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Human urine-derived stem cells seeded in a modified 3D porous small intestinal submucosa scaffold for urethral tissue engineering
Biomaterials 32 (2011) 1317e1326
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Human urine-derived stem cells seeded in a modified 3D porous small intestinal submucosa scaffold for urethral tissue engineering Shaofeng Wu a, b, Yan Liu a, Shantaram Bharadwaj a, Anthony Atala a, Yuanyuan Zhang a, * a b
Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, United States Shanghai Children’s Hospital, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 September 2010 Accepted 3 October 2010 Available online 4 November 2010
The goal of this study was to determine whether urothelial cells (UC) and smooth muscle cells (SMC) derived from the differentiation of urine-derived stem cells (USC) could be used to form engineered urethral tissue when seeded on a modified 3-D porous small intestinal submucosa (SIS) scaffold. Cells were obtained from 12 voided urine samples from 4 healthy individuals. USC were isolated, characterized and induced to differentiate into UC and SMC. Fresh SIS derived from pigs was decellularized with 5% peracetic acid (PAA). Differentiated UC and SMC derived from USC were seeded onto SIS scaffolds with highly porous microstructure in a layered co-culture fashion and cultured under dynamic conditions for one week. The seeded cells formed multiple uniform layers on the SIS and penetrated deeper into the porous matrix during dynamic culture. USC that were induced to differentiate also expressed UC markers (Uroplakin-III and AE1/AE3) or SMC markers (a-SM actin, desmin, and myosin) after implantation into athymic mice for one month, and the resulting tissues were similar to those formed when UC and SMC derived from native ureter were used. In conclusion, UC and SMC derived from USC could be maintained on 3-D porous SIS scaffold. The dynamic culture system promoted 3-D cellematrix ingrowth and development of a multilayer mucosal structure similar to that of native urinary tract tissue. USC may serve as an alternative cell source in cell-based tissue engineering for urethral reconstruction or other urological tissue repair. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Urine Stem cells Biomaterial Urethral stricture Tissue engineering
1. Introduction Urethral strictures can result from congenital defects, injury and infections. Despite considerable progress in the treatment of this condition, management of complicated long segment strictures is often very difficult. Current surgical repair techniques, such as urethrotomy or removal of the stricture with direct end-to-end anastomosis, and various grafting procedures often result in fibrosis and chronic inflammation, and these conditions can lead to stricture recurrence. Cell-based tissue engineering may offer an alternative technique for urethral reconstruction [1]. This process involves the use of biomaterial scaffolds that can be seeded with appropriate cells in the laboratory and implanted in vivo to replace or regenerate damaged tissue. Currently, for urethral tissue engineering, urothelial cells (UC) and smooth muscle cells (SMC) obtained via bladder or ureter biopsy are used as cell sources for the tissue engineering process. However, the biopsy is an invasive approach * Corresponding author. Tel.: þ1 336 713 1189; fax: þ1 336 713 7290. E-mail address: [email protected] (Y. Zhang). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.10.006
and it may lead to donor-site morbidity. A non-invasive procedure to obtain autologous cells for this process would be highly desirable. In our previous study, we demonstrated that progenitor cells can be isolated from voided urine [2]. About 0.2% of cells isolated from urine express markers characteristic of mesenchymal stem cells. These cells can expand extensively in culture and differentiate towards multiple bladder cell lineages as identified by the expression of UC, SMC, endothelial and interstitial cell markers. We initially referred to these cells as urine progenitor cells. However, our recent experiments indicated that urine-derived cells can give rise to additional specialized cell types, including osteocytes, chondrocytes, myocytes and adipocytes. Furthermore, in addition to the ability to differentiate into multiple lineages, these cells also exhibit self-renewal capacity, which is consistent with the definition of stem cells [3]. Thus, urine-derived stem cells (USC) may be an excellent alternative cell source for urological tissue engineering applications, especially because they are easily obtained from a patient using a non-invasive procedure. Another critical element required for successful urethral tissue engineering is the biomaterial scaffold. Three types of scaffolds have
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been introduced for urethral reconstruction. First, living autologous tissues such as buccal mucosa [4,5], skin [6], bladder mucosa [7,8], ureter [9], appendix [10], radial forearm free flap [11] and blood vessel [12] have been used as urethral grafts. Second, synthetic biomaterials, including poly L-polylactic acid [13] and copoly(L-lactide/epsilon-caprolactone) [P(LA/CL)] with a type I collagen sponge [14] have been tested in a number of studies, although synthetic biomaterials are not commonly used in urethral tissue engineering because they lack appropriate biologically active molecules that allow tissue regeneration across the graft to proceed quickly. Finally, natural collagen-based materials have been used. These are mostly derived from allogeneic or xenogenic sources and include small intestinal submucosa (SIS) [15e24], bladder “submucosa” (BSM) [25e28], demineralized bone matrix [29], urethral extracellular matrix [30], acellular artery matrix [31] and amniotic membranes [32]. Although living tissue grafts provide a favorable outcome, this procedure may increase hospital stays and lead to donorsite morbidity. As an alternative, natural collagen-based acellular matrices possess advantages for urethral tissue engineering because these materials promote early wound coverage, accelerate tissue formation and improve early functional reconstruction [25,33,34]. However, natural collagen-based materials have certain disadvantages that limit their clinical application. It has been shown that the high density of collagen, particularly on the mucosal side of the matrix, can block cell penetration into the collagen matrix when cells are seeded onto the material. In addition, despite decellularization procedures, these matrices can retain heterogenic cellular compounds that can result in chronic immunoreactions, fibrosis or calcification, all of which can lead to stricture recurrence. Finally, collagen materials often display wide batch-to-batch variations, leading to variable results. Therefore, a 3-dimensional (3-D) porous acellular scaffold that is free of native cellular compounds and that retains its mechanical strength during decellularization and reseeding procedure is desirable. Our recent study [35] demonstrated that a modified BSM scaffold that is free of cellular compounds possesses a 3-D porous microstructure after decellularization and oxidation with 5% peracetic acid (PAA). This 3-D porous BSM scaffold promoted cellematrix penetration and the formation of a multilayer of uroepithelial structure in vitro, and it supported cell growth in vivo when UC and SMC were seeded and grown in dynamic culture on the porous matrix. In this study, we used this well-established decellularization/oxidation strategy as described [35] to generate a tissue-engineered urethral construct using autologous USC seeded on 3-D porous SIS scaffold. This cell-based tissue-engineered urethra may be useful for patients with complicated long strictures who need urethral reconstruction. 2. Materials and methods 2.1. SIS scaffold preparation Fresh porcine small intestines were provided by Ni Sausage farm (Lexington, NC, USA). The mucosa were manually removed and washed in distilled water on a rotary shaker at 200 RPM and 4 C for 2 days (distilled water decellularization of SIS). For further decellularization, samples of SIS were oxidized by soaking in peracetic acid (PAA) at a concentration of 0 (without PAA, as control), 1, 2, 3, 5 or 10% (v/v) for 4 h. This was followed by a similar treatment with 1% Triton X-100 solution for another 2 days. The SIS was then washed with distilled water for 2 more days. Finally, the SIS scaffold was disinfected using 0.1% PAA in 20% ethanol for 2 h, rinsed three times with sterile distilled water for 10 min each and stored in sterile distilled water at 4 C until needed [35]. 2.2. Histological analysis of decellularized SIS Fresh and decellularized SIS samples were fixed in 10% buffered formalin and embedded in paraffin. Consecutive 5.0 mm sections were prepared for histological analysis. For detection and identification of cellular material, sections were
stained with hematoxylin and eosin (H&E) (Sigma, St. Louis, MO, USA) and 4,6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA, USA). Light micrographs were taken at 100 magnification. For measurement of residual DNA, decellularized SIS samples treated with the different concentrations of PAA (1, 3, 5 and 10%, n ¼ 4 for each concentration), distilled water (0% PAA) and fresh SIS samples (n ¼ 4) were dabbed dry with tissue paper and weighed. Samples were minced and any remaining nucleic acids were extracted using a commercially available kit (DNeasyÔ, Qiagen, Valencia, CA). The DNA concentration in each sample was calculated using a UV-spectrophotometer (ND-1000 NanoDrop Technologies, Wilmington, DE) to measure the absorbance at 260 nm. The DNA yield was normalized to the initial wet weight of the samples. 2.3. Scanning electron microscopy Fresh SIS and decellularized/oxidized SIS were fixed in 2.5% glutaraldehyde for at least 2 h at room temperature before washing with distilled water (3 min). The SIS was incubated at 80 C for 24 h, sputter coated with gold, and mounted for analysis. Electron micrographs of mucosa slides were obtained at 25.0 kV, 50 Pa, 500 magnifications using a Hitachi S-2600 Scanning Electron Microscope (Hitachi Technologies America, Pleasanton, CA). 2.4. Mechanical testing For mechanical testing, fresh SIS and decellularized/oxidized SIS samples were mechanically loaded onto a uniaxial load frame (Instron Corporation, Issaquah, WA, USA). A short strip of SIS was clamped at the ends for axial testing. The crosshead speed was set at 0.1 mm/s and the test was stopped when the force decreased by 10% after the onset of failure. At least ten specimens were tested for each treatment with or without PAA (6 groups: fresh SIS, 0%, 1%, 3%, 5% and 10% PAA) and the mean and standard deviation were calculated. Based on the stressestrain profile of the SIS samples, tensile elongation (at break) was also calculated. 2.5. Culture of human ureter cells and USC Samples of normal human ureter tissue were obtained from kidney transplant donors and used for isolating UC and SMC as described previously [35]. Briefly, the samples were digested with 1 mg/ml collagenase type IV at 37 C for 1 h. The UC were released by gentle scraping from the stroma and cultured in keratinocyteserum free medium (KFSM, Invitrogen, Carlsbad, CA, USA). For SMC isolation, the remaining tissues were minced finely into small pieces about 0.1 cm3 in size, and these pieces were plated in high glucose DMEM containing 10% fetal bovine serum (FBS). Culture medium was changed every 2 days until the cells reached 80%e90% confluence. Twelve voided urine samples (average about 200 ml/sample) were collected from 4 healthy male individuals. Each urine sample was centrifuged at 500 g for 5 min to collect cells. The supernatant was carefully aspirated and the cell pellet was gently resuspended in initiation medium, a 1:1 mixture of KSFM and embryonic fibroblast medium (EFM) [2]. Media was changed every other day after 48 h of initiation and cells were split at 70e80% confluence. 2.6. Urothelial and smooth muscle differentiation of USC For urothelial differentiation of USC, USC were plated at 3000 cells/cm2 in a mixture of medium containing KSFM and EFM at a ratio of 4:1 (2% FBS at final concentration) and 30 ng/ml epidermal growth factor (EGF; six times the EGF concentration of normal UC culture medium [36]) for 14 days. Media was replaced every 3 days and the cells were analyzed on days 7 and 14. For SMC differentiation, USC were plated at a density of 2000 cells/cm2 in myogenic differentiation media containing DMEM and EFM (without EGF, 10% FBS) at a ratio of 1:1, as well as TGF-b1 and PDGF-BB (R&D systems, Minneapolis, MN) at concentrations of 2.5 ng/ml and 5 ng/ml, respectively. Cell morphology changes were monitored. Media was replaced every 3 days and the cells were analyzed on days 7 and 14. Percentages of USC that had differentiated into UC and SMC were calculated based on immunofluorescent staining for UC and SMC-specific markers (Table 1).
