Int Urol Nephrol (2015) 47:1303–1310 DOI 10.1007/s11255-015-1025-x
UROLOGY - ORIGINAL PAPER
Decellularized dermal strip as a suburethral sling in a rat model of stress urinary incontinence Abdol‑Mohammad Kajbafzadeh1 · Sarah Mozafarpour1 · Seyedeh Sanam Ladi Seyedian1 · Reza Khorramirouz1 · Haleh Nasser Hojjati2
Received: 23 March 2015 / Accepted: 1 June 2015 / Published online: 20 June 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Introduction To report the feasibility, efficacy and safety of decellularized dermal strip as a sling material in the pudendal denervated stress urinary incontinence (SUI) in an animal model. Methods Thirty female Sprague–Dawley rats were randomly allocated into three equal groups (n = 10). In group 1, rats underwent bilateral pudendal nerve transection (BPNT) with no vaginal sling placement. Group 2 underwent BPNT with homologous decellularized dermal strip placement as vaginal sling 12 weeks later. Group 3 was considered as controls to evaluate baseline leak point pressure (LPP). Leak point pressure was measured 18 weeks after BPNT in group 1 and 6 weeks after sling implantation in group 2. Sling and sphincter samples were prepared for histological and immunohistochemistry (IHC) staining. Results H&E staining of external urethral sphincter showed decrease in striated muscle layer in group 1 (BPNT) compared to control confirmed by desmin staining (45 % decrease in desmin staining). Sling implantation caused significant increase in mean LPP from 11.6 ± 2.8 cm H2O in group 1 (BPNT) to 27.2 ± 5.4 cm H2O in group 2 (BPNT/sling). There was no significant difference between LPP after the sling placement in group 2 and LPP in group
* Abdol‑Mohammad Kajbafzadeh [email protected] 1
Section of Tissue Engineering and Stem Cells Therapy, Pediatric Urology Research Center, Children’s Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Qarib’s Street, Keshavarz Boulevard, 1419433151 Tehran, Iran (IRI)
2
Department of Urology, Ziaeian Hospital, Tehran University of Medical Sciences, Tehran, Iran (IRI)
3 (27.2 ± 5.4 cm H2O vs. 27.6 ± 5.9, P = 0.832). IHC staining was positive for α-SMA and CD34. Conclusions Based on present results, this dermal strip could be considered as a potential sling material for treatment of SUI in this animal model. This study provides the basis for further investigation of the efficacy of biological decellularized scaffold as suburethral sling material. Keywords Leak point pressure · Pudendal nerve · Stress urinary incontinence · Sling · Urethra
Introduction Stress urinary incontinence (SUI), the most common type of urinary incontinence, is a major urological issue affecting millions of women worldwide [1]. It is defined as the involuntary leakage of urine during increased intra-abdominal pressure [2]. Therapeutic options for SUI range from conservative measurements such as pelvic floor exercise to medical treatment and surgical interventions. Suburethral slings remain the mainstay of surgical treatment for SUI. In this procedure, a strip of tissue or mesh material is inserted in order to support the bladder neck [3]. However, this procedure is sometimes accompanied by bothersome and serious complications and sometimes needs reoperations [4]. Various sling materials have been studied in anti-incontinence surgeries with variable degrees of local inflammatory reactions [5]. Selection of the sling material is a key issue to circumvent postsurgical complications. The ideal prosthesis for pelvic reconstructive surgeries should be a safe, mechanically stable, biocompatible material devoid of eliciting allergic or inflammatory reactions, which could be obtained in a sterile fashion with “off the shelf” availability [6]. Artificial sling materials have shown to be associated with higher complication rates such
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as infection and urethral erosion over biological materials, which have subsequently limited their use in selective populations [7]. Biological prosthesis including autologous materials (rectus fascia, vaginal wall, etc.), allografts (cadaveric tissues including dura mater and dermis) or xenografts (porcine small intestinal mucosa and porcine dermis) has their own limitations [8]. Pelvic tissue of patients with SUI usually does not have appropriate supporting quality. Additionally, autologous grafts require extra surgical incisions that further weaken the pelvic structures and increase morbidity [6]. It has been reported that in a substantial percent of women, usage of pelvic fascia as a suburethral sling material has caused comorbid pelvic organ dysfunction such as vaginal or rectal prolapse [9, 10]. Also, comparable results have been reported regarding patients’ satisfaction and improved quality of life with dermal allografts [11]. On the other hand, allografts and xenografts have retained antigenicity and harbor a risk of disease transmission [8, 12]. Recently, preliminary reports of tissue-engineered materials have emerged in the literature as potential alternatives for suburethral slings [13–15]. However, there are very limited data on their application in preclinical settings. In the present study, we seek to explore the biocompatibility and efficacy of decellularized dermal strip as a sling material in a sphincter-damaged rat model of SUI. The aim of this study was to find out whether a decellularized dermal strip can be used as a suburethral sling material and also to evaluate leak point pressure (LPP) and histological changes of this decellularized dermal strip after placement.
