- Email: [email protected]
Biomechanical and Biochemical Assessment of Properties of the Anterior Urethra After Hypospadias Repair in a Rabbit Model
Biomechanical and Biochemical Assessment of Properties of the Anterior Urethra After Hypospadias Repair in a Rabbit Model Marianna Lalla,* Carl Christian Danielsen, Helene Austevoll, Lars Henning Olsen and Troels Munch Jørgensen From the Institute of Clinical Medicine (ML, HA, LHO, TMJ) and Section of Paediatric Urology, Department of Urology (ML, LHO, TMJ) Aarhus University Hospital-Skejby Sygehus and Department of Connective Tissue Biology, Institute of Anatomy (CCD, HA), University of Aarhus, Aarhus, Denmark
Purpose: We created a rabbit model to test hypospadias operations and investigate the biomechanical properties of the urethra at long-term followup using biomechanical and biochemical assessments. Materials and Methods: A total of 38 New Zealand White rabbits were randomized into 4 groups, including controls, sham operation and 2 operation groups (experimental creation of a hypospadias-like defect and acute repair, respectively). In operation group 1 the ventral urethral wall and dorsal plate were longitudinally incised, half of the ventral urethral wall was excised (hypospadias-like defect) and the incised urethra was tubularized (tubularized incised posterior plate urethroplasty group). In operation group 2 the urethra was mobilized from the corpora cavernosa, excised in its distal end (hypospadias-like defect) and advanced to the glanular tip (mobilization and advancement group). At 23 weeks postoperatively biochemical and biomechanical assessments were performed. Results: Maximum urethral strength and stiffness, strain at maximum load and the collagen weight fraction were not significantly different among the groups. Urethral diameter was larger and the total amount of collagen was higher in the mobilization and advancement group only (p ⬍0.05). The mechanical quality of urethral collagen was decreased in the 2 operation groups (p ⬍0.05). Conclusions: This animal model proved to be useful for testing hypospadias operations and urethral mechanical properties. At long-term followup after experimental hypospadias repair biochemical and biomechanical assessments showed no differences among the groups in mechanical strength, strain and stiffness, and no indication of fibrosis. Consequently testing new hypospadias repair techniques and evaluating their biomechanical long-term results could be performed using hypospadiac animal models before clinical use. Key Words: biomechanics, urethra, rabbits, hypospadias, hydroxyproline
ypospadias repair is usually performed in children at an early age.1 The validity of hypospadias techniques is traditionally assessed by the fistula and stenosis rates. Therefore, the success or failure of a new hypospadias repair technique is based on complication rates that are usually assessed after a short followup. However, a final outcome assessment of urethra during puberty, when the urethra is subjected to maximum growth, would be optimal. Moreover, testing a new surgical technique for hypospadias repair in animal models before taking it into clinical use is not standard practice. Biomechanical testing and collagen content analysis are useful for clarifying the structure-function relationship in a tubular organ such as the urethra and for quantifying the success/failure rate of a hypospadias repair technique in terms of urethral mechanical strength and luminal dimension. We created an animal
H
Submitted for publication September 5, 2006. Study received approval from the Danish Animal Ethical Committee. Supported by ANNUUM Klinisk Institut-Skejby Sygehus and Professor J. C. Christoffersens Mindefond. * Correspondence: Institute of Clinical Medicine-Klinisk Institut, Aarhus University Hospital-Skejby Sygehus, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark (telephone: ⫹45-8949 5511; FAX: ⫹45-8949 6011; e-mail: [email protected]).
0022-5347/07/1776-2375/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION
model in which it is possible to perform new hypospadias repair techniques and assess the biomechanical properties of the urethra and its collagen content after long-term followup.
