Development of a porcine renal extracellular matrix scaffold as a platform for kidney regeneration Seock Hwan Choi,1* So Young Chun,2* Seon Yeong Chae,2 Jin Rae Kim,3 Se Heang Oh,4 Sung Kwang Chung,1 Jin Ho Lee,3 Phil Hyun Song,5 Gyu-Seog Choi,6 Tae-Hwan Kim,1* Tae Gyun Kwon1,2* 1
Department of Urology, School of Medicine, Kyungpook National University, Daegu, Korea Joint Institute for Regenerative Medicine, Kyungpook National University Hospital, Daegu, Korea 3 Department of Advanced Materials, Hannam University, Daejeon, Korea 4 Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Chungnam, Korea 5 Department of Urology, College of Medicine, Yeungnam University, Daegu, Korea 6 Department of Colorectal Cancer Center, School of Medicine, Kyungpook National University, Daegu, Korea 2
Received 14 April 2014; revised 8 June 2014; accepted 3 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35274 Abstract: Acellular scaffolds, possessing an intact threedimensional extracellular matrix (ECM) architecture and biochemical components, are promising for regeneration of complex organs, such as the kidney. We have successfully developed a porcine renal acellular scaffold and analyzed its physical/biochemical characteristics, biocompatibility, and kidney reconstructive potential. Segmented porcine kidney cortexes were treated with either 1% (v/v) Triton X-100 (Triton) or sodium dodecyl sulfate (SDS). Scanning electron microscopy showed both treatments preserved native tissue architecture, including porosity and composition. Swelling behavior was higher in the Triton-treated compared with the SDS-treated scaffold. Maximum compressive strength was lower in the Triton-treated compared with the SDS-treated scaffold. Attenuated total reflective-infrared spectroscopy showed the presence of amide II (ANH) in both scaffolds.
Furthermore, richer ECM protein and growth factor contents were observed in the Triton-treated compared with SDStreated scaffold. Primary human kidney cell adherence, viability, and proliferation were enhanced on the Triton-treated scaffold compared with SDS-treated scaffold. Following murine in vivo implantation, tumorigenecity was absent for both scaffolds after 8 weeks and in the Triton-treated scaffold only, glomeruli-like structure formation and neovascularity were observed. We identified 1% Triton X-100 as a more suitable decellularizing agent for porcine renal ECM scaffolds C 2014 Wiley Periodicals, Inc. J prior to kidney regeneration. V Biomed Mater Res Part A: 00A:000–000, 2014.
Key Words: kidney reconstruction, extracellular matrix scaffold, Triton X-100, sodium dodecyl sulfate, partial nephrectomy
How to cite this article: Choi SH, Chun SY, Chae SY, Kim JR, Oh SH, Chung SK, Lee JH, Song PH, Choi G-S, Kim TH, Kwon TG. 2014. Development of a porcine renal extracellular matrix scaffold as a platform for kidney regeneration. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
From the United Network for Organ Sharing (UNOS) data, it is very striking that every year kidney donation numbers remain relatively flat, but the waiting list continues to grow.1 As a consequence, numbers of donated organs are insufficient and it is essential to find potential sources of organs or alternative measures to prevent patients from falling into chronic renal failure (CRF). Generally, renal surgery deteriorates global kidney function proportionately to the volume of renal parenchyma loss.2 Even partial nephrectomy causes progressive glomerulo-sclerosis and increases single nephron glomerular filtration rate, which could even-
tually lead to CRF and the unfortunate requirement for dialysis.2,3 A primary challenge of kidney regeneration after partial nephrectomy is to maintain normal renal function by applying alternative tissues with functional complexity. However, to the best of our knowledge, an efficient method for kidney regeneration after partial nephrectomy is yet to be developed. In this study, we investigated the use of porcine renal cortex extracellular matrix (ECM) scaffolds for reconstruction of the resected portion of the kidney. There are several reasons why we choose porcine renal cortex ECM. First, the renal cortex contains renal tubular and glomerular
*These authors contributed equally to this work. Correspondence to: T.H. Kim; e-mail: [email protected] or T.G. Kwon; e-mail: [email protected] Contract grant sponsors: Korea Health technology R&D Project, Ministry of Health & Welfare, and Republic of Korea; contract grant number: A111345