Table 1 Antibodies used in this study. Primary antibody
Host
Dilution
Catalog #/Company
a-SM actin
Mice Mice Mice Mice Mice Mice
1:200 1:100 1:200 1:20 1:100 1:100
A5228/Sigma M0760/Dako M7786/Sigma AB78196/Abcam M3515/Dako NA09L/Calbiochem
Desmin Myosin Uroplakin-III AE1/AE3 NuMA
S. Wu et al. / Biomaterials 32 (2011) 1317e1326 2.7. Cell-seeded SIS scaffold in vitro To assess the effect of PAA treatment on scaffold porosity and the role of dynamic culture conditions on cell growth and cellematrix penetration, ureter SMC and UC were seeded on decellularized SIS in the wells of a 6-well plate. The SIS scaffold was firmly held over a sterile silicone insert for ease of cell seeding. For layered co-cultures, SMC were seeded on the serosal side of the SIS at a density of 2 105 cells/cm2/day in DMEM with 10% FBS under static culture for 3 days. Next, UC were mixed with KSFM-DMEM (1:1) and seeded on top of the muscle cells at similar cell numbers. The seeded scaffolds were then cultured under static (control) or 3-D dynamic culture conditions for 7 more days. After these 14 days of culture, the cellseeded SIS was released from the silicone insert and cut into 1 1 cm2 pieces for histological and immunohistochemical analysis or for implantation in vivo. To analyze the viability and proliferative capacity of the cells grown on the seeded decellularized SIS, MTT assays were performed. SMC and UC co-cultured on either 0% PAA or 5% PAA-treated SIS and grown under static or 3-D culture conditions (10 RPM and 40 RPM) were compared. Cells cultured in 96-well plates were incubated with MTT reagent as a control (Sigma, St. Louis, MO, USA) for 30 min and the optical density read using a SpectraMax-M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). 2.8. Implantation of cell-seeded SIS grafts in vivo SIS scaffolds seeded with cells obtained through urothelial and myogenic differentiation of USC (UC-USC and SMC-USC) were kept in static culture for 6e7 days and in dynamic culture for 7 more days as described at Section 2.7. USC seeded SIS constructs (n ¼ 12) were subcutaneously implanted into the flanks of athymic nude mice (4 weeks of age; 2 grafts/animal). SIS seeded with UC and SMC derived from ureteral samples (n ¼ 10) were used as controls and were implanted in a similar manner. All eleven animals were followed for 1 month. At this time, all animals were euthanized and the implants were removed for analysis and evaluation of cell infiltration and differentiation. 2.9. Immunohistochemical analysis To identify urothelial and myogenic differentiation of USC seeded SIS scaffolds in vitro and in vivo, the tissue graft samples were analyzed using immunohistochemistry. The tissue samples were embedded in OCT freezing media and immediately floated onto liquid nitrogen for 15 s. The frozen SIS samples were cut into 5 mm continuous sections using a cryostat (Leica, Bannockburn, IL USA). Cryosections (5 mm) were incubated overnight at 4 C with the following primary antibodies (see Table 1 for details): human-specific monoclonal antibody NuMA, for detection of grafted human cells within the implants in the mouse model; anti-Uroplakin-III and anti-cytokeratin (AE1/AE3) for visualization of UC; and anti a-SM actin, anti-desmin and anti-myosin for visualization of SMC. Slides were then washed with PBS and incubated with the appropriate fluorescence-conjugated secondary antibody (FITC/ TRITC conjugated goat anti mouse, Jackson Immuno Research) diluted at 1:200 for 30 min at room temperature. Finally, the slides were rinsed twice with PBS and counterstained with DAPI (Vectashield Vector, Burlingame, CA, USA) or Propidium Iodine (PI, Vectashield Vector, Burlingame, CA, USA) containing mounting media.
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in SIS that had been decellularized but not treated with PAA (Fig. 1Ab,e). 3.2. DNA content analysis In order to quantify the extent of decellularization in each group, the DNA content of decellularized SIS scaffolds was measured. It was observed that DNA content significantly decreased compared to fresh SIS when increasing concentrations of PAA were used. The DNA content of the decellularized SIS that had been treated with 0%, 1%, 3%, 5% and 10% PAA decreased dramatically with increasing PAA concentration (i.e. 2.86 0.05, 1.40 0.04, 0.73 0.02, 0.27 0.17, 0.15 0.01 and 0.04 0.01 mg DNA/mg tissue, respectively) (Fig. 1B). This result indicates that increasing the concentration of PAA leads to more effective removal of DNA from the matrix. In addition, mechanical testing results indicated that there was no significant difference in tensile elongation at break between each PAA treatment group. The percentage elongation at break is as follows: 0% PAA ¼ 198.1 24.8, 1% PAA ¼ 200 23.2, 3% PAA ¼ 213.9 12.0 and 5% PAA ¼ 191.3 51.1 (n ¼ 10 for each treatment, p > 0.05). 3.3. Scanning electron microscopy Scanning electron micrographs showed the ultrastructure of the smooth and water-tight mucosa basal side of fresh SIS (Fig. 2a). Following 1% triton decellularization (0% PAA) treatment, porosity or cracks appeared on the surface of the basal membrane (Fig. 2b). After 5% PAA treatment, the extent of porosity and the porous size were considerably increased compared to fresh SIS and 0% PAA treatment (Fig. 2c). 3.4. Effect of porous SIS scaffold on cellematrix penetration The number of ureter-derived SMC that penetrated into the porous SIS matrix increased when the matrix was treated with PAA (Fig. 3). In addition, more layers of ureter-dervied UC were formed on the top of the ureter-derived SMC layers during co-culture on the decellularized/oxidized SIS treated with 5% PAA compared to non-PAA treated SIS (Fig. 3). This indicates that PAA treatment enhances the porosity of the collagen matrix and promotes cellular penetration into the matrix.
2.10. Statistical analysis For DNA content, MTT and tensile strength analysis, means and standard errors of the mean (SEM) were calculated for the groups (n ¼ 4 for DNA content, n ¼ 6 for MTT test, n ¼ 10 for tensile strength each PAA treatment) and a comparison was made between groups using a two tailed, Student’s t-test with unequal variances. Differences were considered significant at p < 0.05.
3. Results 3.1. Histological analysis of decellularized SIS A cross section of fresh SIS stained with H&E (Fig. 1Aa) and DAPI (Fig. 1Ad) shows that without decellularization, dense cellular components are seen within the SIS. Even after treatment with distilled water decellularization procedures (0% PAA), a certain amount of nuclear material was still retained in the matrix (Fig. 1Ab and e). In contrast, decellularization of SIS following 5% PAA treatment was nearly complete, and nuclear material could not be seen after the process (Fig. 1Ac,f). However, the tissue structure in the 5% PAA treatment group (Fig. 1Ac) become visibly more porous, and the intra- and inter-fascicular space was increased in the lamina propria that appear more extended compared to that seen
3.5. Effect of dynamic culture on ingrowth of ureter cells To optimize the culture conditions, ureter UC and SMC were grown under static (control) or dynamic culture conditions at different rotational speeds. During static culture, the seeded ureter UC formed 2e3 cell layers on the surface of the scaffold. In addition, when ureter SMC were seeded onto the SIS, the SMC penetrated the scaffold to a depth of 3e5 cell layers after static culture (Fig. 4A, left column). However, use of 3-D dynamic culture conditions (10 RPM and 40 RPM) led to growth of at least 3e6 layers of urothelial cells on top of 10e12 layers of SMC on the serosal side of the SIS scaffold (Fig. 4A middle and right column). In addition, SMC also infiltrated the matrix to a larger extent (50e60% of the matrix) when this dynamic co-culture method was used. Next, an MTT assay was performed to measure the cell growth pattern of both UC and SMC within the SIS. The data from this analysis corroborates the finding that a significant increase in cell number was observed in the scaffolds treated with 5% PAA compared to those treated with 0% PAA (Fig. 4B). In addition, the cell number significantly increased under dynamic culture (10 or 40 RPM) compared to static culture conditions (Fig. 4A,B).