MATERIALS AND METHODS Materials and Surgical Procedures The study was reviewed and approved by the Danish Animal Ethical Committee. A total of 38 New Zealand White male rabbits at a mean ⫾ SEM age of 9 weeks ⫾ 4 days weighing 1.867 ⫾ 32 gm were randomly allocated into 4 groups, including 2 operation groups of 10 each, a sham operation group of 9 and 9 controls. The hypospadias repair techniques performed in the 2 operation groups were TIP,2,3 and the urethral mobilization and advancement procedure.4,5 All procedures were performed at 10⫻ magnification with the animals placed supine under general anesthesia, which was induced by intramuscular injection of midazolam (2 mg/kg) and fentanyl-fluanisone (0.3 ml/kg), and maintained with half doses of the anesthetic mix at 1:1 every 20 to 30 minutes. The surgical procedures started with a midline penile skin incision from anus to glans with mobilization of the
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Vol. 177, 2375-2380, June 2007 Printed in U.S.A. DOI:10.1016/j.juro.2007.01.139
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preputial glands until the urethra was exposed and the corpora cavernosa were visible. In the sham operation group the skin was closed again. In the TIP group the ventral wall of the penile urethra was longitudinally opened for 1 cm in the midline between 2 holding stitches. Thus, the dorsal urethral plate was visible and incised longitudinally in the midline for 1 cm without involving the underlying tunica albuginea. Urethral circumference was measured and the hypospadias-like defect was created by a 1 cm long excision of the ventral urethra, reducing it to half of its circumference. Randomly in half of the cases a pair of 7-zero polyglactin rapid stitches between the tunica albuginea and the incised dorsal urethra were placed at a distance of 1/8 of the urethral circumference to keep the incision open. In the other half the dorsal urethra was left unfixed. Finally, the urethra was tubularized using an 8-zero polyglactin running suture. In the urethral mobilization and advancement group the urethra was mobilized from the corpora cavernosa in a distal and proximal direction without involving the ventral glans to advance freely without tension. Great care was taken to avoid damage to the dorsal urethra, which appeared to be thin and easy to damage. When the urethra was sufficiently mobilized, transection of the urethra at the junction between the glans and anterior urethra was performed and 0.5 cm of the distal anterior urethra was excised to create a hypospadias-like defect. The length of the remaining urethra was measured and dissection was continued until a 4:1 ratio was achieved between the length of the mobilized anterior urethra and the length of the glans. The urethra was then advanced through the glans, the mucosa was resected and the urethra was sutured to the tip of the glans using an interrupted suture of 8-zero Polysorb™. No stents or catheters were used. Preoperatively subcutaneous administration of sulfadiazine plus trimethoprim (48 mg/kg) was followed by postoperative administration daily for 3 days. Buprenorphine (0.01 to 0.05 mg/kg) was administered subcutaneously 2 to 3 times daily for 3 days. The rabbits were kept in regular cages, fed a standard rabbit diet and inspected daily for complications. After 23 weeks at ages 32 weeks ⫾ 6 days and weighing 3.677 ⫾ 60 gm the rabbits were sacrificed under general anesthesia by an intracardial overdose of pentobarbital (200 mg/ml). The urethras were harvested.
Biomechanical and Biochemical Methods Six parallel, transverse, 1 mm high ring specimens were systematically sampled from the middle three-fourths of the anterior urethra by cutting the urethra at right angles along the longitudinal axis. The specimens were immediately frozen and stored at – 80C in 50 mM tris HCl buffer (pH 7.4) until analysis. At biomechanical assessment the frozen specimens were thawed, cleaned of surrounding loose connective tissue and tested within a few minutes after thawing. Each specimen was mounted in an Alwetron TCT5 horizontal material testing machine (Lorentzen and Wettre, Stockholm, Sweden). With 2 L-shaped wires (diameter 0.55 mm) through the lumen and immersed in tris HCl buffer at room temperature the specimen was stretched at a constant deformation rate of 10 mm per minute until rupture during video recording. From the recorded load deformation data the maximum load (N), the original luminal diameter (d0) of the ring specimen, that is the urethral diameter at the
smallest recordable load value in mm, and the diameter at maximum load (dmax) in mm were determined. From calculated load-strain curves the strain at maximum load (max) and the maximum stiffness (maximum slope of the loadstrain curve in N) were derived.6,7 Following mechanical testing the specimens were examined under a microscope to detect the point of rupture with reference to the corporeal attachment. Rupture patterns were later compared to those seen in the video recordings. Because the dorsal urethra was defined as the portion of the urethra adjacent to the corpora cavernosa, rupture patterns were classified as the dorsal urethra, the lateral and anterior ventral urethra, and the transition between the dorsal and ventral urethra, where a V-shaped loose connective tissue laterally anchors the corpora to the lateral ventral urethra. Dry defatted weight was determined and collagen content in the ring specimen was estimated by hydroxyproline determination (1 mg hydroxyproline ⬇ 7.46 mg collagen).7 Collagen content per ring specimen in mg and collagen weight fraction in mg collagen per mg dry defatted weight were determined. The mechanical quality of collagen was estimated by normalizing maximum load and maximum stiffness with regard to mg collagen per mm circumference of the ring specimens in N/mg/mm. Statistics Statistical analysis was performed using Stata® 9.2. Data on an average of 6 specimens per animal were obtained and statistical analysis was performed on the group mean. Results are shown as the mean ⫾ SEM. Logarithmic transformation was applied when appropriate to achieve a normal distribution. The groups were compared by ANOVA and the homogeneity of variance was tested by Bartlett’s test. If ANOVA showed a statistically significant difference, all groups were post hoc compared in pairwise fashion. Significance for all statistical tests was considered at 5%. Because the TIP group was subdivided into 2 subgroups by performing a modification in the operation in half of the cases, the t test was done between these 2 subgroups for all variables. All ANOVA tests were performed using 4 and 5 groups. Urethral rupture patterns were analyzed with the chi-square test.
FIG. 1. Mean ⫾ SEM load-strain curves with max and maximum load shown as scatterplots.
BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR
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RESULTS Data on 32 rabbits were analyzed. Four rabbits in the mobilization and advancement group were lost due to anesthesia complications in 3 and suspected infection in 1 at 2 months postoperatively. Two rabbits in the control group were excluded due to technical errors. The t test comparing the 2 TIP subgroups did not show any statistical difference for any variables. The ANOVA tests for 4 and 5 groups were similar and, therefore, results are only shown for the 4 group analysis. Biomechanical analysis showed no statistically significant differences between the groups in maximum load, maximum stiffness or strain at maximum load (p ⫽ 0.202, 0.285 and 0.172, respectively, figs. 1 and 2). Urethral dimension results showed that diameter values at start load (d0) and at maximum load (dmax) were statistically significantly larger in the mobilization and advancement group compared to those in the other 3 groups (p ⬍0.05, fig. 3). Analysis of d0 showed the smallest value in the TIP group and the largest value in the mobilization and advancement group. The same test for dmax showed that diameter values in the urethral advancement group were significantly larger than those in the other groups (p ⬍0.05).
FIG. 3. Mean ⫾ SEM diameter at load start and at maximum load. Single asterisk indicates p ⬍0.05 vs sham operation. Double asterisks indicate p ⬍0.01 vs sham operation. Single curly indicates p ⬍0.05 vs controls. Double curlies indicate p ⬍0.01 vs controls. Boxed circle indicates p ⬍0.01 vs TIP.
Biochemical analysis showed that collagen content per specimen was higher in the 2 hypospadias repair groups than in the sham operation group, whereas the collagen weight fraction was not found to be different (p ⬍0.05 and 0.559, respectively, fig. 4). The mechanical quality of urethral collagen was decreased in the 2 operation groups compared to that in controls and the sham operation group (p ⬍0.05, fig. 5). Analysis of urethral failure patterns during mechanical testing showed no statistical difference among the groups (p ⫽ 0.2, see table). DISCUSSION
FIG. 2. Mean ⫾ SEM maximum load and maximum stiffness
The history of hypospadias repair is characterized by several proposals for surgical repair techniques, of which many were proposed without previous experimental studies. Only after considerable followup was the validity of these techniques assessed with consequent success or failure in daily clinical life. In recent times the TIP procedure2 was proposed, raising some concerns about stricture development during puberty, when the genitalia undergo pronounced growth.
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BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR of glands in the subcutaneous tissue, any technique involving the use of skin or vascular subcutaneous tissues in the repair was discarded. Therefore, the TIP repair technique elaborated for a rabbit model,3 and the mobilization and advancement technique comparable to that described in humans by Atala5 were the best choices for our purposes, although the latter is technically demanding due to the extreme fragility of the rabbit dorsal urethral plate. The timing of operations and biomechanical assessments was set at prepubertal/peripubertal and at postpubertal ages in rabbits, respectively.16 Biochemical and biomechanical assessments were performed to test the quantitative long-term functional outcome after operation with regard to the risk of stricture, signs of fibrosis and urethral strength or stiffness. Because the urethra is mostly subjected to circular stresses during urine flow and it may share some of the biomechanical characteristics of other tubular organs, it seemed appropriate to test urethral biomechanical properties in a circular direction. To our knowledge no other groups have investi-
FIG. 4. Total collagen per ring specimen and collagen weight fraction (geometric mean [ ⫽ exp (mean (log-data))] ⫾ geometric SEM [ ⫽ exp (SEM (log-data))]). Single asterisk indicates p ⬍0.05 vs sham operation. Double asterisks indicate p ⬍0.01 vs sham operation.
Essentially this procedure does not differ from internal urethrotomy. Therefore, many clinical studies have been done and no urethral strictures have been reported at early followup, although they occurred 6 to 27 months after the operation.8 –11 Although this technique is widely accepted, the mechanism of posterior urethral healing is generally still obscure. Experimental studies of acute urethral healing after TIP repair histologically showed re-epithelialization without scarring or excess collagen deposition of the incised urethral plate.3,12–14 Furthermore, estimation of the collagen content in acute healing after TIP repair showed no signs of fibrosis.15 The results of these experimental acute urethral healing studies were obtained at a maximum followup of 3 months with no stricture and no fibrosis but none of these investigators assessed the stricture risk during and around puberty. In the current study we evaluated different hypospadias repair techniques in an animal model and tested long-term results in a period of animal life corresponding to puberty. Because of the hairy skin over the urethra and the presence
FIG. 5. Normalized maximum load and normalized maximum stiffness (geometric mean [ ⫽ exp (mean (log-data))] ⫾ geometric SEM [ ⫽ exp (SEM (log-data))]). Single asterisk indicates p ⬍0.05 vs sham operation. Double asterisks indicate p ⬍0.01 vs sham operation. Single curly indicates p ⬍0.05 vs controls. Double curlies indicate p ⬍0.01 vs controls.
BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR
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Urethral rupture position after mechanical testing No. Urethral Rupture Position/Total No. (%) Groups
Dorsal
Dorsoventral
Lateral Ventral
Anteroventral
Control Sham operation TIP Mobilization ⫹ advancement
1/7 (14.3) 2/9 (22.2) 4/10 (40) 2/6 (33.3)
2/7 (28.6) 5/9 (55.6) 1/10 (10) 4/6 (66.7)
3/7 (42.9) 2/9 (22.2) 4/10 (40) —
1/7 (14.3) — 1/10 (10) —
9/32 (28.1)
12/32 (37.5)
9/32 (28.1)
2/32 (6.3)
Totals Chi-square test p ⫽ 0.2.
gated the biomechanical properties of the urethra in a hypospadias animal model. Our results showed that 23 weeks after hypospadias repair the 2 operation groups showed urethral mechanical strength and stiffness comparable to those in the sham operation group and controls (figs. 1 and 2). These biomechanical findings indicated that hypospadias repaired urethras had sufficient elasticity to avoid voiding problems and the fully functional capacity to resist circular stresses during urine flow. Fibrosis or excessive scar tissue formation leading to stricture could be a serious adverse effect after hypospadias repair. The current results did not indicate any urethral stricture following the 2 hypospadias operations. On the contrary, in the mobilization and advancement group the diameter was even larger than that in controls and the sham operation group (fig. 3). Urethral attenuation due to advancement in this group would expectedly result in a decreased diameter. The enlarged diameter observed instead at load start and at maximum load could have been a consequence of urethral mobilization from the corpora with loss of the natural connective tissue connections between corpora and urethra, of a changed orientation of collagen fibrils or of the advancement of a more proximal urethra with different biomechanical properties. The decreased diameter in the TIP group could suggest a risk of stricture, although there were no statistical differences compared with controls and the sham operation group (fig. 3). Biochemical analysis showed that the operations did not change the collagen weight fraction at long-term followup. Therefore, no indication of fibrosis was present. The collagen content per ring specimen was increased in the 2 operation groups compared to that in the sham operation group (fig. 4). In the TIP group this indicates that some collagen present in the granulation tissue formed during healing of the dorsal urethral plate incision may have persisted at followup. In the mobilization and advancement group the higher amount of collagen may have been a consequence of the increased diameter. Urethral advancement may result in increased longitudinal stress and promote increased collagen formation with fibrillar orientation to resist the imposed longitudinal stress. This would be expected to give a higher collagen content of lower mechanical quality in the circular direction and possibly contribute to the larger diameter. The lower mechanical quality of the collagen found in the 2 hypospadias groups could indicate that the collagen formed after the operation was not fully integrated or circularly aligned in the urethral wall (fig. 5). The natural adjacency of the dorsal urethra to the corpora left intact in controls and the sham operation group was expected to provide resistance to circular stresses, such as during voiding. At long-term followup
urethral incision or mobilization in the repair groups had only minor nonsignificant effects on circular mechanical strength but they may have contributed to the significant deterioration in collagen mechanical quality. Thus, the orientation and integration of collagen fibrils in the urethral wall and the disruption of the natural adjacency of the dorsal urethra to the corpora following hypospadias repairs seemed only partially recovered 23 weeks after the operations. Only extended followup could disclose when or whether repaired urethras are fully recovered. CONCLUSIONS The 2 reconstructive operation techniques in the rabbit model allowed simulation of the hypospadias defect and repair and resulted in fully functional urethras with apparently no adverse effect after 23 weeks of followup. Biomechanics appears to be a valuable tool that provides a functional idea of the physiological behavior of the urethra at long-term followup. ACKNOWLEDGMENTS Marianne Axelsen, Peter-Martin Krarup and Eva Kjeld Mikkelsen provided technical assistance, Henrik Frederik Thomsen provided statistical advice and Merete Fisher provided linguistic revision.
Abbreviations and Acronyms TIP ⫽ tubularized incised posterior plate urethroplasty
REFERENCES 1.