C 2014 WILEY PERIODICALS, INC. V
1
structures, which are essential elements for functional recovery of the kidney and to maintain the complex micro network as a natural kidney. Second, the tissue has low immunogenicity and provides an abundant source of porcine supplements.4,5 Our primary hypothesis was that renal cortex ECM scaffold implantation could be a feasible approach for kidney restoration, which could ultimately solve the problem regarding deterioration of global kidney function after partial nephrectomy. Before scaffold implantation, optimal preparation is a key step for success. The characteristics of ECM scaffolds, including composition, structure, and biocompatibility, are reportedly different according to the type of agent used for decellularization. These differences are largely owing to the distinction of mechanical integrity, chemical composition, and biological activity of the ECM and growth factors, which are affected by the detergent during decellularization.6 When scaffolds are recellularized, the retained ECM and growth factor constituents act as morphogenic modulators, and are responsible for mediating cellular organization, regulating signal transduction pathways, and controlling cell growth and proliferation. For these reasons, it is essential to prepare scaffolds using the most suitable agent.6,7 The main three types of decellularization methods include physical, chemical, or enzymatic protocols. Chemical treatments are used for the majority of decellularization studies,8 with the most frequently used being Triton X-100 (Triton) and sodium dodecyl sulfate (SDS).6 However, debate exists over which agent is best for optimal decellularization of kidney tissue for tissue engineering purposes.6–10 Therefore, it is necessary to select an optimum agent to produce an effective renal ECM scaffold for partial kidney reconstruction. The primary goal of the current study was to compare previously described agents used for decellularization and identify the most suitable one for porcine renal tissue decellularization. Intensive analyses were carried out for physical, biochemical, and biocompatible aspects. After identification of the optimum agent for renal cortex decellularization, the secondary objective was to analyze the scaffold reconstructive potential in vivo, following implantation into the kidney after partial nephrectomy. In this article, we report the data of our investigations, demonstrating that (1) Triton-treated scaffolds are better at maintaining their mechanical integrity, biochemical components, and biocompatibility, compared with SDS-treated scaffolds; and (2) following implantation of scaffolds into the injured site after partial nephrectomy, glomerulus-, and vessel-like structures were regenerated more effectively in Triton-treated compared with SDS-treated scaffolds. MATERIALS AND METHODS
Preparation of acellular renal cortex scaffolds Cortical sections (10 3 10 3 2 mm3) of kidney were collected from female Yorkshire porcine animals (2–3 months, 22–30 kg). Decellularization was performed using a modified, previously published protocol.10 Kidney sections were washed twice with phosphate buffered saline (PBS), followed by decellularization in a solution of either 1% (v/v)
2
CHOI ET AL.
Triton X-100 (Sigma-Aldrich, St. Louis, MO) or 1% (v/v) SDS (Invitrogen, Carlsbad, CA) (containing 100 U/mL penicillin and 100 lg/mL streptomycin), both diluted in distilled water. Sample were decellularized at 4 C in a shaking incubator (200 rpm). The decellularization solution, using 15–20 fold of sample volume (weight), was changed 4 h after initial tissue harvest and then every 24 h until tissues were transparent (for 10–14 days). Tissues were then treated with DNase (30 lg/mL; Sigma-Aldrich) in PBS for 1 h. Finally, tissues were rinsed with PBS to flush out residual DNase. Decellularized kidney scaffolds were cryoembedded in optimum cutting temperature compound (Sakura Finetek/Tissue Tek, Torrance, CA). After confirmation of decellularization with hematoxylin and eosin (H&E) and 4’,6-diamidino-2-phenylindole (DAPI) stain analysis, scaffolds were sterilized with gradient ethanol and lyophilized, then stored at 4 C until analysis. Physical characterization: Morphology, swelling behavior, and mechanical property in vitro For morphological analysis, both native and acellular sections of kidney were formalin fixed, paraffin embedded, sectioned at 5 lm thickness, and prepared with standard H&E