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Keywords:

  • Bladder;
  • transplantation;
  • homologous;
  • graft survival;
  • canine model

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Objective To determine the functional potential and antigenicity of the homologous bladder acellular matrix graft (BAMG) in a dog model.

Materials and methods Seven mongrel dogs underwent partial cystectomy (20–50%) and grafting with an equal-sized BAMG; two control animals underwent partial cystectomy (40%) only. The dogs were killed after 30 (one), 120 (one) and 210 days (five dogs). Blood samples were obtained before and at 1, 2, 4, 7, 14, 30, 90 and 210 days after surgery. The dogs underwent cystography, intravenous pyelography and ultrasonography before and after surgery, and on the day they were killed, with cystoscopy carried out just before death. The grafted tissue was assessed using routine and immunohistochemical techniques.

Results All the dogs survived surgery; a complete blood cell count, chemical panel and white blood cell count showed no significant difference between the experimental and control animals. Cystography, cystoscopy and ultrasonography revealed no pathological changes in the upper urinary tract. After 7 months, the mean bladder capacity in the augmented dogs was significantly higher (P = 0.035) than in the controls (264 vs 172 mL). Histological evaluation showed an invasion of all bladder wall components during the first month; at 7 months, the morphological examination showed essentially complete regeneration.

Conclusion In this dog model, the potential of the BAMG as a bladder augmentation graft was confirmed, having minimal antigenicity with maximal acceptance. The reconstructed bladder matched the morphological and functional properties of the normal bladder.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

In the search for a material for urinary bladder augmentation that can regenerate fully with no adverse side-effects, researchers have used several approaches over the last 90 years to avoid enterocystoplasty. Since Neuhof transplanted autologous fascia as an augmentation material in the urinary bladder in the dog [1], many investigators have sought to achieve their goal with a variety of synthetic and natural tissues. We adopted a different approach by producing a full acellular matrix graft for partial organ substitution [2], according to a method adapted from Meezan et al.[3]. This material showed potential for homologous and heterologous bladder augmentation, and for partial ureteric substitution in the rat model [4–7]. In both organs, regeneration was complete.

To investigate the potential of the bladder acellular matrix graft (BAMG) for clinical use, we studied the larger and more relevant canine model, focusing on the functional and immunological aspects of the graft.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Graft production

Fresh bladder segments from two partially cystectomized control dogs were placed in a Petri dish containing 200 mL of 10 mmol/L PBS (pH 7.0) and 0.1% sodium azide, and stirred for 10 h to produce partial cell lysis. The bladder segments were washed with 200 mL of PBS and treated with 200 mL of 1 mol/L sodium chloride containing 2000 Kunitz units of DNase (Sigma Chemical Co., St. Louis, MO) and stirred for 10 h. Lysis was then complete and all the intracellular components were released. The samples were treated with 100 mL of 4% desoxycholate containing 0.1% sodium azide, and stirred for 10 h to solubilize the lipid cell membrane and intracellular membrane lipids. This treatment was repeated twice. The resultant BAMG was washed five times with 100 mL PBS and stored in 10% neomycin sulphate at 4°C until grafted.

Experimental protocol

Nine healthy male mongrel dogs (two control and seven experimental) were used (one year old, body weight 20.4–29.6 kg). All were vaccinated against the common infectious diseases and the health status of each was verified by a veterinarian (general physical examination, neurological examination, urine analysis and aerobic bacteriological culture of a urine sample) before the experiment began. Cefoxitin (11–22 mg/kg) was administered before surgery and at 15, 24, 36 and 48 h afterwards.

Blood samples (for a complete blood cell count, chemical panel and white blood cell count) were obtained before and at 1, 2, 4, 7, 14, 30, 90 and 210 days after surgery. The urine was analysed before and 1 month after surgery, and at death. The bladder and the kidneys were assessed radiologically (intravenous pyelography, cystography) and by ultrasonography (Picker International, W3000, probe 3.5 MHz) before and 1 month after surgery, and on the day the dogs were killed. The bladder capacity was determined cystometrically before and 7 months after surgery; studies were repeated twice to verify the findings. The distance between the identification sutures (anterior to posterior, left to right; see below) was measured at a bladder pressure of 30 cmH2O, and the dogs underwent cystoscopy just before death.