Manzoni G, Bracka A, Palminteri E and Marrocco G: Hypospadias surgery: when, what and by whom? BJU Int 2004; 94: 1188. 2. Snodgrass W: Tubularized, incised plate urethroplasty for distal hypospadias. J Urol 1994; 151: 464. 3. Hafez AT, Herz D, Bagli D, Smith CR, McLorie G and Khoury AE: Healing of unstented tubularized incised plate urethroplasty: an experimental study in a rabbit model. BJU Int 2003; 91: 84. 4. Koff SA: Mobilization of the urethra in the surgical treatment of hypospadias. J Urol 1981; 125: 394. 5. Atala A: Urethral mobilization and advancement for midshaft to distal hypospadias. J Urol 2002; 168: 1738. 6. Knudsen UB, Spanggaard H, Uldbjerg N and Danielsen CC: Biomechanical analysis of cervix uteri in nonpregnant,
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pregnant and antigestagen (ZK 98 299) treated pregnant rats. Connect Tissue Res 1994; 31: 67. 7. Danielsen CC and Andreassen TT: Mechanical properties of rat tail tendon in relation to proximal-distal sampling position and age. J Biomech 1988; 21: 207. 8. Snodgrass W, Koyle M, Manzoni G, Hurwitz R, Caldamone A and Ehrlich R: Tubularized incised plate hypospadias repair: results of a multicenter experience. J Urol 1996; 156: 839. 9. Scherz HC, Kaplan GW, Packer MG and Brock WA: Posthypospadias repair urethral strictures: a review of 30 cases. J Urol 1988; 140: 1253. 10. Stehr M, Lehner M, Schuster T, Heinrich M and Dietz HG: Tubularized incised plate (TIP) urethroplasty (Snodgrass) in primary hypospadias repair. Eur J Pediatr Surg 2005; 15: 420. 11. Palmer LS, Palmer JS, Franco I, Friedman SC, Kolligian ME, Gill B et al: The “long Snodgrass”: applying the tubularized
incised plate urethroplasty to penoscrotal hypospadias in 1-stage or 2-stage repairs. J Urol 2002; 168: 1748. 12. Genc A, Taneli C, Gunsar C, Turkdogan P, Yilmaz O, Arslan OA et al: Histopathological evaluation of the urethra after the Snodgrass operation: an experimental study in rabbits. BJU Int 2002; 90: 950. 13. Lopes JF, Schned A, Ellsworth PI and Cendron M: Histological analysis of urethral healing after tubularized incised plate urethroplasty. J Urol 2001; 166: 1014. 14. Bleustein CB, Esposito MP, Soslow RA, Felsen D and Poppas DP: Mechanism of healing following the Snodgrass repair. J Urol 2001; 165: 277. 15. Taneli F, Ulman C, Genc A, Yilmaz O and Taneli C: Biochemical analysis of urethral collagen content after tubularized incised plate urethroplasty: an experimental study in rabbits. Urol Res 2004; 32: 219. 16. Skinner JD: Puberty in the male rabbit. J Reprod Fertil 1967; 14: 151.
Purpose: We created a rabbit model to test hypospadias operations and investigate the biomechanical properties of the urethra at long-term followup using biomechanical and biochemical assessments. Materials and Methods: A total of 38 New Zealand White rabbits were randomized into 4 groups, including controls, sham operation and 2 operation groups (experimental creation of a hypospadias-like defect and acute repair, respectively). In operation group 1 the ventral urethral wall and dorsal plate were longitudinally incised, half of the ventral urethral wall was excised (hypospadias-like defect) and the incised urethra was tubularized (tubularized incised posterior plate urethroplasty group). In operation group 2 the urethra was mobilized from the corpora cavernosa, excised in its distal end (hypospadias-like defect) and advanced to the glanular tip (mobilization and advancement group). At 23 weeks postoperatively biochemical and biomechanical assessments were performed. Results: Maximum urethral strength and stiffness, strain at maximum load and the collagen weight fraction were not significantly different among the groups. Urethral diameter was larger and the total amount of collagen was higher in the mobilization and advancement group only (p ⬍0.05). The mechanical quality of urethral collagen was decreased in the 2 operation groups (p ⬍0.05). Conclusions: This animal model proved to be useful for testing hypospadias operations and urethral mechanical properties. At long-term followup after experimental hypospadias repair biochemical and biomechanical assessments showed no differences among the groups in mechanical strength, strain and stiffness, and no indication of fibrosis. Consequently testing new hypospadias repair techniques and evaluating their biomechanical long-term results could be performed using hypospadiac animal models before clinical use. Key Words: biomechanics, urethra, rabbits, hypospadias, hydroxyproline
ypospadias repair is usually performed in children at an early age.1 The validity of hypospadias techniques is traditionally assessed by the fistula and stenosis rates. Therefore, the success or failure of a new hypospadias repair technique is based on complication rates that are usually assessed after a short followup. However, a final outcome assessment of urethra during puberty, when the urethra is subjected to maximum growth, would be optimal. Moreover, testing a new surgical technique for hypospadias repair in animal models before taking it into clinical use is not standard practice. Biomechanical testing and collagen content analysis are useful for clarifying the structure-function relationship in a tubular organ such as the urethra and for quantifying the success/failure rate of a hypospadias repair technique in terms of urethral mechanical strength and luminal dimension. We created an animal
H
Submitted for publication September 5, 2006. Study received approval from the Danish Animal Ethical Committee. Supported by ANNUUM Klinisk Institut-Skejby Sygehus and Professor J. C. Christoffersens Mindefond. * Correspondence: Institute of Clinical Medicine-Klinisk Institut, Aarhus University Hospital-Skejby Sygehus, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark (telephone: ⫹45-8949 5511; FAX: ⫹45-8949 6011; e-mail: [email protected]).
0022-5347/07/1776-2375/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION
model in which it is possible to perform new hypospadias repair techniques and assess the biomechanical properties of the urethra and its collagen content after long-term followup.
