Tunica Repair with Acellular Bladder Matrix Maintains Corporal Tissue Function

ORIGINAL ARTICLE Tunica repair with acellular bladder matrix maintains corporal tissue function

D Eberli, R Susaeta, JJ Yoo and A Atala

Department of Urology and Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA

Penile conditions, such as Peyronie’s disease or tumor resection may require surgical reconstruction of the tunica albuginea. Various materials have been proposed, as a biomaterial for tunica albuginea repair, however, little functional data are available. We examined the applicability and functional outcome of a collagen-based matrix derived from the bladder (acellular bladder matrix (ABM)), as a biomaterial for tunica repair. Biocompatibility testing was performed on the matrix, which included mitochondrial metabolic activity, cell viability and apoptosis. Approximately 50% of the dorsal penile tunica albuginea was replaced with the collagen-based matrix patch after surgical removal in 24 New Zealand White rabbits. Cavernosometry and cavernosography were performed. The animals were killed 1, 2 and 3 months after surgery for analyses. The matrix showed excellent biocompatibility. All animals implanted with the matrix survived without any noticeable untoward effects. There was no evidence of inflammation or infection at the time of retrieval. Cavernosometry of the implanted animals demonstrated normal intracavernosal pressures with visual erections. Cavernosography of the repaired corpora showed a normal anatomical configuration. Biomechanical analysis of the retrieved matrices demonstrated similar tensile strengths as native tunica. Histologically, there was only a minimal inflammatory response, which gradually decreased over time. These results show that ABM is biocompatible, durable and effective when used as a tunica substitute. The matrix may be useful as an off-the-shelf biomaterial for patients requiring tunica albuginea repair. International Journal of Impotence Research (2007) 19, 602–609; doi:10.1038/sj.ijir.3901587; published online 2 August 2007

