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Journal of Pediatric Urology (2015) xx, 1.e1e1.e6
The effect of penile urethral fat graft application on urethral angiogenesis M. Cakmak a, I. Yazıcı b, O. Boybeyi c, S. Ayva d, M.K. Aslan c, M.F. Senyucel c, T. Soyer e a
Ankara University, Medical Faculty, Department of Pediatric Surgery, Ankara, Turkey
b
Kırıkkale University, Medical Faculty, Department of Reconstructive Surgery, Kırıkkale, Turkey
c Kırıkkale University, Medical Faculty, Department of Pediatric Surgery, Kırıkkale, Turkey
d
Baskent University, Medical Faculty, Department of Pathology, Ankara, Turkey
e
Hacettepe University, Medical Faculty, Department of Pediatric Surgery, Ankara, Turkey Correspondence to: M. Cakmak, Ankara University, Faculty of Medicine, Department of Pediatric Surgery, Ankara, Turkey, Tel.: þ90 532 3510811 [email protected] (M. Cakmak) [email protected] (I. Yazıcı) [email protected] (O. Boybeyi) [email protected] (S. Ayva) [email protected] (M.K. Aslan) [email protected] (M.F. Senyucel) [email protected] (T. Soyer) Keywords Hypospadias; Urethra; Fat graft; Angiogenesis Received 18 October 2014 Accepted 17 March 2015 Available online xxx
Summary Background Autologous fat grafts are rich in adipose-derived stem cells, providing optimal soft-tissue replacement and significant quantities of angiogenic growth factor. Although fat grafts (FG) are used in several clinical conditions, the use of FG in urethral repairs and the effects of FG to urethral repairs have not yet been reported. Objective An experimental study was performed to evaluate the effect of FG on urethral angiogenesis and tissue growth factor (GF) levels. Study design Sixteen Wistar albino, adult, male rats were allocated into two groups: the control group (CG) (n Z 8) and the experiment group (EG) (n Z 8). After anesthetization of all rats, 3-mm vertical incisions were made on the urethras, and then sutured with interrupted 5/0 vicryl sutures. The operations were performed under a stereo dissecting microscope under magnification (20). In the CG, no additional procedure was performed. In the EG after the same surgical procedure, 1 mm3 FG was removed from the inguinal region by sharp dissection with a knife. The grafts were trimmed to 1 1 mm dimensions on millimeter paper. The FGs were placed on the repaired urethras. The skin was then closed. Samples from urethral and penile skin were taken 21 days after surgery in both groups. Density and intensity of staining with vascular-endothelial GF (VEGF), VEGF-receptor, and endothelial-GF receptor
(EGFR) in the endothelial and mesenchymal cells of the penile urethral vessels were immunohistochemically evaluated. Data obtained from immunohistochemical evaluations were analyzed with SPSS 15.0. The P-values lower than 0.05 were considered as significant. Results Density of VEGF staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). Density of the EGFR staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05) (Table). Intensity of VEGF, VEGF-R and EGFR staining was not significantly different between the two groups. There were no significant differences between groups regarding to VEGFR staining and mesenchymal examination. Discussion Decreased density was found in the VEGF staining in the vascular endothelium. This could be explained by the day that the tissues were harvested or because autologous fat grafts might cause decreased growth factor levels, which is contrary to the literature data. Conclusion Fat grafting has an immunohistochemical effect on the growth factor levels that are related to angiogenesis after urethral repair. It is difficult to make a firm conclusion about the role of fat grafting on urethral healing. Therefore, future studies are needed to see if FG can be used as an alternative to other procedures in order to avoid complications.
Table The median values of density grades of VEGF, VEGF-R, and EGF staining in the vascular endothelium (interquartile ranges within brackets).
VEGF VEGF-R EGF
Control group
Experimental group
2.0 (1.75e2.0) a 1.50 (0e2.25) 2.0 (1.75e2.0) b
1.0 (0e1.0) a 0.50 (0e1.0) 1.0 (0e1.25) b
a, b, P < 0.05. vascular-endothelial growth factor: VEGF, VEGF-receptor: VEGF-R, endothelial-growth factor receptor: EGF. http://dx.doi.org/10.1016/j.jpurol.2015.03.014 1477-5131/ª 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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Introduction Reconstruction of the urethra is one of the most challenging issues in pediatric urology. It is mostly required to treat congenital urinary anomalies e mostly hypospadias e and less commonly in traumatic defects or urethral strictures [1]. A variety of surgical techniques for urethral repair, especially in hypospadias, have been introduced in order to get satisfying results after surgery. Besides some wellknown issues, including tension-free anastomosis, gentle tissue handling, using absorbable suture materials and covering with well-vascularized tissue, several surgical grafts or flaps have also been developed to minimize the complication rates [2,3]. Despite all of the advances in surgical techniques, complications after urethral repair are still problematic, indicating the presence of congenital or structural anomalies in the penile urethra and prepuce of hypospadiac children [4e7]. Autologous fat transfers have been widely used for improving the quality of underlying soft tissue, especially in plastic surgery [8]. These fat grafts (FG) can be injected into another tissue/organ as a free graft. They are rich in adipose-derived stem cells, providing optimal soft-tissue replacement. Fat grafts have also been reported to contain significant quantities of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), which have a possible role in wound healing [8,9]. Although these grafts are used in several clinical conditions, the use and effects of FG in urethral repair have not yet been reported. Therefore, an experimental study was performed to evaluate the effect of FG on urethral angiogenesis and tissue growth factor (GF) levels.
