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Biotechnology Letters 25: 1505–1508, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

1505

Renal tissue reconstitution by the implantation of renal segments on biodegradable polymer scaffolds Sang-Soo Kim1,2 , Heung Jae Park3 , Joungho Han4 , Cha Yong Choi2 & Byung-Soo Kim1,∗ 1 Department of

Chemical Engineering, Hanyang University, Seoul, Korea; Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul,

2 Interdisciplinary

Korea 3 Department

of Urology, Kangbuk Samsung Hospital and 4 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea ∗ Author for correspondence (Fax: +82-2-2298-4101; E-mail: [email protected]) Received 2 May 2003; Revisions requested 29 May 2003; Revisions received 25 June 2003; Accepted 3 July 2003

Key words: biodegradable polymer scaffold, renal reconstitution, tissue engineering

Abstract Renal units were created in vivo by transplanting isolated renal segments on three-dimensional, biodegradable polymer scaffolds. Renal segments, freshly isolated from rat kidneys, were seeded on polymer scaffolds and subcutaneously implanted in athymic mice for two and four weeks. Three-dimensional renal reconstructs were formed with glomeruli and tubules, showing a possibility of reconstituting renal structures by transplanting renal segments.

Introduction End-stage renal failure is a devastating disease that afflicts more than 300 000 individuals in the USA (Hammerman 2002) and 1.1 million worldwide (Lysaght 2002). Dialysis and renal transplantation are currently available treatments of this disease. However, these treatments have severe limitations. Dialysis does not replace the whole renal functions and often brings complications. Although renal transplantation can restore the whole renal functions, many limitations still remain, such as donor shortage, allograft failure, and long-term immunosuppression (Amiel & Atala 1999, Hammerman 2002, Kim et al. 2000, Yoo et al. 2002). There have been efforts directed towards the development of extracorporeal bioartificial renal units. A bioartificial renal tubule device has been developed by culturing renal proximal tubule cells on the inner surface of hollow fibers (MacKay et al. 1998). Recently, it was demonstrated that the combination of a synthetic filtration device and a renal tubule cell therapy device in an extracorporeal perfusion circuit replaces certain physiologic functions of the kidney in uremic

dogs (Humes et al. 1999). However, the application of these extracorporeal devices may be best reserved for temporary situations rather than a permanent solution. Transplantation of renal cells or segments has been investigated to develop a new therapy for renal failure. In a study, renal cells, which were isolated from early-stage cloned bovine fetus, seeded on polymer scaffolds, and implanted into the nuclear donor animal, regenerated glomeruli and tubule structures and excreted urine-like fluid in vivo (Lanza et al. 2002). In another study, the implantation of kidney precursor cells derived from early embryo resulted in the renal structure formation and dilute urine excretion (Dekel et al. 2003). In the present study, we investigated the possibility of creating an artificial renal unit in vivo by transplanting isolated postnatal renal segments on three-dimensional, biodegradable polymer scaffolds. Renal segments were isolated from postnatal rat kidneys, seeded on to polyglycolic acid scaffolds, and implanted into subcutaneous spaces of athymic mice. Two and four weeks after implantation, renal structures of the newly-formed tissues were examined by histological and immunohistochemical analyses.

1506 Materials and methods Renal segment isolation Kidneys were surgically removed from 1-d-old Sprague–Dawley rats (SLC, Tokyo, Japan) under a sterile condition. The renal capsule and collecting systems were removed, and the kidneys were washed with ice-cold phosphate buffered saline (PBS) thoroughly. The kidneys were minced very well into small pieces, resuspended in ice cold PBS with rough pipetting, and strained through a 200 µm sieve to remove large tissue fragments. The cell type in the renal segments includes nephron epithelial cells, endothelial cells, vascular smooth muscle cells, and stromal cells.

Fig. 1. Scanning electron microscopic photograph of biodegradable polymer scaffold. The porous mesh was fabricated from biodegradable polyglycolic acid fibers, which provides cell or tissue segment adhesion surface and tissue formation space. Scale bar = 100 µm.