Keywords: penile reconstruction; tunica albuginea; acellular bladder matrix

Introduction However, some of these materials have been asso- ciated with donor site morbidity, extensive inflam- Penile conditions, such as Peyronie’s disease, mation resulting in fibrosis and erectile dysfunction fibrosis, tumor resection or augmentation surgery, due to venous leak.1,4,5 often require reconstruction of the tunica albuginea. An ideal biomaterial for tunica repair should be Surgical reconstruction of these conditions has been biocompatible, elicit minimal inflammation, pro- a challenge due to the limited availability of tissues mote new tissue formation, and be able to withstand suitable for tunica albuginea replacement. A variety adequate tensile forces for maintaining structural of materials has been investigated over the years, integrity.6 Acellular bladder matrix (ABM) is a which include the skin, muscular aponeurosis, collagen-based biomaterial processed from the blad- tunica vaginalis, human dura mater, pericardium, der submucosa, known as the lamina propria. The polyethylene, temporalis fascia-free graft, vein patch matrix has shown to be biocompatible, is processed graft, fascia lata graft, Dacron and Goretex.1–3 easily, and has good characteristics for tissue handling. This matrix has been used experimentally and clinically for several applications, including urethra and bladder reconstruction procedures.7–9 Correspondence: Dr A Atala, Wake Forest University In this study, we investigated whether ABM School of Medicine, Medical Center Boulevard, Winston- Salem, NC 27157 USA. possesses the ideal material characteristics required E-mail: [email protected] for penile reconstruction. We examined the applic- Received 13 February 2007; revised 11 May 2007; ability and functional outcome of ABM for tunica accepted 3 June 2007; published online 2 August 2007 repair. Tunica repair with ABM maintains corporal tissue function D Eberli et al 603 Materials and methods MTT (mitochondrial metabolic activity) The mitochondrial metabolic activity of the bioma- Study design terial was determined by the MTT (3-(4,5-di- Acellular bladder matrices, processed from porcine methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) bladders, were examined for biocompatibility using assay using the direct contact method.13 The cell mitochondrial metabolic activity, cell viability and layers were rinsed with PBS, and 125 mlofMTT apoptosis assays. The matrix was tested in 24 New (Sigma, Saint Louis, MI, USA, 1 mg/ml MTT in PBS Zealand White rabbits weighing approximately containing 1 g/l glucose) was added to each well. 3.5 kg (Millbrook Farms, Concord, MA, USA). After After a 3-h incubation period at 371C, the MTT performing functional assessments, the animals solution was removed, and the insoluble Formazan were killed after 1, 2 and 3 months. The tissue crystals formed were dissolved using 100 mlof samples were retrieved and analyzed histologically, dimethylsulfoxide (Sigma). The absorbance was ultrastructurally and biomechanically. The findings measured at 540 nm using a 96-well plate spectro- were compared to normal controls (four animals). photometer (ELX 800, BioTek Inc., Winooski, VN, USA). The intensity of the blue color is directly proportional to the metabolic activity of the cells and inversely proportional to the toxicity of the material. Preparation of the ABM Porcine bladders, obtained from IBP Inc. (Logan- sport, IN, USA), were processed at ACell, Inc. (West Lafayette, IN, USA). Briefly, each bladder was rinsed Cell viability assays (Neutral Red) with running tap water, and placed in a À201C The cytotoxic and bioactive effects of the biomater- freezer overnight. The bladder was opened and the ial in direct contact with the cells were tested with 14 muscle layer was excised with sharp scissors. The Neutral Red according to the reported standards. remaining tissue, consisting of the lamina propria or The cell layers were rinsed with PBS, and 100 mlof often referred as the bladder submucosa, was placed Neutral Red Solution (Sigma, 0.005% in culture in 0.9% normal saline. The bladders were disin- medium) was added into each well. After a 3-h fected in a solution consisting of peracetic acid, incubation at 371C, the Neutral Red solution was alcohol, and water for 2 h followed by repeated removed, and dye extraction was performed by cycles of sterile phosphate-buffered saline (PBS) adding 100 ml of 1% acetic acid in 50% ethanol water rinses. solution into each well. After placing the wells on a Cells within the ABM were removed using the cell platform shaker for 5 min, the absorbance was lyses technique described previously.10 Briefly, measured at 540 nm by using a 96-well plate bladder lamina propria was treated with distilled spectrophotometer. The intensity of the red color is water in a stirring flask at 41C for 1 day to lyse the directly proportional to the viability of the cells and cells in the tissue. Subsequently, the tissues were inversely proportional to the toxicity of the material. treated with 1% Triton X100 and 0.1% ammonium hydroxide in a stirring flask at 41C for 14 days. The collagen matrix was then rinsed with distilled water Apoptosis index at 41C for 2 days followed by a treatment with PBS The quantification of apoptosis was measured with for 24 h. The bladder matrix was lyophilized, an in situ detection of DNA fragmentation in the cell trimmed to size, packaged, E-beam sterilized stored cultures using a commercially available kit (Titer in a desiccator until use. TACSs, Trevigen, Helgermann, MD, USA) according to the manufacturer’s instructions. Apoptosis was assessed after 3 days of cell-material contact Biomaterials testing using 96-well plates. No media change was per- Biocompatibility of the ABM was assessed using the formed. Unlabeled cells indicated the level of previously described methods, which include direct background labeling, and TACS-nuclease generated contact cell viability assay, mitochondrial metabolic DNA breaks served as a positive control. The results activity assay and quantification of apoptosis (n ¼ 8 were adjusted for cell density in the monolayer to per group per test).11 Primary mouse dermal fibro- take the high toxicity of latex into account. blasts were used for the biocompatibility testing. ABM fragments (0.03 cm2/well) were placed in the center of subconfluent fibroblast monolayers in 96- Anesthesia and surgery well plates, according to the established stan- All experimental procedures were reviewed and dards.12 Cells on plates not exposed to the bioma- approved by the Institutional Animal Care and Use terial served as a positive control and latex Committee, and were performed in compliance with fragments as a negative control. The material-cell the Animal Welfare Act and the Guide for the Care contact was maintained for 3 days at 371C and 5% and Use of Laboratory Animals. The animals were CO2, followed by each assay. sedated with an intramuscular injection of 10 mg/kg

