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Bladder Reconstruction by Tissue Engineering—With or Without Cells?
Bladder Reconstruction by Tissue Engineering—With or Without Cells? ladder augmentation serves to restore bladder capacity and compliance, and prevent vesicoureteral reflux and renal damage. Currently augmentation cystoplasty is usually accomplished using intestinal segments or gastric flaps, but this can result in certain severe complications including urinary tract infection, intestinal obstruction, mucus production, electrolyte abnormalities, perforation and carcinogenesis. Tissue engineering technology has been proposed as an alternative approach to generate bladder tissues for reconstruction. Since the introduction of the concept of building bladder tissues using this technology in the early 1990s, significant progress has been made toward the goal of clinical translation. In fact, this technology is currently in clinical trials involving multiple centers. Two types of tissue engineering technologies have been used to build bladder tissue, namely cell seeded and nonseeded. Cell seeded technology uses scaffolds that are seeded in vitro with primary cultured cells obtained from a bladder biopsy. This composite graft is then implanted back in the host for carryover of the regenerative process.1– 6 The nonseeded technology uses cell-free biodegradable scaffolds to allow the natural process of regeneration to occur in vivo and was successfully applied experimentally in partial (30% to 40% tissue replacement) cystoplasty models.7,8 One may wonder which approach is preferred for large bladder tissue replacement as numerous investigations involving both technologies have been actively conducted over the years. In this issue of The Journal Jayo et al (page 392) report the results of a comparative study in which bladder shaped biodegradable scaffolds were seeded with or without cells for large bladder replacement procedures in a canine subtotal (more than 70% tissue replacement) cystectomy model. The authors demonstrate that the cell seeded scaffold group outperformed the nonseeded group, resulting in a larger bladder capacity and near normal bladder compliance. Histological analyses of the cell seeded group showed the presence of organized tissues consisting of urothelial, suburothelial and smooth muscle layers in the regenerated bladder wall 9 months after augmentation. In contrast, bladder augmentation with unseeded scaffolds led to inadequate tissue development with reepithelialization on the graft lumen. The scaffolds implanted without cells showed fibrosis, graft contraction, and small bladder capacity and dysfunction. These results are consistent with the outcomes of a previous investigation in which seeded and nonseeded scaffolds were used to reconstruct the bladder in a canine model of subtotal cystectomy.2 These independent studies demonstrate that cells are necessary to achieve improved bladder tissue function when large bladder tissue is in demand. Further validation of the cell seeded technology was demonstrated through a clinical trial involving bladder augmentation in patients with myelomeningocele.5
In cell based bladder tissue engineering, autologous bladder cells are a gold standard cell source for bladder regeneration. To provide functional cells for tissue reconstruction and remodeling cultured cells at early passage (under passage 5) provide the optimal results. In addition, cells should be cultured on the scaffold for a short time (less than 2 weeks) since smooth muscle cells lose their phenotype after long-term static culture.9 Mechanical preconditioning of muscle cells might be an alternative culture method that would allow the maintenance of phenotype and functional characteristics before implantation.10 Immortalized muscle cell lines may also generate a large amount of functional cells. However, this type of cell line has limited clinical application, as immortalized cells carry the risk of tumor formation.11 When normal bladder cells are not available in patients due to bladder cancer, an alternative cell source needs to be found. Several cell types are being investigated for this purpose