MATERIALS AND METHODS Materials and Surgical Procedures The study was reviewed and approved by the Danish Animal Ethical Committee. A total of 38 New Zealand White male rabbits at a mean ⫾ SEM age of 9 weeks ⫾ 4 days weighing 1.867 ⫾ 32 gm were randomly allocated into 4 groups, including 2 operation groups of 10 each, a sham operation group of 9 and 9 controls. The hypospadias repair techniques performed in the 2 operation groups were TIP,2,3 and the urethral mobilization and advancement procedure.4,5 All procedures were performed at 10⫻ magnification with the animals placed supine under general anesthesia, which was induced by intramuscular injection of midazolam (2 mg/kg) and fentanyl-fluanisone (0.3 ml/kg), and maintained with half doses of the anesthetic mix at 1:1 every 20 to 30 minutes. The surgical procedures started with a midline penile skin incision from anus to glans with mobilization of the
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Vol. 177, 2375-2380, June 2007 Printed in U.S.A. DOI:10.1016/j.juro.2007.01.139
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BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR
preputial glands until the urethra was exposed and the corpora cavernosa were visible. In the sham operation group the skin was closed again. In the TIP group the ventral wall of the penile urethra was longitudinally opened for 1 cm in the midline between 2 holding stitches. Thus, the dorsal urethral plate was visible and incised longitudinally in the midline for 1 cm without involving the underlying tunica albuginea. Urethral circumference was measured and the hypospadias-like defect was created by a 1 cm long excision of the ventral urethra, reducing it to half of its circumference. Randomly in half of the cases a pair of 7-zero polyglactin rapid stitches between the tunica albuginea and the incised dorsal urethra were placed at a distance of 1/8 of the urethral circumference to keep the incision open. In the other half the dorsal urethra was left unfixed. Finally, the urethra was tubularized using an 8-zero polyglactin running suture. In the urethral mobilization and advancement group the urethra was mobilized from the corpora cavernosa in a distal and proximal direction without involving the ventral glans to advance freely without tension. Great care was taken to avoid damage to the dorsal urethra, which appeared to be thin and easy to damage. When the urethra was sufficiently mobilized, transection of the urethra at the junction between the glans and anterior urethra was performed and 0.5 cm of the distal anterior urethra was excised to create a hypospadias-like defect. The length of the remaining urethra was measured and dissection was continued until a 4:1 ratio was achieved between the length of the mobilized anterior urethra and the length of the glans. The urethra was then advanced through the glans, the mucosa was resected and the urethra was sutured to the tip of the glans using an interrupted suture of 8-zero Polysorb™. No stents or catheters were used. Preoperatively subcutaneous administration of sulfadiazine plus trimethoprim (48 mg/kg) was followed by postoperative administration daily for 3 days. Buprenorphine (0.01 to 0.05 mg/kg) was administered subcutaneously 2 to 3 times daily for 3 days. The rabbits were kept in regular cages, fed a standard rabbit diet and inspected daily for complications. After 23 weeks at ages 32 weeks ⫾ 6 days and weighing 3.677 ⫾ 60 gm the rabbits were sacrificed under general anesthesia by an intracardial overdose of pentobarbital (200 mg/ml). The urethras were harvested.
Biomechanical and Biochemical Methods Six parallel, transverse, 1 mm high ring specimens were systematically sampled from the middle three-fourths of the anterior urethra by cutting the urethra at right angles along the longitudinal axis. The specimens were immediately frozen and stored at – 80C in 50 mM tris HCl buffer (pH 7.4) until analysis. At biomechanical assessment the frozen specimens were thawed, cleaned of surrounding loose connective tissue and tested within a few minutes after thawing. Each specimen was mounted in an Alwetron TCT5 horizontal material testing machine (Lorentzen and Wettre, Stockholm, Sweden). With 2 L-shaped wires (diameter 0.55 mm) through the lumen and immersed in tris HCl buffer at room temperature the specimen was stretched at a constant deformation rate of 10 mm per minute until rupture during video recording. From the recorded load deformation data the maximum load (N), the original luminal diameter (d0) of the ring specimen, that is the urethral diameter at the
smallest recordable load value in mm, and the diameter at maximum load (dmax) in mm were determined. From calculated load-strain curves the strain at maximum load (max) and the maximum stiffness (maximum slope of the loadstrain curve in N) were derived.6,7 Following mechanical testing the specimens were examined under a microscope to detect the point of rupture with reference to the corporeal attachment. Rupture patterns were later compared to those seen in the video recordings. Because the dorsal urethra was defined as the portion of the urethra adjacent to the corpora cavernosa, rupture patterns were classified as the dorsal urethra, the lateral and anterior ventral urethra, and the transition between the dorsal and ventral urethra, where a V-shaped loose connective tissue laterally anchors the corpora to the lateral ventral urethra. Dry defatted weight was determined and collagen content in the ring specimen was estimated by hydroxyproline determination (1 mg hydroxyproline ⬇ 7.46 mg collagen).7 Collagen content per ring specimen in mg and collagen weight fraction in mg collagen per mg dry defatted weight were determined. The mechanical quality of collagen was estimated by normalizing maximum load and maximum stiffness with regard to mg collagen per mm circumference of the ring specimens in N/mg/mm. Statistics Statistical analysis was performed using Stata® 9.2. Data on an average of 6 specimens per animal were obtained and statistical analysis was performed on the group mean. Results are shown as the mean ⫾ SEM. Logarithmic transformation was applied when appropriate to achieve a normal distribution. The groups were compared by ANOVA and the homogeneity of variance was tested by Bartlett’s test. If ANOVA showed a statistically significant difference, all groups were post hoc compared in pairwise fashion. Significance for all statistical tests was considered at 5%. Because the TIP group was subdivided into 2 subgroups by performing a modification in the operation in half of the cases, the t test was done between these 2 subgroups for all variables. All ANOVA tests were performed using 4 and 5 groups. Urethral rupture patterns were analyzed with the chi-square test.