stain. Samples were then observed using a scanning electron microscope (SEM, S-4300 & EDX-350; Hitachi, Tokyo, Japan) operated at an accelerating voltage of 20 kV, to elucidate the influence of decellularization agents on scaffold micro-architecture. Before morphological observations, all scaffolds were sputter coated with gold in an argon atmosphere. Swelling behavior of the scaffolds (5 3 5 3 3 mm3) in PBS (pH 7.4, room temperature), was determined by scaffold weight change at predetermined periods for up to 48 h. A known weight of lyophilized scaffold material was placed in PBS, and wet weight was determined at each time period. The percentage of swelling by water of the scaffold was calculated from the following equation: Swelling ratio (%) 5 [(Wt–W0)/W0] 3 100, where Wt represents the wet weight of scaffold for each time point of incubation, and W0 is the initial weight of the scaffold at dry state. Values were expressed as means 6 standard deviation (SD) (n 5 5). The compressive mechanical properties were tested with a mechanical tester (M500-25KN; Testometric, Lancashire, England) and 50 kg kgf load cell. Scaffolds were cut at a dimension of 7 3 7 3 1 mm3, in triplicate. A rod with a ball-shaped (diameter, 4.7 mm) tip was hammered vertically at a crosshead speed of 1 mm/min on the scaffold specimen. The maximum compressive strength was determined from the stress-strain curve. Values were expressed as means 6 standard error of the mean (SEM) (n 5 5). Biochemical characterization: Attenuated total reflective-infrared (ATR-IR) spectroscopy, ECM protein, and growth factor identification Scaffold surfaces were characterized by ATR-IR spectroscopy (Spectrum GX & AutoImage; Perkin Elmer, Santa Clara, CA). The sample compartment of the ATR-IR machine was
PORCINE RENAL EXTRACELLULAR MATRIX SCAFFOLD FOR KIDNEY REGENERATION
ORIGINAL ARTICLE
TABLE I. Primer Sequences for Real-time PCR Sequences Genes Wnt4 Pax2 Wt1 Emx2 Krt10 KSP-Cadherin vWf Laminin Collagen 4 Gapdh
Forward
Reverse
0
5 -GGAGCCTAGCCCGGACTTC 50 -AAATCTCTATGCAAAATGACGAACAT 50 -GGTCCGCCATCACAACATG 50 -TGGCCAGAAAGCCAAAGC 50 -TGGCCTCCTACATGGACAAAG 50 -CTCAGCCCCCCAGGAACT 50 -GCTGTGCGGTGATTTTAACATC 50 -GGCCACAACTGCGAAAGGT 50 -TTGCTCATAGGCCCCTTTCTT 50 -TGTGTCCGTCGTGGATCTGA
purged with dry air for 1 h to remove moisture before characterization. When cells are seeded onto acellular scaffolds, ECM proteins in the scaffold play a major role in cell behavior. Consequently, investigation of the ECM protein component of the scaffold is important.11 For ECM protein analysis, tissue samples (10 3 10 3 3 mm3) were frozen in liquid nitrogen and ground to a fine powder using a pre-chilled mortar and pestle. The powdered tissue was suspended in RadioImmunoprecipitation Assay buffer containing 13 protease inhibitor cocktail (Thermo Scientific, Waltham, MA). The lysate was incubated overnight at 4 C with gentle rotation and centrifuged at 12,000 rpm for 10 min at 4 C. Supernatants were collected and the bicinchoninic acid assay (Pierce, Rockford, IL) was used to determine protein concentration, prior to storage at 280 C. Equal amounts (30 lg per sample) of native and de-cellularized tissue samples were separated on 10% SDS-PAGE and on NuPage 4–12% Bis-Tris pre-cast gels, using MOPS SDS running buffer (Invitrogen). Membranes were incubated for 2 h with collagen I, collagen IV, laminin, and fibronectin antibodies (dilution 1:100; Sigma-Aldrich). Detection was performed following 1-h incubation at room temperature with secondary antibodies coupled with horseradish peroxidase and SuperSignal West Dura Detection System (Pierce). Densitometry was performed using Quantity One software (Bio-Rad, Hercules, CA). For quantification of growth factors in the scaffold, enzyme-linked immunosorbent assay (ELISA) was performed using vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF1), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) ELISA kits (R&D system, Minneapolis, MN). Optical density was determined using a microplate reader (ELx800; BIO-TEK INSTRUMENTS, Winooski, VT). Biocompatibility: Cell adhesion, viability, proliferation, and tumorigenicity This experiment was approved by the Institutional Review Board of Kyungpook National University Hospital. Primary cultured human renal cells were used to determine scaffold biocompatibility. For cell preparation, renal cortical tissue
0