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Fig. 1. Comparison of gross morphology of decellularized SIS with PAA treatment. (A) H&E (a,b,c) and DAPI staining (d,e,f) on cross sections of fresh SIS (a,d), 0% PAA (i.e. distill water with non-PAA treated) treated SIS (b,e) and 5% PAA-treated SIS (c,f). Cells and cell nuclei are visible in fresh SIS tissue (arrows; a,d) whereas a rapid decrease in cellular contents was visible with 0% PAA treatment (decellularization alone) (arrows; b,e). However, complete removal of all cellular components was achieved using 5% PAA treatment (c,f). An increase in porosity of the micro-architecture of SIS was visible after PAA treatment (c,f). Scale bar represents 200 um. (B) Effect of PAA on DNA clearance. DNA was extracted from fresh SIS and decellularized SIS prior to and after PPA treatment. The DNA values were plotted after normalizing to initial wet weight of the sample. A significant clearance of DNA content was observed with increased PAA concentrations.
Fig. 2. Electron micrograph of mucosal side of decellularized SIS with PAA. (a) Untreated (fresh) SIS, (b) Decellularized SIS (0% PAA treatment) and (c) Decellularized SIS (5% PAA). A remarkable high level of porosity was achieved after treatment with 5% PAA when compared to the 0% PAA treatment. Scale bar ¼ 100 um.
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Fig. 3. Effect of PAA concentration on cell penetration and proliferation. SIS were treated with 0% and 5% PAA prior to seeding with ureter SMC and UC for 14 Days. Sections were stained with Masson’s Trichrome (top panel) and DAPI (bottom panel). Cellematrix ingrowth in 5% PAA-treated SIS were significantly higher (arrows, indicating presence of cells at different depths) than that of the 0% PAA treatment. UC and SMC layers are marked. The vertical black line in the top panels on the left side indicates the scaffold thickness. Scale bar represents 100 um.
3.6. Induced USC in a porous SIS scaffold in vitro
3.8. Urothelial and myogenic-differentiated USC in vivo
Following in vitro differentiation of USC with myogenic growth factors, 50e70% cells at passage 3 expressed SMC markers (SMCUSC) compared to less than 1/4 of the cells in the untreated control group. Similarly, up to 90% of the USC expressed UC markers (UCUSC) at passage 3 when high doses of EGF (30 ng/ml) were used, compared to only 20% of the cells in the control group, which was treated with 7.5 ng/ml EGF. When both SMC-USC and UC-USC were seeded onto a SIS matrix in a layered co-culture fashion under dynamic culture conditions (40 RPM), SMC-USC were able to penetrate the SIS matrix treated with 5% PAA to a greater depth than SMC-USC seeded onto SIS matrices that were not treated with PAA (Fig. 5A). A few cell layers of UC-USC were formed on top of the SMC-USC, and the resulting urethral-like structure was similar to that seen when ureter UC were co-cultured with ureter SMC in vitro (Fig. 3).
Immunofluorescent studies confirmed that the urothelial cell differentiated USC were layered in a well-organized manner on the top of the myogenic-differentiated USC on the luminal side of the SIS scaffold when the layered co-culture system was used in vitro. The UC-USC stained positive for both AE1/AE3 (Fig. 6A, top right) and uroplakin-III (Fig. 6B, top right). One month after implantation in vivo, differentiation of USC to urothelial-like cells was demonstrated by immunofluorescent staining for the expression of urothelial-specific antigens (Fig. 6A and B, bottom right). These staining patterns were similar to those seen in native urothelium and when ureter-derived UC was co-cultured with SMC on SIS in vitro and in vivo (Fig. 6 A and B, left panel). SMC-specific staining for a-SM actin, desmin, and myosin in the seeded scaffolds in vitro indicated well-organized layers of SMC-like cells on and partially within the SMC-USC seeded SIS scaffold (Fig. 7AeC, top right). This pattern was similar to that seen when ureter-derived SMC were seeded on SIS in vitro (Fig. 7A, B, C, top left). Moreover, SMC-USC seeded on SIS and implanted in vivo stained positive for the same SMC markers (Fig. 7A, B, C, bottom right).
3.7. Implantation of USC seeded SIS in vivo All animals used for in vivo studies survived and remained healthy during the course of the experiment. No evidence of inflammatory reactions, infections, or implant extrusion was observed at the sites of implantation in the seeded grafts. Microscopically, progressive tissue regeneration was observed in the cellseeded porous grafts. The use of an antibody specific for human nuclei (Nuclear Mitotic apparatus), which stained bright green and appeared yellow when merged with the red PI nuclear stain (Fig. 5B), indicated that the implanted human USC-derived cells survived up to one month after implantation in vivo. Cellular penetration into the matrix was also evident (arrows) when both H&E and Masson’s trichrome staining were performed on scaffolds that had been grown in vitro and in vivo (Fig. 5A). Small blood vessel penetration into the SIS matrix could also be seen in vivo, and the onset of some graft degradation was also observed at the edges of each decellularized/oxidized SIS scaffold after one month in vivo.
4. Discussion This study indicates that using autologous USC as an alternative cell source for urethral tissue engineering may be advantageous. The cells can be efficiently differentiated into UC and SMC using epithelial and myogenic growth factors, respectively, and these can then be seeded on a modified 3-D SIS scaffold. One voided urine sample of about 200 ml can generate at least 3.2 108 viable cells at early passages (p4), and this provides enough cells to create a tissueengineered urethral construct. Additionally, fully-decellularized SIS provides a 3-D porous collagen matrix, which encourages cell repopulation within the scaffold. The modified SIS scaffold retains majority of its original mechanical strength, with removal of all of the heterogeneous cellular compounds. A combination of this
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Fig. 4. Effect of culture condition on cell penetration and proliferation. A) Masson’s Trichrome staining (top panel) and DAPI staining (bottom panel) performed on 5% PAA-treated SIS, seeded with ureter SMC and UC under static or dynamic (10 or 40 RPM) culture condition for 14 d. Cell penetration (lower arrow) and proliferation (upper arrow) was significantly improved with dynamic culture condition as compared to static culture condition. The vertical black line in the top panels denotes scaffold thickness. Inset images shows lower magnification of the entire thickness of the scaffold. Scale bar represents 100 um. B) Analysis of cell growth. Ureter SMC and UC seeded SIS were analyzed for cell growth using the MTT assay. OD values were recorded after reading the plate at 450 nm. The graph shows cells grown in the PAA-treated (5%) and the untreated (0%) under different static and dynamic (10 and 40 RPM) culture condition. Irrespective of the culture conditions, the treatment with PAA caused a significant growth of cells compared to the nontreatment (0%) (p < 0.05).
porous matrix, dynamic culture conditions and a system to co-culture epithelial and stromal cells significantly enhances cellematrix infiltration by SMC as well as urothelial mucosa formation by UC. Both of these are required for the creation of a cell-seeded SIS scaffold for urethral tissue engineering. The use of uroepithelial cells isolated from urine or bladder washes for urological tissue engineering applications has been reported [37,38]. However, the success rate of cultures of these cells is low (55%), and they also have limited expansion capability in culture. In a recent study, we were able to successfully isolate and culture USC from almost every urine sample tested. USC are not
mature UC or SMC, and they express pericyte/mesenchymal stem cell markers as well as detectable levels of telomerase [3]. Importantly, these cells possess both self-renewal and multilineage differentiation capabilities. They can give rise to osteocytes, chrodrocytes, adipocytes, skeletal myocytes [3], interstitial cells and endothelial cells [2]. They differentiate particularly well into bladder cells, i.e. UC and SMC [2]. The numbers of USC clones that can be consistently obtained from each freshly voided urine sample (200e300 ml) ranges from 10 to 15. The average population doubling (PD) of USC in these urine samples was about 34 (ranging from 32e46, n ¼ 9). In other words, a single cell clone of USC could
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Fig. 5. Tracking of human cell-seeded SIS in vitro and in vivo. A) Masson’s trichrome staining, as shown on the left at 200, depicts cellular (SMC) penetration (black arrows) and proliferation (black arrowheads) as well as UC layers (white arrows). The in vivo image at 400 shows appearance of vessel-like structures (black arrows). B) Detection of human ureter cells by immunostaining with human nuclear antibody (NuMA, in green) in layered co-culture of ureter UC/SMC (as controls in the left column) and SMC-USC along with UCUSC in the right column) in vitro (top panel) as well as in vivo (bottom panel), 4 weeks after implantation. Arrows indicate representative strong positively labeled implanted human cells. As the cell nuclei were counterstained with PI, human cells appear yellow due to merging of the green and red signal. Scale bar represents 50 um.