FIG. 1. Mean ⫾ SEM load-strain curves with max and maximum load shown as scatterplots.
BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR
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RESULTS Data on 32 rabbits were analyzed. Four rabbits in the mobilization and advancement group were lost due to anesthesia complications in 3 and suspected infection in 1 at 2 months postoperatively. Two rabbits in the control group were excluded due to technical errors. The t test comparing the 2 TIP subgroups did not show any statistical difference for any variables. The ANOVA tests for 4 and 5 groups were similar and, therefore, results are only shown for the 4 group analysis. Biomechanical analysis showed no statistically significant differences between the groups in maximum load, maximum stiffness or strain at maximum load (p ⫽ 0.202, 0.285 and 0.172, respectively, figs. 1 and 2). Urethral dimension results showed that diameter values at start load (d0) and at maximum load (dmax) were statistically significantly larger in the mobilization and advancement group compared to those in the other 3 groups (p ⬍0.05, fig. 3). Analysis of d0 showed the smallest value in the TIP group and the largest value in the mobilization and advancement group. The same test for dmax showed that diameter values in the urethral advancement group were significantly larger than those in the other groups (p ⬍0.05).
FIG. 3. Mean ⫾ SEM diameter at load start and at maximum load. Single asterisk indicates p ⬍0.05 vs sham operation. Double asterisks indicate p ⬍0.01 vs sham operation. Single curly indicates p ⬍0.05 vs controls. Double curlies indicate p ⬍0.01 vs controls. Boxed circle indicates p ⬍0.01 vs TIP.
Biochemical analysis showed that collagen content per specimen was higher in the 2 hypospadias repair groups than in the sham operation group, whereas the collagen weight fraction was not found to be different (p ⬍0.05 and 0.559, respectively, fig. 4). The mechanical quality of urethral collagen was decreased in the 2 operation groups compared to that in controls and the sham operation group (p ⬍0.05, fig. 5). Analysis of urethral failure patterns during mechanical testing showed no statistical difference among the groups (p ⫽ 0.2, see table). DISCUSSION
FIG. 2. Mean ⫾ SEM maximum load and maximum stiffness
The history of hypospadias repair is characterized by several proposals for surgical repair techniques, of which many were proposed without previous experimental studies. Only after considerable followup was the validity of these techniques assessed with consequent success or failure in daily clinical life. In recent times the TIP procedure2 was proposed, raising some concerns about stricture development during puberty, when the genitalia undergo pronounced growth.
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BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR of glands in the subcutaneous tissue, any technique involving the use of skin or vascular subcutaneous tissues in the repair was discarded. Therefore, the TIP repair technique elaborated for a rabbit model,3 and the mobilization and advancement technique comparable to that described in humans by Atala5 were the best choices for our purposes, although the latter is technically demanding due to the extreme fragility of the rabbit dorsal urethral plate. The timing of operations and biomechanical assessments was set at prepubertal/peripubertal and at postpubertal ages in rabbits, respectively.16 Biochemical and biomechanical assessments were performed to test the quantitative long-term functional outcome after operation with regard to the risk of stricture, signs of fibrosis and urethral strength or stiffness. Because the urethra is mostly subjected to circular stresses during urine flow and it may share some of the biomechanical characteristics of other tubular organs, it seemed appropriate to test urethral biomechanical properties in a circular direction. To our knowledge no other groups have investi-
FIG. 4. Total collagen per ring specimen and collagen weight fraction (geometric mean [ ⫽ exp (mean (log-data))] ⫾ geometric SEM [ ⫽ exp (SEM (log-data))]). Single asterisk indicates p ⬍0.05 vs sham operation. Double asterisks indicate p ⬍0.01 vs sham operation.