5 -GCAGCCGTCAATGGCTTTAG 50 -GAGAGATGCAGGGCGATGAA 50 -CTTTCCTGCCTGGGATGCT 50 -TCCGCTCCCACCACGTAAT 50 -TGGCTTGAGTTGCCATGCT 50 -CAGGGCTCAACAACTGATATACAATG 50 -CCGTTTACACCGCTGTTCCT 50 -ACTGTCAGGACCATCAGGACAA 50 -GACTGACTGTTGATATGGCTTCTTTG 50 -CCTGCTTCACCACCTTCTTGA
from a consented patient was collected in Krebs-Henseleit Buffer Solution (Sigma-Aldrich) containing 10% antibiotic/ antimycotic solution (Invitrogen). The renal tissue was digested with collagenase type I (Worthington, Lakewood, NJ) for 1 h at 37 C in a water bath. The cell pellet was passed through a 40-lm cell strainer and plated at a density of 5 3 105 cells/mL in tissue culture plates. Cell culture medium comprised Dulbecco’s Modified Eagle’s Medium:Nutrient Mixture F-12 (Ham‘s) (1:1) (Invitrogen). Medium preparation was followed according to a previously published protocol.12 The cells were incubated at 37 C in a 5% CO2 air environment with a change of medium every 3 days. Cells were subcultured for expansion at a ratio of 1:3 on reaching confluence. Cells at passage 3 were used for experimentation. To determine cell adhesion, scaffolds (5 3 5 3 1 mm3) were treated with ethanol gradient grade for sterilization and soaked in 1 mL of culture medium for 24 h in advance. Cells (1 3 106) were then seeded onto scaffolds at 37 C in a shaking incubator (75 rpm) for 24 h. Adhesion was assessed with Hoechst 33258 (2 ug/mL, Sigma-Aldrich) and values were expressed by DNA concentration (n 5 3). We also performed SEM analysis to observe cell morphology on the scaffold. To measure cell viability and proliferation, samples of 1 3 105 cells/scaffold were placed in wells of a 96well plate. The number of cells was counted using the Cell Counting kit-8 (CCK-8; Dojindo, Kumatoto, Japan) at 3, 6, 24, and 48 h to determine cell viability, and 1, 3, 5, 7, and 10 days to determine cell proliferation. Solution absorbance was measured at 450 nm using a microplate reader. Experiments were run in triplicate per sample. All data were expressed as means 6 SD (n 5 3). To evaluate acellular and renal cell-seeded scaffolds safety in vivo, scaffolds seeded with primary cultured human renal cells (1 3 104 cells/scaffold, 5 3 2 3 1 mm3) were inserted into the renal subcapsule of male ICR mice (5 weeks old, male, Hyochang science, Korea) (n 5 5/group). Animals were sacrificed 4 and 8 weeks post-implantation and kidneys were harvested for routine H&E histologic confirmation. For analysis of scaffold immunogenicity, routine immunohistochemical staining with a cytotoxic T-cell marker (CD8; BD Pharmigen, San Jose, CA) was carried out.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
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FIGURE 1. Histological analysis of porcine renal extracellular matrix (ECM) scaffolds treated with Triton X-100 (Triton) or SDS decellularization agents. Gross findings of the native (a) and de-cellularized (b,c) renal tissue. H&E and DAPI stain showed that scaffolds treated with decellularization agents preserved their native architecture without cellular components. Native porcine renal tissue (d), the architecture of decelluarized scaffolds (e–f, H&E). Nuclear label (g–I, DAPI), and porous structure with SEM (j–l). H&E; hematoxylin and eosin stain, DAPI; 40 ,6-diamidino-2-phenylindole; SEM; scanning electron microscopy. Scale bars in d–i are 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Reconstruction of injured kidney cortex after partial nephrectomy To determine the tissue reconstruction potential of implanted scaffolds, the right kidney of 15 mice (ICR, 5 weeks old, 20 g, male) were subjected to partial nephrectomy (excision volume 4 3 4 3 1 mm3). Ten mice were implanted with a scaffold (Triton X or SDS, 5 3 5 3 1 mm3) and five mice remained scaffold free (control). Mice were euthanized at 6 and 12 weeks post-operation for morphologic analysis. Following tissue/scaffold excision, half of each sample was fixed in 4% paraformaldehyde for histology processing. The remaining half was prepared for realtime polymerase chain reaction (PCR) analysis. Primer sequences of the candidate genes and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, internal control) are shown in Table I. SYBR Green PCR conditions were 95 C for 10 min, followed by 45 cycles of 95 C for 10 s, 58 C for 50 s, and 72 C for 20 s. To analyze the relative changes in gene expression, the 2–DDCt method was used. Regeneration
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of the injury region in the scaffold-treated groups was compared with the control group (incision only). Statistics All values are given as means 6 SD or means 6 SEM. The results were analyzed by a Student’s t-test and one-way analysis of variance (ANOVA). A p-value of