International Journal of Impotence Research Tunica repair with ABM maintains corporal tissue function D Eberli et al 604 ketamine and an intravenous injection of 0.5 mg/ filling in both corpora and by determining the kg acepromazine. After an induction with propofol corporal anatomical contour. Special attention was (4–8 mg/kg, IV), the animals were intubated and the given to identify the contrast media passing under anesthesia was maintained with 2–3% isoflurane. the replaced tunica to the distal end of the penis. The animals were placed in the supine position. The The findings were compared to normal controls. area of the surgery was shaved and disinfected with iodine solution. After placing a tourniquet around the base of the Implant retrieval and gross examination penis, a longitudinal dorsal incision was made. The Immediately after euthanasia, the implant site was connective tissue was dissected and the tunica inspected. Parameters assessed for gross examina- albuginea was exposed. A defect sized 3.5 Â 7mm tion included the integrity, size and integration of was created on the tunica albuginea. The ABM was the biomaterial into surrounding connective tissue, soaked in saline for 5 min and trimmed to fit the fibrosis, infection and inflammatory response. The defect (3.5 Â 7 mm). The biomaterial was sutured to penis was surgically explored through a dorsal the tunica albuginea with 5–0 polydioxanone (PDS) incision. The site of the surgery was marked with running sutures (absorbable) to cover the defect. 4–0 non-absorbable sutures at each corner of the After allowing arterial inflow to the penis, the suture implants as a reference. The dimension of each line was confirmed for water tightness. The four biomaterial segment was measured and the average corners of the implanted biomaterial were marked change was expressed in percent. The size of the with 5–0 Prolene (non-absorbable) sutures. The biomaterials before surgery was defined as 100%. wound closure was performed in a running manner with subcutaneous 3–0 Vicryl (absorbable) and 4–0 PDS (absorbable) sutures for the skin. At the end of the procedure, the animals were extubated and Histology recovered. The retrieved tissue specimens were fixed in 10% neutral Formalin. All specimens were rinsed three times in PBS and placed in the tissue processor (Citadel 1001, Thermo-Shandon). Five micrometer Cavernosometry thick sections were obtained from the paraffin- Under general anesthesia, the penis was surgically embedded blocks (RM2145, Leica) followed by exposed dorsally, showing the site of penis repair hematoxylin and eosin, a staining method widely and the proximal corpora cavernosa. Two 26-gauge accepted for the assessment of local inflammatory venous catheters were placed into the proximal response. Elastin and collagen stains (Elastin-kit, corpora. Correct placement was confirmed by an Sigma) were performed. erection with saline injection. One catheter was To determine the density of cellular infiltration, connected to an infusion pump system with the the number of nuclei from eight randomly selected infusion rate of 0.7 ml of saline per minute. The high power fields were counted for each group and inflow alone did not lead to an erection. The second for each time point. catheter was connected to a pressure sensor (Urodyn 5001, Dantec, Denmark), measuring the intracavernosal pressure in real time.15 The measurements were recorded (Urodyn 5001, Dantec, Denmark). Scanning electron microscopy After confirming the recording quality, 5 mg papa- For ultrastructural analysis implants, obtained at verine was injected into the corpora. Continuous each time point, were fixed in a 2.5% glutaralde- pressure measurements were performed. The inflow hyde solution containing 0.085 M cacodylate buffer of saline was stopped when the pressure reached for 1 h. All samples were dehydrated through a over 250 cm H2O. A spontaneous natural erection graded series of ethanol and were eventually stored due to surgical manipulation was rated as the best at 41C. After a critical point drying and sputtering possible outcome. All findings were compared to using a gold platinum target, the samples were normal controls. analyzed using a scanning electron microscope (s.e.m., Leo 1450 VP) using a 500 Â , 1500 Â and 5000 Â magnification. Cavernosography A 26-gage venous catheter was placed into the proximal corpora. The correct placement was con- Biomechanical studies firmed by an erection with saline injection. Contrast Rectangular tissue strips measuring 16 mm Â 6mm media (Optiray 240, Malinckrodt Inc., St Louis, MO, were used. Tensile tests (Instron model 5544, MA, USA) was then injected into the corpora cavernosa USA) were performed by elongating the tissue strips using the established access. Radiography (Trex-net longitudinally at a speed of 0.05 mm/s with a HR, Littleton, MA, USA) assessed the degrees of preload of 0.2 N until failure. The grip-to-grip

