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Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissue-engineered repair
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2016 www.elsevier.com/locate/jbiosc
Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissue-engineered repair Shu-wei Xiao,1 Peng-chao Wang,1 Wei-jun Fu,1, * Zhong-xin Wang,1 Gang Li,3 and Xu Zhang2 Department of Urology, Hainan Branch of Chinese People’s Liberation Army General Hospital, Hainan 572013, PR China,1 Department of Urology, General Hospital of Chinese People’s Liberation Army, Beijing 100853, PR China,2 and Department of Urology, 309 Hospital of Chinese People’s Liberation Army, Beijing 100091, PR China3 Received 6 January 2016; accepted 15 June 2016 Available online xxx
As the endoscopic technique is widely used in the diagnosis and treatment of diseases, the incidence of ureteral injuries increases annually. The classical surgical therapies are not always satisfactory. With the constant development of the tissue engineering technology in the field of urinary reconstruction, the ureteral reconstruction has become possible technology. In this study, a novel perfusion-decellularized protocol, which combined a perfusion system with the commonly used physical and chemical methods, was used to prepare the decellularized ureters for ureteral reconstruction and the urinary tract-derived cells (UDCs) were seeded on the surface of the perfusion-decellularized ureters (PDUs) in order to observe the cells survival, adhesion, proliferation and distribution. The data of H&E staining, DAPI staining, and the agarose gel electrophoresis demonstrated that the cellular components of PDUs were removed, and the decellularized time was shorter than previous study. In addition, compared with the native ureters, the DNA content of the PDUs was significantly decreased just two percent residue (P < 0.05). Scanning electron microscopy, collagen and glycosaminoglycan content assay showed that the three-dimensional (3D) ultrastructure and the compositions of the extracellular matrix (ECM)of PDUs were well preserved. When the UDCs were seeded onto the PDUs, the UDCs formed multilayer structure on the surface of the PDUs, infiltrated into the deep layer of the decellularized ureters and then formed laminated structure. In conclusion, the decellularized ureters prepared by the novel perfusion-decellularized method may be the potential surrogate for ureteral tissue-engineered repair. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Ureter; Decellularized; Perfusion system; Tissue engineering; Repair]
Ureter, as a retroperitoneal organ, is located in the deep of the body, therefore the incidence of ureteral injuries is low (1). In recent years, with the increasing use of endoscopic technique to diagnose and treat the diseases, the risks of ureteral injures were increasing. The classical surgical therapies, such as colon substitution, bladder flaps, transureteroureterostomy and renal autotransplantation could not achieve entirely functional recovery and they also might induce a series of clinical complications such as recurrent strictures, urinary leakage, even renal damages (2,3). However, tissue engineering technology could reconstruct the ureter and solve these clinical problems. With the advance of tissue engineering technology, the exploration of ureteral regeneration has never stopped. Various types of synthetic or biological scaffolds were introduced in ureteral reconstruction (3). However, synthetic scaffolds often had a lack of in biocompatibility, peristaltic motion, and incrustation (4). Unlike the synthetic scaffolds, the biological scaffolds had unique advantages, especially when the natural acellular matrix materials removed the antigenic cellular components, the native 3D ultrastructure and the compositions of the ECM were preserved. The remained ECM had a close relationship with the cell attachment, migration, proliferation and three-dimensional spatial
* Corresponding author. Tel./fax: þ86 0898 38830531. E-mail address: [email protected] (W.-j. Fu).
arrangement (5). The acellular matrix derived from different tissues was used to study the ureter regeneration, such as bladder, vessel, and ureter itself (6e9). The continuous stirring and the chemical detergents were widely used to prepare the ECM for ureteral tissueengineered repair (4,10,11). This decellularized process took several days or even a week, and the process which was continuous stirring and the effect of detergents could destroy the organizational structure and cause the loss of bioactive components of ECM. Besides, the prepared ECM has never been evaluated systematically. Moreover, the prepared ECM should be conducive to the cells adhesion and proliferation, which was beneficial for its survival in vivo and functional repair. In this study, we combined the perfusion system and the commonly used physical and chemical methods scientifically, and then explored the best decellularized time by trial and error. At last, a novel perfusion-decellularized protocol was established. Compared with the traditional methods, this perfusion process would take less time and remove more cellular components. Meanwhile the 3D structure was well preserved. The characteristics of the PDUs were also described in detail, including the structural integrity and the quantification of the biological components. Moreover, the feasibility of the PDUs for the tissue-engineered repair was evaluated clearly through seeding the UDCs on the surface of the PDUs. The UDCs could survive and proliferate in the decellularized ureters, which was beneficial for the tissueengineered repair.