Department of Urology, School of Medicine, Kyungpook National University, Daegu, Korea Joint Institute for Regenerative Medicine, Kyungpook National University Hospital, Daegu, Korea 3 Department of Advanced Materials, Hannam University, Daejeon, Korea 4 Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Chungnam, Korea 5 Department of Urology, College of Medicine, Yeungnam University, Daegu, Korea 6 Department of Colorectal Cancer Center, School of Medicine, Kyungpook National University, Daegu, Korea 2
Received 14 April 2014; revised 8 June 2014; accepted 3 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35274 Abstract: Acellular scaffolds, possessing an intact threedimensional extracellular matrix (ECM) architecture and biochemical components, are promising for regeneration of complex organs, such as the kidney. We have successfully developed a porcine renal acellular scaffold and analyzed its physical/biochemical characteristics, biocompatibility, and kidney reconstructive potential. Segmented porcine kidney cortexes were treated with either 1% (v/v) Triton X-100 (Triton) or sodium dodecyl sulfate (SDS). Scanning electron microscopy showed both treatments preserved native tissue architecture, including porosity and composition. Swelling behavior was higher in the Triton-treated compared with the SDS-treated scaffold. Maximum compressive strength was lower in the Triton-treated compared with the SDS-treated scaffold. Attenuated total reflective-infrared spectroscopy showed the presence of amide II (ANH) in both scaffolds.
Furthermore, richer ECM protein and growth factor contents were observed in the Triton-treated compared with SDStreated scaffold. Primary human kidney cell adherence, viability, and proliferation were enhanced on the Triton-treated scaffold compared with SDS-treated scaffold. Following murine in vivo implantation, tumorigenecity was absent for both scaffolds after 8 weeks and in the Triton-treated scaffold only, glomeruli-like structure formation and neovascularity were observed. We identified 1% Triton X-100 as a more suitable decellularizing agent for porcine renal ECM scaffolds C 2014 Wiley Periodicals, Inc. J prior to kidney regeneration. V Biomed Mater Res Part A: 00A:000–000, 2014.
Key Words: kidney reconstruction, extracellular matrix scaffold, Triton X-100, sodium dodecyl sulfate, partial nephrectomy
How to cite this article: Choi SH, Chun SY, Chae SY, Kim JR, Oh SH, Chung SK, Lee JH, Song PH, Choi G-S, Kim TH, Kwon TG. 2014. Development of a porcine renal extracellular matrix scaffold as a platform for kidney regeneration. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
From the United Network for Organ Sharing (UNOS) data, it is very striking that every year kidney donation numbers remain relatively flat, but the waiting list continues to grow.1 As a consequence, numbers of donated organs are insufficient and it is essential to find potential sources of organs or alternative measures to prevent patients from falling into chronic renal failure (CRF). Generally, renal surgery deteriorates global kidney function proportionately to the volume of renal parenchyma loss.2 Even partial nephrectomy causes progressive glomerulo-sclerosis and increases single nephron glomerular filtration rate, which could even-
tually lead to CRF and the unfortunate requirement for dialysis.2,3 A primary challenge of kidney regeneration after partial nephrectomy is to maintain normal renal function by applying alternative tissues with functional complexity. However, to the best of our knowledge, an efficient method for kidney regeneration after partial nephrectomy is yet to be developed. In this study, we investigated the use of porcine renal cortex extracellular matrix (ECM) scaffolds for reconstruction of the resected portion of the kidney. There are several reasons why we choose porcine renal cortex ECM. First, the renal cortex contains renal tubular and glomerular
*These authors contributed equally to this work. Correspondence to: T.H. Kim; e-mail: [email protected] or T.G. Kwon; e-mail: [email protected] Contract grant sponsors: Korea Health technology R&D Project, Ministry of Health & Welfare, and Republic of Korea; contract grant number: A111345
C 2014 WILEY PERIODICALS, INC. V
1