including bone marrow stem cells,3,4 skeletal myoblasts12 and adipose progenitor cells.13 The bio-scaffold selected is another critical factor for a large bladder tissue replacement with tissue engineering. The ideal bio-scaffolds for bladder or other hollow organ tissue engineering should posses the proper biomechanical and physical properties so that the organ can develop and form the correct shape during tissue regeneration. Currently the 2 types of bio-scaffolds commonly used in experimental and clinical applications for bladder tissue engineering are natural collagen acellular matrices such as small intestinal submucosa,3,4,6 – 8 bladder lamina propria (bladder submucosa)1 and bladder acellular matrix graft,14 and synthetic polymers such as polyglycolic acid (PGA) and polylactic-coglycolic acid (PLGA).2,5 Natural collagen acellular matrixes are produced by removing all cellular remnants from a piece of tissue to obtain a biological scaffold. This material is biocompatible and contains numerous growth factors that promote tissue regeneration.7,15 In partial cystoplasty biological material alone (without seeded cells) can facilitate tissue regeneration. Smooth muscle cell infiltration, vascularization and innervation can be observed at early stages, and muscle bundle formation is observed later after surgery in the animal study.7,8 However, the use of natural collagen matrices for bladder regeneration is limited by the size of the bladder defect to be replaced. A recent study showed that a large bladder augmentation with cell seeded or cell-free natural collagen matrix did not improve bladder regeneration.6 The results from seeded and nonseeded groups showed graft shrinkage, low bladder capacity, poor vascularization and disorganized muscle development. One of reasons is that the collagen matrix may be too soft to maintain the shape of the organ during tissue remodeling and the scaffold collapses after implantation as a result. Other concerns about the use of natural collagen matrices center on the
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0022-5347/08/1801-0010/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
10
Vol. 180, 10-11, July 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.04.068
BLADDER RECONSTRUCTION BY TISSUE ENGINEERING possibility of residual porcine cellular and DNA content, which could still cause immunological reactions in vivo after implantation.16 For replacement of large amounts of tissue PGA-PLGA with cells is preferred. PGA-PLGA can provide a 3-dimensional space to allow the loading of more cells during the in vitro phase, and this can lead to the formation of complete tissues in vivo. Importantly PGA/PLGA provides adequate physical properties to hold the shape of the implant and prevent collapse during tissue healing processes. Additionally, this material is highly porous which facilitates gas and nutrition exchange, and promotes cell metabolism within the cell-scaffold composite. PGA/PLGA is Food and Drug Administration approved as a scaffold material and has been used for many applications. In conclusion, Jayo et al demonstrate that current data on the use of engineered bladder tissue for cystoplasty are repeatable, and this study supports the clinical feasibility of using autologous bladder cells to seed a biodegradable polymer and create a large replacement bladder. This technology may become the preferred method that replaces the current practice of bladder tissue reconstruction using gastrointestinal tissues. Yuanyuan Zhang Wake Forest Institute for Regenerative Medicine Wake Forest University School of Medicine Winston Salem, North Carolina
5.
6.
7.
8.
9. 10.
11.
12.
13.
REFERENCES 1.
Yoo JJ, Meng J, Oberpenning F and Atala A: Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 1998; 51: 221. 2. Oberpenning F, Meng J, Yoo JJ and Atala A: De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999; 17: 149. 3. Chung SY, Krivorov NP, Rausei V, Thomas L, Frantzen M, Landsittel D et al: Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. J Urol 2005; 174: 353. 4. Zhang Y, Lin HK, Frimberger D, Epstein RB and Kropp BP: Growth of bone marrow stromal cells on small intestinal
14.
15.
16.