For surgery, the dogs were sedated with acepromazine (0.05 mg/kg, intramuscular) and anaesthesia induced with ketamine (5 mg/kg, intravenous) and diazepam (0.25 mg/kg, intravenous). The animals were intubated and isoflurane (1–2%) used to maintain anaesthesia, with spontaneous breathing. Saline (10 mL/kg/h) was substituted through an intravenous catheter. The postoperative analgesic treatment included one subcutaneous injection with buprenorphine (0.01–0.02 mg/kg) and oxymorphone (0.1–0.2 mg/kg intravenous or intramuscular) every 2–4 h for 2 days.

The bladder was emptied through an 8 F single-use catheter (CR Bard Inc., Covington, GA) and exposed through a midline incision. The control animals underwent a 40–50% cystectomy; in the experimental animals, the bladder was filled through the catheter with sterile saline, fixed at 30 cm above bladder level, and 20–50% of the bladder removed (20% in two, 30–40% in three and 50% in two dogs). A 10 F balloon catheter (CR Bard Inc) inflated with 50 mL saline was placed in the remnant bladder to maintain an appropriate shape for the correct adaptation of the BAMG to the host. The catheter was removed before closure was completed.

In the experimental dogs, a BAMG about 1.5 times larger than the resected area was fixed in place using a double-row of sutures—5/0 plain catgut in the inner circle and 2/0 chromic catgut on the outside ( Fig. 1). In addition, four transmural sutures of nonabsorbable Dermalon 2/0 were placed to identify the matrix borders (anterior, posterior, left and right). The distance between the identification sutures was measured at a bladder pressure of 30 cmH2O. In four dogs 20–40% of the ventral bladder was augmented and in three 30–50% of the bladder dome. The augmented bladder was not covered with additional tissue such as omentum or perivesical fat. An 8 F single-use catheter was inserted into the bladder and sutured to the glans penis for postoperative drainage. The bladder was filled with 50 mL saline to test for leakage. When closure was satisfactory the abdominal wall and the skin were closed. Urine was diverted for 6 days.

image

Figure 1. Six stages in bladder augmentation with the BAMG: (a) normal bladder, maximally distended; (b) empty bladder with the BAMG placed over an inverted cup; (c) hemicystectomy of the dome; (d) coaptation of the graft to the urothelium of the bladder; (e) grafted bladder; (f) appearance after 7 months (black arrows indicate sutures identifying the BAMG margins).

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Before death the animals were anaesthetized as described, the bladder exposed through a midline incision and needles placed through the bladder wall where the identification sutures were found; the distances were again measured. For cystoscopy, the cystoscope was inserted through a perineal incision of the urethra. When all the examinations were completed, the bladder was excised, distended over a balloon catheter filled with 30 mL saline and processed for further examination.

Staining

The specimens were fixed in the distended state described above for 24 h, by immersion overnight in 10% buffered formalin. After dehydration in graded ethanol solutions, clearing in Histoclear and embedding in paraffin, sections of 5 mm were cut and air-dried onto precoated glass slides. They were stained with trichrome for muscle and collagen, haematoxylin and eosin (H&E) for nuclei, α-actin for smooth muscle, and protein gene product (PGP) 9.5 as a general marker for nerves.

The data are presented as the mean ( sem); Student’s t-test was used to compare means, with P < 0.05 considered to indicate statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

All the dogs survived surgery in both the control and experimental groups. The mean weight decreased from 22.15 to 21.70 (1.95) kg in the control group and increased from 24.59 to 24.83 (1.92) kg in the experimental group. The mean operative duration for the control animals was 1.25 (0.05) h and for the grafted dogs 3.30 (0.32) h.

