key: cord-0851337-vkw9dpd9
authors: Dahms, Stefan E.; Piechota, Hans J.; Nunes, Lora; Dahiya, Rajvir; Lue, Tom F.; Tanagho, Emil A.
title: Free ureteral replacement in rats: regeneration of ureteral wall components in the acellular matrix graft
date: 1997-11-30
journal: Urology
DOI: 10.1016/s0090-4295(97)00391-9
sha: e8783fc13569f93fe28583406ca08bfdbc9ec2fb
doc_id: 851337
cord_uid: vkw9dpd9

Abstract Objectives To evaluate ureteral replacement by a free homologous graft of acellular matrix in a rat model. Methods In 30 male Sprague-Dawley rats, a 0.3 to 0.8-cm midsegment of the left ureter was resected and replaced with an acellular matrix graft of equal length placed on a polyethylene stent. The animals were killed at varying intervals, and the grafted specimens were prepared for light and electron microscopy. Results In all animals, the acellular matrix graft remained in its original position without evidence of incrustation or infection, and histologic examination showed complete epithelialization and progressive infiltration by vessels. At 10 weeks, smooth muscle fibers were observed; at 12 weeks, nerve fibers were first detected; at 4 months, smooth muscle cells had assumed regular configuration. Conclusions The ureteral acellular matrix graft appears to promote the regeneration of all ureteral wall components.

with an acellular matrix graft of equal length placed on a polyethylene stent. The animals were killed at varying intervals, and the grafted specimens were prepared for light and electron microscopy.

In all animals, the acellular matrix graft remained in its original position without evidence of incrustation or infection, and histologic examination showed complete epithelialization and progressive infiltration by vessels. At 10 weeks, smooth muscle fibers were observed; at 12 weeks, nerve fibers were first detected; at 4 months, smooth muscle cells had assumed regular configuration. Conclusions.

The ureteral acellular matrix graft appears to promote the regeneration of all ureteral wall components. U reteral replacement has long been a subject of interest to researchers, and many surgical procedures and materials have been tried.le4

However, an entirely satisfactory method has yet to be found. Previous research has demonstrated that collagen-based materials, such as porcine small intestine submucosa (SIS), have the best potential regenerative capability.5 In this group of biomaterials, a new acellular matrix has recently been shown, in the bladder of the rat model, to serve as a scaffold consisting of collagen and elastin fibers for the ingrowth of all bladder wall components.6'7 In addition, the contractility of graftregenerated bladders has been observed in vivo (preliminary results). We designed the present study to determine whether this acellular matrix could be used as a free ureteral graft in a rat model. and the skin were closed. For microsurgery, an Olympus under a dissecting microscope, and collected. All specibinocular operating microscope (10X to 40X) was used. No mens were rinsed with saline solution to remove excess drugs were administered postoperatively.

intraluminal urine. Tissues were fixed at the time of accession and processed for light and transmission electron mi-LIGHTMICROSCOPICAND ULTRASTRUCTURAL croscopy.

Light Microscopy. Specimens were fixed in 10% buffered EVALUATION formalin for at least 24 hours. After dehydration in graded The animals were killed at the following times: 4 days (n ethanol solutions, the specimens were embedded in paraffin, = 3), 3 weeks (n = 5), 6 weeks (n = 31, 10 weeks (n = sectioned (5 pm), and stained with trichrome for collagen 7), 3 months (n = 5), and 4 months (n = 5). Host ureter and smooth muscle, hematoxylin and eosin (H Q E) for nuand graft were identified, freed from the surrounding tissue clei, alpha-actin for smooth muscle, and protein gene prod- 

Of the 30 animals, 2 died 6 weeks after surgery from a coronavirus infection associated with severe respiratory tract obstruction and were not used for further microscopic evaluation. In contrast to our reported experience with the matrix grafted to the bladder,7 stone formation was not observed in either the upper or lower urinary tract.

Light microscopy of the pure ureteral matrix demonstrated the effectiveness of the matrix preparation process. The acellularity of the graft as an intact framework consisting of elastin and collagen fibers was apparent (Fig. 3A) . Scanning electron microscopy showed the intact nature of the ure- Step-by-step regeneration of ureteral wall components (Masson's trichrome stain, original magnification x200). After infiltration of erythrocytes and mononuclear ceils (arrow) (A), urothelium (arrow) begins to develop in the first weeks (B). At 10 weeks (C), the first few muscle fibers are seen (arrow), and after 4 months (D), a complete regeneration of urothelium and muscularization can be observed (arrow marks borderline (suture] between the matrix graft [left] and the host ureter [right]) (U-AMG, ureteral acellular matrix graft).

teral matrix surface and confirmed the scaffoldlike structure of the graft without evidence of cellular elements (Fig. 3B, C) .