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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Animals and decellularization processes Animal experiments were approved by the Animal Experimental Ethics Committee of People’s Liberation Army General Hospital. The ureters were harvested from the adult beagles (7e10 kg) euthanized in connection with other experimentation, and then were kept at 80 C for at least 24 h. A perfusion system was used throughout the whole decellularization process. The ureters were thawed at room temperature. A 16-gauge needle was inserted into the ureteral lumen and then connected to a perfusion pump using silicon tubing. The ureters were flushed with 1% TritonX-100 for 7 h, and then with the deionized water for 1 h. Subsequently, the ureters were treated with 1% Sodium dodecyl sulfate (SDS) for 1 h. Finally, the ureters were rinsed with deionized water for 24 h, in order to remove the detergent completely. During the decellularization process, the perfusion speed was controlled at 1.5 ml/ min. Meanwhile, the harvested ureters were treated by the commonly traditional methods (11). TritonX-100 treatment (TritonX-100 group): Ureters were placed in a 1% TritonX-100 solution with 0.02% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) together with DNase and RNase and shaken for 48 h. The solution was changed every 24 h. After the decellularized procedures, the decellularized tissue was washed with deionized water for 24 h. SDS treatment (SDS group): The ureters were treated in a solution of 0.5% SDS, 150 mM NaCl, and 10 mM EDTA in 10 mM tris-HCL (pH 8.0) and shaken for 48 h. Then the decellularized ureters were washed with deionized water for 24 h. Histological and immunohistochemical (IHC) analysis Native or decellularized ureters were fixed in 10% neutral buffered formalin, dehydrated with a graded ethanol and embedded in paraffin, sectioned into 5 mm slices. Haematoxylin and eosin (H&E) staining was used to obverse the cellular content and general structure of the decellularized ureters. Nucleic acids were stained with 4,6diamidino-2-phenylindole (DAPI). Masson’s trichrome staining was used to detect the collagen distribution and orientation. Alcian Blue staining was carried out for qualitative analysis of glycosaminoglycan (GAG) (12). In the IHC staining, we placed the slides into antigen retrieval solution and heated them until the temperature reached 95e100 C for 30 min. Endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide solution. We blocked slides with 4% goat serum. Sections were incubated with primary antibody at 4 C overnight. The secondary antibody was applied for 30 min. The slides were treated with streptavidin-horseradish peroxidase complex, diaminobenzidine (DAB) solution and counterstaining with hematoxylin, mounted and imaged using microscopy (Olympus, Tokyo, Japan). The primary antibody used was rabbit anti-Collagen type I antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:200. The secondary antibody was goat anti-rabbit IgG (Abcam, Cambridge, MA) at a dilution of 1:100. Quantification of total genomic DNA The total genomic DNA of the samples was extracted using a Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Both native ureters (n ¼ 6) and decellularized ureters (n ¼ 6, each group) were lyophilized and digested with proteinase K and RNase for 5 h until no visible material remained. The remaining genomic DNA was collected and quantified using the spectrophotometer (Bio-Rad, CA, USA). The size of the collected DNA fragments was exhibited using 2% agarose gel electrophoresis. The DNA quantity was express as ng/mg dry weight of the samples. Collagen and GAG content assay The collagen content was determined on the content of hydroxyproline (Hyp) (13). Both native ureters (n ¼ 6) and decellularized ureters (n ¼ 6, each group) were lyophilized and acid-hydrolyzed. The amount of total collagen content per mg dry weight of the decellularized ureters was calculated using a Hyp-to-collagen ratio of 1:7.2. The GAG content was quantified using a GAG assay kit (Bangyi, Shanghai, China) according to the manufacturer’s instructions. Final values were expressed as mg of GAG per dry weight. Scanning electron microscopy and porosity measurement The native ureters (n ¼ 4) and PDUs (n ¼ 4) were cut longitudinally, and expanded into slices. The slices were fixed and dehydrated through an ethanol gradient, sputter coated with gold and mounted for analysis (14). The tissue samples were then viewed under the scanning electron microscope (SEM, Zeiss, Germany). The swelling ratio can be used to reflect the porosity of native ureters and PDUs (15). Both native ureters (n ¼ 6) and PDUs (n ¼ 6) were immersed in PBS at 25 C for 24 h to achieve fully swollen, and measured the weight (Ws). Then, the samples were lyophilized and measured the weight (Wd). The swelling ratio was expressed as (Ws-Wd)/Wd. Isolation and culture of the UDCs Portions of the bladder (2.5 cm 2.5 cm) were removed from beagles. The muscular layer was minced and digested with 1 mg/ml collagenase type IV and dispase at 37 C for 30 min (16). The enzymatic activity was neutralized by high glucose-Dulbecco’s modified Eagle’s medium (HDMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco). Dissociated tissue was filtered to remove debris and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended and washed twice. The remaining cells were plated onto 100-mm culture dish and cultured in H-DMEM with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cultures were maintained at 37 C with 5% CO2. The medium was replaced every 3 days. The adherent cells were more than 80% confluent, they were digested with 0.25% trypsin-0.02% EDTA, and re-plated at a dilution of 1:2. Third-passage cells were used in the subsequent experiments.
Cytotoxicity assay of the PDUs The PDUs were incubated in standard HDMEM for 72 h, and the supernatant was collected as a test extract for later use (17). The UDCs were seeded in 24-well plates at a concentration of 2 104 cells/well, and incubated in standard H-DMEM for 24 h. The medium was then removed and replaced with the test extract. Cells cultured in standard medium served as the control group. The cell viability was observed by acridine orange and propidium iodide (AO/PI) staining, on days 1, 3 and 7. Five replicates were conducted per sample. At the same time, proliferative activity of the cells was determined by MTT assay on days 1, 3, 5, and 7. Recellularization of the PDUs with UDCs The PDUs were scissored longitudinally and cut into thin slices (1 cm 1 cm). The samples were immersed in HDMEM for 24 h and dried using sterile filter paper. The UDCs were seeded on the thin slices at a density of106 cells/cm2 in the 24-well plates. The seeded slices were incubated for 2 h before the supplemented culture medium was slowly added. The culture medium was changed every 3 days. The slices were collected and examined on days 3, 7, 14 after cell seeding. Three replicates were conducted each time. The samples were washed with PBS, fixed for 48 h in 10% formalin solution, embedded in paraffin and sectioned into 5 mm sections. The sections were stained with H&E and DAPI to observe the cells distribution in the PDUs. By IHC, the sections were stained with proliferating cell nuclear antigen (PCNA) to evaluate the cells’ proliferative ability. The primary antibody was rabbit anti-PCNA antibody (Boster, Wuhan, China) at a dilution of 1:200. The secondary antibody was goat anti-rabbit IgG (Abcam, Cambridge, MA) at a dilution of 1:100. Statistical analysis Statistical analysis was performed using SPSS (17.0, USA). The results were expressed as mean standard deviation. The differences between groups were estimated using Student’s t test. P values of less than 0.05 were considered significant.