expand to 234, or 1.7 10 exp10 cells, by passage 6e8 within 5 weeks [39]. However, in order to maintain the maximal multipotent capability of USC, we used them at earlier passages (p3e4). A single cell clone can generate 32e48 106 cells (Population doublings ¼ 25) at passage 4 and this occurs within 3e4 weeks. To prepare a cell-seeded scaffold for use in urological tissue regeneration, the cell concentration for seeding must be about 50 106 cells/cm3 [1]. Thus, the number of cells from one urine sample of about 200 ml volume can provide enough cells to create a piece of cell-seeded scaffold about 6.5e10 cm3 in size, and USC from multiple urine samples could easily meet the required cell numbers for constructing larger cell-scaffold constructs for repair of longer urethral segments. Importantly, USC are capable of efficiently differentiating into both UC and SMC with the application of defined growth factors. Up to 70% of USC induced with muscle-specific growth factors express SMC markers (alpha-smooth muscle actin, desmin, and myosin) compared to 10e25% of cells induced in DMEM with 10% FBS in vitro [2]. Similarly, the majority of USC can give rise to urotheliallike cells displaying the morphology and cellular markers (AE1/AE3 and Uroplakin-III) of UC when induced using 4 times the dose of EGF used during standard proliferation. However, USC start to senesce and a decrease in differentiation potential becomes apparent at higher passages (p10). For USC to have a robust potential, it is recommended that they be used at passages younger than 6 for cell-based therapy. Although natural collagen matrices such as SIS or BSM are commonly used for cell-based tissue engineering, fibrosis formation and inflammatory reactions have limited their clinical application to date. These complications of collagen matrix use are caused by xenogenic proteins that remain within the matrix and create immunogenicity. This immune response eventually leads to fibrosis or calcification of the biomaterial. Additionally, the high density of collagen within the matrix inhibits cellematrix infiltration. Our recent study on the decellularization of another natural collagen matrix, BSM [35], demonstrates that oxidation procedures provide a more optimal decellularization protocol for use in the fabrication of biological collagen matrices with minimal retention of cellular components. Oxidation also leads to higher scaffold porosity and increased pore size, which should increase cellematrix infiltration.
Both a porous matrix and a dynamic culture system play important roles in cellematrix infiltration. The dynamic culture system promotes 3-D cell ingrowth within the highly porous matrix, thereby making it potentially useful in cell-based tissue engineering for hollow organ tissue engineering or other soft tissue repair [35]. In this study, we used the same approaches to generate a 3-D SIS scaffold with porous microstructure. For fully-decellularized SIS, we combined mechanical agitation (physical) and Triton X-100, along with high concentrations (5%) of PAA (chemical). We found that PAA at a concentration of 5% increased porosity within the matrix compared to untreated SIS. Additionally, more cells infiltrated into the matrix on the serosal side rather than the urothelial side. Better cellematrix infiltration was observed when the combination of both a highly porous matrix and dynamic culture conditions were used than with any of the other approaches alone. Dynamic culture within a bioreactor enhances the efficiency not only of cellematrix infiltration but also of the homogeneity of bladder cell differentiation. Furthermore, decellularized SIS treated with 5% PAA retained similar mechanical strength as the non-PAA treated matrix. Thus, the culture system described here utilizes simple, efficient and low cost equipment to improve the culture environment for use in urological tissue engineering. SIS possesses a unique property, in which its permeability is “sided”, or direction-dependent. The mucosal to serosal direction is less permeable than the serosal to mucosal direction. When nonseeded SIS is used in urological applications, this property should be considered because it can assist in preventing urine leakage from the lumen of the urethra or bladder into surrounding tissues. However, in cell-based tissue engineering, this direction-dependent permeability appears less important in preventing urine leakage, because heavy cellular infiltration “fills up” the pores within the matrix to prevent leakage. Therefore, the porous SIS scaffold is suitable for cell-seeded tissue engineering in urethral reconstruction. In vivo data in this study demonstrated that the urothelial and smooth muscle differentiated USC survive and maintain both cell type markers one month after implantation, suggesting that differentiated grafted cells could retain urological cell phenotypes in the in vivo environment. The urethral tissues regenerated from differentiated USC using this 3D porous collagen scaffold will be investigated in a large animal model in a future study.
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Fig. 6. Immunofluorescent staining of urothelial-differentiated USC in vitro and in vivo. Tissue sections of in vivo and in vitro cultured cells were immunostained (green) using A) pancytokeratin (AE1/AE3) and B) Uroplakin-III antibodies. USC were differentiated in vitro with EGF for 14 days by using dynamic culture condition (10 RPM) followed by subcutaneous implantation into nude mice for a further 4 weeks. The nuclei were counterstained with PI (red). The left panel indicates in vitro and in vivo co-cultures of UC and SMC as a positive control. Arrows show a few representative cells that are positively stained. Scale bar represents 50 um.
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Fig. 7. Immunofluorescent staining of myogenic-differentiated USC in vitro and in vivo. Tissue sections of in vitro (top panel) and in vivo (bottom panel) cultured cells that were immunostained (green or red) using A) a-SM actin, B) Desmin and C) Myosin antibodies. USC were differentiated in vitro using PDGF and TGF for 14 days under dynamic condition (10 RPM), following by subcutaneously implantation into nude mice for a further 4 weeks (right column). In vitro and in vivo seeded UC/SMC scaffolds (left column) were used as controls. Positively stained cells for a SM actin and Myosin appear green (FITC), whereas staining for Desmin appears red (Rhodamine). Nuclei were counterstained with PI (red) or DAPI (blue). Arrows show a few representative cells that are positively stained. Scale bar represents 50 um.
5. Conclusions USC can be easily and consistently obtained by a non-invasive approach and extensively expanded in vitro. USC efficiently give rise to UC and SMC in appropriate induction conditions. In addition, oxidized SIS scaffolds have higher porosity and larger pore size than non-oxidized scaffolds, and they are almost completely free of cellular compounds that could lead to immune reactions in vivo. Thus, their use allows abundant graft cells to be loaded onto a biological scaffold. This contributes to a higher cellular proliferation rate and increased cellematrix infiltration in vitro, and may also enhance the ability of host cells to infiltrate into the matrix when the scaffold is implanted in vivo, thereby leading to better in vivo tissue regeneration. Using these modified decellularization procedures, 3-D porous SIS that possesses the highly interconnected macro- or micro-porous structures to allow cell infiltration into the matrix is created. Importantly, heterogeneous cellular proteins are removed, which may eliminate inflammatory reactions, fibrosis, calcification and graft shrinkage that are seen
when other types of scaffolds are used for urological tissue engineering. Finally, dynamic culture conditions maximize cellematrix infiltration and cell growth. This study indicated that use of urothelial and smooth muscle cells derived from the differentiation of USC and seeded on a 3D porous biological collagen matrix could be used to construct a tissue-engineered urethra. Acknowledgements The authors would like to thank Dr. Karl-Erik Andersson for valuable comments and Dr. Jennifer Olson and Ms. Carmen Pruitt for editorial assistance with this manuscript. Appendix Figures with essential colour discrimination. Figs. 1 and 3e7 in this article are difficult to interpret in black and white. The full colour images can be found in the online version, at doi:10.1016/j. biomaterials.2010.10.006.