Moderate adhesions were noted to the surrounding retroperitoneal fat. The graft remained in its original position in all animals, without evidence of incrustation or infection (Fig. 4) , whereas the polyethylene tube migrated to the distal ureter. For this reason, gross examination of all surgical specimens revealed varying degrees of hydroureteronephrosis to the level of the graft. There was no evidence of postoperative urinary leakage in any of the animals at death. Histologic examination of all graft-regenerated ureters showed step-by-step regeneration of urothelium and smooth muscle fibers (Fig. 5) . At 4 days, the graft showed an infiltration of erythrocytes and mononuclear cells, and the urothelial lining appeared to begin to bridge the matrix graft. At 10 weeks, the graft was composed of several layers of urothelium and some capillaries, and characteristic arrangements of smooth muscle fibers (Fig. 6) were first observed. The number of vascular elements (capillaries) had increased. At 12 weeks, nerve regeneration was first detected by PGP 95positive staining (Fig. 7) . At 4 months, neomuscularization was well developed. The months p.0.) smooth muscle cells were arranged in parallel rows in the longitudinal direction. The thickness of these muscle bundles seemed to decrease in the central part of the graft. There was no sign of degenerative change such as calcification or necrosis of the smooth muscle layer. The urothelial lining, differentiated muscularization, and surrounding fibrous adventitia appeared qualitatively similar to normal ureteral wall components (see below). In contrast, we noted that the number of nerve fibers was less than in the normal ureter.

Smooth muscle regeneration in the grafted ureteral matrix was confirmed by transmission electron microscopy (Fig. 8) . At 3 months, specimens demonstrated a lower density of myofilaments in the graft than in the normal rat ureter. At 4 months, the number of myofilaments was significantly increased. These observations corresponded to the light microscopic findings at the same time. Nerve regeneration was confirmed in 4-month specimens studied by electron microscopy (Fig. 9) . Although the number of nerve fibers in the matrix graft was notably less than in the normal ureter, their morphologic characteristics were similar.

COMMENT Ureteral replacement has long been a challenge for urologists.

In other specialties, the success of acellular matrix grafts for organ substitution has been reported (eg, replacement of heart valves, coronary artery bypass, and, in particular, skin).'l-14 Although the concept of free grafts to treat ureteral diseases is not new, none of the materials previously reported has been able to satisfy all the criteria for the ideal substitute (Table I) .15-24 In the present study, complete epithelialization, angiogenesis, and regeneration of smooth muscle fibers and nerves were observed with no signs of rejection.

The reasons for the improved acceptance of the ureteral acellular matrix graft over that of other free ureteral transplants are still unknown. In accordance with our previous experience, we used extremely fine nonabsorbable suture material to minimize immediate postoperative inflammatory reactions, which can result in incrustation or stone formation.

Rapid epithelialization, progressive A. :ively (C : and : ureter than neoangiogenesis, and continuous smooth muscle regeneration, apparently arising from the adjacent edges of the host ureter, are possible factors responsible. Expression of growth factors seems to play a key role in the mechanisms controlling epithelialization.25

In our research, studies of transforming growth factor (TGF) expression in the rat bladder acellular matrix graft showed variable induction of TGFa, TGFPr, TGFP2, and TGF& transcription, with prominent mRNA expression of TGFa and TGF& (preliminary results). In time, early smooth muscle cells matured into normal-appearing smooth muscle bundles. Because smooth muscle production has been re-UROLOGY 50 (51, 1997 ported from pericytes after capillary neovascularization,26 we hypothesize on the basis of our studies that the source of smooth muscle fibers in the acellular matrix graft is the adjacent edges of the host ureter. Regulating mechanisms of smooth muscle differentation are poorly understood. Besides growth factor expression (as just mentioned), epithelial-mesenchymal interactions are thought to be important for smooth muscle regeneration. In 1961, Taderera2' demonstrated that the absence of lung epithelium causes failure of both smooth muscle and cartilage differentation.

Consistent with these results, Baskin et aL2* reported in 1996 that intact bladder as well as isolated blad- der mesenchyme recombined with bladder urothelium from rat fetuses, when grafted under the renal capsule of adult rats, demonstrated expression of smooth muscle differentiation; however, grafts of bladder mesenchyme alone failed to induce smooth muscle differentiation. Therefore, early epithelialization in the graft may be highly important for smooth muscle differentation.

The observation that the overall (PGP 95positive) density of the reinnervation of the ureteral acellular matrix graft was significantly lower than that of the normal ureter may indicate that cellderived factors are needed to achieve normal lev- A similar experience has been described by Gavazzi et a1.29 They showed that, after grafting of frozen and thawed acellular cerebral blood vessels in oculo, the reinnervation of the graft was less than that of the control, and PGP 95positive nerves appeared less dense on the transplants as well. However, in the present study regeneration of nerves was confirmed by light and electron microscopy and may increase over a longer period of time to achieve functional capacities.

In conclusion, the ureteral acellular matrix graft appears to promote the regeneration of all ureteral wall components.

These results may indicate that the graft has a potential for functional neomuscularization that will result in its maintenance as a physiologic ureteral wall. Tube migration with consequent obstruction at the level of the stent, causing hydroureteronephrosis, was not avoidable. It would thus be reasonable to undertake further study in a larger animal model. Detailed functional and molecular biologic experiments are needed to evaluate whether the ureteral acellular matrix graft as a ureteral substitute can conduct peristaltic activity in coordination with the host components to preserve the functional integrity of the renal parenchyma. The findings of regenerated ureteral wall components are encouraging and support the clinical potential of the acellular matrix graft in genitourinary tract reconstruction.

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