RESULTS Evaluation of the decellularization efficiency The macroscopic images show the native ureters and PDUs. During the perfusion process, the ureters changed into white gradually (Fig. S1). H&E and DAPI staining revealed the absence of cell nuclei in the PDUs compared with the native ureters. The cellular nuclei could be found in the TritonX-100 group, while there was nothing in the SDS group (Fig. 1A). The DNA content was measured to evaluate the decellularized effect quantitatively. From the data, the significant difference could be lightly discovered between the PDUs and the native ureters (1760.8 190.1 ng/mg for the native ureters and 40.3 3.5 ng/mg for the decellularized ureters, P < 0.05). Also, there were not visible DNA bands on a 2% agarose gel (Fig. 1B). In the SDS group, the nuclei were almost completely removed (39.7 4.6 ng/mg), while a number of nuclei could be detected in the TritonX-100 group (158.5 27.8 ng/mg). There was no significant difference between the PDUs and the SDS group (Fig. 1C) (P > 0.05). Biologic components of the decellularized ureters Collagen and GAG, the two main biological components of ECM, had been detected. Masson’s trichrome and Alcian Blue staining showed that the collagen fiber and GAG were remained in the PDUs. In the TritonX-100 group, the collagen structure and GAG distribution became loose, while the collagen structure was severely damaged and GAG distribution was disordered in the SDS group (Fig. 2A). The IHC staining implied that the collagen type I was the main ingredient of collagen structure (Fig. S1). We used the Hyp content to calculate the collagen content. The collagen content in the PDUs did not reduced compared with that in the native ureters (284.47 19.60 mg/mg dry weight in the native ureters and 265.89 17.74 mg/mg dry weight in the PDUs, P > 0.05). However, the collagen content in the TritonX-100 group (196.57 10.44 mg/mg) and the SDS group (155.82 8.56 mg/mg) was significantly decreased, compared with the content of PDUs(Fig. 2B). The GAG content was tested by a GAG assay kit. The results showed that the GAG content in the PDUs decreased significantly (3.61 0.16 mg/mg) dry weight in the native ureters and 1.73 0.15 mg/mg dry weight in the PDUs, (P < 0.05). The GAG content had little difference between the TritonX-100 group (0.928 0.067 mg/mg) and the SDS group (0.890 0.024 mg/mg)
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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FIG. 1. (A) H&E and DAPI staining of the native ureters and the decellularized ureters. DAPI stained the nuclei. Scale bars are 100 mm. (B) Agarose gel electrophoresis showed the DNA bands of the native ureters and the decellularized ureters. (C) The DNA content of the native ureters and the decellularized ureters.*P < 0.05.
FIG. 2. (A) Masson trichrome staining and Alcian Blue staining of the native ureters and the decellularized ureters. Masson trichrome and Alcian Blue stained the distribution of collagen and GAG respectively. Scale bars are 100 mm. (B) The collagen content of the native ureters and the decellularized ureters. (C) The quantitative analysis of GAG of the native ureters and the decellularized ureters. *P < 0.05
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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(P > 0.05), while there was significant difference between the PDUs and the TritonX-100 group or SDS group (Fig. 2C).
DISCUSSION
SEM and porosity SEM indicated the ultrastructure of intraluminal surface and wall surface of the native ureters and the PDUs (Fig. 3). In the PDUs, the massive pores could be observed clearly. Nevertheless, the surfaces of the native ureters were very compact. The swelling ratio of the native ureters and the PDUs has obvious distinction (9.56 0.46 VS. 15.49 0.43 mg water/mg sample dry weight respectively, P < 0.05).
The native ECM, a unique niche of composition and form, serves as a foundational scaffold that supports organ-specific cell types and enables normal organ function (18). The goal of different decellularization protocols is to obtain the ECM, which completely remove the cellular components and preserve its unique structural and biochemical properties as much as possible. With the continuous development of decellularization techniques, perfusion decellularization have been widely used in the creation of wholeorgan scaffolds, such as heart (19), lung (20), liver (21), kidney (22). The principle of the perfusion decellularization is that the decellularized solution flows through the vascular structure of organs continuously, while taking away the cellular components and preserving the 3D structure of organs. Based on these, the perfusion system was introduced into our decellularized process. In this study, we had established a novel perfusiondecellularized protocol by combining a perfusion system with the commonly used physical and chemical methods (23). By utilizing this approach, intracellular ice crystals will disrupt the cellular membrane by rapidly freezing the ureter, which help to facilitate the infiltration of the decellularization solutions into the cells and preserve the ECM ultrastructure as far as possible (24). Moreover, the perfusion system with greatly increase the efficiency of decellularization. In this protocol, the effects of both detergents were taken into consideration respectively. Firstly, TritonX-100 is the most widely used non-ionic detergent in traditional protocols of decellularization with excellent infiltration capability but with poor cleaning effect (23). Conversely, SDS is a strong detergent which can effectively remove cellular components from tissue. However, SDS tends to disrupt the native tissue structure, and decreases the concentration of GAG (25). A number of cellular nuclei could be found in the decellularized ureters by applying TritonX-100 separately for 48 h. In addition, the collagen structure and GAG distribution became loose. In another control group, by using SDS separately for 48 h, the nuclei could not be detected in the decellularized ureters, but the collagen structure had suffered from severe destruction and the GAG distribution was disordered. Based our results, it was suggested that prolonged time of decellularization will destroy the structure and the bioactive ingredients of the decellularized ureters. In our modified method, firstly, the TritonX-100 was used for 7 h to loose the ECM structure and improve the cell permeability. Secondly, the SDS was used for just 1 h to remove the cellular nuclei rapidly. Moreover, the perfusion system kept the perfusion fluid through the ureteral tissue continuously, facilitated the circulation of detergent, and effectively took away the cell debris. The prepared decellularized ureters were evaluated in detail from the following three aspects: nuclear residues; structural integrity; retention of biological components. In tissue engineering research, residual nucleus is one of the most important evaluation indicators for the decellularized scaffolds, because the residual cellular material within ECM could elicit a predominantly M1 type macrophage response and result in the deposition of dense connective tissue and/or scarring (26). In addition, DNA is directly correlated to the adverse host reactions (27,28). Therefore, it was important that the cellular components were removed completely. There were objective criteria to evaluate the efficacy of the decellularization. The criteria included the following factors: 1) the DNA content, per mg ECM dry weight, less than 50 ng; 2) the DNA fragment length less than 200 bp; 3) lack of visible nuclear material in the tissue sections stained with DAPI and H&E (25). In this study, the nuclear material could not be observed in the PDUs by DAPI and H&E staining, while there were nuclear residues in the TritonX-100 treatment group. Besides, the DNA
Cytotoxicity analysis Comparing with the OD values in the extracts of the PDUs, the MTT test suggested that there was no difference in that when the cells cultured in standard medium (P > 0.05) (Fig. 4A). The AO/PI staining also confirmed that the extract of the PDUs was not toxic (Fig. 4B). Evaluation of UDCs integration with the PDUs It was observed that the UDCs seeded on the surface of the PDUs showed significant migration and proliferation properties on days 3, 7 and 14 by H&E and DAPI staining. On the third day, the UDCs formed multilayer structure on the surface of the PDUs, and a small amount of the UDCs infiltrated into the PDUs. On the seventh day, the multilayer structure became thin, and the UDCs gradually infiltrated into the deep of the PDUs. On the fourteenth day, the multilayer structure almost disappeared, while the UDCs continually proliferated, and formed laminated structure. The PCNA staining directly confirmed that the seeded cells maintained the proliferative ability (Fig. 5). Overall, the results demonstrated that the UDCs could survive, adhere, proliferate, and infiltrate into the deep of the PDUs.