structures, which are essential elements for functional recovery of the kidney and to maintain the complex micro network as a natural kidney. Second, the tissue has low immunogenicity and provides an abundant source of porcine supplements.4,5 Our primary hypothesis was that renal cortex ECM scaffold implantation could be a feasible approach for kidney restoration, which could ultimately solve the problem regarding deterioration of global kidney function after partial nephrectomy. Before scaffold implantation, optimal preparation is a key step for success. The characteristics of ECM scaffolds, including composition, structure, and biocompatibility, are reportedly different according to the type of agent used for decellularization. These differences are largely owing to the distinction of mechanical integrity, chemical composition, and biological activity of the ECM and growth factors, which are affected by the detergent during decellularization.6 When scaffolds are recellularized, the retained ECM and growth factor constituents act as morphogenic modulators, and are responsible for mediating cellular organization, regulating signal transduction pathways, and controlling cell growth and proliferation. For these reasons, it is essential to prepare scaffolds using the most suitable agent.6,7 The main three types of decellularization methods include physical, chemical, or enzymatic protocols. Chemical treatments are used for the majority of decellularization studies,8 with the most frequently used being Triton X-100 (Triton) and sodium dodecyl sulfate (SDS).6 However, debate exists over which agent is best for optimal decellularization of kidney tissue for tissue engineering purposes.6–10 Therefore, it is necessary to select an optimum agent to produce an effective renal ECM scaffold for partial kidney reconstruction. The primary goal of the current study was to compare previously described agents used for decellularization and identify the most suitable one for porcine renal tissue decellularization. Intensive analyses were carried out for physical, biochemical, and biocompatible aspects. After identification of the optimum agent for renal cortex decellularization, the secondary objective was to analyze the scaffold reconstructive potential in vivo, following implantation into the kidney after partial nephrectomy. In this article, we report the data of our investigations, demonstrating that (1) Triton-treated scaffolds are better at maintaining their mechanical integrity, biochemical components, and biocompatibility, compared with SDS-treated scaffolds; and (2) following implantation of scaffolds into the injured site after partial nephrectomy, glomerulus-, and vessel-like structures were regenerated more effectively in Triton-treated compared with SDS-treated scaffolds. MATERIALS AND METHODS
Preparation of acellular renal cortex scaffolds Cortical sections (10 3 10 3 2 mm3) of kidney were collected from female Yorkshire porcine animals (2–3 months, 22–30 kg). Decellularization was performed using a modified, previously published protocol.10 Kidney sections were washed twice with phosphate buffered saline (PBS), followed by decellularization in a solution of either 1% (v/v)
2
CHOI ET AL.
Triton X-100 (Sigma-Aldrich, St. Louis, MO) or 1% (v/v) SDS (Invitrogen, Carlsbad, CA) (containing 100 U/mL penicillin and 100 lg/mL streptomycin), both diluted in distilled water. Sample were decellularized at 4 C in a shaking incubator (200 rpm). The decellularization solution, using 15–20 fold of sample volume (weight), was changed 4 h after initial tissue harvest and then every 24 h until tissues were transparent (for 10–14 days). Tissues were then treated with DNase (30 lg/mL; Sigma-Aldrich) in PBS for 1 h. Finally, tissues were rinsed with PBS to flush out residual DNase. Decellularized kidney scaffolds were cryoembedded in optimum cutting temperature compound (Sakura Finetek/Tissue Tek, Torrance, CA). After confirmation of decellularization with hematoxylin and eosin (H&E) and 4’,6-diamidino-2-phenylindole (DAPI) stain analysis, scaffolds were sterilized with gradient ethanol and lyophilized, then stored at 4 C until analysis. Physical characterization: Morphology, swelling behavior, and mechanical property in vitro For morphological analysis, both native and acellular sections of kidney were formalin fixed, paraffin embedded, sectioned at 5 lm thickness, and prepared with standard H&E stain. Samples were then observed using a scanning electron microscope (SEM, S-4300 & EDX-350; Hitachi, Tokyo, Japan) operated at an accelerating