Materials
Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Bio-Idea (Tehran, Iran). Triton X-100 (TX) and sodium dodecyl sulfate (SDS) were procured from Merck (New Jersey, USA). 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) was obtained from Bio Basic (Markham, Canada). Peracetic acid was provided from NanoGenDarou (Semnan, Iran). Masson’s trichrome and orcein staining kit was obtained from Asia Pajoohesh (Mazandaran, Iran). The DNA extraction kit and the glycosaminoglycan (GAG) assay kit were from Pars Kia Gene (Kerman, Iran) and Kiazist (Hamedan, Iran), respectively. Apelin-13 enzyme-linked immunosorbent assay (ELISA) was purchased from Eastbiopharm (Zhejiang, China). EDC, NHS, Bio-Beads SM-2, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma-Aldrich (Massachusetts, USA). Human dermal fibroblasts, vascular smooth muscle cells (VSMCs), and human umbilical vein endothelial cells (HUVECs) were obtained from the Pasteur Institute of Iran (Tehran, Iran).
Animals and harvesting the vessels
A total number of 80 adult male Wistar rats aged 4 months old were purchased from the animal house of Kerman University of Medical Sciences and used in this study for setting up the tests as well as performing assays. All animal procedures were approved by the Ethics Committee of Kerman University of Medical Sciences, Kerman, Iran (IR.KMU.AH.REC.1400.007) and performed in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines of laboratory animal care. Animals were housed 2–3/cage in an animal room with a constant temperature (20–22 °C) and humidity (50–60%) under a 12-h interval light/dark cycle with free access to standard food. Next, the animals were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine before euthanasia, and subsequently, the aorta was harvested. The harvested vessels were then placed in the phosphate-buffered saline (PBS) medium supplemented with an antibiotic/antimycotic cocktail and kept at -20 °C overnight to facilitate the decellularization process.
Decellularization protocols
Previously published protocols used several decellularizing agents including detergents and enzymes such as TX, SDS, sodium deoxycholate (SDC), and CHAPS [24, 25]. In this study, the decellularization process with different combinations of detergents was carried out using a perfusion peristaltic pump (Peristaltic Pump P-1, Pharmacia Biotech, Sweden) at a 1.5 mL/min rate. Figure 1A depicts the decellularization protocols of the present study in detail. Briefly, vessels were decellularized by either detergent perfusion or shaking in the detergent solution. In the perfusion group, vessels were perfused with 50 mL of 0.5% SDS or 1% TX or a combination of 0.25% SDS and 0.5% TX for 16 h at room temperature. In the shaking group, the vessels were shaken at 37 °C in 50 mL of a detergent solution including 8 mM CHAPS, 1 M NaCl, and 25 mM EDTA for 2 h. Next, the tissues were washed by shaking in the PBS solution for 30 min, and then they were shaken at 37 °C in a solution of 1.8 mM SDS, 1 M NaCl, and 25 mM EDTA for 2 h. Afterward, the scaffolds were perfused with 0.1% peracetic acid for an hour and then rinsed with sterile PBS containing antibiotic/antimycotic. A cocktail was utilized to neutralize the acid and remove the residual detergent components from the vascular scaffold. The decellularized vessels were then stored in sterile PBS at 4 °C for further investigations including the evaluation of DNA, collagen, and GAG content as well as histological analysis. Moreover, all the experiments performed in the present study were carried out in three independent runs [13].
Histological analysis
To investigate the efficacy of the decellularization process, samples were fixed in 10% formalin solution for 24 h. The paraffin-embedded samples were stained with hematoxylin and eosin (H&E), Masson’s trichrome for distinguishing collagen, and orcein for the determination of elastic fibers according to the standard protocols before being examined under a light microscope (Olympus BX53, Japan).
DNA quantification
To evaluate the quality of decellularization, the total DNA content of native and decellularized vessels was measured. Firstly, the samples were homogenized in the lysis buffer provided in the DNA extraction kit. Total DNA content was extracted according to the manufacturer’s instructions, and the DNA concentration was quantified using a spectrophotometer (ND-1000, Thermo Fisher Scientific, USA) at the 260 nm absorbance wavelength and was normalized according to the wet tissue weight. The results are expressed as ng/mg wet weight of samples.
Hydroxyproline quantification
A colorimetric assay was employed to assess the collagen content of native and decellularized samples through the measurement of hydroxyproline [26]. Concisely, homogenized samples were incubated with cupric sulfate, sodium hydroxide, and hydrogen peroxide at 80 °C for 5 min and then were cooled. Afterward, sulfuric acid and ρ-dimethylaminobenzaldehyde in 1-propanol were added to the samples, which were then incubated at 80 °C for 30 min, and absorption was read at 560 nm using a spectrophotometer (Epoch, BioTek, USA). Moreover, the hydroxyproline level was directly determined using a hydroxyproline standard curve and normalized using wet tissue weight.