FIG. 3. The 3D ultrastructure of the intraluminal surface (A) and the wall surface (B) of the native ureters and the PDUs. Scale bars are 20 mm, for magnified figures, scale bars are 10 mm.
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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FIG. 4. (A) Cytotoxicity analysis of the PDUs. The cellular proliferative activity of the cells cultured in standard medium and the extracts of the PDUs. (B) AO/PI staining showed the UDCs viability cultured in standard medium and extracts of the PDUs. The vital cells presented green fluorescence. Scale bars are 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
content (40.3 3.5 ng/mg) was less than 50 ng and the agarose gel electrophoresis also confirmed that there were no visible DNA bands in the PDUs. The prepared PDUs met the objective criteria for evaluating the efficacy of the decellularization, which indicated that the PDUs could be used to the further study. Collagen is the most abundant and ubiquitous protein in tissues, at the same time it provides strength to the ECM structures (29). By H&E and Masson staining, the structure of the PDUs was well preserved, but the collagen structure of the decellularized ureters was badly destroyed in the SDS treatment group. Collagen quantification showed that the collagen content had no significant difference between the PDUs and the native ureters, while the collagen content of the traditional treatment groups was decrease, especially the SDS treatment group. The SEM images indicated that the PDUs maintained the 3D network structure and had plentiful pores. Comparing with the native ureters, a significant increase in the swelling ratio was discovered in the PDUs, which also proved the existence of porous structure indirectly. We also confirmed that the collagen type I was the main ingredients of the 3D framework of
the PDUs. GAG, one of the most important components in the ECM, is able to bind growth factors and contribute to the cells adhesion and proliferation (30). The GAG content showed the decellularized ureters of the three groups had significant decline (P < 0.05), but repeated tests confirmed that the GAG content of the PDUs was nearly half of the native ureters, while the GAG content of the traditional treatment groups was little. The loss of the GAG may be due to its high sensitivity to the detergents, especially the SDS solution (31). Based on these detections, it was believed that the resulting PDUs well retained the framework and the majority of activity components. Furthermore, testing the biocompatibility of treated specimens was the most important feature of decellularized scaffolds for tissue engineering (32). MTT results and AO/PI confirmed that the PDUs were non-cytotoxic. Seeding the UDCs on the surface of the PDUs and then observing the cells distribution on different times were used to evaluate the feasibility of the PDUs for the tissueengineered repair. On the third day, the UDCs formed multilayer structure on the surface of the PDUs, and a small amount of the
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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FIG. 5. H&E, DAPI and PCNA staining of the PDUs seeded with the UDCs on days 3, 7, and 14. H&E showed the distribution of the UDCs in the PDUs. DAPI stained the nuclei clearly. PCNA stained the cells with the proliferative activity. The region between the white dotted lines represented the distribution of the UDCs in the decellularized ureters. The arrow represented the seeded surface. Scale bars are 100 mm.
UDCs infiltrate into the PDUs. As time goes on, the surface multilayer structure became thin, while the UDCs continually proliferated and infiltrated into the deep layer of the PDUs then formed laminated structure. All of these results suggested that the decellularized ureters prepared by the novel perfusion-decellularized protocol may be the potential surrogate for ureteral tissueengineered repair. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2016.06.009. ACKNOWLEDGMENTS This work was supported by Beijing Natural Science Foundation (7142145), The Scientific Research Foundation for the Returned Overseas Chinese Scholars, Natural Science Foundation of Hainan Province (20158299), Key Science Research Project of Medical and Health in Hainan Province (14A110062). References 1. Elliott, S. P. and Mcaninch, J. W.: Ureteral injuries: external and iatrogenic, Urol. Clin. North Am., 33, 55e66 (2006). 2. Kloskowski, T., Kowalczyk, T., Nowacki, M., and Drewa, T.: Tissue engineering and ureter regeneration: is it possible? Int. J. Artif. Organs, 36, 392e405 (2013). 3. Simaioforidis, V., De Jonge, P., Sloff, M., Oosterwijk, E., Geutjes, P., and Feitz, W. F.: Ureteral tissue engineering: where are we and how to proceed? Tissue Eng. Part B Rev., 19, 413e419 (2013). 4. Koch, H., Hammer, N., Ossmann, S., Schierle, K., Sack, U., Hofmann, J., Wecks, M., and Boldt, A.: Tissue engineering of ureteral grafts: preparation of biocompatible crosslinked ureteral scaffolds of porcine origin, Front. Bioeng. Biotechnol., 3, 89 (2015). 5. Badylak, S. F.: Xenogeneic extracellular matrix as a scaffold for tissue reconstruction, Transpl. Immunol., 12, 367e377 (2004). 6. Liao, W., Yang, S., Song, C., Li, X., Li, Y., and Xiong, Y.: Construction of ureteral grafts by seeding bone marrow mesenchymal stem cells and smooth muscle cells into bladder acellular matrix, Transpl. Proc., 45, 730e734 (2013).
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26. Brown, B. N., Valentin, J. E., Stewart-Akers, A. M., Mccabe, G. P., and Badylak, S. F.: Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component, Biomaterials, 30, 1482e1491 (2009). 27. Nagata, S., Hanayama, R., and Kawane, K.: Autoimmunity and the clearance of dead cells, Cell, 140, 619e630 (2010). 28. Zheng, M. H., Chen, J., Kirilak, Y., Willers, C., Xu, J., and Wood, D.: Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation, J. Biomed. Mater Res. B Appl. Biomater., 73, 61e67 (2005). 29. Badylak, S. F.: The extracellular matrix as a scaffold for tissue reconstruction, Semin. Cell Dev. Biol., 13, 377e383 (2002). 30. Yang, B., Zhang, Y., Zhou, L., Sun, Z., Zheng, J., Chen, Y., and Dai, Y.: Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering, Tissue Eng. Part C Methods, 16, 1201e1211 (2010). 31. Chen, K., Lin, X., Zhang, Q., Ni, J., Li, J., Xiao, J., Wang, Y., Ye, Y., Chen, L., Jin, K., and Chen, L.: Decellularized periosteum as a potential biologic scaffold for bone tissue engineering, Acta Biomater., 19, 46e55 (2015). 32. Xu, H., Xu, B., Yang, Q., Li, X., Ma, X., Xia, Q., Zhang, Y., Zhang, C., Wu, Y., and Zhang, Y.: Comparison of decellularization protocols for preparing a decellularized porcine annulus fibrosus scaffold, PLoS One, 9, e86723 (2014).
