FIBRIN GLUE AS A DELIVERY VEHICLE FOR AUTOLOGOUS UROTHELIAL CELL TRANSPLANTATION ONTO A PREFABRICATED POUCH

0022-5347/98/1602-0583$03.00/0 THEJOURNAL OF UROLOGY Copyright 0 1998 by AMERICANUROLOGICAL ASSOCIATION,INC

Vol. 160,583-586, August 1998 Printed in U.S.A.

FIBRIN GLUE AS A DELIVERY VEHICLE FOR AUTOLOGOUS UROTHELIAL CELL TRANSPLANTATION ONTO A PREFABRICATED POUCH GOTTFRIED WECHSELBERGER,* THOMAS SCHOELLER, ARNULF STENZL, MILOMIR NINKOVIC, SEAN LILLE AND ROBERT C. RUSSELL From the Department of Plastic and Reconstructive Surgery and the Department of Urology, Leopold-Franzens Uniuersity, Innsbruck, Austria and the Institute for Plastic and Reconstructive Surgery, Department of Surgery, Southern Illinois University School of Medicine, Springfield, Illinois

ABSTRACT

Purpose: The purpose of the study was to investigate how in vivo expanded urothelium can be transplanted onto a prefabricated capsule-pouch for urinary reconstruction. Materials a n d Methods: Urothelial cells from 40 rats were harvested for culture. A tissue expander was used to induce a capsule-pouch on which the cell culture were reimplanted ten days later. As delivery vehicle we compared standard culture media and fibrin glue. Results: The histology demonstrated viable, multilayered clusters of urothelium cells only in the group using t h e fibrin glue delivery vehicle. Conclusion: We conclude that cultured urothelial cells can be successfully reimplanted onto a prefabricated capsule-pouch via fibrin glue, showing potential for urinary reconstructions. KEY WORDS: urinary reconstruction, tissue engineering, flap prefabrication, autologous cell culture, fibrin glue

Due to the limited supply of urothelium, urinary tract reconstruction using bowel, skin or other biological or synthetic materials has been employed. These materials, however, cannot always satisfactorily replace urothelium. Metabolic abnormalities, infection, perforation, stone formation, and increased mucus production may occur. In addition, an increased rate of diverticular formation and malignancy have been associated with intestinal segments incorporated into the urinary tract.13 An ideal solution would be to use a de novo prefabricated tissue that is well-vascularized, transferable on a single pedicle and lined with urothelium for urinary reconstructions. Human urothelial cells have been successfully cultured from tissue explants to produce enough cells for reimplantation to engineer functional urinary t i ~ s u e .However, ~ few reports in the literature address reimplantation of urothelial cell cultures.sg A proven and successful graft take for reconstructive purposes has not yet been described because simple reimplantation into the organism and integration through revascularisation as used in skin grafts or ceratinocyte sheets has consistently failed with urothelial cells. Among other things the highly differentiated cell type and the specific membrane protein complexes may require a special delivery vehicle. Different delivery vehicles such as biodegradable polyglactin 910 scaffolds, coated and uncoated with collagen, and other biodegradable materials such as collagen sponge have been tried with limited success.6.lo,l1 Because cultured autologous keratinocytes have been successfully transplanted as single cells suspended in fibrin glue in bum patients, we hypothesized that fibrin glue could be successfully used as the delivery vehicle for transplanting urothelium cells in an immunologically intact animal model.12 To determine the effectiveness of fibrin glue as a delivery vehicle, we transplanted urothelial cells onto a prefabricated capsule-pouch created by a tissue expander since previous in

vivo studies demonstrated successful results of skin grafting on induced capsular tissue.13.14 MATERIALS AND METHODS