Materials and methods This experimental animal study protocol was approved by the committee on animal welfare in Tehran University of Medical Sciences. All animals were treated based on the recommendations of the animal care committee of the university and in accordance with the Guide for Care and Use of Laboratory Animals (National Research Council, revised in 1996). The animals were placed in a temperature-controlled room, with free access to food and water. Thirty female Sprague–Dawley rats weighing 210– 240 g were randomly allocated into three groups (n = 10). In group 1, rats underwent bilateral pudendal nerve transection (BPNT) with no vaginal sling implantation. Group 2 underwent BPNT with homologous decellularized dermal strip placement as a vaginal sling. Group 3 considered as controls to evaluate baseline LPP with no intervention. Decellularized skin preparation The slings were prepared using decellularized rat skin. Female rats were anesthetized with ketamine and used as
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Int Urol Nephrol (2015) 47:1303–1310
skin donors. Dorsal hair was first shaved and then removed more completely by surgical hair removal cream, the skin was scrubbed by Betadine® and sections with the size of 0.5 × 2 cm and 3-mm thickness were removed. Then, the adjacent parts were sutured. The removed skins were washed with sterile phosphatebuffered saline (PBS) and then treated by 0.25 % sodium dodecyl sulfate (SDS; Sigma, Germany) at room temperature for 48 h with continuous shaking. Subsequently, the sections were washed with PBS and then stored in sterile PBS solution at 4 °C before implantation. All the solutions contained 1 % antibiotics (i.e., penicillin, gentamycin and amphotericin), and all the procedures were done aseptically. Surgical procedure For BPNT, the rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (15 mg/kg). In a prone position, ischiorectal fossa was exposed bilaterally by a dorsal midline incision over the lumbosacral area. Fascia and coccygeus muscles were dissected, and the sciatic nerve was exposed crossing the overlying fascia superomedially to inferolaterally. Using a surgical microscope, BPNT was performed by concomitant transection of pudendal nerve trunk and anastomotic branch of the sciatic nerve. A 2-cm segment of the proximal pudendal nerve trunk was removed on each side. Coccygeus muscle, fascia and skin were closed one by one. The animals received preoperative and postoperative cefazolin to prevent infection. After 18 weeks of BPNT, rats in group 1 underwent LPP measurement. For group 2, vaginal sling placement was performed 12 weeks after BPNT. For the sling procedure, the rats were anesthetized as described before and placed in a supine position. The urethra was catheterized with a PE-50 catheter. The urethra–vaginal space was dissected horizontally with 1-cm incision, and the space was dissected more deeply, and the decellularized strip was placed without any attachments to the adjacent bone or fascia. The graft was marked with a 5-0 Prolene at each corner of the decellularized skin (0.5 × 2 cm), and the sling was placed below the midurethra. The fascia between the vagina and urethra was closed using 5-0 Vicryl. All of the slings had the same size and were placed in the same position in all rats. The animals were evaluated every day for possible signs of urinary retention and given an intramuscular injection of cefazolin daily for 3 days. At 6 weeks after sling implantation, the rats underwent LPP measurement. All the surgical procedures were performed under sterile condition (Fig. 1).