Severe postoperative complications only occurred in the first experimental dog, which developed an intra-abdominal urinoma. This was treated by needle aspiration and prolonged catheterization (10 days). One control dog had an infected seroma. All other dogs recovered well from the procedure. Only minor adhesions were noted during exposure of the augmented bladder ( Fig. 1f). At 7 months the BAMG had maintained 70 (16.8)% of its initial size and the graft covered 105.2 (30.4)% of the resected area.

The mean ( sem, range) preoperative bladder capacity in the nine dogs was 244 (123.8, 85–445) mL; at 7 months it had decreased in the two controls to 171.5 (28.5, 143–200) mL and in the five augmented dogs it had increased to 263.6 (103.7, 95–440) mL, a statistically significant difference (P = 0.035).

Preoperatively the urine was sterile; the analysis at one month showed a urinary infection in six dogs (five study and one control). At death, four dogs (three study and one control) showed significant bacterial contamination. The preoperative and final complete blood cell count was not significantly different between the experimental and control animals. Creatinine was in the reference range (4–18 mg/L) in all animals, except the dog that had developed an abdominal urinoma, although this returned to normal and remained so until death. The white blood cell count (6–17 × 103/mL) exceeded the upper reference level for 1 day in the controls and for 2 days in the experimental animals. The development of neutrophil segment cells (reference 60–77%) was similar. In contrast, lymphocytes (reference 12–30%) decreased for the first 2 days after surgery and reference range on the fourth day. Monocytes (reference 3–10%) decreased for the first 2 days and then exceeded the reference range for the next 4 days in the augmented animals and for 14 days in the controls.

Cystography before, immediately after surgery and at 7 months ( Fig. 2) showed integration of the BAMG into the host bladder. While the normal dog bladder is oval, the cystographic appearance immediately after surgery was more mushroom-shaped. At 7 months, the bladder was smooth and round, closely resembling the initial state. Ultrasonography showed no pathological changes in the upper urinary tract. At 7 months the bladder wall had a similar morphological appearance to its preoperative state and the wall thickness had recovered. In addition, there were no signs of scar formation, with only a slight heterogeneity apparent at the dome.

image

Figure 2. Cystogram from an experimental dog with half the dome augmented: (a) before surgery; (b) directly after surgery (note the mushroom-like shape of the expanded BAMG); (c) at 7 months after surgery (note the smooth and round configuration closely resembling the preoperative state).

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Cystoscopy after 7 months in the grafted dogs showed an almost perfect inner lumen. There were no signs of hyperplasia, tumour or scarring at the graft margins in any animal ( Fig. 3a,b). The only notable area was in the centre of the graft, where there was a less vascularized, small, irregular spot (≈5 mm in diameter) covered with urothelium ( Fig. 3c).

image

Figure 3. Cystoscopy in a BAMG-augmented dog 7 months after surgery. The broken white circles indicate the area enlarged in the succeeding image. (a) The black arrowheads mark the identification sutures for the matrix margins. (b) The broken black line marks the graft, with the graft margins on the right. (c) The centre of the graft appears slightly irregular and less vascularized.

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Histology

To verify the effectiveness of the BAMG production process, and consequently the acellularity of the graft, histological sections were assessed by light microscopy. These showed an intact fibre framework with no evidence of remaining nuclei. The structure, density and thickness of the collagen differed from the luminal side to the outside ( Fig. 4). The former urothelial and muscularis mucosa area consisted mostly of small and tightly packed fibres; in contrast, the detrusor muscle zone contained thicker and loosely oriented collagen.

image

Figure 4. BAMG stained with trichrome, H&E, PGP 9.5 and α-actin (all × 400). Note the preserved layered structure, and the different fibre sizes and distribution towards the luminal side (black stars).

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At 1 month all bladder wall components were present in the BAMG. Only a few mononuclear cells were apparent. The whole inner surface of the graft was covered by urothelium and there was pronounced capillary infiltration in the entire matrix. Muscle invaded from the border zone and even small muscle bundles formed ( Fig. 5). Nerves were detectable not only close to the suture line but also submucosally ( Fig. 6).

image

Figure 5. An overview of the right side of the grafted area at one month (arrows indicate the margin sutures). The magnified section (× 200, α-actin) shows a small muscle bundle (black arrowhead) with some remaining connective tissue. The star indicates a vessel.