11
submucosa: an alternative cell source for tissue engineered bladder. BJU Int 2005; 96: 1120. Atala A, Bauer SB, Soker S, Yoo JJ and Retik AB: Tissueengineered autologous bladders for patients needing cystoplasty. Lancet 2006; 367: 1241. Zhang Y, Frimberger D, Cheng EY, Lin HK and Kropp BP: Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model. BJU Int 2006; 98: 1100. Badylak SF, Kropp B, McPherson T, Liang H and Snyder PW: Small intestinal submucosa: a rapidly resorbed bioscaffold for augmentation cystoplasty in a dog model. Tissue Eng 1998; 4: 379. Kropp BP, Rippy MK, Badylak SF, Adams MC, Keating MA, Rink RC et al: Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J Urol 1996; 155: 2098. Chamley-Campbell J, Campbell GR and Ross R: The smooth muscle cell in culture. Physiol Rev 1979; 59: 1. Akhyari P, Fedak PW, Weisel RD, Lee TY, Verma S, Mickle DA et al: Mechanical stretch regimen enhances the formation of bioengineered autologous cardiac muscle grafts. Circulation 2002; 106: I137. Koh GY, Klug MG, Soonpaa MH and Field LJ: Differentiation and long-term survival of C2C12 myoblast grafts in heart. J Clin Invest 1993; 92: 1548. Lu SH, Sacks MS, Chung SY, Gloeckner DC, Pruchnic R, Huard J et al: Biaxial mechanical properties of musclederived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials 2005; 26: 443. Jack GS, Almeida FG, Zhang R, Alfonso ZC, Zuk PA and Rodriguez LV: Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction. J Urol 2005; 174: 2041. Piechota HJ, Gleason CA, Dahms SE, Dahiya R, Nunes LS, Lue TF et al: Bladder acellular matrix graft: in vivo functional properties of the regenerated rat bladder. Urol Res 1999; 27: 206. Chun SY, Lim GJ, Kwon TG, Kwak EK, Kim BW, Atala A et al: Identification and characterization of bioactive factors in bladder submucosa matrix. Biomaterials 2007; 28: 4251. Feil G, Christ-Adler M, Maurer S, Corvin S, Rennekampff HO, Krug J et al: Investigations of urothelial cells seeded on commercially available small intestine submucosa. Eur Urol 2006; 50: 1330.
In cell based bladder tissue engineering, autologous bladder cells are a gold standard cell source for bladder regeneration. To provide functional cells for tissue reconstruction and remodeling cultured cells at early passage (under passage 5) provide the optimal results. In addition, cells should be cultured on the scaffold for a short time (less than 2 weeks) since smooth muscle cells lose their phenotype after long-term static culture.9 Mechanical preconditioning of muscle cells might be an alternative culture method that would allow the maintenance of phenotype and functional characteristics before implantation.10 Immortalized muscle cell lines may also generate a large amount of functional cells. However, this type of cell line has limited clinical application, as immortalized cells carry the risk of tumor formation.11 When normal bladder cells are not available in patients due to bladder cancer, an alternative cell source needs to be found. Several cell types are being investigated for this purpose including bone marrow stem cells,3,4 skeletal myoblasts12 and adipose progenitor cells.13 The bio-scaffold selected is another critical factor for a large bladder tissue replacement with tissue engineering. The ideal bio-scaffolds for bladder or other hollow organ tissue engineering should posses the proper biomechanical and physical properties so that the organ can develop and form the correct shape during tissue regeneration. Currently the 2 types of bio-scaffolds commonly used in experimental and clinical applications for bladder tissue engineering are natural collagen acellular matrices such as small intestinal submucosa,3,4,6 – 8 bladder lamina propria (bladder submucosa)1 and bladder acellular matrix graft,14 and synthetic polymers such as polyglycolic acid (PGA) and polylactic-coglycolic acid (PLGA).2,5 Natural collagen acellular matrixes are produced by removing all cellular remnants from a piece of tissue to obtain a biological scaffold. This material is biocompatible and contains numerous growth factors that promote tissue regeneration.7,15 In partial cystoplasty biological material alone (without seeded cells) can facilitate tissue regeneration. Smooth muscle cell infiltration, vascularization and innervation can be observed at early stages, and muscle bundle formation is observed later after surgery in the animal study.7,8 However, the use of natural collagen matrices for bladder regeneration is limited by the size of the bladder defect to be replaced. A recent study showed that a large bladder augmentation with cell seeded or cell-free natural collagen matrix did not improve bladder regeneration.6 The results from seeded and nonseeded groups showed graft shrinkage, low bladder capacity, poor vascularization and disorganized muscle development. One of reasons is that the collagen matrix may be too soft to maintain the shape of the organ during tissue remodeling and the scaffold collapses after implantation as a result. Other concerns about the use of natural collagen matrices center on the
B
0022-5347/08/1801-0010/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
10
Vol. 180, 10-11, July 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.04.068
BLADDER RECONSTRUCTION BY TISSUE ENGINEERING possibility of residual porcine cellular and DNA content, which could still cause immunological reactions in vivo after implantation.16 For replacement of large amounts of tissue PGA-PLGA with cells is preferred. PGA-PLGA can provide a 3-dimensional space to allow the loading of more cells during the in vitro phase, and this can lead to the formation of complete tissues in vivo. Importantly PGA/PLGA provides adequate physical properties to hold the shape of the implant and prevent collapse during tissue healing processes. Additionally, this material is highly porous which facilitates gas and nutrition exchange, and promotes cell metabolism within the cell-scaffold composite. PGA/PLGA is Food and Drug Administration approved as a scaffold material and has been used for many applications. In conclusion, Jayo et al demonstrate that current data on the use of engineered bladder tissue for cystoplasty are repeatable, and this study supports the clinical feasibility of using autologous bladder cells to seed a biodegradable polymer and create a large replacement bladder. This technology may become the preferred method that replaces the current practice of bladder tissue reconstruction using gastrointestinal tissues. Yuanyuan Zhang Wake Forest Institute for Regenerative Medicine Wake Forest University School of Medicine Winston Salem, North Carolina
5.