GAG quantification
Native and decellularized samples were first digested in papain overnight at 65 °C to assess the GAG content using the GAG assay kit (Kiazist, Iran). Then, the samples were centrifuged (Sigma 3-16PK, Germany) at 8000 g for 15 min, the protein precipitant solution was added to the supernatant, and the samples were centrifuged again at 8000 g for 15 min. Subsequently, a GAG reagent was added to the samples and total absorption was measured at 560 nm using a spectrophotometer (Epoch, BioTek, USA). Moreover, the chondroitin sulfate standard curve was used to directly determine the GAG content and the total tissue GAG content was normalized using wet tissue weight.
Field emission scanning electron microscopy (FESEM)
To characterize the topography of the luminal surface, native and decellularized tissues were freeze-dried (Eleya, Japan), mounted on aluminum stubs, and coated with a thin layer of gold. The analysis of morphology and sample structure was performed at a voltage of 15 kV by FESEM (TESCAN MIRA3, TESCAN, the Czech Republic) [27].
Immobilization of apelin-13 on the scaffold
In the present study, apelin-13 was attached to the scaffold via passive and active absorption to investigate the efficacy of EDC-NHS linkers in the attachment of apelin-13. Decellularized vessels were perfused with 6 μg/mL apelin-13 for 4 h at a rate of 0.5 mL/min for passive absorption. However, in the EDC-NHS conjugated group, the vascular scaffold was first perfused with 4.8 mg/mL EDC and 12 mg/mL NHS in PBS for 1 h and then perfused with 6 μg/mL apelin-13 for 4 h at a rate of 0.5 mL/min. The efficacy of apelin-13 attachment to the scaffold was assessed using an apelin-13 ELISA kit. Moreover, no apelin-13 treatment was performed for the vascular scaffold in the negative control group. The group with higher apelin-13 attachment efficacy was selected for further studies.
Viability and proliferation of cells in the decellularized scaffold
The viability of cells in acellular scaffolds was assessed using two different washing approaches. In the first approach, the viability of seeded cells in the presence of acellular scaffolds was assessed by the MTT assay according to the manufacturer’s instructions after washing them with the PBS solution for 48 h without Bio-Beads SM-2 incubation. In brief, decellularized scaffolds (with and without apelin-13) were placed in 12-well tissue culture plates and cells were seeded at a density of 2.5 × 105 cells/well for 1, 3, and 7 days.
In the second approach, the MTT assay was performed to evaluate the viability and proliferation of seeded cells in the acellular scaffolds incubated with 1 g/mL (dry weight) Bio-Beads SM-2 for 48 h as described in the previous section using the same cell density. At the end of the 1st, 3rd, and 7th days, the MTT solution was added to the wells and the cells were incubated at 37 °C for 3 h in the dark (cell culture incubator, Jal Teb, Iran). Finally, the absorbance of the produced color was evaluated at 570 nm using a spectrophotometer (Epoch, BioTek, USA). Higher color production indicates a higher number of viable cells.
Recellularization of acellular vascular scaffold
In the present study, the recellularization process was performed following the sterilization of the vascular scaffold using an antibiotic/antimycotic cocktail. Afterward, decellularized scaffolds (with and without apelin-13) were rinsed with PBS. The recellularization process was carried out on three consecutive days by the perfusion of 1.5 million cells/mL human fibroblast cells, HUVECs, and VSMCs at 37 °C and a perfusion rate of 0.5 mL/min using a peristaltic pump. Next, the efficacy of the recellularization process was evaluated 7 days after the end of the procedure via histological, fluorescent (DP71, Olympus fluorescent microscope, Japan), and FESEM analyses as well as the measurement of the DNA content.
Functional study
To assess the mechanical properties and uniaxial tensile strength of decellularized and recellularized vessels compared to the native ones, a Testometric tensile testing machine (Testometric Machine M350-10CT, Rochdale, UK) was used, and tests were performed at room temperature. Due to variant aorta wall thickness, the results were adjusted according to the tension per unit thickness of the specimen to avoid the effect of aorta wall thickness on the calculation of tensile strength. The calculation of tension was based on the formula T = F/I in which T denotes tension in N/mm, F denotes the acquired force in N, and I indicates the specimen width in mm.
Statistical analysis
One-way analysis of variance (ANOVA) and TUKEY post hoc tests were performed to compare groups using GraphPad Prism software version 8 for Windows (GraphPad Software Inc., San Diego, CA, USA), and P-values less than 0.05 were considered statistically significant.