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[20] Gilbert TW, Wognum S, Joyce EM, Freytes DO, Sacks MS, Badylak SF. Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix. Biomaterials 2008;29:4775e82. [21] Chen RN, Ho HO, Tsai YT, Sheu MT. Process development of an acellular dermal matrix (ADM) for biomedical applications. Biomaterials 2004;25:2679e86. [22] Sacks MS, Gloeckner DC. Quantification of the fiber architecture and biaxial mechanical behavior of porcine intestinal submucosa. J Biomed Mater Res 1999;46:1e10. [23] Raghavan D, Kropp BP, Lin HK, Zhang Y, Cowan R, Madihally SV. Physical characteristics of small intestinal submucosa scaffolds are location-dependent. J Biomed Mater Res A 2005;73:90e6. [24] Zhang Y, Kropp BP, Lin HK, Cowan R, Cheng EY. Bladder regeneration with cell-seeded small intestinal submucosa. Tissue Eng 2004;10:181e7. [25] Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible “off the shelf” biomaterial for urethral repair. Urology 1999;54:407e10. [26] Fu Q, Deng CL, Liu W, Cao YL. Urethral replacement using epidermal cell-seeded tubular acellular bladder collagen matrix. BJU Int 2007;99: 1162e5. [27] Chen F, Yoo JJ, Atala A. Experimental and clinical experience using tissue regeneration for urethral reconstruction. World J Urol 2000;18:67e70. [28] Pariente JL, Kim BS, Atala A. In vitro biocompatibility evaluation of naturally derived and synthetic biomaterials using normal human bladder smooth muscle cells. J Urol 2002;167:1867e71. [29] el-Kassaby A, AbouShwareb T, Atala A. Randomized comparative study between buccal mucosal and acellular bladder matrix grafts in complex anterior urethral strictures. J Urol 2008;179:1432e6. [30] Hu YF, Yang SX, Wang LL, Jin HM. Curative effect and histocompatibility evaluation of reconstruction of traumatic defect of rabbit urethra using extracellular matrix. Chin J Traumatol 2008;11:274e8. [31] Parnigotto PP, Gamba PG, Conconi MT, Midrio P. Experimental defect in rabbit urethra repaired with acellular aortic matrix. Urol Res 2000;28:46e51. [32] Koziak A, Marcheluk A, Dmowski T, Szczesniewski R, Kania P, Dorobek A. Reconstructive surgery of male urethra using human amnion membranes (grafts)efirst announcement. Ann Transplant 2004;9:21e4. [33] Fiala R, Vidlar A, Vrtal R, Belej K, Student V. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol 2007;51:1702e8. discussion 1708. [34] Feng C, Xu YM, Fu Q, Zhu WD, Cui L, Chen J. Evaluation of the biocompatibility and mechanical properties of naturally derived and synthetic scaffolds for urethral reconstruction. J Biomed Mater Res A 2010;94:317e25. [35] Liu Y, Bharadwaj S, Lee SJ, Atala A, Zhang Y. Optimization of a natural collagen scaffold to aid cell-matrix penetration for urologic tissue engineering. Biomaterials 2009;30:3865e73. [36] Zhang YY, Frey P. Growth of cultured human urothelial cells into stratified urothelial sheet suitable for autografts. Adv Exp Med Biol 2003;539: 907e20. [37] Nagele U, Maurer S, Feil G, Bock C, Krug J, Sievert KD, et al. In vitro investigations of tissue-engineered multilayered urothelium established from bladder washings. Eur Urol 2008;54:1414e22. [38] Fossum M, Gustafson CJ, Nordenskjold A, Kratz G. Isolation and in vitro cultivation of human urothelial cells from bladder washings of adult patients and children. Scand J Plast Reconstr Surg Hand Surg 2003;37:41e5. [39] Bharadwaj S, Wu S, Rohozinski J, Furth M, Atala A, Zhang Y. Multipotent stem cells from urine for tissue engineered bladder. American Urology Associate (AUA); 2010. Abstract: E61.
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Human urine-derived stem cells seeded in a modified 3D porous small intestinal submucosa scaffold for urethral tissue engineering Shaofeng Wu a, b, Yan Liu a, Shantaram Bharadwaj a, Anthony Atala a, Yuanyuan Zhang a, * a b
Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, United States Shanghai Children’s Hospital, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 September 2010 Accepted 3 October 2010 Available online 4 November 2010
The goal of this study was to determine whether urothelial cells (UC) and smooth muscle cells (SMC) derived from the differentiation of urine-derived stem cells (USC) could be used to form engineered urethral tissue when seeded on a modified 3-D porous small intestinal submucosa (SIS) scaffold. Cells were obtained from 12 voided urine samples from 4 healthy individuals. USC were isolated, characterized and induced to differentiate into UC and SMC. Fresh SIS derived from pigs was decellularized with 5% peracetic acid (PAA). Differentiated UC and SMC derived from USC were seeded onto SIS scaffolds with highly porous microstructure in a layered co-culture fashion and cultured under dynamic conditions for one week. The seeded cells formed multiple uniform layers on the SIS and penetrated deeper into the porous matrix during dynamic culture. USC that were induced to differentiate also expressed UC markers (Uroplakin-III and AE1/AE3) or SMC markers (a-SM actin, desmin, and myosin) after implantation into athymic mice for one month, and the resulting tissues were similar to those formed when UC and SMC derived from native ureter were used. In conclusion, UC and SMC derived from USC could be maintained on 3-D porous SIS scaffold. The dynamic culture system promoted 3-D cellematrix ingrowth and development of a multilayer mucosal structure similar to that of native urinary tract tissue. USC may serve as an alternative cell source in cell-based tissue engineering for urethral reconstruction or other urological tissue repair. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Urine Stem cells Biomaterial Urethral stricture Tissue engineering
1. Introduction Urethral strictures can result from congenital defects, injury and infections. Despite considerable progress in the treatment of this condition, management of complicated long segment strictures is often very difficult. Current surgical repair techniques, such as urethrotomy or removal of the stricture with direct end-to-end anastomosis, and various grafting procedures often result in fibrosis and chronic inflammation, and these conditions can lead to stricture recurrence. Cell-based tissue engineering may offer an alternative technique for urethral reconstruction [1]. This process involves the use of biomaterial scaffolds that can be seeded with appropriate cells in the laboratory and implanted in vivo to replace or regenerate damaged tissue. Currently, for urethral tissue engineering, urothelial cells (UC) and smooth muscle cells (SMC) obtained via bladder or ureter biopsy are used as cell sources for the tissue engineering process. However, the biopsy is an invasive approach * Corresponding author. Tel.: þ1 336 713 1189; fax: þ1 336 713 7290. E-mail address: [email protected] (Y. Zhang). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.10.006
and it may lead to donor-site morbidity. A non-invasive procedure to obtain autologous cells for this process would be highly desirable. In our previous study, we demonstrated that progenitor cells can be isolated from voided urine [2]. About 0.2% of cells isolated from urine express markers characteristic of mesenchymal stem cells. These cells can expand extensively in culture and differentiate towards multiple bladder cell lineages as identified by the expression of UC, SMC, endothelial and interstitial cell markers. We initially referred to these cells as urine progenitor cells. However, our recent experiments indicated that urine-derived cells can give rise to additional specialized cell types, including osteocytes, chondrocytes, myocytes and adipocytes. Furthermore, in addition to the ability to differentiate into multiple lineages, these cells also exhibit self-renewal capacity, which is consistent with the definition of stem cells [3]. Thus, urine-derived stem cells (USC) may be an excellent alternative cell source for urological tissue engineering applications, especially because they are easily obtained from a patient using a non-invasive procedure. Another critical element required for successful urethral tissue engineering is the biomaterial scaffold. Three types of scaffolds have
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been introduced for urethral reconstruction. First, living autologous tissues such as buccal mucosa [4,5], skin [6], bladder mucosa [7,8], ureter [9], appendix [10], radial forearm free flap [11] and blood vessel [12] have been used as urethral grafts. Second, synthetic biomaterials, including poly L-polylactic acid [13] and copoly(L-lactide/epsilon-caprolactone) [P(LA/CL)] with a type I collagen sponge [14] have been tested in a number of studies, although synthetic biomaterials are not commonly used in urethral tissue engineering because they lack appropriate biologically active molecules that allow tissue regeneration across the graft to proceed quickly. Finally, natural collagen-based materials have been used. These are mostly derived from allogeneic or xenogenic sources and include small intestinal submucosa (SIS) [15e24], bladder “submucosa” (BSM) [25e28], demineralized bone matrix [29], urethral extracellular matrix [30], acellular artery matrix [31] and amniotic membranes [32]. Although living tissue grafts provide a favorable outcome, this procedure may increase hospital stays and lead to donorsite morbidity. As an alternative, natural collagen-based acellular matrices possess advantages for urethral tissue engineering because these materials promote early wound coverage, accelerate tissue formation and improve early functional reconstruction [25,33,34]. However, natural collagen-based materials have certain disadvantages that limit their clinical application. It has been shown that the high density of collagen, particularly on the mucosal side of the matrix, can block cell penetration into the collagen matrix when cells are seeded onto the material. In addition, despite decellularization procedures, these matrices can retain heterogenic cellular compounds that can result in chronic immunoreactions, fibrosis or calcification, all of which can lead to stricture recurrence. Finally, collagen materials often display wide batch-to-batch variations, leading to variable results. Therefore, a 3-dimensional (3-D) porous acellular scaffold that is free of native cellular compounds and that retains its mechanical strength during decellularization and reseeding procedure is desirable. Our recent study [35] demonstrated that a modified BSM scaffold that is free of cellular compounds possesses a 3-D porous microstructure after decellularization and oxidation with 5% peracetic acid (PAA). This 3-D porous BSM scaffold promoted cellematrix penetration and the formation of a multilayer of uroepithelial structure in vitro, and it supported cell growth in vivo when UC and SMC were seeded and grown in dynamic culture on the porous matrix. In this study, we used this well-established decellularization/oxidation strategy as described [35] to generate a tissue-engineered urethral construct using autologous USC seeded on 3-D porous SIS scaffold. This cell-based tissue-engineered urethra may be useful for patients with complicated long strictures who need urethral reconstruction. 2. Materials and methods 2.1. SIS scaffold preparation Fresh porcine small intestines were provided by Ni Sausage farm (Lexington, NC, USA). The mucosa were manually removed and washed in distilled water on a rotary shaker at 200 RPM and 4 C for 2 days (distilled water decellularization of SIS). For further decellularization, samples of SIS were oxidized by soaking in peracetic acid (PAA) at a concentration of 0 (without PAA, as control), 1, 2, 3, 5 or 10% (v/v) for 4 h. This was followed by a similar treatment with 1% Triton X-100 solution for another 2 days. The SIS was then washed with distilled water for 2 more days. Finally, the SIS scaffold was disinfected using 0.1% PAA in 20% ethanol for 2 h, rinsed three times with sterile distilled water for 10 min each and stored in sterile distilled water at 4 C until needed [35]. 2.2. Histological analysis of decellularized SIS Fresh and decellularized SIS samples were fixed in 10% buffered formalin and embedded in paraffin. Consecutive 5.0 mm sections were prepared for histological analysis. For detection and identification of cellular material, sections were