Animal model. 40 male Wistar rats (12-16 weeks old, 350-500 gm.) were obtained from Harlan Sprague Dawley Laboratories and housed in the animal facilities at SKJ School of Medicine. The animals were handled in accordance within the guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and all protocols were approved by the Southern Illinois School of Medicine Animal Care and Use Committee. Intraperitoneal pentobarbital at 45 mg./kg. was administered for anesthesia. All SINgical procedures were done under sterile conditions. A 3 cm. longitudinal skin incision was made in the lower abdomen to expose the bladder. A 0.5 by 0.5 cm. area from the dome of the bladder was excised for urothelial tissue culturing. The created bladder defect was closed in one layer using a running 8-0 nylon suture. A sterile spherical tissue expander (2.7 cm. dia., Volume: 10 cc., CUI-Corp., Santa Barbara, CA) was placed underneath the right lateral abdominal skin and epigastric vessels through the same skin incision to induce a capsule formation for the later capsule-pouch. The implant was secured by suturing the subcutaneous tissue to the muscle with absorbable suture. The filler port was advanced subcutaneously and secured between the shoulder blades. The expander was filled intra-operatively and on days 2,4,6 and 8 postoperatively with 2 ml. of saline solution to reach the final volume of 10 ml. After 10 days, all animals were reanesthetized and a second laparotomy was performed. To compare the delivery vehicles we divided our animals into two groups. In group I (n = 20) the cultured cells were suspended and transferred in Dulbecco’s modified Eagle medium (DMEM), (Gibco, Grand Island, NY) and in group I1 (n = 20) we used fibrin glue (Immuno AG, Vienna, Austria) as the delivery vehicle. In each animal the capsule was incised, and cultured urothelial cells in one of the two suspensions were injected in the space between the capsule and the Accepted for publication February 20, 1998. in its appropriate group. The capsules and skin * Re uests for reprints: Univ.-Klinik fur Plastisehe und Wieder- expander were then closed using sutures. herstefiungchirurgie, Anichstrasse 35, A-6020 Innsbruck, Austria. 583