International Journal of Impotence Research Tunica repair with ABM maintains corporal tissue function D Eberli et al 605 spacing was approximately 1 cm. All specimens (10072.9%, n ¼ 8). The assays showed complete were tested at room temperature and kept moist. cell viability for the ABM (10771.4%, n ¼ 8), when The maximum tensile stress and strain were compared to the positive control (Po0.020) (Fig- assessed. The results of the 0, 1, 2 and 3 months ure 1). The apoptotic activity of the fibroblasts time points were compared and analyzed (n ¼ 8). cultured in direct contact with the ABM for 3 days The tendenous portion of the native external was measured. The apoptotic activity of the cells in abdominal muscle served as a normal control. direct contact with the ABM was minimal (9.671.3%, n ¼ 6, Po0.001), when compared to the nuclease treated positive control (10070.3%, n ¼ 6). Latex significantly increased the level of Statistical analysis 7 The results of the MTT, Neutral Red assay and the apoptosis (39.3 6.8%, n ¼ 6, Po0.001). Untreated samples used as negative controls for background quantification of apoptosis are expressed as the 7 percentage of the corresponding positive controls. labeling were 2.2 0.7%. All presented data are expressed as averages and the corresponding standard error of the mean (s.e.m.). For statistical analysis, we used SPSS v11 (SPSS Gross examination Inc.). Outliers were defined as having a z-score The presented data are based on findings of 24 larger than þ 3.0 or smaller than À3.0 using rabbits. Two rabbits had to be replaced due to modified z-score (median of the absolute deviation). unrelated medical problems. Four rabbits were used One-way analysis of the variance and post hoc as normal controls. analysis (Bonferroni) were used for all analysis with The implants and the surrounding tissues were exception of the biomechanical testing were the inspected grossly after euthanasia. The implanted independent t-test comparing each time point with biomaterial was palpable in all cases. Macroscopically, native tissue was used. An alpha of P40.05 was there was no evidence of infection or gross fibrosis considered significant. and the biomaterial was seamlessly integrated into surrounding tunica albuginea. No significant changes in size were observed, when compared to 100% at the Results time of implantation. The size of the ABM was 96.3711.6, 96.377.6 and 97.4%716.1% after 1, 2 Biocompatibility testing and 3 months, respectively (n ¼ 8). The mitochondrial activity was significantly less (Po0.001) with latex (5.770.9%, n ¼ 8) when compared to the positive control (10072.8%, Cavernosography n ¼ 8). The assays showed a mitochondrial activity Cavernosography was performed in two animals per of 8871.9% (n ¼ 8) for the ABM, when compared to group in all time points. The radiography showed a the positive controls (Po0.001) (Figure 1). watertight repair without any evidence of leakage in Direct contact with latex induced significant all time points. The contrast media filled the entire decrease in cell viability (5.070.5%, n ¼ 8, corpora cavernosa from the glans to the proximal Po0.001) when compared to the positive controls two crura. Cavernosography of the corpora showed a

Figure 1 Biocompatibility testing: (a) direct mitochondrial metabolic activity (MTT) assay showed viability of 8871.9% (n ¼ 8, Po0.001) for the ABM and 5.770.9% (n ¼ 8, Po0.001) for latex, when compared to cells without treatment. (b) Cell Viability Assays using Neutral Red showed complete cell viability for the ABM (10771.4%, n ¼ 8, Po0.020), when compared to the positive controls (10072.9%, n ¼ 8). Cells in direct contact with latex significantly decreased viability to 5.070.5% (n ¼ 8, Po0.001). Latex, latex-treated cells, ABM, acellular bladder matrix, Control, untreated cells.

International Journal of Impotence Research Tunica repair with ABM maintains corporal tissue function D Eberli et al 606

Figure 2 Histomorphologic analysis. The cellular infiltration observed at 1 month gradually decreased over time. Infiltration of lymphocytes was predominant at 1 month, however, these cells were gradually replaced by fibroblasts. Tissue organization with directional alignment of fibroblasts was observed within the ABM by 3 months. (a) Native Tissue, (b) 1 month, (c) 2 months, (d) 3months. (hematoxylin and eosin, 200 Â ).