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image

Figure 6. An overview of the right side of the grafted area at 1 month (arrows indicate the margin sutures). The magnified section (× 400, PGP 9.5) shows small nerves (black arrowheads) close to the urothelium (black star).

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After 4 months, all bladder wall components were present throughout the entire graft; there was an increase in amount and size of smooth muscle. In addition, an organized muscularis mucosa ( Fig. 7) and a well-defined serosa were apparent ( Fig. 8).

image

Figure 7. An overview of the grafted area at 4 months (arrows indicate the margin sutures). The magnified section (× 200, α-actin) shows the muscularis mucosa of the host bladder wall (left) and of the middle of the graft (right). Note the similar appearance of the layered structure and distribution.

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image

Figure 8. An overview of the grafted area at 4 months (arrows indicate the margin sutures). The magnified sections (× 40 and × 200, trichrome) show the centre of the graft (left) and the serosa (right) covering the outside of the BAMG.

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At 7 months, there were no changes in the urothelial lining and especially no signs of hyperplasia. The regeneration process was nearly complete and only a small part of the original graft was identifiable. Nerve fibres were present with a distribution and size similar to the host’s ( Fig. 9). In Fig. 10, muscle bundles from the centre of the graft are imaged both horizontally and vertically to show that there was slightly more collagen in the regeneration area than in the more highly developed muscle structures toward the host bladder. In the control animals, there was slight bladder-wall hypertrophy after 7 months; in the area of the cystotomy there was considerable fibrotic tissue.

image

Figure 9. An overview of the grafted area at 7 months (arrows indicate the margin sutures). The magnified sections (×100 and × 400, PGP 9.5) show (left) a muscle bundle with a normal neural distribution of tiny branches in the connective tissue and (right) the small submucosal nerve fibres.

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image

Figure 10. An overview of the grafted area at 7 months (arrows indicate the margin sutures). The magnified sections (× 100 and × 200, trichrome) show the central part of the graft, with the enlarged area showing that there was more collagen inside these bundles than in the host muscles.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

The goal of bladder augmentation is reinstate, with no side-effects, the ability to store urine and to evacuate it under low pressure [8, 9]. Promising results have been reported by Atala [10], using a polymer scaffold seeded with autologous smooth muscle and urothelial cells, and by Kropp [11], working with intestinal submucosa from the pig. Recent studies emphasize that connective tissue, and specifically the collagen content and ratio, are important in the pathogenesis of a compliant and contractile urinary bladder [12–14]. Hypothesizing that the cells (muscle, urothelium and nerve) remain capable of faithful regeneration, we adapted a method that retains this collagen mesh from the desired tissue to provide a scaffold for host tissue regeneration [2, 15]. This acellular matrix graft was used successfully for homologous and heterologous bladder augmentation, and for homologous partial ureteric replacement in the rat [2, 4, 6, 7].

The present study was designed to confirm the promising results from the rat model, and to focus on the functional and immunological aspects in the more relevant dog model. In contrast to the graft preparation method described previously, the bladder mucosa was not scraped off in an attempt to reduce the mechanical stress on the scaffold structure ( Fig. 4), which is considered important for bladder reconstruction and compliance [13].

The present study showed that the BAMG can be implanted safely into the dog bladder with no graft rejection or upper urinary tract complications (dilation, fistulae, calculi formation). The result was a morphologically similar reconstructed bladder wall that stored significantly more urine than in the control dogs. All additional clinical examinations (ultrasonography, cystography and cystoscopy) confirmed the promising outcome of bladder augmentation with the BAMG. In contrast, the outcome in the control animals was poor; as others have reported, scarring in the area of the cystotomy and the rigidity of fibrotic tissue are probably the cause [16, 17].