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One and four weeks after urothelial cell grafting, 10 rats Mannheim, Indianapolis, IN) to detect the reimplanted were removed from each group and histologic sections from xothelium on the capsule surface. The rat-specific streptathe capsule-pouch were taken for hematoxylin-eosin (H&E) vidin biotin (LSAB) 2 Kit (DAKO, Carpintene, CAI in which and immunohistochemical staining. The animals were eutha- a biotinylated secondary antibody reacts with several nized using a 1 ml. intracardiac dose of 26% sodium pento- peroxidase-conjugated streptavidin molecules was used for antibody detection.16 barbital. Cell preparations. Primary cell cultures were established RESULTS from fresh bladder tissue specimens from the rats. Urothelium tissue was carefully removed aseptically under a stereoConsistently in all 40 animals a highly vascularized capmicroscope using fine iridectomy scissors and forceps. Sec- sule in the shape of a hollow viscus was induced by the tissue tions of urothelium were washed two times in 0.02% expansion process (fig. 1). ethylenediaminetetraaceticacid (EDTA) (Sigma, St. Louis, Experimental groups MO) and then incubated at 37C for 1.5 hours with 0.25% Group Z (n = 20): Transplantation of cultured urothelial trypsin/O.02% EDTA solution to create a single-cell suspen- cells suspended in DMEM. Of the 20 rats that underwent sion. grafting of cultured urothelium cells via the standard culture Tissue debris was removed from the dish and the trypsin medium, none had evidence of viable urothelial epithelium was inactivated with fetal calf serum (FCS). The cell suspen- along the capsule surface documented by staining with H&E. sion was centrifuged for 10 minutes at 1000 rpm. The cell Immunohistochemical staining with AE1 & AE3 anticytokpellet was resuspended in Hanks Balanced Saline Solution eratin antibodies one and four weeks postoperatively was (Gibco, Grand Island, NY) and recentrifuged. Final cell sus- performed and could not identify any epithelial cells on the pensions containing urothelial cells were plated in Ham’s capsule surface. H&E staining revealed a mild inflammatory F-l2/DMEM. response consistent with a foreign-body reaction and dense The portion of bladder dissected yielded 7 X lo4 to 2 X lo5 fibrous tissue and vascular proliferation. cells counted by haemocytometer (Bright-line, Cambridge Group II (n = 20): Transplantation of cultured urothelial Instruments, Buffalo, NY, USA). The cells obtained from cells suspended in fibrin glue. All 20 rats had evidence of each sample were plated in one T25 flask with 5 X lo5 viable urothelial cells demonstrated by staining with H&E or irradiated 3T3 mouse fibroblasts (ATCC No. CCL92). 3T3 AE1 & AE3 anticytokeratin antibodies one and four weeks cells were maintained in DMEM with 10% FCS. The cultures postoperatively (figs. 2, 3).After one week several cells were were passaged when the cells were sub-confluent using starting to cluster on the capsule surface. The fibrin clot in 0.25% trypsin. To use as a feeder layer, the cells were which the cultures were embedded was already dissolved and trypsinized, resuspended in media, counted, and irradiated replaced by connective tissue. After four weeks the cells with 6000 rads from a 137Cs source. formed a more dense cluster, but did not show a confluent Cultures were incubated at 37C in 5% CO,. The medium urothelial cell lining along the capsule. As in group I, H&E was changed three times a week. The primary cultures be- staining showed a mild inflammatory response, dense fibrous came confluent after four to eight days then the cells were capsule tissue and vascular proliferation. The cell populastripped using the trypsin/EDTA solution and resuspended tions appeared to expand from a single layer to several layers in 0.6 ml. DMEM (n = 20) or in the thrombin component of of thickness with progressive cell organization following exfibrin glue in group I1 (n = 20). tended implantation times. At that time quantitation of cell recovery by cell counting using a haemocytometer indicated that this method resulted DISCUSSION in an average recovery of 2.6 X lo6 viable cells (5 X lo5-6 X Our study demonstrates that urothelial cell cultures can be lo6) per sample of bladder tissue (n = 40). Medium and reagem. Cell extraction washes contained 0.02% expanded ex vivo with an average expansion factor of 10, to EDTA and 0.25% trypsin (Gibco, Grand Island,Ny)/0.02% EDTA produce a sufficient quantity of cells for a successful autolosolution to create a singk-mll suspension for cell plating. The gous reimplantation onto a prefabricated capsule-pouch usincubation medium for mothelid cells contained Ham’s F-12 ing fibrin glue as the delivery vehicle. Cells pre-treated in medium/DMEM, (Gibco, Grand Island, NY) in a 1:l ratio sup- identical fashion to the fibrin glue group but with standard plemented with 10%fetal calfserum (Gibco, Grand Island, NY), culture media used as the delivery conduit failed to produce 10 nglml. cholera toxin (Sigma, St. Louis, MO), 0.4 uglml. an adherent and viable cell layer. A capsule-pouch was hydrocortisone (Sigma, St. Louis, MO), 5.0 uglml.insulin (Sigma, St. Louis, MO), 5.0 uglml. transferrin (Sigma, St. Louis, MO), 10 ngJml. epidermal growth factor (CollaborativeBiomedical), lOOU/ml. penicillin and 100 uglml. streptomycin solution (Sigma, St. Louis, MO), and 0.25 ugJml. Fungizone (Gibco, Grand Island, NY). The (human) freeze-dried sealer protein concentrate (provided by Immuno AG Vienna, Austria) contained 100to 130 mglml. total protein (70 to 110 mgJml. fibrinogen, 2 to 9 mglml. plasmafibronectin (CIG), 10 to 50 U/ml. Factor XIII, and 0.04 to 0.12 mglml. plasminogen), and was dissolved with a bovine aprotinin solution (3000 KIU/ml.). The thrombin component 500 IU/ml. was mixed with a 40 mmollml. calcium chloride solution and fibrinogen with 3000 KIE/ml. aprotinin. The urothelid cells were homogeneously suspended in the thrombinICaC1, solution which was then mixed with the fibrinogen solution to form a clot on the capsule-pouch surface. Histomorphologic studies. Samples were fmed with 10% formalin and routinely processed, embedded in parafin and cut vertically for hematoxylin-eosin (H&E) staining. Additionally, immunohistochemical staining specific for epithelial cells was performed on paraffin-embedded tissues with AE1 FIG. 1. Capsule-pouch formed under right lateral abdominal skin & AE3 anticytokeratin monoclonal antibodies’” (Boehringer using technique of capsule induction by tissue expander.

UROTHELIAL TISSUE ENGINEERING

FIG. 2. Clusters of urothelial cells four weeks after grafting on prefabricated capsule-pouch. H & E, reduced from X10.