Materials and methods The experiments were performed after approval by the Local Ethical Committee and the study was supported by Kırıkkale University Scientific Research Council (KU-2011/ 47).
M. Cakmak et al. receptor, endothelial-GF receptor (EGFR) in the endothelial and mesenchymal cells of the penile urethral vessels were immunohistochemically evaluated. Data obtained from the immunohistochemical evaluations were analyzed with SPSS 15.0. The distribution between groups was analyzed with the KruskaleWallis test and the difference between two groups was evaluated with the ManneWhitney U test. The P-values lower than 0.05 were considered as significant.
Operation and postoperative care The rats were placed in the supine position. Their genitalia were prepared with poviodine-iodine. The operations were performed under stereo dissecting microscope (Amscope, China) magnification (20). A 23-gauge angiocath was used to catheterize the urethra. The penile skin was incised circumferentially at the level of the mucosa and hairy skin junction on the penis. In order to reach the urethra it was then degloved up to the glans. A 3-mm length vertical incision was performed on the urethra and then sutured with interrupted 5/0 vicryl sutures (Fig. 1). In the experimental group, the FG was then harvested from the inguinal fat pad. The inguinal regions of the animals were shaved and prepared with poviodine-iodine. The fat grafts were taken by sharp dissection with a knife. The grafts were prepared on cotton gauze before implanting onto the urethras. The excessive connective tissue was cleaned and grafts were trimmed to 1 1 mm dimensions on millimeter paper in order to standardize them (Fig. 2). The FGs were placed onto the repaired regions of the urethras. The skin was closed with interrupted 5/0 silk sutures. The repaired regions on the urethras were marked with non-absorbable sutures in all animals. All animals urinated well after the procedures. They were sacrificed on 21st postoperative day for sampling. The penises were harvested and fixed in 10% formalin.
Immunohistochemical evaluation Animals and experimental design Sixteen Wistar albino, male, adult rats, weighing between 250 and 300 g, were used in the study. They were housed in standard cages under the same environmental conditions. The rats were kept at 22 C room temperature and 12h day/night cycle. They were provided with tap water and standard food ad libitum. The rats were randomly divided into two groups: control group and experimental group. They were anesthetized with intraperitoneal ketamine hydrochloride (50 mg/kg, Ketalar, Eczacıbası, Istanbul, Turkey). In all animals, after anesthetization, 3-mm vertical incisions were performed on the urethra, and then they were sutured with interrupted 5/0 vicryl sutures. In the control group (CG) (n Z 8), no additional procedures were done. In the experiment group (EG) (n Z 8), 1 mm3 FG was taken from the inguinal region and placed onto the repaired urethra. In both groups, samples from the urethra and penile skin were taken 21 days after surgery. Density and intensity of staining with vascular-endothelial GF (VEGF), VEGF-
From paraffin embedded blocks, 4e6 mm sections were obtained. Sections were immunohistochemically stained using an automatic staining machine (Bond System, Leica Microsystems GmbH, Wetzlar, Germany) with a biotin-free Bond Polymer Define Detection System (Leica Microsystems GmbH, catalog no. DS9800, Germany). Vascular endothelial growth factor antibody (VEGF-Abcam; 1/50), vascular endothelial growth factor receptor-1 antibody (VEGFR1-Abcam; 1/50) and endothelial growth factor receptor antibody (EGFR, Abcam; 1/80) were used as primary antibodies. Ethylenediaminetetraacetic acid (EDTA) was used as the epitope retrieval solution for VEGFR-1 and EGFR antibodies. Citrate buffered solution (pH Z 6) was used as the antigen retrieval solution for VEGF antibodies. 3,30 -Diaminobenzidine (DAB) was used as the chromogen. Slides were washed between all steps in phosphate-buffered saline with a pH of 7.4. Placental tissues were used as a positive control for VEGF and VEGFR-1 and breast carcinoma sections were used for EGFR. Phosphate-buffered
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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Figure 1 Urethral repair in the control group. A: degloving to reach the urethra; B and C: 3-mm length vertical incision; D: suturing with interrupted 5/0 vicryl sutures.