Polyglycolic acid (PGA) scaffolds Biodegradable, non-woven meshes fabricated from fibers (12 µm diam.) of 100% PGA were purchased from Albani International Inc. (Mansfield, MA) and utilized as three-dimensional scaffolds for renal tissue reconstitution. The inherent viscosity of PGA was 1.23 dl g−1 . The crystallinity of PGA was 57%, as measured by differential scanning calorimetry. Prior to use, the scaffolds were cut into squares (5 × 5 mm, 2 mm thickness), sterilized using ethanol, and washed with sterile distilled water. Seeding on to scaffolds and implantation Renal segments isolated from 2 kidneys were suspended in 0.3 ml cell culture medium (Dulbecco’s Modified Eagle Medium, 100 units penicillin ml−1 , and 0.1 mg streptomycin ml−1 ) and seeded on to six PGA scaffolds. The renal segment-seeded scaffolds were implanted into subcutaneous dorsal spaces of 6 athymic mice (SLC, Tokyo, Japan). As a control, unseeded scaffolds were implanted into subcutaneous dorsal spaces of 6 athymic mice.

Fig. 2. Scanning electron microscopic photograph of renal segment-seeded scaffold. The scanning electron microscopic photograph shows that the renal segments attached well to the polymer scaffolds. Scale bar = 100 µm.

transplantation was fixed in 10% (v/v) buffered formalin and embedded in paraffin. Tissue sections, 4 µm thick, were processed for standard hematoxylin and eosin staining. For immunohistochemical analyses, 4 µm thick sections were stained using antibodies against CD31 (Dako, Glostrup, Denmark), a glomerulus endothelial specific marker. The staining signal was developed with avidin-peroxidase system (ABC kit, Vector Laboratory, Burlingame, USA).

Analyses For scanning electron microscopic examination, specimens were fixed in 1% (v/v) glutaraldehyde and 0.1% (v/v) formaldehyde for 30 min and 24 h, respectively, dehydrated with a graded ethanol series, and dried. The dried samples were mounted on aluminum supports and sputter coated with gold. A scanning electron microscope (JEOL, Tokyo, Japan) was operated to image samples. For histological analyses, specimens (n = 3) retrieved two and four weeks after

Results and discussion The seeding of renal segments on three-dimensional polymer scaffolds resulted in the formation of threedimensional renal segment-polymer constructs. PGA meshes served as a porous scaffold (Figure 1), which provides a substrate for cell adhesion and threedimensional space for tissue formation upon implantation. One day after seeding, scanning electron micro-

1507 scopic examination of renal segment-seeded scaffolds revealed that the renal segments adhered well on the polymer scaffolds (Figure 2). Two and four weeks after implantation, the implanted renal segments on polymer scaffolds reconstituted renal tissues. Histological analyses of the specimens retrieved at two weeks showed the tubular structures with hollow centers composed of single cell layer and the glomerulus structures with vascular tufts (Figure 3a). Tubular and glomerular structures were also observed in the specimens retrieved at four weeks (Figures 3b, c). In contrast, no renal structures were observed in the unseeded polymer implants (Figure 3d). Fibrotic tissues were formed in the unseeded polymer scaffolds, as host fibroblasts from the surrounding tissues migrated into the unseeded polymer scaffolds. In the seeded scaffolds, tissues were formed mainly by renal cells present in the scaffolds rather than host cells migrating from the surrounding tissues. Biodegradable polymer fibers were degrading incompletely and still present in the implants at four weeks. Degradation of the scaffolds would allow for the removal of synthetic parts from the implants and the subsequent formation of natural tissues. To confirm renal structures in the implants, immunohistochemical analyses were performed using antibodies against CD31, a glomerulus endothelial specific marker. Both of the two- and four-week implants stained positively for CD31, identifying the vascular tufts of glomerular structures in the reconstituted tissues (Figure 4). Instead of cultured renal cells, renal segments were utilized as a cell source in the present study, because renal segments could be more effective than cultured renal cells for renal tissue reconstitution. Kidney is composed of at least 26 terminally differentiated cell types and its structure and function are extremely complex (Al-Awqati & Oliver 2002). This makes kidney one of the most difficult organs to reconstruct (Amiel & Atala 1999). The use of proteolytic enzymes for renal cell isolation from renal tissue biopsy would damage the cells and destroy renal unit structures. In vitro cell cultivation may cause the loss of differentiated renal cell phenotypes. Thus, it would be advantageous to use renal segments, which are prepared without proteolytic enzyme treatment and in vitro cultivation, for renal tissue reconstitution. In summary, we report on the reconstitution of renal tissues by transplanting renal segments isolated from postnatal renal tissues on biodegradable poly-