Int Urol Nephrol (2015) 47:1303–1310
1305
Fig. 1 Sling operation on a female urinary incontinence rat model, a decellularized dermal graft, b sling placement surgery, c pudendal nerve (yellow arrow) and sciatic nerve (blue arrow), d external genitalia following sling placement
Leak point pressure measurement LPP measurement was done 18 weeks after BPNT in group 1, 6 weeks after sling implantation for the group 2 and also group 3. According to the cystometric protocol described in our previous study, rats were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (15 mg/kg). After creating midline abdominal and dorsal upper thoracic incisions, a single-lumen 4.5-Fr catheter (bladder pressure catheter, MED5100; Mediplus, Cressex Business Park, Bucks, UK) was placed subcutaneously. The other end of the catheter was passed through the abdominal musculature and fixed in the bladder dome with purse-string non-absorbable sutures. Before closing the abdominal wall, the catheter was flushed with normal saline to check whether there was any leakage around the catheter. Finally, the incision was closed in a double layer using 5/0 absorbable suture. Then, the bladder catheter was connected to the urodynamic catheter, and the LPP measurement was performed via physiologic filling of the
bladder after overhydrating the animal by intraperitoneal injection of 2-mL normal saline using urodynamic system (F.M. Wiest Medizintechnik, GmbH, Unterhaching, Germany). The measurements were obtained three times for each animal, and the mean was used for the analysis. The LPP measurement was performed by a practitioner who was blinded to group assignment [16]. Histological and immunohistochemistry (IHC) staining For histological evaluations, samples of the urinary sphincter from group 1 (BPNT) and the sling surrounding urethra from group 2 (BPNT/vaginal sling) and group 3 (control) were fixed in 4 % buffered formalin (PH 7.2) for 24 h and then embedded in paraffin. For evaluating tissue structure and organization, hematoxylin and eosin (H&E) and Masson trichrome staining were performed using the standard method. IHC staining was performed using the standard method [17]. Sections from paraffin-embedded samples were prepared, and
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heated slides were submerged in 30 % (v/v) rabbit serum in PBS for 30 min at room temperature and incubated with primary antibodies, which were rinsed in a PBS solution containing 2 % (v/v) bovine serum albumin, in a humidified chamber for 1 h at 37 °C. In the immunohistochemical staining, primary antibodies including desmin for the sphincter samples of group 1 and 3 and CD34 and smooth muscle actin (α-SMA) for the samples of group 2 were applied. CD34 and α-SMA were applied for identification of endothelial progenitor cells and smooth muscle cells, respectively. All of the specimens were reviewed by a pathologist who was blinded to the groups. Images were processed and analyzed using Image J 1.48n software (National Institutes of Health, Bethesda, MD). Analysis was performed using threshold analysis. We used the option measure to calculate the area fraction on the examined areas. The percentage of IHC staining was calculated by dividing the IHC stained area over the total area.