6.
7.
8.
9. 10.
11.
12.
13.
REFERENCES 1.
Yoo JJ, Meng J, Oberpenning F and Atala A: Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 1998; 51: 221. 2. Oberpenning F, Meng J, Yoo JJ and Atala A: De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999; 17: 149. 3. Chung SY, Krivorov NP, Rausei V, Thomas L, Frantzen M, Landsittel D et al: Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. J Urol 2005; 174: 353. 4. Zhang Y, Lin HK, Frimberger D, Epstein RB and Kropp BP: Growth of bone marrow stromal cells on small intestinal
14.
15.
16.
11
submucosa: an alternative cell source for tissue engineered bladder. BJU Int 2005; 96: 1120. Atala A, Bauer SB, Soker S, Yoo JJ and Retik AB: Tissueengineered autologous bladders for patients needing cystoplasty. Lancet 2006; 367: 1241. Zhang Y, Frimberger D, Cheng EY, Lin HK and Kropp BP: Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model. BJU Int 2006; 98: 1100. Badylak SF, Kropp B, McPherson T, Liang H and Snyder PW: Small intestinal submucosa: a rapidly resorbed bioscaffold for augmentation cystoplasty in a dog model. Tissue Eng 1998; 4: 379. Kropp BP, Rippy MK, Badylak SF, Adams MC, Keating MA, Rink RC et al: Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J Urol 1996; 155: 2098. Chamley-Campbell J, Campbell GR and Ross R: The smooth muscle cell in culture. Physiol Rev 1979; 59: 1. Akhyari P, Fedak PW, Weisel RD, Lee TY, Verma S, Mickle DA et al: Mechanical stretch regimen enhances the formation of bioengineered autologous cardiac muscle grafts. Circulation 2002; 106: I137. Koh GY, Klug MG, Soonpaa MH and Field LJ: Differentiation and long-term survival of C2C12 myoblast grafts in heart. J Clin Invest 1993; 92: 1548. Lu SH, Sacks MS, Chung SY, Gloeckner DC, Pruchnic R, Huard J et al: Biaxial mechanical properties of musclederived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials 2005; 26: 443. Jack GS, Almeida FG, Zhang R, Alfonso ZC, Zuk PA and Rodriguez LV: Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction. J Urol 2005; 174: 2041. Piechota HJ, Gleason CA, Dahms SE, Dahiya R, Nunes LS, Lue TF et al: Bladder acellular matrix graft: in vivo functional properties of the regenerated rat bladder. Urol Res 1999; 27: 206. Chun SY, Lim GJ, Kwon TG, Kwak EK, Kim BW, Atala A et al: Identification and characterization of bioactive factors in bladder submucosa matrix. Biomaterials 2007; 28: 4251. Feil G, Christ-Adler M, Maurer S, Corvin S, Rennekampff HO, Krug J et al: Investigations of urothelial cells seeded on commercially available small intestine submucosa. Eur Urol 2006; 50: 1330.