The matrix-grafted area was 30% smaller than the original implant, but covered 105% of the resected area; in the absence of fibrotic tissue, this was probably because the production process removed all contractile elements from the organ [13]. As a result, the BAMG assumes a greater area. We propose that, with the ingrowth of contractile smooth muscle, the framework is reduced to its original size [18].

The differing blood cell counts between the experimental and control dogs (which were not statistically significant) can be explained by the prolonged duration of surgery and catheterization in the former [19–21]. No further clinical signs of rejection (e.g. loss of weight or appetite, fever) occurred, supporting the potential of the BAMG as a biomaterial with only marginal antigenicity.

Histologically, the BAMG-regenerated bladders re-sembled normal bladder wall, in that all four layers (mucosa, muscularis mucosa, detrusor muscle and serosa) were present. In addition, the presence of PGP 9.5-positive nerve fibres throughout the entire graft indicated re-innervation of the reconstructed bladder wall.

The integration progress at 1, 4 and 7 months showed, as in our rat study, the ingrowth of the host detrusor muscle from the edges of the graft. Three mechanisms have been proposed to be responsible for smooth muscle regeneration in the BAMG: (i) migration of neighbouring mature cells; (ii) de-differentiation, migration and re-differentiation of mature cells; and (iii) infiltration of myofibroblasts with subsequent differentiation into smooth muscle cells [22]. The central hypercollagenous area in the graft showed incomplete integration of the BAMG segments ( Fig 3C and Fig. 6). In our rat model these zones were also seen in the earlier states and vanished during completion of the regeneration process [2].

Augmentation cystoplasty with the homologous BAMG is a simple and inexpensive method that provides regeneration of the detrusor. Bladders thus regenerated have histological evidence of innervation that is similar to that in normal tissue. This allows the graft to work in coordination with the host bladder components to generate adequate filling capacity and an intravesical pressure capable of sustained voiding.

Encouraged by these experimental studies and the regenerative potential of the BAMG, we are confident that a clinical application can be attempted. Accordingly, we have submitted a request for approval of a human study; with an appropriate selection of candidates, we hope to confirm the successful use of the BAMG in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Supported in part by the Deutsche Forschungs-gemeinschaft (Grants Pi 272/1–2, Pr 478/1–2) and the United States Department of Health and Human Services (NIH grant RO1 NS 18029)

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors
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    Probst M, Carrier S, Dahiya R, Tanagho EA. Reproduction of functional smooth muscle tissue and partial bladder replacement. Br J Urol 1997; 79: 505 15
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    Piechota HJ, Dahms S, Lue TF, Dahiya R, Nunes LS, Tanagho EA. In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J Urol 1998; 159: 1717 24
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    Dahms SE, Piechota HJ, Hohenfellner M, Gleason CA, Dahiya R, Tanagho EA. Bladder acellular matrix graft in rats: its neurophysiologic properties and mRNA expression of growth factors TGF-alpha and TGF-beta. Neurourol Urodynam 1998; 17: 37 54
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    Chang SL, Howard PS, Koo HP, Macarak EJ. Role of type III collagen in bladder filling. Neurourol Urodynam 1998; 17: 135 45
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    Dahms SE, Piechota HJ, Dahiya R, Lue TF, Tanagho EA. Composition and biomechanical properties of the bladder acellular matrix graft: comparative analysis in rat, pig and human. Br J Urol 1998; 82: 411 9
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    Kropp BP, Rippy MK, Badylak SF 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 104
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    Gasser TC, Larsen EH, England DM et al. Urinary bladder reformation: regeneration or dilatation? Neurourol Urodynam 1987; 6: 129
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    Piechota HJ, Gleason CA, Dahms SE et al. Bladder acellular matrix graft: In vivo functional properties of the regenerated rat bladder. Urol Res 1999;in press
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Authors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

M. Probst, MD, currently Universität Frankfurt, Klinik fürUrologie.

H.J. Piechota, MD, currently Universität Münster, Klinik fürUrologie.

R. Dahiya, PhD, Associate Adjunct Professor.

E.A. Tanagho, MD, Professor.