Int Urol Nephrol (2015) 47:1303–1310 Table 1 Comparison of LPP between groups Group comparison
LPP (cm H2O)
Group 1 Group 2
11.6 ± 2.8 27.2 ± 5.4 0.034
P valuea Group 2 Group 3 P valuea Group 1 Group 3 P valuea
27.2 ± 5.4 27.6 ± 5.9 0.832 11.6 ± 2.8 27.6 ± 5.9 0.027
Group 1 bilateral pudendal nerve transection (BPNT), Group 2 BPNT/sling, Group 3 control The bold numbers are significant P values LPP leak point pressure a
Mann–Whitney test
Histological and immunohistochemistry staining Statistical analysis Statistical analysis was performed with Statistical Package of Social Science software version 17 (SPSS Inc., Chicago, IL). Mann–Whitney test was executed for statistical comparisons of LPP values in different groups. Data were expressed as mean ± SD, and a P value
UROLOGY - ORIGINAL PAPER
Decellularized dermal strip as a suburethral sling in a rat model of stress urinary incontinence Abdol‑Mohammad Kajbafzadeh1 · Sarah Mozafarpour1 · Seyedeh Sanam Ladi Seyedian1 · Reza Khorramirouz1 · Haleh Nasser Hojjati2
Received: 23 March 2015 / Accepted: 1 June 2015 / Published online: 20 June 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Introduction To report the feasibility, efficacy and safety of decellularized dermal strip as a sling material in the pudendal denervated stress urinary incontinence (SUI) in an animal model. Methods Thirty female Sprague–Dawley rats were randomly allocated into three equal groups (n = 10). In group 1, rats underwent bilateral pudendal nerve transection (BPNT) with no vaginal sling placement. Group 2 underwent BPNT with homologous decellularized dermal strip placement as vaginal sling 12 weeks later. Group 3 was considered as controls to evaluate baseline leak point pressure (LPP). Leak point pressure was measured 18 weeks after BPNT in group 1 and 6 weeks after sling implantation in group 2. Sling and sphincter samples were prepared for histological and immunohistochemistry (IHC) staining. Results H&E staining of external urethral sphincter showed decrease in striated muscle layer in group 1 (BPNT) compared to control confirmed by desmin staining (45 % decrease in desmin staining). Sling implantation caused significant increase in mean LPP from 11.6 ± 2.8 cm H2O in group 1 (BPNT) to 27.2 ± 5.4 cm H2O in group 2 (BPNT/sling). There was no significant difference between LPP after the sling placement in group 2 and LPP in group
* Abdol‑Mohammad Kajbafzadeh [email protected] 1
Section of Tissue Engineering and Stem Cells Therapy, Pediatric Urology Research Center, Children’s Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Qarib’s Street, Keshavarz Boulevard, 1419433151 Tehran, Iran (IRI)
2
Department of Urology, Ziaeian Hospital, Tehran University of Medical Sciences, Tehran, Iran (IRI)
3 (27.2 ± 5.4 cm H2O vs. 27.6 ± 5.9, P = 0.832). IHC staining was positive for α-SMA and CD34. Conclusions Based on present results, this dermal strip could be considered as a potential sling material for treatment of SUI in this animal model. This study provides the basis for further investigation of the efficacy of biological decellularized scaffold as suburethral sling material. Keywords Leak point pressure · Pudendal nerve · Stress urinary incontinence · Sling · Urethra
Introduction Stress urinary incontinence (SUI), the most common type of urinary incontinence, is a major urological issue affecting millions of women worldwide [1]. It is defined as the involuntary leakage of urine during increased intra-abdominal pressure [2]. Therapeutic options for SUI range from conservative measurements such as pelvic floor exercise to medical treatment and surgical interventions. Suburethral slings remain the mainstay of surgical treatment for SUI. In this procedure, a strip of tissue or mesh material is inserted in order to support the bladder neck [3]. However, this procedure is sometimes accompanied by bothersome and serious complications and sometimes needs reoperations [4]. Various sling materials have been studied in anti-incontinence surgeries with variable degrees of local inflammatory reactions [5]. Selection of the sling material is a key issue to circumvent postsurgical complications. The ideal prosthesis for pelvic reconstructive surgeries should be a safe, mechanically stable, biocompatible material devoid of eliciting allergic or inflammatory reactions, which could be obtained in a sterile fashion with “off the shelf” availability [6]. Artificial sling materials have shown to be associated with higher complication rates such
13
1304
as infection and urethral erosion over biological materials, which have subsequently limited their use in selective populations [7]. Biological prosthesis including autologous materials (rectus fascia, vaginal wall, etc.), allografts (cadaveric tissues including dura mater and dermis) or xenografts (porcine small intestinal mucosa and porcine dermis) has their own limitations [8]. Pelvic tissue of patients with SUI usually does not have appropriate supporting quality. Additionally, autologous grafts require extra surgical incisions that further weaken the pelvic structures and increase morbidity [6]. It has been reported that in a substantial percent of women, usage of pelvic fascia as a suburethral sling material has caused comorbid pelvic organ dysfunction such as vaginal or rectal prolapse [9, 10]. Also, comparable results have been reported regarding patients’ satisfaction and improved quality of life with dermal allografts [11]. On the other hand, allografts and xenografts have retained antigenicity and harbor a risk of disease transmission [8, 12]. Recently, preliminary reports of tissue-engineered materials have emerged in the literature as potential alternatives for suburethral slings [13–15]. However, there are very limited data on their application in preclinical settings. In the present study, we seek to explore the biocompatibility and efficacy of decellularized dermal strip as a sling material in a sphincter-damaged rat model of SUI. The aim of this study was to find out whether a decellularized dermal strip can be used as a suburethral sling material and also to evaluate leak point pressure (LPP) and histological changes of this decellularized dermal strip after placement.
