Lung transplantation is the last treatment for patients with end-stage lung disorders. Annually, more than 3,700 lung transplantation are performed, with an overall 5-year survival of approximately 53%.1 However, it is limited to popularize due to shortage of donor lungs. Lung tissue engineering (LTE) derived from a patient’s own cells is a promising approach without the need for immunosuppressive therapy.2 However, there are still many challenges for clinical transplantation. A crucial step is to obtain acellular scaffolds that provide hospitable microenvironments to reseed cells and maintain three-dimensional (3D) structure conducive to growth.3 Currently, acellular scaffolds can be produced by detergent-based perfusion protocol in which cells are removed and key extracellular matrix (ECM) proteins are preserved.4–6 The composition of ECM retains a complex mixture of functional and structural molecules that affect cell engraftment after recellularization.7
In recent years, different detergents have been widely described such as sodium dodecyl sulfate (SDS),6 sodium deoxycholate,8 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.9 However, there is no evidence which is better. In fact, acellular scaffolds undergo substantial ECM damage due to detergents.10 Recently, a novel detergent, sodium lauryl ether sulfate (SLES) was reported that could replace SDS in decellularization of rat heart.11 Sodium lauryl ether sulfate is an anionic detergent with mild chemical properties, excellent detergency and dispersion performance, wide compatibility, and high biodegradation ability. However, as a strong anionic detergent, SDS can denature proteins, damage phospholipid membranes, and leading to cell toxicity.12 Since the mild detergent SLES can work well for heart, we hypothesized that it may be suitable for lung as a spacious organ which is composed of abundant loose connective tissues.
In the present study, we assessed the decellularization efficiency of rat lungs based on SDS and SLES. Ultimately, we expected to determine whether SLES is more suitable than SDS for LTE.
Materials and Methods
Sodium lauryl ether sulfate (CAS No.68585-34-2) was made by Hebei Wanye Ltd. (Shijiazhuang, China). A Langendorff perfusion system was purchased from Beijing zhongshidichuang Co. Ltd. (Beijing, China). Male Sprague-Dawley (SD) rats (200–250 g) were obtained from Experimental Animal Center of Fuwai hospital. All animal experiments and protocols were performed by Fuwai hospital Animal Care and Use Committee (No.0074-R-40-GZX).
Groups and Harvest of Lungs
Totally 33 rats were randomly divided into three groups: native group (n = 9, the control group without perfusion decellularization), SDS group (n = 12, decellularization with SDS), and SLES group (n = 12, decellularization with SLES). In both SDS group and SLES group, three rats were used for perfusion studies, three rats for histology, immunohistochemistry, scanning electron microscopy (SEM), western blot, and six rats for DNA quantification, GAGs quantification, subcutaneous implantation. There were no perfusion studies and subcutaneous transplantation in the native group. The samples of each experiment were from different rats.
Rat lungs were harvested as previously described.13 In brief, the rats were injected intraperitoneally with pentobarbital sodium (140 mg/kg, Sigma-Aldrich, St. Louis, MO) for anesthesia and heparin (250 U/kg, Sigma) for anticoagulation. Immediately after euthanasia, the intact heart-lung blocs were removed and stored at −80°C until used.
The decellularization procedures were modified with the protocols described previously.5 The heart-lung blocs were thawed in a water bath at 37°C. The hearts were removed and pulmonary artery (PA) trunks were transected. The lungs were cannulated by the PA with a 16-gauge cannula and decellularized in a Langendorff perfusion system, which controlled perfusion pressure (20 cm H2O) and temperature (25°C).
The rat lungs were first perfused with phosphate-buffered saline (PBS, pH 7.3, Sigma) containing 50 U/mL heparin for 15 minutes. Next, the two detergents were used: 1) SDS group: 0.1% SDS (Sigma) for 2 hours; 2) SLES group: 0.1% SLES for 2 hours. Subsequently, the lungs were perfused with deionized water (DI) for 15 minutes, 1% TritonX-100 for 10 minutes, DI for 15 minutes, 1M NaCl for 1 hour, DNase I (Sigma) solutions (30 µg/mL bovine pancreatic DNase I, 2 mM CaCl2, 1.3 mM MgSO4, in DI) for 1 hour, DI for 15 minutes, 0.1% peracetic acid in 4% ethanol wash for 1 hour. Finally, PBS with 100 U/ml Pen/Strep (Gibco, GrandIsland, NY) and 25 ug/ml amphotericin B (Sigma) were perfused for 2 hours to remove resident cellular debri. Decellularized lung scaffolds were stored in PBS at 4°C.
To test microvasculature networks integrity of decellularized scaffolds, 1% methylene blue (Sigma) solution was infused through PA after decellularization. The whole process was recorded using a camera (Canon EOS 700D, Tokyo).