Fig. 3. Histological analyses of the implants (hematoxyline and eosin staining). The seeded implants two (a) and four weeks (b, c) after implantation contained tubule structures (open arrowhead) with hollow centers, vascular tufts of glomerulus-like structures (closed arrowhead) and neovessels (open arrow). In contrast, no renal-like structures were observed from the unseeded control polymer implants at two weeks (d). Polyglycolic acid fibers (closed arrow) degraded incompletely in the implants at four weeks. Scale bars = 30 µm (a, b, d) or 15 µm (c).

1508 References

Fig. 4. Immunohistochemical analysis using CD31 antibodies identifies vascular tufts of glomerular structure (closed arrowhead). Scale bar = 30 µm.

mer scaffolds. The tissue reconstructs contained the structures of glomeruli and tubules. However, further studies are necessary to assess the clinical usage of this approach. The renal cell transplantation experiments need to be repeated in animal models with fully functional immune systems. The effects of immunosuppressive treatments also remain to be investigated. To avoid potential immune problems, transplantation of autologous bone marrow-derived stem cells, which are capable of differentiate into renal cells (Poulsom et al. 2003), could be attempted. Implantation of renal segment-polymer constructs into kidney in renal failure animal models would be critical to evaluate the therapeutic potential of this approach.

Acknowledgement This work was supported by a grant (No. R01-2001000-00491-0) from the Basic Research Program of the Korea Science & Engineering Foundation.

Al-Awqati Q, Oliver JA (2002) Stem cells in the kidney. Kidney Int. 61: 387–395. Amiel GE, Atala A (1999) Current and future modalities for functional renal replacement. Urol. Clin. North Am. 26: 235–246. Dekel B, Burakova T, Arditti FD, Reich-Zeliger S, Milstein O, Aviel-Ronen S, Rechavi G, Friedman N, Kaminski N, Passwell JH, Reisner Y (2003) Human and porcine early kidney precursors as a new source for transplantation. Nat. Med. 9:53–60. Hammerman MR (2002) Transplantation of developing kidneys. Transplant. Rev. 16: 62–71. Humes DH, Buffington DA, MacKay SM, Funke AJ, Weitzel WF (1999) Replacement of renal function in uremic animals with a tissue engineered kidney. Nat. Biotechnol. 17: 451–455. Kim BS, Mooney DJ, Atala A (2000) Genitourinary system. In: Lanza RP, Langer R, Vacanti J, eds. Principles of Tissue Engineering, 2nd edn. San Diego: Academic Press, pp. 655–667. Lanza RP, Chung HY et al. (2002) Generation of histocompatible tissues using nuclear transplantation. Nat. Biotechnol. 20: 689– 696. Lysaght MJ (2002) Maintenance dialysis population dynamics: current trends and long-term implications. J. Am. Soc. Nephrol. 13: S37–S40. MacKay SM, Funke AJ, Buffington DA, Humes HD (1998) Tissue engineering of a bioartificial renal tubule. Am. Soc. Artif. Intern. Organs J. 44: 179–183. Poulsom R, Alison MR, Cook T, Jeffery R, Ryan E, Forbes SJ, Hunt T, Wyles S, Wright NA (2003) Bone marrow stem cells contribute to healing of the kidney. J. Am. Soc. Nephrol. 14 (Suppl. 1): S48–S54. Yoo JJ, Kwon TG, Atala A (2002) Intracorporeal kidney. In: Atala A, Lanza RP, eds. Methods of Tissue Engineering. San Diego: Academic Press, pp. 999–1003.

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