1.5 ml of Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, USA) solution. The negative control was the wells with DMEM without scaffold and cells. The optical density (OD) of the solution at a wavelength of 570 nm was observed by a spectrophotometer (Cecil BioQuest CE 2501, UK). The blank OD was subtracted from the OD of the other groups. MTT assay was done in 24 h, 48 h, and 7 days of culture.
5.8. Scanning Electron Microscope
Scanning electron microscope (SEM) (TECAN-VEGA-II, Czech Republic) was used to investigate the amnionic and chorionic basement membrane after the decellularization process and also endothelial cell adhesion to the scaffold. Tissue samples were prepared for SEM as previously described. 44 Briefly, dbACM scaffolds and cell seeded dbACM scaffold were fixed using paraformaldehyde 10% solution for one hour and then dehydrated using an ethanol graded concentration of 30%, 50%, 70%, 80%, 90%, and twice of 100% for 10 min. After coating samples with gold by sputtering, SEM images were taken and analyzed at acceleration voltage of 15 kV. For investigating the morphology of the cells adhered to the scaffold, HUVECs were cultured with a density of 4⋅104 per cm2 on both the chorionic side and amniotic side of the scaffold in a growth media consists of DMEM/F12 + 10% FBS + 90 U/ml heparin and 1% penicillin-streptomycin and incubated in 95% air and 5% CO2 at 37°C for five days. The culture media was changed every 48–72 h.
5.9. Immunohistochemistry
For evaluating the capability of the amniochorionic membrane to act as a scaffold for supporting cells and endothelial cell adhesion, the cell seeded dbACM scaffolds were assayed by immunohistochemistry (IHC). For this purpose, HUVECs with a density of 4⋅104 per well were seeded on both the chorionic side and amniotic sides of the scaffold in a 24-well plate with a growth media of DMEM/F12 + 10% FBS + 90 U/ml heparin and 1% penicillin-streptomycin and incubated in 95% air and 5% CO2 at 37°C. The culture media was changed every 2–3 days. After five days of culture, samples were fixed with paraformaldehyde 10% solution, and the scaffold was processed and stained with anti-von Willebrand factor (vWF) for assessing endothelial characteristic.
5.10. In-vivo Implantation in Dorsal Skinfold Chamber
For evaluating pro-angiogenic capability of the scaffold and its the ability to support endothelial proliferation in-vivo, we utilized the dorsal skinfold chamber model. All of the animal surgeries and in-vivo procedures were approved and conducted under the supervision of SBMU ethics committee policies on animal research (Code: IR.SBMU.MSP.REC.1399.466) and also according to Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. Dorsal skinfold chamber analysis was conducted as we described previously. 45 Briefly, 4-6-week-old male rats weighing between 180–200 g were selected and anesthetized using intraperitoneal injection of Ketamine with a dose of 80 mg/kg and Xylazine with a dose of 10 mg/kg. After sedation, the dorsal skin of the rats was shaved at the site of the surgery. Sterilized custom-made platinum chambers were mounted on the rats’ dorsal skinfold and stabilized. One side of the fold’s skin was removed in a circle with 1 cm diameter using a scalpel with blade No.15. After removing the skin, the dorsal skinfold window site was prepared and covered by a sterile glass to make a dorsal skinfold chamber. 24 h after surgery, both amniotic side-down and chorionic side-down dbACM with a size of 5⋅5 mm was implanted in the dorsal skinfold chamber and observed for 10 days. In each dorsal skinfold chamber, four regions with a size of 0.1⋅0.1 mm was randomly selected from the central part of the model where the dbACM was previously implanted. The final analysis was conducted using the open-source ImageJ software with the Fiji plugin package, which is based on ImageJ2 core. After the initial enhancement of images, the pictures turned into binary pictures, and after skeletonizing process, the Analyze Skeleton plugin was used for evaluating the number of branches and the total length of vessels in the selected regions.
5.11. Statistical Methods
All analyses were done using GraphPad Prism version 8. We used one-way ANOVA followed by Tukey’s post-Hoc test for statistical analysis. P value less than 0.05 was considered significant.