Decellularized scaffolds and native lungs were fixed with 4% paraformaldehyde for 4 hours, dehydrated, embedded in paraffin, and 5 μm sections mounted on glass slides. Hematoxylin and eosin (H&E), Verhoeff’s Van Gieson (EVG, for elastin, Sigma), Masson’s trichrome (for collagen, Sigma), or Alcian blue (for glycosaminoglycans, GAGs, Sigma) were performed with standard protocols.4 Representative images were obtained with a microscope (Leica, Heidelberg). The staining intensity of elastin, collagen, and GAGs was quantified and compared between groups by three independent and blinded pathologists who assigned a score from 0 to 3 (0, negative; 1, equivocal; 2, weak; 3, strong).14
For immunohistochemistry, antigen retrieval was performed in citrate buffer at 98°C for 30 minutes, rinsed in PBS, and then blocked with 10% normal goat serum (Sigma) and 0.3% Triton X-100 for 90 minutes. Primary antibodies were used against collagen I (1:200, Abcam, Cambridge), collagen IV (1:200, Abcam), fibronectin (1:100, Abcam), laminin (1:50, Abcam), elastin (1:200, Abcam), and sections were incubated overnight at 4°C. Secondary antibodies (Alexa fluor 594, Abcam) were added at 1:300 dilution for 1 hour at 37°C. Finally, the sections were mounted using mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, Sigma). The experiments were repeated at least three times and confocal microscope images were recorded using a fluorescence microscope (Leica). The mean optical density of the images was measured by Image-Pro Plus (IPP) 6.0 software to assess immunostaining quantification.15
Scanning Electron Microscopy
Decellularized scaffold and native lung samples (1 × 1 cm) were fixed using 2.5% glutaraldehyde at 4°C for 1 hour, rinsed in PBS, and then immersed in 1% osmium tetroxide for 1 hour, rinsed in PBS, and dehydrated through an ethanol series (50%, 70%, 90%, 100%) for 5 minutes each. Although the samples were coated with a thin layer of gold under vacuum using aion beam coater (HITACHI, Tokyo). Imaging was performed at 20 kV with a microscope (FEI, Hillsboro, OR).
Decellularized scaffolds and native lungs were lyophilized and dried in a vacuum freezing dryer (Labconco, Kansas City, MO). DNA content of samples was extracted and quantified identically using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Fluorescence was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Pittsburg, PA) at 260 nm, and DNA concentration was calculated.
Sulfated GAGs Assay
Sulfated GAGs were quantified using the Blyscan GAG assay kit (Biocolor, Carrickfergus). Tissues were lyophilized and weighed and assayed according to the manufacturer’s instructions. Absorption was measured at 650 nm, and results were normalized to tissue dry weight.
One hundred milligrams of minced decellularized scaffolds and native lungs was homogenized at 6,000 rpm for 30 seconds with radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor cocktail (Thermo Scientific), subsequently centrifuged at 14,000 g for 15 minutes. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Thermo Scientific), and then boiled at 70°C for 10 minutes with LDS Sample Buffer (Invitrogen, Carlsbad, CA). Twenty-five micrograms of protein lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting as described previously.16 The proteins were transferred to membranes (Invitrogen) and blocked with 5% nonfat dry milk in Tris-buffered saline Tween for 2 hours. The membranes were incubated overnight at 4°C with primary antibodies against collagen IV (1:1,000, Abcam), laminin (1:1,000, Abcam), elastin (1:1,000, Abcam), glyceraldehyde 3-phosphate dehydrogenase (GADPH) (1:1,000, Abcam), and β-actin (1:1,000, Abcam). We used horseradish peroxidase conjugated goat anti-rabbit (1:5,000, Abcam) and goat anti-mouse (1:5,000, Abcam) secondary antibodies detected by enhanced chemiluminescence and visualized with a Alpha Innotech-FCE imager (ProteinSimple, Santa Clara, CA). Densitometry was performed using Quantity One software (BioRad, Hercules, CA).
The upper right lobe from SDS and SLES decellularized scaffolds were subcutaneously implanted into recipient SD rats as previously described.13 After 6 weeks, the implants were retrieved and assessed by H&E and Masson staining. All operative procedures were performed using aseptic technique. Cell infiltration and blood vessel formation were quantified by using a previously reported method.17 The number of cells per each field of view (0.5 × 0.4 mm) and the largest vessels diameter per field were measured using IPP software.
Statistical analysis was performed on each experimental measure with multiple groups with one-way ANOVA (with post hoc pairwise multiple comparison Tukey test). When comparing two groups, independent sample t-test was performed (SPSS statistics software v.19, IBM). Results were expressed as mean ± SD. p < 0.05 was considered statistically significant.