stained with hematoxylin and eosin (H&E) (Sigma, St. Louis, MO, USA) and 4,6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA, USA). Light micrographs were taken at 100 magnification. For measurement of residual DNA, decellularized SIS samples treated with the different concentrations of PAA (1, 3, 5 and 10%, n ¼ 4 for each concentration), distilled water (0% PAA) and fresh SIS samples (n ¼ 4) were dabbed dry with tissue paper and weighed. Samples were minced and any remaining nucleic acids were extracted using a commercially available kit (DNeasyÔ, Qiagen, Valencia, CA). The DNA concentration in each sample was calculated using a UV-spectrophotometer (ND-1000 NanoDrop Technologies, Wilmington, DE) to measure the absorbance at 260 nm. The DNA yield was normalized to the initial wet weight of the samples. 2.3. Scanning electron microscopy Fresh SIS and decellularized/oxidized SIS were fixed in 2.5% glutaraldehyde for at least 2 h at room temperature before washing with distilled water (3 min). The SIS was incubated at 80 C for 24 h, sputter coated with gold, and mounted for analysis. Electron micrographs of mucosa slides were obtained at 25.0 kV, 50 Pa, 500 magnifications using a Hitachi S-2600 Scanning Electron Microscope (Hitachi Technologies America, Pleasanton, CA). 2.4. Mechanical testing For mechanical testing, fresh SIS and decellularized/oxidized SIS samples were mechanically loaded onto a uniaxial load frame (Instron Corporation, Issaquah, WA, USA). A short strip of SIS was clamped at the ends for axial testing. The crosshead speed was set at 0.1 mm/s and the test was stopped when the force decreased by 10% after the onset of failure. At least ten specimens were tested for each treatment with or without PAA (6 groups: fresh SIS, 0%, 1%, 3%, 5% and 10% PAA) and the mean and standard deviation were calculated. Based on the stressestrain profile of the SIS samples, tensile elongation (at break) was also calculated. 2.5. Culture of human ureter cells and USC Samples of normal human ureter tissue were obtained from kidney transplant donors and used for isolating UC and SMC as described previously [35]. Briefly, the samples were digested with 1 mg/ml collagenase type IV at 37 C for 1 h. The UC were released by gentle scraping from the stroma and cultured in keratinocyteserum free medium (KFSM, Invitrogen, Carlsbad, CA, USA). For SMC isolation, the remaining tissues were minced finely into small pieces about 0.1 cm3 in size, and these pieces were plated in high glucose DMEM containing 10% fetal bovine serum (FBS). Culture medium was changed every 2 days until the cells reached 80%e90% confluence. Twelve voided urine samples (average about 200 ml/sample) were collected from 4 healthy male individuals. Each urine sample was centrifuged at 500 g for 5 min to collect cells. The supernatant was carefully aspirated and the cell pellet was gently resuspended in initiation medium, a 1:1 mixture of KSFM and embryonic fibroblast medium (EFM) [2]. Media was changed every other day after 48 h of initiation and cells were split at 70e80% confluence. 2.6. Urothelial and smooth muscle differentiation of USC For urothelial differentiation of USC, USC were plated at 3000 cells/cm2 in a mixture of medium containing KSFM and EFM at a ratio of 4:1 (2% FBS at final concentration) and 30 ng/ml epidermal growth factor (EGF; six times the EGF concentration of normal UC culture medium [36]) for 14 days. Media was replaced every 3 days and the cells were analyzed on days 7 and 14. For SMC differentiation, USC were plated at a density of 2000 cells/cm2 in myogenic differentiation media containing DMEM and EFM (without EGF, 10% FBS) at a ratio of 1:1, as well as TGF-b1 and PDGF-BB (R&D systems, Minneapolis, MN) at concentrations of 2.5 ng/ml and 5 ng/ml, respectively. Cell morphology changes were monitored. Media was replaced every 3 days and the cells were analyzed on days 7 and 14. Percentages of USC that had differentiated into UC and SMC were calculated based on immunofluorescent staining for UC and SMC-specific markers (Table 1).
Table 1 Antibodies used in this study. Primary antibody
Host
Dilution
Catalog #/Company
a-SM actin
Mice Mice Mice Mice Mice Mice
1:200 1:100 1:200 1:20 1:100 1:100
A5228/Sigma M0760/Dako M7786/Sigma AB78196/Abcam M3515/Dako NA09L/Calbiochem
Desmin Myosin Uroplakin-III AE1/AE3 NuMA
S. Wu et al. / Biomaterials 32 (2011) 1317e1326 2.7. Cell-seeded SIS scaffold in vitro To assess the effect of PAA treatment on scaffold porosity and the role of dynamic culture conditions on cell growth and cellematrix penetration, ureter SMC and UC were seeded on decellularized SIS in the wells of a 6-well plate. The SIS scaffold was firmly held over a sterile silicone insert for ease of cell seeding. For layered co-cultures, SMC were seeded on the serosal side of the SIS at a density of 2 105 cells/cm2/day in DMEM with 10% FBS under static culture for 3 days. Next, UC were mixed with KSFM-DMEM (1:1) and seeded on top of the muscle cells at similar cell numbers. The seeded scaffolds were then cultured under static (control) or 3-D dynamic culture conditions for 7 more days. After these 14 days of culture, the cellseeded SIS was released from the silicone insert and cut into 1 1 cm2 pieces for histological and immunohistochemical analysis or for implantation in vivo. To analyze the viability and proliferative capacity of the cells grown on the seeded decellularized SIS, MTT assays were performed. SMC and UC co-cultured on either 0% PAA or 5% PAA-treated SIS and grown under static or 3-D culture conditions (10 RPM and 40 RPM) were compared. Cells cultured in 96-well plates were incubated with MTT reagent as a control (Sigma, St. Louis, MO, USA) for 30 min and the optical density read using a SpectraMax-M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). 2.8. Implantation of cell-seeded SIS grafts in vivo SIS scaffolds seeded with cells obtained through urothelial and myogenic differentiation of USC (UC-USC and SMC-USC) were kept in static culture for 6e7 days and in dynamic culture for 7 more days as described at Section 2.7. USC seeded SIS constructs (n ¼ 12) were subcutaneously implanted into the flanks of athymic nude mice (4 weeks of age; 2 grafts/animal). SIS seeded with UC and SMC derived from ureteral samples (n ¼ 10) were used as controls and were implanted in a similar manner. All eleven animals were followed for 1 month. At this time, all animals were euthanized and the implants were removed for analysis and evaluation of cell infiltration and differentiation. 2.9. Immunohistochemical analysis To identify urothelial and myogenic differentiation of USC seeded SIS scaffolds in vitro and in vivo, the tissue graft samples were analyzed using immunohistochemistry. The tissue samples were embedded in OCT freezing media and immediately floated onto liquid nitrogen for 15 s. The frozen SIS samples were cut into 5 mm continuous sections using a cryostat (Leica, Bannockburn, IL USA). Cryosections (5 mm) were incubated overnight at 4 C with the following primary antibodies (see Table 1 for details): human-specific monoclonal antibody NuMA, for detection of grafted human cells within the implants in the mouse model; anti-Uroplakin-III and anti-cytokeratin (AE1/AE3) for visualization of UC; and anti a-SM actin, anti-desmin and anti-myosin for visualization of SMC. Slides were then washed with PBS and incubated with the appropriate fluorescence-conjugated secondary antibody (FITC/ TRITC conjugated goat anti mouse, Jackson Immuno Research) diluted at 1:200 for 30 min at room temperature. Finally, the slides were rinsed twice with PBS and counterstained with DAPI (Vectashield Vector, Burlingame, CA, USA) or Propidium Iodine (PI, Vectashield Vector, Burlingame, CA, USA) containing mounting media.
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in SIS that had been decellularized but not treated with PAA (Fig. 1Ab,e). 3.2. DNA content analysis In order to quantify the extent of decellularization in each group, the DNA content of decellularized SIS scaffolds was measured. It was observed that DNA content significantly decreased compared to fresh SIS when increasing concentrations of PAA were used. The DNA content of the decellularized SIS that had been treated with 0%, 1%, 3%, 5% and 10% PAA decreased dramatically with increasing PAA concentration (i.e. 2.86 0.05, 1.40 0.04, 0.73 0.02, 0.27 0.17, 0.15 0.01 and 0.04 0.01 mg DNA/mg tissue, respectively) (Fig. 1B). This result indicates that increasing the concentration of PAA leads to more effective removal of DNA from the matrix. In addition, mechanical testing results indicated that there was no significant difference in tensile elongation at break between each PAA treatment group. The percentage elongation at break is as follows: 0% PAA ¼ 198.1 24.8, 1% PAA ¼ 200 23.2, 3% PAA ¼ 213.9 12.0 and 5% PAA ¼ 191.3 51.1 (n ¼ 10 for each treatment, p > 0.05). 3.3. Scanning electron microscopy Scanning electron micrographs showed the ultrastructure of the smooth and water-tight mucosa basal side of fresh SIS (Fig. 2a). Following 1% triton decellularization (0% PAA) treatment, porosity or cracks appeared on the surface of the basal membrane (Fig. 2b). After 5% PAA treatment, the extent of porosity and the porous size were considerably increased compared to fresh SIS and 0% PAA treatment (Fig. 2c). 3.4. Effect of porous SIS scaffold on cellematrix penetration The number of ureter-derived SMC that penetrated into the porous SIS matrix increased when the matrix was treated with PAA (Fig. 3). In addition, more layers of ureter-dervied UC were formed on the top of the ureter-derived SMC layers during co-culture on the decellularized/oxidized SIS treated with 5% PAA compared to non-PAA treated SIS (Fig. 3). This indicates that PAA treatment enhances the porosity of the collagen matrix and promotes cellular penetration into the matrix.