voltage of 20 kV, to elucidate the influence of decellularization agents on scaffold micro-architecture. Before morphological observations, all scaffolds were sputter coated with gold in an argon atmosphere. Swelling behavior of the scaffolds (5 3 5 3 3 mm3) in PBS (pH 7.4, room temperature), was determined by scaffold weight change at predetermined periods for up to 48 h. A known weight of lyophilized scaffold material was placed in PBS, and wet weight was determined at each time period. The percentage of swelling by water of the scaffold was calculated from the following equation: Swelling ratio (%) 5 [(Wt–W0)/W0] 3 100, where Wt represents the wet weight of scaffold for each time point of incubation, and W0 is the initial weight of the scaffold at dry state. Values were expressed as means 6 standard deviation (SD) (n 5 5). The compressive mechanical properties were tested with a mechanical tester (M500-25KN; Testometric, Lancashire, England) and 50 kg kgf load cell. Scaffolds were cut at a dimension of 7 3 7 3 1 mm3, in triplicate. A rod with a ball-shaped (diameter, 4.7 mm) tip was hammered vertically at a crosshead speed of 1 mm/min on the scaffold specimen. The maximum compressive strength was determined from the stress-strain curve. Values were expressed as means 6 standard error of the mean (SEM) (n 5 5). Biochemical characterization: Attenuated total reflective-infrared (ATR-IR) spectroscopy, ECM protein, and growth factor identification Scaffold surfaces were characterized by ATR-IR spectroscopy (Spectrum GX & AutoImage; Perkin Elmer, Santa Clara, CA). The sample compartment of the ATR-IR machine was
PORCINE RENAL EXTRACELLULAR MATRIX SCAFFOLD FOR KIDNEY REGENERATION
ORIGINAL ARTICLE
TABLE I. Primer Sequences for Real-time PCR Sequences Genes Wnt4 Pax2 Wt1 Emx2 Krt10 KSP-Cadherin vWf Laminin Collagen 4 Gapdh
Forward
Reverse
0
5 -GGAGCCTAGCCCGGACTTC 50 -AAATCTCTATGCAAAATGACGAACAT 50 -GGTCCGCCATCACAACATG 50 -TGGCCAGAAAGCCAAAGC 50 -TGGCCTCCTACATGGACAAAG 50 -CTCAGCCCCCCAGGAACT 50 -GCTGTGCGGTGATTTTAACATC 50 -GGCCACAACTGCGAAAGGT 50 -TTGCTCATAGGCCCCTTTCTT 50 -TGTGTCCGTCGTGGATCTGA
purged with dry air for 1 h to remove moisture before characterization. When cells are seeded onto acellular scaffolds, ECM proteins in the scaffold play a major role in cell behavior. Consequently, investigation of the ECM protein component of the scaffold is important.11 For ECM protein analysis, tissue samples (10 3 10 3 3 mm3) were frozen in liquid nitrogen and ground to a fine powder using a pre-chilled mortar and pestle. The powdered tissue was suspended in RadioImmunoprecipitation Assay buffer containing 13 protease inhibitor cocktail (Thermo Scientific, Waltham, MA). The lysate was incubated overnight at 4 C with gentle rotation and centrifuged at 12,000 rpm for 10 min at 4 C. Supernatants were collected and the bicinchoninic acid assay (Pierce, Rockford, IL) was used to determine protein concentration, prior to storage at 280 C. Equal amounts (30 lg per sample) of native and de-cellularized tissue samples were separated on 10% SDS-PAGE and on NuPage 4–12% Bis-Tris pre-cast gels, using MOPS SDS running buffer (Invitrogen). Membranes were incubated for 2 h with collagen I, collagen IV, laminin, and fibronectin antibodies (dilution 1:100; Sigma-Aldrich). Detection was performed following 1-h incubation at room temperature with secondary antibodies coupled with horseradish peroxidase and SuperSignal West Dura Detection System (Pierce). Densitometry was performed using Quantity One software (Bio-Rad, Hercules, CA). For quantification of growth factors in the scaffold, enzyme-linked immunosorbent assay (ELISA) was performed using vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF1), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) ELISA kits (R&D system, Minneapolis, MN). Optical density was determined using a microplate reader (ELx800; BIO-TEK INSTRUMENTS, Winooski, VT). Biocompatibility: Cell adhesion, viability, proliferation, and tumorigenicity This experiment was approved by the Institutional Review Board of Kyungpook National University Hospital. Primary cultured human renal cells were used to determine scaffold biocompatibility. For cell preparation, renal cortical tissue
0
5 -GCAGCCGTCAATGGCTTTAG 50 -GAGAGATGCAGGGCGATGAA 50 -CTTTCCTGCCTGGGATGCT 50 -TCCGCTCCCACCACGTAAT 50 -TGGCTTGAGTTGCCATGCT 50 -CAGGGCTCAACAACTGATATACAATG 50 -CCGTTTACACCGCTGTTCCT 50 -ACTGTCAGGACCATCAGGACAA 50 -GACTGACTGTTGATATGGCTTCTTTG 50 -CCTGCTTCACCACCTTCTTGA