In our study, macroscopically, both detergents eventually produced transparent white lung scaffolds after perfusion. The images for earlier stages of the process (0.5–2 hours) showed that the SDS lung appeared to be swollen. However, SLES lung closely resembled native lung and had smaller changes in morphology. The dye dispersed through to the terminal lung areas without extravasation. Compared with SDS scaffold, the dye dispersion of SLES scaffold was more homogeneous. The whole process of rat lungs decellularization was recorded for the first time (Figure 1).
Hematoxylin and eosin staining analysis demonstrated that intact cells were absent in both decellularized scaffolds compared with native lungs. The normal architecture of airways and blood vessels was well preserved after decellularization. However, there was significantly higher retention of collagen, elastin, and GAGs in SLES scaffolds than SDS scaffolds by quantitative analysis of staining scores (Figure 2).
Immunofluorescence staining results revealed removal of cell nuclei and retention of key ECM proteins, including collagen I, collagen IV, laminin, fibronectin, and elastin. However, SLES resulted in the better retention of ECM structure integrity and continuity than SDS. Furthermore, the results of immunostaining quantification showed that the expression of ECM proteins in SLES scaffolds was significantly higher than SDS scaffolds (Figure 3).
Scanning electron microscopy imaging showed that cells were effectively removed from lungs by the two protocols. There was no visible difference in surface view between SDS and SLES scaffolds. Notably, internal view demonstrated that the ultrastructures of SLES scaffolds preserved more integrity than SDS scaffolds (Figure 4).
The DNA content of both decellularized scaffolds was significantly decreased compared with native lungs (n = 6; p < 0.01). But there was no significant difference between SDS and SLES scaffolds (n = 6; p > 0.05). Native lungs = 1,073.8 ± 87.5, SDS scaffolds = 44.2 ± 3.3, and SLES scaffolds = 34.1 ± 3.2 ng/mg dry tissue (Figure 5A).
The sulfated GAGs content was significantly diminished in both decellularized scaffolds compared with native lungs (n = 6; p < 0.01). However, the sulfated GAGs content was significantly higher in SLES scaffolds than SDS scaffolds (n = 6; p < 0.01). Native lungs = 63.9 ± 3.6, SDS scaffolds = 8.8 ± 0.6, and SLES scaffolds = 14.6 ± 0.6 ug/mg dry tissue (Figure 5B).
The two protocols were effective to remove cytosolic proteins as noted by the absence of detectable GAPDH and β-actin relative to native lungs. Meanwhile, Western blot analysis revealed that the representative ECM proteins including collagen IV, laminin, and elastin were preserved after decellularization. The results indicated more ECM proteins in SLES scaffolds than SDS scaffolds: collagen IV, 51.5% vs. 34.2% (n = 3; p < 0.01); laminin, 71.5% vs. 47.9% (n = 3; p < 0.05); elastin, 44.6% vs. 32.7% (n = 3; p < 0.01; Figure 6).
After 6 weeks of subcutaneous implantation, all implants were well integrated with the host tissues without rupture or breakdown. Moreover, new blood vessels were visible clearly. However, SLES implants showed a significantly greater number of infiltrating cells than SDS implants by H&E staining: 695 ± 76 cells vs. 304 ± 43 cells per 0.2 mm2 (n = 6; p < 0.01), whereas the distribution of cells in the SLES implants was well proportioned. Red blood cells were present by H&E staining and Masson staining, which demonstrated blood vessel formation of the implants in vivo. Vessel diameter of the largest vessels was quantified to assess the capacity for blood vessel formation. The SLES implants had greater vessel diameter than SDS implants: 50.7 ± 1.9 um vs. 31.2 ± 1.6 um (n = 6; p < 0.01; Figure 7).
Lung tissue engineering is an exciting and emerging solution that has the ultimate aim of generating lungs for transplantation. However, there are three important demands to successfully create bioengineered lungs: the type of scaffolds, the composition of ECM, and the source of cells.18 It is widely believed that the native ECM scaffold prepared by decellularization is a logical and ideal scaffold for organ and tissue regeneration.19 In the current study, we compared two decellularized scaffolds using detergents SDS and SLES. So far, this is the first study of SLES in lung decellularization.