Essentially this procedure does not differ from internal urethrotomy. Therefore, many clinical studies have been done and no urethral strictures have been reported at early followup, although they occurred 6 to 27 months after the operation.8 –11 Although this technique is widely accepted, the mechanism of posterior urethral healing is generally still obscure. Experimental studies of acute urethral healing after TIP repair histologically showed re-epithelialization without scarring or excess collagen deposition of the incised urethral plate.3,12–14 Furthermore, estimation of the collagen content in acute healing after TIP repair showed no signs of fibrosis.15 The results of these experimental acute urethral healing studies were obtained at a maximum followup of 3 months with no stricture and no fibrosis but none of these investigators assessed the stricture risk during and around puberty. In the current study we evaluated different hypospadias repair techniques in an animal model and tested long-term results in a period of animal life corresponding to puberty. Because of the hairy skin over the urethra and the presence
FIG. 5. Normalized maximum load and normalized maximum stiffness (geometric mean [ ⫽ exp (mean (log-data))] ⫾ geometric SEM [ ⫽ exp (SEM (log-data))]). Single asterisk indicates p ⬍0.05 vs sham operation. Double asterisks indicate p ⬍0.01 vs sham operation. Single curly indicates p ⬍0.05 vs controls. Double curlies indicate p ⬍0.01 vs controls.
BIOMECHANICAL PROPERTIES OF ANTERIOR URETHRA AFTER HYPOSPADIAS REPAIR
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Urethral rupture position after mechanical testing No. Urethral Rupture Position/Total No. (%) Groups
Dorsal
Dorsoventral
Lateral Ventral
Anteroventral
Control Sham operation TIP Mobilization ⫹ advancement
1/7 (14.3) 2/9 (22.2) 4/10 (40) 2/6 (33.3)
2/7 (28.6) 5/9 (55.6) 1/10 (10) 4/6 (66.7)
3/7 (42.9) 2/9 (22.2) 4/10 (40) —
1/7 (14.3) — 1/10 (10) —
9/32 (28.1)
12/32 (37.5)
9/32 (28.1)
2/32 (6.3)
Totals Chi-square test p ⫽ 0.2.
gated the biomechanical properties of the urethra in a hypospadias animal model. Our results showed that 23 weeks after hypospadias repair the 2 operation groups showed urethral mechanical strength and stiffness comparable to those in the sham operation group and controls (figs. 1 and 2). These biomechanical findings indicated that hypospadias repaired urethras had sufficient elasticity to avoid voiding problems and the fully functional capacity to resist circular stresses during urine flow. Fibrosis or excessive scar tissue formation leading to stricture could be a serious adverse effect after hypospadias repair. The current results did not indicate any urethral stricture following the 2 hypospadias operations. On the contrary, in the mobilization and advancement group the diameter was even larger than that in controls and the sham operation group (fig. 3). Urethral attenuation due to advancement in this group would expectedly result in a decreased diameter. The enlarged diameter observed instead at load start and at maximum load could have been a consequence of urethral mobilization from the corpora with loss of the natural connective tissue connections between corpora and urethra, of a changed orientation of collagen fibrils or of the advancement of a more proximal urethra with different biomechanical properties. The decreased diameter in the TIP group could suggest a risk of stricture, although there were no statistical differences compared with controls and the sham operation group (fig. 3). Biochemical analysis showed that the operations did not change the collagen weight fraction at long-term followup. Therefore, no indication of fibrosis was present. The collagen content per ring specimen was increased in the 2 operation groups compared to that in the sham operation group (fig. 4). In the TIP group this indicates that some collagen present in the granulation tissue formed during healing of the dorsal urethral plate incision may have persisted at followup. In the mobilization and advancement group the higher amount of collagen may have been a consequence of the increased diameter. Urethral advancement may result in increased longitudinal stress and promote increased collagen formation with fibrillar orientation to resist the imposed longitudinal stress. This would be expected to give a higher collagen content of lower mechanical quality in the circular direction and possibly contribute to the larger diameter. The lower mechanical quality of the collagen found in the 2 hypospadias groups could indicate that the collagen formed after the operation was not fully integrated or circularly aligned in the urethral wall (fig. 5). The natural adjacency of the dorsal urethra to the corpora left intact in controls and the sham operation group was expected to provide resistance to circular stresses, such as during voiding. At long-term followup
urethral incision or mobilization in the repair groups had only minor nonsignificant effects on circular mechanical strength but they may have contributed to the significant deterioration in collagen mechanical quality. Thus, the orientation and integration of collagen fibrils in the urethral wall and the disruption of the natural adjacency of the dorsal urethra to the corpora following hypospadias repairs seemed only partially recovered 23 weeks after the operations. Only extended followup could disclose when or whether repaired urethras are fully recovered. CONCLUSIONS The 2 reconstructive operation techniques in the rabbit model allowed simulation of the hypospadias defect and repair and resulted in fully functional urethras with apparently no adverse effect after 23 weeks of followup. Biomechanics appears to be a valuable tool that provides a functional idea of the physiological behavior of the urethra at long-term followup. ACKNOWLEDGMENTS Marianne Axelsen, Peter-Martin Krarup and Eva Kjeld Mikkelsen provided technical assistance, Henrik Frederik Thomsen provided statistical advice and Merete Fisher provided linguistic revision.
Abbreviations and Acronyms TIP ⫽ tubularized incised posterior plate urethroplasty
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