2.10. Statistical analysis For DNA content, MTT and tensile strength analysis, means and standard errors of the mean (SEM) were calculated for the groups (n ¼ 4 for DNA content, n ¼ 6 for MTT test, n ¼ 10 for tensile strength each PAA treatment) and a comparison was made between groups using a two tailed, Student’s t-test with unequal variances. Differences were considered significant at p < 0.05.
3. Results 3.1. Histological analysis of decellularized SIS A cross section of fresh SIS stained with H&E (Fig. 1Aa) and DAPI (Fig. 1Ad) shows that without decellularization, dense cellular components are seen within the SIS. Even after treatment with distilled water decellularization procedures (0% PAA), a certain amount of nuclear material was still retained in the matrix (Fig. 1Ab and e). In contrast, decellularization of SIS following 5% PAA treatment was nearly complete, and nuclear material could not be seen after the process (Fig. 1Ac,f). However, the tissue structure in the 5% PAA treatment group (Fig. 1Ac) become visibly more porous, and the intra- and inter-fascicular space was increased in the lamina propria that appear more extended compared to that seen
3.5. Effect of dynamic culture on ingrowth of ureter cells To optimize the culture conditions, ureter UC and SMC were grown under static (control) or dynamic culture conditions at different rotational speeds. During static culture, the seeded ureter UC formed 2e3 cell layers on the surface of the scaffold. In addition, when ureter SMC were seeded onto the SIS, the SMC penetrated the scaffold to a depth of 3e5 cell layers after static culture (Fig. 4A, left column). However, use of 3-D dynamic culture conditions (10 RPM and 40 RPM) led to growth of at least 3e6 layers of urothelial cells on top of 10e12 layers of SMC on the serosal side of the SIS scaffold (Fig. 4A middle and right column). In addition, SMC also infiltrated the matrix to a larger extent (50e60% of the matrix) when this dynamic co-culture method was used. Next, an MTT assay was performed to measure the cell growth pattern of both UC and SMC within the SIS. The data from this analysis corroborates the finding that a significant increase in cell number was observed in the scaffolds treated with 5% PAA compared to those treated with 0% PAA (Fig. 4B). In addition, the cell number significantly increased under dynamic culture (10 or 40 RPM) compared to static culture conditions (Fig. 4A,B).
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Fig. 1. Comparison of gross morphology of decellularized SIS with PAA treatment. (A) H&E (a,b,c) and DAPI staining (d,e,f) on cross sections of fresh SIS (a,d), 0% PAA (i.e. distill water with non-PAA treated) treated SIS (b,e) and 5% PAA-treated SIS (c,f). Cells and cell nuclei are visible in fresh SIS tissue (arrows; a,d) whereas a rapid decrease in cellular contents was visible with 0% PAA treatment (decellularization alone) (arrows; b,e). However, complete removal of all cellular components was achieved using 5% PAA treatment (c,f). An increase in porosity of the micro-architecture of SIS was visible after PAA treatment (c,f). Scale bar represents 200 um. (B) Effect of PAA on DNA clearance. DNA was extracted from fresh SIS and decellularized SIS prior to and after PPA treatment. The DNA values were plotted after normalizing to initial wet weight of the sample. A significant clearance of DNA content was observed with increased PAA concentrations.
Fig. 2. Electron micrograph of mucosal side of decellularized SIS with PAA. (a) Untreated (fresh) SIS, (b) Decellularized SIS (0% PAA treatment) and (c) Decellularized SIS (5% PAA). A remarkable high level of porosity was achieved after treatment with 5% PAA when compared to the 0% PAA treatment. Scale bar ¼ 100 um.
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Fig. 3. Effect of PAA concentration on cell penetration and proliferation. SIS were treated with 0% and 5% PAA prior to seeding with ureter SMC and UC for 14 Days. Sections were stained with Masson’s Trichrome (top panel) and DAPI (bottom panel). Cellematrix ingrowth in 5% PAA-treated SIS were significantly higher (arrows, indicating presence of cells at different depths) than that of the 0% PAA treatment. UC and SMC layers are marked. The vertical black line in the top panels on the left side indicates the scaffold thickness. Scale bar represents 100 um.
3.6. Induced USC in a porous SIS scaffold in vitro
3.8. Urothelial and myogenic-differentiated USC in vivo
Following in vitro differentiation of USC with myogenic growth factors, 50e70% cells at passage 3 expressed SMC markers (SMCUSC) compared to less than 1/4 of the cells in the untreated control group. Similarly, up to 90% of the USC expressed UC markers (UCUSC) at passage 3 when high doses of EGF (30 ng/ml) were used, compared to only 20% of the cells in the control group, which was treated with 7.5 ng/ml EGF. When both SMC-USC and UC-USC were seeded onto a SIS matrix in a layered co-culture fashion under dynamic culture conditions (40 RPM), SMC-USC were able to penetrate the SIS matrix treated with 5% PAA to a greater depth than SMC-USC seeded onto SIS matrices that were not treated with PAA (Fig. 5A). A few cell layers of UC-USC were formed on top of the SMC-USC, and the resulting urethral-like structure was similar to that seen when ureter UC were co-cultured with ureter SMC in vitro (Fig. 3).
Immunofluorescent studies confirmed that the urothelial cell differentiated USC were layered in a well-organized manner on the top of the myogenic-differentiated USC on the luminal side of the SIS scaffold when the layered co-culture system was used in vitro. The UC-USC stained positive for both AE1/AE3 (Fig. 6A, top right) and uroplakin-III (Fig. 6B, top right). One month after implantation in vivo, differentiation of USC to urothelial-like cells was demonstrated by immunofluorescent staining for the expression of urothelial-specific antigens (Fig. 6A and B, bottom right). These staining patterns were similar to those seen in native urothelium and when ureter-derived UC was co-cultured with SMC on SIS in vitro and in vivo (Fig. 6 A and B, left panel). SMC-specific staining for a-SM actin, desmin, and myosin in the seeded scaffolds in vitro indicated well-organized layers of SMC-like cells on and partially within the SMC-USC seeded SIS scaffold (Fig. 7AeC, top right). This pattern was similar to that seen when ureter-derived SMC were seeded on SIS in vitro (Fig. 7A, B, C, top left). Moreover, SMC-USC seeded on SIS and implanted in vivo stained positive for the same SMC markers (Fig. 7A, B, C, bottom right).
3.7. Implantation of USC seeded SIS in vivo All animals used for in vivo studies survived and remained healthy during the course of the experiment. No evidence of inflammatory reactions, infections, or implant extrusion was observed at the sites of implantation in the seeded grafts. Microscopically, progressive tissue regeneration was observed in the cellseeded porous grafts. The use of an antibody specific for human nuclei (Nuclear Mitotic apparatus), which stained bright green and appeared yellow when merged with the red PI nuclear stain (Fig. 5B), indicated that the implanted human USC-derived cells survived up to one month after implantation in vivo. Cellular penetration into the matrix was also evident (arrows) when both H&E and Masson’s trichrome staining were performed on scaffolds that had been grown in vitro and in vivo (Fig. 5A). Small blood vessel penetration into the SIS matrix could also be seen in vivo, and the onset of some graft degradation was also observed at the edges of each decellularized/oxidized SIS scaffold after one month in vivo.
4. Discussion This study indicates that using autologous USC as an alternative cell source for urethral tissue engineering may be advantageous. The cells can be efficiently differentiated into UC and SMC using epithelial and myogenic growth factors, respectively, and these can then be seeded on a modified 3-D SIS scaffold. One voided urine sample of about 200 ml can generate at least 3.2 108 viable cells at early passages (p4), and this provides enough cells to create a tissueengineered urethral construct. Additionally, fully-decellularized SIS provides a 3-D porous collagen matrix, which encourages cell repopulation within the scaffold. The modified SIS scaffold retains majority of its original mechanical strength, with removal of all of the heterogeneous cellular compounds. A combination of this
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Fig. 4. Effect of culture condition on cell penetration and proliferation. A) Masson’s Trichrome staining (top panel) and DAPI staining (bottom panel) performed on 5% PAA-treated SIS, seeded with ureter SMC and UC under static or dynamic (10 or 40 RPM) culture condition for 14 d. Cell penetration (lower arrow) and proliferation (upper arrow) was significantly improved with dynamic culture condition as compared to static culture condition. The vertical black line in the top panels denotes scaffold thickness. Inset images shows lower magnification of the entire thickness of the scaffold. Scale bar represents 100 um. B) Analysis of cell growth. Ureter SMC and UC seeded SIS were analyzed for cell growth using the MTT assay. OD values were recorded after reading the plate at 450 nm. The graph shows cells grown in the PAA-treated (5%) and the untreated (0%) under different static and dynamic (10 and 40 RPM) culture condition. Irrespective of the culture conditions, the treatment with PAA caused a significant growth of cells compared to the nontreatment (0%) (p < 0.05).