Low concentration of SDS at 0.1% was reported in the decellularization of rat lungs.6 Herein, we chose the same concentration of SLES in our study. After decellularization, 0.1% SLES produced a transparent white scaffold without significant changes to the native lung architecture. Furthermore, the microvasculature and ultrastructure of SLES scaffolds were maintained at normal levels, which have been shown by perfusion of dyes and SEM analysis. What’s more, our studies demonstrated that the airways and blood vessels were preserved well using SLES by histological staining. As the airways and blood vessels networks are essential part of the respiratory system, preserving the integrity of networks is crucial for successful recellularization.20
It is well known that DNA can be a strong immunoactivator. Balestrini et al. 21 reported that the DNA removal data ranged from 75–98% in acellular lungs. In our study, more than 95.3% (SDS scaffolds) and 96.3% (SLES scaffolds) of DNA were removed compared with native lungs. Both the decellularized scaffolds met the criteria for tissue engineering purposes: < 50 ng dsDNA per mg dry weight and lack of visible nuclear materials in tissue sections stained with DAPI or H&E.22 Additionally, the efficiency was also evident based on SEM and Western blot for cytoskeletal proteins (GAPDH and β-actin).
To obtain an ideal 3D scaffold, it is important to identify the optimal decellularization protocol that preserves key ECM proteins such as collagen, laminin, elastin, fibronectin, and GAGs, which maintain the structure of the lung. After decellularization, residual Collagen I and Collagen IV are critical components of fibrillar basement membrane for lung transplantation.23 Laminin is another component of basement membrane and play a key role in epithelial cell matrix adhesion and motility after recellularization.7 Elastin is an important protein that impacts lung development predominantly during the alveolar stage and impacts the vessels’ ability to recoil.23 Fibronectin plays a critical role in cell adhesion, growth, migration, and differentiation.24 GAGs contribute to endothelial cell migration and adhesion and play an important role in angiogenesis upon reendothelialization.25 All these ECM proteins in SLES scaffolds were preserved more intact than SDS scaffolds. Petersen et al.26 reported that only about 20% of collagen, 15% of elastin, and 5% of sulfated GAGs were retained in rat lungs with SDS-based decellularization. In this study, decellularization using SLES, the ECM proteins of collagen IV, laminin, elastin, and sulfated GAGs were significantly higher than SDS scaffolds.
The important property of decellularized lung scaffolds is the ability to integrate with the host and induce blood vessel formation of the implants.27 In the current study, SDS scaffolds and SLES scaffolds were subcutaneously implanted for 6 weeks. We found that SLES implants were superior to promote incorporation. The decellularized scaffolds provided a compatible ECM microenvironment for host cells infiltrating. Interestingly, SLES implants demonstrated a greater potential for cell infiltration than SDS implants. One limitation of our study is that the types of infiltrating cells were not identified. Tsuchiya et al.13 believed that the majority of infiltrating cells were red blood cells. The infiltration of host cells into the implants induced blood vessel formation. The quantitative results of vessel diameter indicated that blood vessel formation was better in the SLES implants than SDS implants. One possible reason is that, as a strong detergent, SDS might result in depletion of growth factors and negatively impact recellularization and outcomes of implantation.28 Another shortcoming of our study is the implantation of scaffolds without cell seeding. Ling et al.29 reported more blood vessel formation in cell-seeded scaffolds than in acellular scaffolds after subcutaneous implantation. Actually, significant challenges remain concerning the optimum cell choice and the methodologies for recellularization.30 Several groups have generated bioengineered lungs for orthotopic implantation in rats, but there is still a long way from clinical application.2 , 6 , 9 , 31 , 32 Our results indicated that the performance of SLES scaffolds had superiority over SDS scaffolds in subcutaneous implantation, and future studies will be needed to evaluate SLES scaffolds in orthotopic implantation.
In this study, we obtained the acellular rat lung scaffolds by perfusion of detergents and enzymes using a Langendorff perfusion system, which has shown to be effective for generating whole porcine lungs.33 In general, DNase I is used to facilitate dislodgement of the cells at 25°C,34 and perfusion pressure of ordinary decellularization is 20 cm H2O.35 Our study showed that DNA was almost completely removed, and the ECM integrity was preserved well in SLES scaffolds. In the past, lung decellularization protocols can be accomplished ranging from 24 to 72 hours.21 , 27 , 36 Recently, Khalpey et al. 37 reported a 7-hour porcine lung decellularization protocol. In our study, the whole process of decellularization was not more than 9 hours. Remarkably, the time-efficient and reproducible decellularization protocols may be favorable for organ regeneration. It is a limitation that we did not measure the mechanical properties of decellularized lungs. In future studies, the effect of SLES needs to be assessed in decellularization of human and large-animal lungs.
Our results demonstrated that the mild anionic detergent SLES can be used to produce an acellular lung scaffold, and the ECM architecture and proteins were preserved effectively compared with strong anionic detergent SDS. Furthermore, SLES scaffolds had a greater capability of cell infiltration and blood vessel formation than SDS scaffolds using a rat to rat subcutaneous model. In summary, SLES is a promising detergent in lung decellularization for LTE.
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