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissue-engineered repair Shu-wei Xiao,1 Peng-chao Wang,1 Wei-jun Fu,1, * Zhong-xin Wang,1 Gang Li,3 and Xu Zhang2 Department of Urology, Hainan Branch of Chinese People’s Liberation Army General Hospital, Hainan 572013, PR China,1 Department of Urology, General Hospital of Chinese People’s Liberation Army, Beijing 100853, PR China,2 and Department of Urology, 309 Hospital of Chinese People’s Liberation Army, Beijing 100091, PR China3 Received 6 January 2016; accepted 15 June 2016 Available online xxx
As the endoscopic technique is widely used in the diagnosis and treatment of diseases, the incidence of ureteral injuries increases annually. The classical surgical therapies are not always satisfactory. With the constant development of the tissue engineering technology in the field of urinary reconstruction, the ureteral reconstruction has become possible technology. In this study, a novel perfusion-decellularized protocol, which combined a perfusion system with the commonly used physical and chemical methods, was used to prepare the decellularized ureters for ureteral reconstruction and the urinary tract-derived cells (UDCs) were seeded on the surface of the perfusion-decellularized ureters (PDUs) in order to observe the cells survival, adhesion, proliferation and distribution. The data of H&E staining, DAPI staining, and the agarose gel electrophoresis demonstrated that the cellular components of PDUs were removed, and the decellularized time was shorter than previous study. In addition, compared with the native ureters, the DNA content of the PDUs was significantly decreased just two percent residue (P < 0.05). Scanning electron microscopy, collagen and glycosaminoglycan content assay showed that the three-dimensional (3D) ultrastructure and the compositions of the extracellular matrix (ECM)of PDUs were well preserved. When the UDCs were seeded onto the PDUs, the UDCs formed multilayer structure on the surface of the PDUs, infiltrated into the deep layer of the decellularized ureters and then formed laminated structure. In conclusion, the decellularized ureters prepared by the novel perfusion-decellularized method may be the potential surrogate for ureteral tissue-engineered repair. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Ureter; Decellularized; Perfusion system; Tissue engineering; Repair]
Ureter, as a retroperitoneal organ, is located in the deep of the body, therefore the incidence of ureteral injuries is low (1). In recent years, with the increasing use of endoscopic technique to diagnose and treat the diseases, the risks of ureteral injures were increasing. The classical surgical therapies, such as colon substitution, bladder flaps, transureteroureterostomy and renal autotransplantation could not achieve entirely functional recovery and they also might induce a series of clinical complications such as recurrent strictures, urinary leakage, even renal damages (2,3). However, tissue engineering technology could reconstruct the ureter and solve these clinical problems. With the advance of tissue engineering technology, the exploration of ureteral regeneration has never stopped. Various types of synthetic or biological scaffolds were introduced in ureteral reconstruction (3). However, synthetic scaffolds often had a lack of in biocompatibility, peristaltic motion, and incrustation (4). Unlike the synthetic scaffolds, the biological scaffolds had unique advantages, especially when the natural acellular matrix materials removed the antigenic cellular components, the native 3D ultrastructure and the compositions of the ECM were preserved. The remained ECM had a close relationship with the cell attachment, migration, proliferation and three-dimensional spatial
* Corresponding author. Tel./fax: þ86 0898 38830531. E-mail address: [email protected] (W.-j. Fu).
arrangement (5). The acellular matrix derived from different tissues was used to study the ureter regeneration, such as bladder, vessel, and ureter itself (6e9). The continuous stirring and the chemical detergents were widely used to prepare the ECM for ureteral tissueengineered repair (4,10,11). This decellularized process took several days or even a week, and the process which was continuous stirring and the effect of detergents could destroy the organizational structure and cause the loss of bioactive components of ECM. Besides, the prepared ECM has never been evaluated systematically. Moreover, the prepared ECM should be conducive to the cells adhesion and proliferation, which was beneficial for its survival in vivo and functional repair. In this study, we combined the perfusion system and the commonly used physical and chemical methods scientifically, and then explored the best decellularized time by trial and error. At last, a novel perfusion-decellularized protocol was established. Compared with the traditional methods, this perfusion process would take less time and remove more cellular components. Meanwhile the 3D structure was well preserved. The characteristics of the PDUs were also described in detail, including the structural integrity and the quantification of the biological components. Moreover, the feasibility of the PDUs for the tissue-engineered repair was evaluated clearly through seeding the UDCs on the surface of the PDUs. The UDCs could survive and proliferate in the decellularized ureters, which was beneficial for the tissueengineered repair.