Figure 3 (Left) Cavernosography of the corpora showed a smooth contour surrounding the tunica albuginea, indicating the maintenance of structural integrity of the penis. The two needles indicate the margins of the implanted ABM. (Right) Cavernosometry of the implanted corpora shows that the intracavernosal pressure reached over 250 cm H2O and decreased slowly after the cessation of saline infusion, similar to normal controls. There was no occurrence of rupture or leakage during the testing. (a) Native, (b) ABM 3 months. Arrows are indicating time of papaverine injection.

smooth contoured tunica albuginia similar to native testing. None of the animals tested showed any penile controls, indicating the maintenance of structural shaft curvature grossly. One rabbit in the 2-month integrity of the penis (Figure 3). group and one rabbit in the 3-month group had a spontaneous erection after manipulation, which indicated normal cavernosal tissue function. Cavernosomety All animals tested showed a physiological response to papaverine with prompt erection after injection. The Ultrastructural analysis (s.e.m.) intracavernosal pressure reached over 250 cm H2O Scanning electron microscopy clearly showed the and decreased slowly after stopping the saline infu- integrity of the collagen matrix throughout all time sion, matching the findings of the normal controls points (Figure 4). There were no differences in the (Figure 3). No rupture or leakage occurred during the collagen structures at the different time points.

International Journal of Impotence Research Tunica repair with ABM maintains corporal tissue function D Eberli et al 607

Figure 4 Scanning electron microscopy shows the maintenance of the ultrastructural architecture similar to native tissue, consisting of organized dense collagen bundles, at all time points. Scale bar is 25 mm. (a) Native Tissue, (b) 1 month, (c) 2 months, (d) 3 months.

Biomechanical studies The biomechanical properties of the ABM were similar to the normal controls (Figure 5). We measured eight samples of all groups. The ABM showed a maximum tensile stress of 2.670.40 MPa before implantation, 1.970.27 MPa after 1 month, 2.470.22 MPa after 2 months and 3.070.29 MPa after 3 months, respectively. These results were comparable to the normal controls (3.670.69 MPa). One month after implantation the maximal strength of the biomaterial reached its weakest point (P ¼ 0.016). A trend towards improvement of strength over time could be observed. There was no significant difference in the maximum tensile stress (MPa) noted between the 0, 2 and 3 months time points and native tissue (P ¼ 0.196, 0.050, 0.295, respectively). The ABM showed a maximum tensile strain of 39.773.4% 7 Figure 5 Biomechanical testing: biomechanical studies of the before implantation, 43.6 2.8% after 1 month, ABM showed an average tensile stress of 2.670.40 MPa before 52.474.0% after 2 months (P ¼ 0.48) and 51.176.9% implantation, 1.970.27 MPa after 1 month (*P ¼ 0.016), after 3 months, respectively. These results were 2.470.22 MPa after 2 months and 3.070.29 MPa after 3 months. comparable to the normal controls (39.074.9%). Native tissue showed a maximal tensile stress of 3.670.69 MPa. There was no significant difference in the maximum tensile stress (MPa) noted between the 0, 2 and 3 months time points and native tissue (P ¼ 0.196, 0.050, 0.295, respectively). Sample size was n ¼ 8 per group. Histology The matrix structure was intact demonstrating longitudinal collagen bundles at all the three time calcification within the septa of the corpora caver- points. The amount of collagen did not change over nosa were found at all time points. Lymphoid the study period (Figure 2). The 1-month implants follicle formation was observed only in the 1-month exhibited a mixed cell infiltration, composed of implants. Generous angiogenesis with vascular lymphocytes, plasma cells, polymorphonuclear infiltration was observed throughout the implanted cells and foreign body granulomatous reactions with matrices at all time points. The cell density giant cells. Giant cells and sporadic dystrophic decreased significantly over time from 15874.4 at