from a consented patient was collected in Krebs-Henseleit Buffer Solution (Sigma-Aldrich) containing 10% antibiotic/ antimycotic solution (Invitrogen). The renal tissue was digested with collagenase type I (Worthington, Lakewood, NJ) for 1 h at 37 C in a water bath. The cell pellet was passed through a 40-lm cell strainer and plated at a density of 5 3 105 cells/mL in tissue culture plates. Cell culture medium comprised Dulbecco’s Modified Eagle’s Medium:Nutrient Mixture F-12 (Ham‘s) (1:1) (Invitrogen). Medium preparation was followed according to a previously published protocol.12 The cells were incubated at 37 C in a 5% CO2 air environment with a change of medium every 3 days. Cells were subcultured for expansion at a ratio of 1:3 on reaching confluence. Cells at passage 3 were used for experimentation. To determine cell adhesion, scaffolds (5 3 5 3 1 mm3) were treated with ethanol gradient grade for sterilization and soaked in 1 mL of culture medium for 24 h in advance. Cells (1 3 106) were then seeded onto scaffolds at 37 C in a shaking incubator (75 rpm) for 24 h. Adhesion was assessed with Hoechst 33258 (2 ug/mL, Sigma-Aldrich) and values were expressed by DNA concentration (n 5 3). We also performed SEM analysis to observe cell morphology on the scaffold. To measure cell viability and proliferation, samples of 1 3 105 cells/scaffold were placed in wells of a 96well plate. The number of cells was counted using the Cell Counting kit-8 (CCK-8; Dojindo, Kumatoto, Japan) at 3, 6, 24, and 48 h to determine cell viability, and 1, 3, 5, 7, and 10 days to determine cell proliferation. Solution absorbance was measured at 450 nm using a microplate reader. Experiments were run in triplicate per sample. All data were expressed as means 6 SD (n 5 3). To evaluate acellular and renal cell-seeded scaffolds safety in vivo, scaffolds seeded with primary cultured human renal cells (1 3 104 cells/scaffold, 5 3 2 3 1 mm3) were inserted into the renal subcapsule of male ICR mice (5 weeks old, male, Hyochang science, Korea) (n 5 5/group). Animals were sacrificed 4 and 8 weeks post-implantation and kidneys were harvested for routine H&E histologic confirmation. For analysis of scaffold immunogenicity, routine immunohistochemical staining with a cytotoxic T-cell marker (CD8; BD Pharmigen, San Jose, CA) was carried out.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
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FIGURE 1. Histological analysis of porcine renal extracellular matrix (ECM) scaffolds treated with Triton X-100 (Triton) or SDS decellularization agents. Gross findings of the native (a) and de-cellularized (b,c) renal tissue. H&E and DAPI stain showed that scaffolds treated with decellularization agents preserved their native architecture without cellular components. Native porcine renal tissue (d), the architecture of decelluarized scaffolds (e–f, H&E). Nuclear label (g–I, DAPI), and porous structure with SEM (j–l). H&E; hematoxylin and eosin stain, DAPI; 40 ,6-diamidino-2-phenylindole; SEM; scanning electron microscopy. Scale bars in d–i are 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Reconstruction of injured kidney cortex after partial nephrectomy To determine the tissue reconstruction potential of implanted scaffolds, the right kidney of 15 mice (ICR, 5 weeks old, 20 g, male) were subjected to partial nephrectomy (excision volume 4 3 4 3 1 mm3). Ten mice were implanted with a scaffold (Triton X or SDS, 5 3 5 3 1 mm3) and five mice remained scaffold free (control). Mice were euthanized at 6 and 12 weeks post-operation for morphologic analysis. Following tissue/scaffold excision, half of each sample was fixed in 4% paraformaldehyde for histology processing. The remaining half was prepared for realtime polymerase chain reaction (PCR) analysis. Primer sequences of the candidate genes and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, internal control) are shown in Table I. SYBR Green PCR conditions were 95 C for 10 min, followed by 45 cycles of 95 C for 10 s, 58 C for 50 s, and 72 C for 20 s. To analyze the relative changes in gene expression, the 2–DDCt method was used. Regeneration
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of the injury region in the scaffold-treated groups was compared with the control group (incision only). Statistics All values are given as means 6 SD or means 6 SEM. The results were analyzed by a Student’s t-test and one-way analysis of variance (ANOVA). A p-value of