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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Animals and decellularization processes Animal experiments were approved by the Animal Experimental Ethics Committee of People’s Liberation Army General Hospital. The ureters were harvested from the adult beagles (7e10 kg) euthanized in connection with other experimentation, and then were kept at 80 C for at least 24 h. A perfusion system was used throughout the whole decellularization process. The ureters were thawed at room temperature. A 16-gauge needle was inserted into the ureteral lumen and then connected to a perfusion pump using silicon tubing. The ureters were flushed with 1% TritonX-100 for 7 h, and then with the deionized water for 1 h. Subsequently, the ureters were treated with 1% Sodium dodecyl sulfate (SDS) for 1 h. Finally, the ureters were rinsed with deionized water for 24 h, in order to remove the detergent completely. During the decellularization process, the perfusion speed was controlled at 1.5 ml/ min. Meanwhile, the harvested ureters were treated by the commonly traditional methods (11). TritonX-100 treatment (TritonX-100 group): Ureters were placed in a 1% TritonX-100 solution with 0.02% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) together with DNase and RNase and shaken for 48 h. The solution was changed every 24 h. After the decellularized procedures, the decellularized tissue was washed with deionized water for 24 h. SDS treatment (SDS group): The ureters were treated in a solution of 0.5% SDS, 150 mM NaCl, and 10 mM EDTA in 10 mM tris-HCL (pH 8.0) and shaken for 48 h. Then the decellularized ureters were washed with deionized water for 24 h. Histological and immunohistochemical (IHC) analysis Native or decellularized ureters were fixed in 10% neutral buffered formalin, dehydrated with a graded ethanol and embedded in paraffin, sectioned into 5 mm slices. Haematoxylin and eosin (H&E) staining was used to obverse the cellular content and general structure of the decellularized ureters. Nucleic acids were stained with 4,6diamidino-2-phenylindole (DAPI). Masson’s trichrome staining was used to detect the collagen distribution and orientation. Alcian Blue staining was carried out for qualitative analysis of glycosaminoglycan (GAG) (12). In the IHC staining, we placed the slides into antigen retrieval solution and heated them until the temperature reached 95e100 C for 30 min. Endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide solution. We blocked slides with 4% goat serum. Sections were incubated with primary antibody at 4 C overnight. The secondary antibody was applied for 30 min. The slides were treated with streptavidin-horseradish peroxidase complex, diaminobenzidine (DAB) solution and counterstaining with hematoxylin, mounted and imaged using microscopy (Olympus, Tokyo, Japan). The primary antibody used was rabbit anti-Collagen type I antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:200. The secondary antibody was goat anti-rabbit IgG (Abcam, Cambridge, MA) at a dilution of 1:100. Quantification of total genomic DNA The total genomic DNA of the samples was extracted using a Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Both native ureters (n ¼ 6) and decellularized ureters (n ¼ 6, each group) were lyophilized and digested with proteinase K and RNase for 5 h until no visible material remained. The remaining genomic DNA was collected and quantified using the spectrophotometer (Bio-Rad, CA, USA). The size of the collected DNA fragments was exhibited using 2% agarose gel electrophoresis. The DNA quantity was express as ng/mg dry weight of the samples. Collagen and GAG content assay The collagen content was determined on the content of hydroxyproline (Hyp) (13). Both native ureters (n ¼ 6) and decellularized ureters (n ¼ 6, each group) were lyophilized and acid-hydrolyzed. The amount of total collagen content per mg dry weight of the decellularized ureters was calculated using a Hyp-to-collagen ratio of 1:7.2. The GAG content was quantified using a GAG assay kit (Bangyi, Shanghai, China) according to the manufacturer’s instructions. Final values were expressed as mg of GAG per dry weight. Scanning electron microscopy and porosity measurement The native ureters (n ¼ 4) and PDUs (n ¼ 4) were cut longitudinally, and expanded into slices. The slices were fixed and dehydrated through an ethanol gradient, sputter coated with gold and mounted for analysis (14). The tissue samples were then viewed under the scanning electron microscope (SEM, Zeiss, Germany). The swelling ratio can be used to reflect the porosity of native ureters and PDUs (15). Both native ureters (n ¼ 6) and PDUs (n ¼ 6) were immersed in PBS at 25 C for 24 h to achieve fully swollen, and measured the weight (Ws). Then, the samples were lyophilized and measured the weight (Wd). The swelling ratio was expressed as (Ws-Wd)/Wd. Isolation and culture of the UDCs Portions of the bladder (2.5 cm 2.5 cm) were removed from beagles. The muscular layer was minced and digested with 1 mg/ml collagenase type IV and dispase at 37 C for 30 min (16). The enzymatic activity was neutralized by high glucose-Dulbecco’s modified Eagle’s medium (HDMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco). Dissociated tissue was filtered to remove debris and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended and washed twice. The remaining cells were plated onto 100-mm culture dish and cultured in H-DMEM with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cultures were maintained at 37 C with 5% CO2. The medium was replaced every 3 days. The adherent cells were more than 80% confluent, they were digested with 0.25% trypsin-0.02% EDTA, and re-plated at a dilution of 1:2. Third-passage cells were used in the subsequent experiments.
Cytotoxicity assay of the PDUs The PDUs were incubated in standard HDMEM for 72 h, and the supernatant was collected as a test extract for later use (17). The UDCs were seeded in 24-well plates at a concentration of 2 104 cells/well, and incubated in standard H-DMEM for 24 h. The medium was then removed and replaced with the test extract. Cells cultured in standard medium served as the control group. The cell viability was observed by acridine orange and propidium iodide (AO/PI) staining, on days 1, 3 and 7. Five replicates were conducted per sample. At the same time, proliferative activity of the cells was determined by MTT assay on days 1, 3, 5, and 7. Recellularization of the PDUs with UDCs The PDUs were scissored longitudinally and cut into thin slices (1 cm 1 cm). The samples were immersed in HDMEM for 24 h and dried using sterile filter paper. The UDCs were seeded on the thin slices at a density of106 cells/cm2 in the 24-well plates. The seeded slices were incubated for 2 h before the supplemented culture medium was slowly added. The culture medium was changed every 3 days. The slices were collected and examined on days 3, 7, 14 after cell seeding. Three replicates were conducted each time. The samples were washed with PBS, fixed for 48 h in 10% formalin solution, embedded in paraffin and sectioned into 5 mm sections. The sections were stained with H&E and DAPI to observe the cells distribution in the PDUs. By IHC, the sections were stained with proliferating cell nuclear antigen (PCNA) to evaluate the cells’ proliferative ability. The primary antibody was rabbit anti-PCNA antibody (Boster, Wuhan, China) at a dilution of 1:200. The secondary antibody was goat anti-rabbit IgG (Abcam, Cambridge, MA) at a dilution of 1:100. Statistical analysis Statistical analysis was performed using SPSS (17.0, USA). The results were expressed as mean standard deviation. The differences between groups were estimated using Student’s t test. P values of less than 0.05 were considered significant.