porous matrix, dynamic culture conditions and a system to co-culture epithelial and stromal cells significantly enhances cellematrix infiltration by SMC as well as urothelial mucosa formation by UC. Both of these are required for the creation of a cell-seeded SIS scaffold for urethral tissue engineering. The use of uroepithelial cells isolated from urine or bladder washes for urological tissue engineering applications has been reported [37,38]. However, the success rate of cultures of these cells is low (55%), and they also have limited expansion capability in culture. In a recent study, we were able to successfully isolate and culture USC from almost every urine sample tested. USC are not
mature UC or SMC, and they express pericyte/mesenchymal stem cell markers as well as detectable levels of telomerase [3]. Importantly, these cells possess both self-renewal and multilineage differentiation capabilities. They can give rise to osteocytes, chrodrocytes, adipocytes, skeletal myocytes [3], interstitial cells and endothelial cells [2]. They differentiate particularly well into bladder cells, i.e. UC and SMC [2]. The numbers of USC clones that can be consistently obtained from each freshly voided urine sample (200e300 ml) ranges from 10 to 15. The average population doubling (PD) of USC in these urine samples was about 34 (ranging from 32e46, n ¼ 9). In other words, a single cell clone of USC could
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Fig. 5. Tracking of human cell-seeded SIS in vitro and in vivo. A) Masson’s trichrome staining, as shown on the left at 200, depicts cellular (SMC) penetration (black arrows) and proliferation (black arrowheads) as well as UC layers (white arrows). The in vivo image at 400 shows appearance of vessel-like structures (black arrows). B) Detection of human ureter cells by immunostaining with human nuclear antibody (NuMA, in green) in layered co-culture of ureter UC/SMC (as controls in the left column) and SMC-USC along with UCUSC in the right column) in vitro (top panel) as well as in vivo (bottom panel), 4 weeks after implantation. Arrows indicate representative strong positively labeled implanted human cells. As the cell nuclei were counterstained with PI, human cells appear yellow due to merging of the green and red signal. Scale bar represents 50 um.
expand to 234, or 1.7 10 exp10 cells, by passage 6e8 within 5 weeks [39]. However, in order to maintain the maximal multipotent capability of USC, we used them at earlier passages (p3e4). A single cell clone can generate 32e48 106 cells (Population doublings ¼ 25) at passage 4 and this occurs within 3e4 weeks. To prepare a cell-seeded scaffold for use in urological tissue regeneration, the cell concentration for seeding must be about 50 106 cells/cm3 [1]. Thus, the number of cells from one urine sample of about 200 ml volume can provide enough cells to create a piece of cell-seeded scaffold about 6.5e10 cm3 in size, and USC from multiple urine samples could easily meet the required cell numbers for constructing larger cell-scaffold constructs for repair of longer urethral segments. Importantly, USC are capable of efficiently differentiating into both UC and SMC with the application of defined growth factors. Up to 70% of USC induced with muscle-specific growth factors express SMC markers (alpha-smooth muscle actin, desmin, and myosin) compared to 10e25% of cells induced in DMEM with 10% FBS in vitro [2]. Similarly, the majority of USC can give rise to urotheliallike cells displaying the morphology and cellular markers (AE1/AE3 and Uroplakin-III) of UC when induced using 4 times the dose of EGF used during standard proliferation. However, USC start to senesce and a decrease in differentiation potential becomes apparent at higher passages (p10). For USC to have a robust potential, it is recommended that they be used at passages younger than 6 for cell-based therapy. Although natural collagen matrices such as SIS or BSM are commonly used for cell-based tissue engineering, fibrosis formation and inflammatory reactions have limited their clinical application to date. These complications of collagen matrix use are caused by xenogenic proteins that remain within the matrix and create immunogenicity. This immune response eventually leads to fibrosis or calcification of the biomaterial. Additionally, the high density of collagen within the matrix inhibits cellematrix infiltration. Our recent study on the decellularization of another natural collagen matrix, BSM [35], demonstrates that oxidation procedures provide a more optimal decellularization protocol for use in the fabrication of biological collagen matrices with minimal retention of cellular components. Oxidation also leads to higher scaffold porosity and increased pore size, which should increase cellematrix infiltration.
Both a porous matrix and a dynamic culture system play important roles in cellematrix infiltration. The dynamic culture system promotes 3-D cell ingrowth within the highly porous matrix, thereby making it potentially useful in cell-based tissue engineering for hollow organ tissue engineering or other soft tissue repair [35]. In this study, we used the same approaches to generate a 3-D SIS scaffold with porous microstructure. For fully-decellularized SIS, we combined mechanical agitation (physical) and Triton X-100, along with high concentrations (5%) of PAA (chemical). We found that PAA at a concentration of 5% increased porosity within the matrix compared to untreated SIS. Additionally, more cells infiltrated into the matrix on the serosal side rather than the urothelial side. Better cellematrix infiltration was observed when the combination of both a highly porous matrix and dynamic culture conditions were used than with any of the other approaches alone. Dynamic culture within a bioreactor enhances the efficiency not only of cellematrix infiltration but also of the homogeneity of bladder cell differentiation. Furthermore, decellularized SIS treated with 5% PAA retained similar mechanical strength as the non-PAA treated matrix. Thus, the culture system described here utilizes simple, efficient and low cost equipment to improve the culture environment for use in urological tissue engineering. SIS possesses a unique property, in which its permeability is “sided”, or direction-dependent. The mucosal to serosal direction is less permeable than the serosal to mucosal direction. When nonseeded SIS is used in urological applications, this property should be considered because it can assist in preventing urine leakage from the lumen of the urethra or bladder into surrounding tissues. However, in cell-based tissue engineering, this direction-dependent permeability appears less important in preventing urine leakage, because heavy cellular infiltration “fills up” the pores within the matrix to prevent leakage. Therefore, the porous SIS scaffold is suitable for cell-seeded tissue engineering in urethral reconstruction. In vivo data in this study demonstrated that the urothelial and smooth muscle differentiated USC survive and maintain both cell type markers one month after implantation, suggesting that differentiated grafted cells could retain urological cell phenotypes in the in vivo environment. The urethral tissues regenerated from differentiated USC using this 3D porous collagen scaffold will be investigated in a large animal model in a future study.
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Fig. 6. Immunofluorescent staining of urothelial-differentiated USC in vitro and in vivo. Tissue sections of in vivo and in vitro cultured cells were immunostained (green) using A) pancytokeratin (AE1/AE3) and B) Uroplakin-III antibodies. USC were differentiated in vitro with EGF for 14 days by using dynamic culture condition (10 RPM) followed by subcutaneous implantation into nude mice for a further 4 weeks. The nuclei were counterstained with PI (red). The left panel indicates in vitro and in vivo co-cultures of UC and SMC as a positive control. Arrows show a few representative cells that are positively stained. Scale bar represents 50 um.
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Fig. 7. Immunofluorescent staining of myogenic-differentiated USC in vitro and in vivo. Tissue sections of in vitro (top panel) and in vivo (bottom panel) cultured cells that were immunostained (green or red) using A) a-SM actin, B) Desmin and C) Myosin antibodies. USC were differentiated in vitro using PDGF and TGF for 14 days under dynamic condition (10 RPM), following by subcutaneously implantation into nude mice for a further 4 weeks (right column). In vitro and in vivo seeded UC/SMC scaffolds (left column) were used as controls. Positively stained cells for a SM actin and Myosin appear green (FITC), whereas staining for Desmin appears red (Rhodamine). Nuclei were counterstained with PI (red) or DAPI (blue). Arrows show a few representative cells that are positively stained. Scale bar represents 50 um.
5. Conclusions USC can be easily and consistently obtained by a non-invasive approach and extensively expanded in vitro. USC efficiently give rise to UC and SMC in appropriate induction conditions. In addition, oxidized SIS scaffolds have higher porosity and larger pore size than non-oxidized scaffolds, and they are almost completely free of cellular compounds that could lead to immune reactions in vivo. Thus, their use allows abundant graft cells to be loaded onto a biological scaffold. This contributes to a higher cellular proliferation rate and increased cellematrix infiltration in vitro, and may also enhance the ability of host cells to infiltrate into the matrix when the scaffold is implanted in vivo, thereby leading to better in vivo tissue regeneration. Using these modified decellularization procedures, 3-D porous SIS that possesses the highly interconnected macro- or micro-porous structures to allow cell infiltration into the matrix is created. Importantly, heterogeneous cellular proteins are removed, which may eliminate inflammatory reactions, fibrosis, calcification and graft shrinkage that are seen
when other types of scaffolds are used for urological tissue engineering. Finally, dynamic culture conditions maximize cellematrix infiltration and cell growth. This study indicated that use of urothelial and smooth muscle cells derived from the differentiation of USC and seeded on a 3D porous biological collagen matrix could be used to construct a tissue-engineered urethra. Acknowledgements The authors would like to thank Dr. Karl-Erik Andersson for valuable comments and Dr. Jennifer Olson and Ms. Carmen Pruitt for editorial assistance with this manuscript. Appendix Figures with essential colour discrimination. Figs. 1 and 3e7 in this article are difficult to interpret in black and white. The full colour images can be found in the online version, at doi:10.1016/j. biomaterials.2010.10.006.
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