Materials and methods This experimental animal study protocol was approved by the committee on animal welfare in Tehran University of Medical Sciences. All animals were treated based on the recommendations of the animal care committee of the university and in accordance with the Guide for Care and Use of Laboratory Animals (National Research Council, revised in 1996). The animals were placed in a temperature-controlled room, with free access to food and water. Thirty female Sprague–Dawley rats weighing 210– 240 g were randomly allocated into three groups (n = 10). In group 1, rats underwent bilateral pudendal nerve transection (BPNT) with no vaginal sling implantation. Group 2 underwent BPNT with homologous decellularized dermal strip placement as a vaginal sling. Group 3 considered as controls to evaluate baseline LPP with no intervention. Decellularized skin preparation The slings were prepared using decellularized rat skin. Female rats were anesthetized with ketamine and used as
13
Int Urol Nephrol (2015) 47:1303–1310
skin donors. Dorsal hair was first shaved and then removed more completely by surgical hair removal cream, the skin was scrubbed by Betadine® and sections with the size of 0.5 × 2 cm and 3-mm thickness were removed. Then, the adjacent parts were sutured. The removed skins were washed with sterile phosphatebuffered saline (PBS) and then treated by 0.25 % sodium dodecyl sulfate (SDS; Sigma, Germany) at room temperature for 48 h with continuous shaking. Subsequently, the sections were washed with PBS and then stored in sterile PBS solution at 4 °C before implantation. All the solutions contained 1 % antibiotics (i.e., penicillin, gentamycin and amphotericin), and all the procedures were done aseptically. Surgical procedure For BPNT, the rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (15 mg/kg). In a prone position, ischiorectal fossa was exposed bilaterally by a dorsal midline incision over the lumbosacral area. Fascia and coccygeus muscles were dissected, and the sciatic nerve was exposed crossing the overlying fascia superomedially to inferolaterally. Using a surgical microscope, BPNT was performed by concomitant transection of pudendal nerve trunk and anastomotic branch of the sciatic nerve. A 2-cm segment of the proximal pudendal nerve trunk was removed on each side. Coccygeus muscle, fascia and skin were closed one by one. The animals received preoperative and postoperative cefazolin to prevent infection. After 18 weeks of BPNT, rats in group 1 underwent LPP measurement. For group 2, vaginal sling placement was performed 12 weeks after BPNT. For the sling procedure, the rats were anesthetized as described before and placed in a supine position. The urethra was catheterized with a PE-50 catheter. The urethra–vaginal space was dissected horizontally with 1-cm incision, and the space was dissected more deeply, and the decellularized strip was placed without any attachments to the adjacent bone or fascia. The graft was marked with a 5-0 Prolene at each corner of the decellularized skin (0.5 × 2 cm), and the sling was placed below the midurethra. The fascia between the vagina and urethra was closed using 5-0 Vicryl. All of the slings had the same size and were placed in the same position in all rats. The animals were evaluated every day for possible signs of urinary retention and given an intramuscular injection of cefazolin daily for 3 days. At 6 weeks after sling implantation, the rats underwent LPP measurement. All the surgical procedures were performed under sterile condition (Fig. 1).