RESULTS Evaluation of the decellularization efficiency The macroscopic images show the native ureters and PDUs. During the perfusion process, the ureters changed into white gradually (Fig. S1). H&E and DAPI staining revealed the absence of cell nuclei in the PDUs compared with the native ureters. The cellular nuclei could be found in the TritonX-100 group, while there was nothing in the SDS group (Fig. 1A). The DNA content was measured to evaluate the decellularized effect quantitatively. From the data, the significant difference could be lightly discovered between the PDUs and the native ureters (1760.8 190.1 ng/mg for the native ureters and 40.3 3.5 ng/mg for the decellularized ureters, P < 0.05). Also, there were not visible DNA bands on a 2% agarose gel (Fig. 1B). In the SDS group, the nuclei were almost completely removed (39.7 4.6 ng/mg), while a number of nuclei could be detected in the TritonX-100 group (158.5 27.8 ng/mg). There was no significant difference between the PDUs and the SDS group (Fig. 1C) (P > 0.05). Biologic components of the decellularized ureters Collagen and GAG, the two main biological components of ECM, had been detected. Masson’s trichrome and Alcian Blue staining showed that the collagen fiber and GAG were remained in the PDUs. In the TritonX-100 group, the collagen structure and GAG distribution became loose, while the collagen structure was severely damaged and GAG distribution was disordered in the SDS group (Fig. 2A). The IHC staining implied that the collagen type I was the main ingredient of collagen structure (Fig. S1). We used the Hyp content to calculate the collagen content. The collagen content in the PDUs did not reduced compared with that in the native ureters (284.47 19.60 mg/mg dry weight in the native ureters and 265.89 17.74 mg/mg dry weight in the PDUs, P > 0.05). However, the collagen content in the TritonX-100 group (196.57 10.44 mg/mg) and the SDS group (155.82 8.56 mg/mg) was significantly decreased, compared with the content of PDUs(Fig. 2B). The GAG content was tested by a GAG assay kit. The results showed that the GAG content in the PDUs decreased significantly (3.61 0.16 mg/mg) dry weight in the native ureters and 1.73 0.15 mg/mg dry weight in the PDUs, (P < 0.05). The GAG content had little difference between the TritonX-100 group (0.928 0.067 mg/mg) and the SDS group (0.890 0.024 mg/mg)
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FIG. 1. (A) H&E and DAPI staining of the native ureters and the decellularized ureters. DAPI stained the nuclei. Scale bars are 100 mm. (B) Agarose gel electrophoresis showed the DNA bands of the native ureters and the decellularized ureters. (C) The DNA content of the native ureters and the decellularized ureters.*P < 0.05.
FIG. 2. (A) Masson trichrome staining and Alcian Blue staining of the native ureters and the decellularized ureters. Masson trichrome and Alcian Blue stained the distribution of collagen and GAG respectively. Scale bars are 100 mm. (B) The collagen content of the native ureters and the decellularized ureters. (C) The quantitative analysis of GAG of the native ureters and the decellularized ureters. *P < 0.05
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(P > 0.05), while there was significant difference between the PDUs and the TritonX-100 group or SDS group (Fig. 2C).
DISCUSSION
SEM and porosity SEM indicated the ultrastructure of intraluminal surface and wall surface of the native ureters and the PDUs (Fig. 3). In the PDUs, the massive pores could be observed clearly. Nevertheless, the surfaces of the native ureters were very compact. The swelling ratio of the native ureters and the PDUs has obvious distinction (9.56 0.46 VS. 15.49 0.43 mg water/mg sample dry weight respectively, P < 0.05).
The native ECM, a unique niche of composition and form, serves as a foundational scaffold that supports organ-specific cell types and enables normal organ function (18). The goal of different decellularization protocols is to obtain the ECM, which completely remove the cellular components and preserve its unique structural and biochemical properties as much as possible. With the continuous development of decellularization techniques, perfusion decellularization have been widely used in the creation of wholeorgan scaffolds, such as heart (19), lung (20), liver (21), kidney (22). The principle of the perfusion decellularization is that the decellularized solution flows through the vascular structure of organs continuously, while taking away the cellular components and preserving the 3D structure of organs. Based on these, the perfusion system was introduced into our decellularized process. In this study, we had established a novel perfusiondecellularized protocol by combining a perfusion system with the commonly used physical and chemical methods (23). By utilizing this approach, intracellular ice crystals will disrupt the cellular membrane by rapidly freezing the ureter, which help to facilitate the infiltration of the decellularization solutions into the cells and preserve the ECM ultrastructure as far as possible (24). Moreover, the perfusion system with greatly increase the efficiency of decellularization. In this protocol, the effects of both detergents were taken into consideration respectively. Firstly, TritonX-100 is the most widely used non-ionic detergent in traditional protocols of decellularization with excellent infiltration capability but with poor cleaning effect (23). Conversely, SDS is a strong detergent which can effectively remove cellular components from tissue. However, SDS tends to disrupt the native tissue structure, and decreases the concentration of GAG (25). A number of cellular nuclei could be found in the decellularized ureters by applying TritonX-100 separately for 48 h. In addition, the collagen structure and GAG distribution became loose. In another control group, by using SDS separately for 48 h, the nuclei could not be detected in the decellularized ureters, but the collagen structure had suffered from severe destruction and the GAG distribution was disordered. Based our results, it was suggested that prolonged time of decellularization will destroy the structure and the bioactive ingredients of the decellularized ureters. In our modified method, firstly, the TritonX-100 was used for 7 h to loose the ECM structure and improve the cell permeability. Secondly, the SDS was used for just 1 h to remove the cellular nuclei rapidly. Moreover, the perfusion system kept the perfusion fluid through the ureteral tissue continuously, facilitated the circulation of detergent, and effectively took away the cell debris. The prepared decellularized ureters were evaluated in detail from the following three aspects: nuclear residues; structural integrity; retention of biological components. In tissue engineering research, residual nucleus is one of the most important evaluation indicators for the decellularized scaffolds, because the residual cellular material within ECM could elicit a predominantly M1 type macrophage response and result in the deposition of dense connective tissue and/or scarring (26). In addition, DNA is directly correlated to the adverse host reactions (27,28). Therefore, it was important that the cellular components were removed completely. There were objective criteria to evaluate the efficacy of the decellularization. The criteria included the following factors: 1) the DNA content, per mg ECM dry weight, less than 50 ng; 2) the DNA fragment length less than 200 bp; 3) lack of visible nuclear material in the tissue sections stained with DAPI and H&E (25). In this study, the nuclear material could not be observed in the PDUs by DAPI and H&E staining, while there were nuclear residues in the TritonX-100 treatment group. Besides, the DNA
Cytotoxicity analysis Comparing with the OD values in the extracts of the PDUs, the MTT test suggested that there was no difference in that when the cells cultured in standard medium (P > 0.05) (Fig. 4A). The AO/PI staining also confirmed that the extract of the PDUs was not toxic (Fig. 4B). Evaluation of UDCs integration with the PDUs It was observed that the UDCs seeded on the surface of the PDUs showed significant migration and proliferation properties on days 3, 7 and 14 by H&E and DAPI staining. On the third day, the UDCs formed multilayer structure on the surface of the PDUs, and a small amount of the UDCs infiltrated into the PDUs. On the seventh day, the multilayer structure became thin, and the UDCs gradually infiltrated into the deep of the PDUs. On the fourteenth day, the multilayer structure almost disappeared, while the UDCs continually proliferated, and formed laminated structure. The PCNA staining directly confirmed that the seeded cells maintained the proliferative ability (Fig. 5). Overall, the results demonstrated that the UDCs could survive, adhere, proliferate, and infiltrate into the deep of the PDUs.