formed using the technique of capsule induction by a tissue expander, a technique not uncommon in plastic surgery. The capsule induction technique as introduced provides several essential advantages for tissue engineering. The expander creates the shape of a spherical capsule-pouch and the induced foreign body reaction provokes a predictable neovascularisation in the capsule tissue around the silicon expander. By placing the expander in the vicinity of large vessels neovascularisation can be controlled and directed so that the later capsule-pouch will be supplied by a single vascular pedicle. Any pedicle suitable for a later transfer and which can be sacrificed without sequelae can be chosen for this prefabrication. Although the capsule contains mainly fibrous tissue it has been shown that in the initial stage of the foreign-body reaction a highly increased blood flow can be proven, which supports the graft take'3.14 provided a suitable cell delivery vehicle is used. The capsule surface serves as a basal lamina, which is essential for each epithelia development. Immunofluorescence analysis of connective tissue components in such a fibrous capsule demonstrated all essential extracellular matrix molecules of a basement membrane such as laminin, fibronectin, collagens, procollagens, and associated collagen molecules.17 At epithelial-stromal boundaries, this newly induced capsule serves as a specialized area of extracellular matrix molecules for cell attachment. Cells in epithelia are held together tenaciously by a series of lateral junctions, they display a n apical-basal polarity, and their basal surface rests on an organized extracellular ma-

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FIG.3. AE1 and AE3 staining of urothelial cell clusters four weeks after grafting. Reduced from x20.

trix, the collagen network of the capsule. The tissue organization and integrity is maintained by cell-cell and cell-matrix adhesive interactions, although the exact signal transduction pathways are still obscure.18 Sophisticated urinary reconstruction requires a neoreservoir with a n urothelial lining. In urinary tissue engineering we have first to overcome the specific problem of urothelial cell reimplantation. Only few reports in the literature describe reimplantation of urothelial cell cultures.s9 Because simple reimplantation into the organism and integration through revascularisation as used in skin grafts or ceratinocytes sheets has consistently failed, a successful graft take for urothelial cells to be used in reconstruction has not yet been described. The reasons for the failure are not clear; however, the highly differentiated cell type and the specific membrane protein complexes may require a special delivery vehicle. Such a delivery vehicle should contain several important features including high levels of biocompatibility and biodegradability, less cytotoxity, and a high affinity to bind to biological surfaces. Encouraged by several reports of autologous cell transplantations using fibrin glue as a successful vehicle for different tissue types we have compared two different vehicles: the simple standard culture medium DMEM, and fibrin glue. In all animals of the standard medium group the culture transfer constantly failed, whereas only the results of the fibrin glue group could demonstrate a successful urothelial culture reimplantation. Fibrin glue mainly contains fibronectin, a key protein in the extracellular matrix. It is well known that cellular growth and differentiation, in

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two-dimensional cell culture as well as i n the threedimensional space of the developing organism, requires the presence of a structured environment with which the cell can interact. This extracellular matrix is composed of fibrous macroproteins such as collagens, elastin, fibrillin, fibronectin (the main component of fibrin glue), laminin, and hydrophilic heteropolysaccharides. The cells interact with t h e matrix and communicate with each other through the ECM-molecules. Therefore the ECM influences t h e cell shape, fate, metabolism and behavior and is considered essential for tissue a n d organ development. As a delivery vehicle, fibronectin in the form of fibrin glue is obviously t h e structural constituent and regulator of cell behavior i n urothelial culture reimplantation. Anchorage of the transplanted cells to the extracellular matrix depends primarily on a group of surface receptors that a r e specialized to recognize and bind linkers such as fibronectin and laminin. Fibronectin binds to cell surface receptors and regulates cell attachment, cell shape, proliferation, migration a n d differentiation. Furthermore, these receptor sites bind growth factors and serve as co-receptors for growth factor receptor interactions.1S Our experiment could demonstrate t h a t these properties a r e essential in a successful reimplantation of urothelial cell culture. Fibrin glue helps attach t h e transplanted cells to the recipient bed, enhances the migration of growth factors and is a nutrient medium itself. These properties a r e important for reimplantation and might be t h e key to bridge t h e g a p during which the reimplanted cell is nourished by diffusion of extracellular fluid until revascularisation a n d definitive incorporation occur. Although transplanted as single cells, the cells spontaneously reoriented into multilayered structures and formed clusters of urothelial cells on the capsule surface. However, we were unable to produce a n evenly distributed urothelium lining. This may be attributed to the missing contact between the neoreservoir with the original urinary tract. Cell-to-cell communication may be required to reorient the epithelial cells to their original phenotype. Theoretical indications for this introduced tissue engineering technique a r e bladder augmentation, complicated hypospadia repair, and ureterallrenal pelvic reconstructions. I n conjunction with a functional muscle transfer, as already used for atonic bladders,19.20 it is possible that a n entire bladder replacement could be achieved.

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