Int Urol Nephrol (2015) 47:1303–1310
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Fig. 1 Sling operation on a female urinary incontinence rat model, a decellularized dermal graft, b sling placement surgery, c pudendal nerve (yellow arrow) and sciatic nerve (blue arrow), d external genitalia following sling placement
Leak point pressure measurement LPP measurement was done 18 weeks after BPNT in group 1, 6 weeks after sling implantation for the group 2 and also group 3. According to the cystometric protocol described in our previous study, rats were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (15 mg/kg). After creating midline abdominal and dorsal upper thoracic incisions, a single-lumen 4.5-Fr catheter (bladder pressure catheter, MED5100; Mediplus, Cressex Business Park, Bucks, UK) was placed subcutaneously. The other end of the catheter was passed through the abdominal musculature and fixed in the bladder dome with purse-string non-absorbable sutures. Before closing the abdominal wall, the catheter was flushed with normal saline to check whether there was any leakage around the catheter. Finally, the incision was closed in a double layer using 5/0 absorbable suture. Then, the bladder catheter was connected to the urodynamic catheter, and the LPP measurement was performed via physiologic filling of the
bladder after overhydrating the animal by intraperitoneal injection of 2-mL normal saline using urodynamic system (F.M. Wiest Medizintechnik, GmbH, Unterhaching, Germany). The measurements were obtained three times for each animal, and the mean was used for the analysis. The LPP measurement was performed by a practitioner who was blinded to group assignment [16]. Histological and immunohistochemistry (IHC) staining For histological evaluations, samples of the urinary sphincter from group 1 (BPNT) and the sling surrounding urethra from group 2 (BPNT/vaginal sling) and group 3 (control) were fixed in 4 % buffered formalin (PH 7.2) for 24 h and then embedded in paraffin. For evaluating tissue structure and organization, hematoxylin and eosin (H&E) and Masson trichrome staining were performed using the standard method. IHC staining was performed using the standard method [17]. Sections from paraffin-embedded samples were prepared, and
13
1306
heated slides were submerged in 30 % (v/v) rabbit serum in PBS for 30 min at room temperature and incubated with primary antibodies, which were rinsed in a PBS solution containing 2 % (v/v) bovine serum albumin, in a humidified chamber for 1 h at 37 °C. In the immunohistochemical staining, primary antibodies including desmin for the sphincter samples of group 1 and 3 and CD34 and smooth muscle actin (α-SMA) for the samples of group 2 were applied. CD34 and α-SMA were applied for identification of endothelial progenitor cells and smooth muscle cells, respectively. All of the specimens were reviewed by a pathologist who was blinded to the groups. Images were processed and analyzed using Image J 1.48n software (National Institutes of Health, Bethesda, MD). Analysis was performed using threshold analysis. We used the option measure to calculate the area fraction on the examined areas. The percentage of IHC staining was calculated by dividing the IHC stained area over the total area.
Int Urol Nephrol (2015) 47:1303–1310 Table 1 Comparison of LPP between groups Group comparison
LPP (cm H2O)
Group 1 Group 2
11.6 ± 2.8 27.2 ± 5.4 0.034
P valuea Group 2 Group 3 P valuea Group 1 Group 3 P valuea
27.2 ± 5.4 27.6 ± 5.9 0.832 11.6 ± 2.8 27.6 ± 5.9 0.027
Group 1 bilateral pudendal nerve transection (BPNT), Group 2 BPNT/sling, Group 3 control The bold numbers are significant P values LPP leak point pressure a
Mann–Whitney test
Histological and immunohistochemistry staining Statistical analysis Statistical analysis was performed with Statistical Package of Social Science software version 17 (SPSS Inc., Chicago, IL). Mann–Whitney test was executed for statistical comparisons of LPP values in different groups. Data were expressed as mean ± SD, and a P value