FIG. 3. The 3D ultrastructure of the intraluminal surface (A) and the wall surface (B) of the native ureters and the PDUs. Scale bars are 20 mm, for magnified figures, scale bars are 10 mm.
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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FIG. 4. (A) Cytotoxicity analysis of the PDUs. The cellular proliferative activity of the cells cultured in standard medium and the extracts of the PDUs. (B) AO/PI staining showed the UDCs viability cultured in standard medium and extracts of the PDUs. The vital cells presented green fluorescence. Scale bars are 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
content (40.3 3.5 ng/mg) was less than 50 ng and the agarose gel electrophoresis also confirmed that there were no visible DNA bands in the PDUs. The prepared PDUs met the objective criteria for evaluating the efficacy of the decellularization, which indicated that the PDUs could be used to the further study. Collagen is the most abundant and ubiquitous protein in tissues, at the same time it provides strength to the ECM structures (29). By H&E and Masson staining, the structure of the PDUs was well preserved, but the collagen structure of the decellularized ureters was badly destroyed in the SDS treatment group. Collagen quantification showed that the collagen content had no significant difference between the PDUs and the native ureters, while the collagen content of the traditional treatment groups was decrease, especially the SDS treatment group. The SEM images indicated that the PDUs maintained the 3D network structure and had plentiful pores. Comparing with the native ureters, a significant increase in the swelling ratio was discovered in the PDUs, which also proved the existence of porous structure indirectly. We also confirmed that the collagen type I was the main ingredients of the 3D framework of
the PDUs. GAG, one of the most important components in the ECM, is able to bind growth factors and contribute to the cells adhesion and proliferation (30). The GAG content showed the decellularized ureters of the three groups had significant decline (P < 0.05), but repeated tests confirmed that the GAG content of the PDUs was nearly half of the native ureters, while the GAG content of the traditional treatment groups was little. The loss of the GAG may be due to its high sensitivity to the detergents, especially the SDS solution (31). Based on these detections, it was believed that the resulting PDUs well retained the framework and the majority of activity components. Furthermore, testing the biocompatibility of treated specimens was the most important feature of decellularized scaffolds for tissue engineering (32). MTT results and AO/PI confirmed that the PDUs were non-cytotoxic. Seeding the UDCs on the surface of the PDUs and then observing the cells distribution on different times were used to evaluate the feasibility of the PDUs for the tissueengineered repair. On the third day, the UDCs formed multilayer structure on the surface of the PDUs, and a small amount of the
Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009
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FIG. 5. H&E, DAPI and PCNA staining of the PDUs seeded with the UDCs on days 3, 7, and 14. H&E showed the distribution of the UDCs in the PDUs. DAPI stained the nuclei clearly. PCNA stained the cells with the proliferative activity. The region between the white dotted lines represented the distribution of the UDCs in the decellularized ureters. The arrow represented the seeded surface. Scale bars are 100 mm.
UDCs infiltrate into the PDUs. As time goes on, the surface multilayer structure became thin, while the UDCs continually proliferated and infiltrated into the deep layer of the PDUs then formed laminated structure. All of these results suggested that the decellularized ureters prepared by the novel perfusion-decellularized protocol may be the potential surrogate for ureteral tissueengineered repair. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2016.06.009. ACKNOWLEDGMENTS This work was supported by Beijing Natural Science Foundation (7142145), The Scientific Research Foundation for the Returned Overseas Chinese Scholars, Natural Science Foundation of Hainan Province (20158299), Key Science Research Project of Medical and Health in Hainan Province (14A110062). References 1. Elliott, S. P. and Mcaninch, J. W.: Ureteral injuries: external and iatrogenic, Urol. Clin. North Am., 33, 55e66 (2006). 2. Kloskowski, T., Kowalczyk, T., Nowacki, M., and Drewa, T.: Tissue engineering and ureter regeneration: is it possible? Int. J. Artif. Organs, 36, 392e405 (2013). 3. Simaioforidis, V., De Jonge, P., Sloff, M., Oosterwijk, E., Geutjes, P., and Feitz, W. F.: Ureteral tissue engineering: where are we and how to proceed? Tissue Eng. Part B Rev., 19, 413e419 (2013). 4. Koch, H., Hammer, N., Ossmann, S., Schierle, K., Sack, U., Hofmann, J., Wecks, M., and Boldt, A.: Tissue engineering of ureteral grafts: preparation of biocompatible crosslinked ureteral scaffolds of porcine origin, Front. Bioeng. Biotechnol., 3, 89 (2015). 5. Badylak, S. F.: Xenogeneic extracellular matrix as a scaffold for tissue reconstruction, Transpl. Immunol., 12, 367e377 (2004). 6. Liao, W., Yang, S., Song, C., Li, X., Li, Y., and Xiong, Y.: Construction of ureteral grafts by seeding bone marrow mesenchymal stem cells and smooth muscle cells into bladder acellular matrix, Transpl. Proc., 45, 730e734 (2013).
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Please cite this article in press as: Xiao, S., et al., Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissueengineered repair, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.06.009