International Journal of Impotence Research Tunica repair with ABM maintains corporal tissue function D Eberli et al 608 1 month to 12171.9 (P ¼ 0.024) at 2 months and to have evaluated corporal tissue function using 9373.4 (Po0.001) at 3 months. Further, a shift from cavernosography and cavernosometry at each time dominance of lymphocytes to dominance of spin- point. All animals tested had normal erectile dle-shaped cells was observed (Figure 2). Similar function with the maintenance of corporal tissue levels of lymphocytes were observed at the junction integrity and were indistinguishable form normal between biomatrix and the surrounding tissues. The controls. ABM biomatrix appeared to have a fewer number of Tunica albuginea is an important component of elastin fibers when compared with the native tunica. erectile tissue, which maintains a high-chamber pressure during the erectile phase. To adequately perform its function, the tunica needs to withstand Discussion high degrees of pressure to maintain the corporal tissue integrity. In our study, we examined the Although a variety of biomaterials has been tried as biomechanical properties of the implanted ABM a tunica substitute, none has shown to be entirely grafts at each time point and show that the levels of satisfactory. Search for an ideal material that is tensile stress and strain remained within normal suitable for tunica substitute has been a challenge. limits during the entire study period. Moreover, the In this study, we tested the collagen-based matrix strength of ABM showed a tendency to increase over processed from the bladder in a rabbit tunica defect time. This may be due to the infiltration of model. We show that ABM is biocompatible, fibroblastic host cells that produce extracellular durable and effective when used as a tunica matrix. substitute. In this study, we have used a single layer of One of the single most important factors consid- processed matrix for the replacement of rabbit ered in the selection of biomaterials for clinical tunica albuginea. The matrix is easily processed applications is biocompatibility. Acellular matrix and has a good tissue handling for surgical proce- derived from the bladder tissue are known for dures. One of the attractive characteristics of ABM is excellent biocompatibility and have been used the feasibility to configure its thickness and size as experimentally and clinically in several applica- needed. These properties contribute to the strength tions.11,16,17 Since this biomaterial was processed in of the matrix, which can be easily configured by an industrial facility using adapted protocols, it was bonding multiple layers of the processed ABM for necessary to confirm the biocompatibility of the various applications. A single layer ABM used in processed materials using standard techniques.11 In this study may not be suitable for tunica repair in our study, ABM demonstrated an excellent biocom- patients. The ability to control matrix configurations patibility when in contact with the living cells, as would add an increased value to the ‘off the shelf’ indicated by the MTT and Neutral Red assays. biomaterial. Furthermore, the apoptotic activity of the cells in In conclusion, the present study demonstrates that contact with ABM was minimal. These results have ABM is biocompatible, durable and effective in the reflected the outcome of the in vivo studies, where repair of tunica albuginea in a rabbit model. The none of the animals showed erosion, infection or implanted matrix is able to restore and maintain graft rejection. All of the replaced tunica implants corporal tissue function. Further studies are necessary were palpable and securely embedded within the to determine the applicability for human application. surrounding native tunica. The ABM used showed excellent stability in size with a minimal contrac- ture of less then 3% after 3 months. Histologically, the ABM grafts successfully inte- Acknowledgments grated into native tissue. Infiltration of host inflam- We thank Evelyn Flynn, Arthur Nedder, DVM and matory cells was observed during the early phase of Laurie Leeds for their technical assistance. We also implantation, which is known as normal tissue thank Karl-Erik Andersson, MD, PhD for his editor- response. However, the number of lymphoid cells ial assistance. This research was supported, in part, decreased over time. Generous neovascularization by Acell Inc., the Swiss National Research Founda- was observed in all of the implants, indicating the tion and Swiss Urological Association. survival of implanted grafts. A potential limitation in relating this work to humans is the graft size. However, the graft size used in this study comprises a proportional surface area comparable to that of References human conditions. The graft size used in this study was 3.5 Â 7 mm, which is a considerable amount of 1 Dean RC, Lue TF. Peyronie’s disease: advancements in recent tissue in the rabbit. surgical techniques. Curr Opin Urol 2004; 14: 339–343. 2 Carson CC, Chun JL. Peyronie’s disease: surgical management: The prime goal of tunica reconstruction is to autologous materials. Int J Impot Res 2002; 14: 329–335. restore corporal function, while maintaining the 3 Egydio PH, Lucon AM, Arap S. Treatment of Peyronie’s tissue’s structural integrity. Toward this goal, we disease by incomplete circumferential incision of the tunica

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