Figure 2 Fat grafting procedure. A: the 1 1 mm fat graft preparation on millimeter paper; B: fat graft placement onto the repaired region of the urethra; C: fat graft stabilization.
saline (PBS) was performed instead of using a primary antibody as a negative control. Brown staining was considered as positive. During immunohistochemical evaluation, growth factor expressions in the penile tissues were examined in the subepithelium. Density and intensity of staining with VEGF, VEGF-R, EGFR in endothelial and mesenchymal cells of the penile urethral vessels were evaluated semi-quantitatively, as described by Hai et al. [10]. Grading was as follows: Intensity Grade 1: Mild (+) Grade 2: Moderate (++) Grade 3: High (+++) Density Degree 0: No staining Degree 1: 1e10% of the field stained Degree 2: 11e50% of the field stained
Degree 3: 51e75% of the field stained Degree 4: 76e100% of the field stained
Results In the harvested samples, all grafts survived. The immunohistochemical examination was performed at the previously marked grafting region with non-absorbable sutures. Density and intensity of VEGF, VEGF-R and EGFR staining were evaluated separately in the vascular endothelial and mesenchymal tissue samples. The results are shown in Table 1. The density of the VEGF staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). The density of EGFR staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). There were no significant between-group differences regarding the density of VEGF-R staining. The intensity of the VEGF, VEGF-R and
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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M. Cakmak et al.
Table 1 The median values of density and intensity grades of VEGF, VEGF-R, and EGF staining (interquartile ranges within brackets). Density in vascular endothelium Control Group VEGF 2 VEGF-R 1.50 EGF 2 Experimental Group VEGF 1 VEGF-R 0.50 EGF 1
Intensity in vascular endothelium
(1.75e2) a (0e2.25) (1.75e2) b
1 (1e2) 1 (0e2) 1 (1e1)
(0e1) a (0e1) (0e1.25) b
1 (0e1.25) 0.50 (0e1) 1 (0e1)
Density in mesenchymal tissue
Intensity in mesenchymal tissue
1 (1e1.25) 1.50 (0.75e2) 1 (0.75e1.25) 0.50 (0e1) 1 (1e2) 0.50 (0e1)
1 (1e1) 1 (0.75e1) 1 (0.75e1) 0.50 (0e1) 1 (1e1) 0.50 (0e1)
a, b: P < 0.05. vascular-endothelial growth factor: VEGF, VEGF-receptor: VEGF-R, endothelial-growth factor receptor: EGF.
EGFR staining was not significantly different between the groups. In the mesenchymal tissue examinations, there were no significant differences between the groups regarding both density and intensity of VEGF, VEGF-R and EGFR staining (Fig. 3).
Discussion Several surgical techniques have been used for successful urethral reconstruction. Although it is accepted that a wellvascularized flap is the best technique, this is not always possible due to lack of tissue around the urethra [1]. Therefore, free grafts are commonly required from several donor sites. The donor tissues used for urethral repair include: free penile or preputial skin, hairless skin, bladder mucosa, buccal mucosa, tunica vaginalis, peritoneum, intestinal mucosa and deep fascia [1e3]. There are also some reports using other possible choices, such as: acellular collagen matrix [11], cyanoacrylates [12], fibrin sealant [13], and platelet-rich fibrin [14]. However, it is unclear as to which one of these is ideal. The advances in urethral reconstruction techniques remain insufficient enough to decrease the complications, which are usually urethrocutaneous fistulas [3]. It is not clear as to why fistulas do or do not develop in some people. Some reports have revealed that a local deficiency of growth factor, such as vascular endothelial growth factor (VEGF) and endothelial growth factor (EGF), may play a role besides surgical techniques [3,4,7]. Local or systemic hormone therapy has controversial results in decreasing complication rates [3]. Soyer et al. used platelet-rich fibrin, which is platelet concentrate containing several growth factors that have role in wound healing and angiogenesis [14]. They suggested that it could be a successful alternative in urethral repair [14]. However, free fat grafts have not been used in urethral repair so far. Autologous fat is commonly used for soft tissue replacement in several conditions associated with soft tissue defects [8]. From literature searches, it is believed that it has not yet been used in urethral repairs. The advantages of using autologous tissue are that it causes less inflammation and allergic reactions. Additionally, fat grafts are rich in adipose-derived stem cells and angiogenic growth
factors [8,9]. They cause more-rapid revascularization in the grafted region [15,16]. Despite widespread usage of fat grafts, the long-term viability of transferred fat remains uncertain. Several studies have revealed that volume loss of grafted fat tissue is inevitable, despite apparently increased vascularization [9]. On the other hand, some studies have revealed that fibrosis is less commonly seen in fat grafted tissues [15]. However, the effect of fat graft on urethral repair is also unknown. The success of urethral reconstruction depends on the viability of the engrafted tissue, with excellent wound healing preventing recurrence of complications. A graft’s survival strictly depends on revascularization. Therefore, examination of angiogenesis, which is an important indication of wound healing, can be a predictor for engrafted tissue viability [15,16]. Vascular endothelial growth factor (VEGF) and endothelial growth factor (EGF) are some of the growth factors that have a role in wound healing. The vascular endothelial growth factor, angiogenic peptide, enhances angiogenesis and endothelial cell growth [4,9]. The VEGF is released in response to tissue hypoxia after grafting and causes revascularization, which is increased by the seventh day of wound healing [9]. It was reported in a study that VEGF release decreases after the 30th day of wound healing, but neo-vessels continue to survive [9]. Endothelial growth factor also plays an important role in cellular proliferation and wound healing [4]. In the present study, autologous fat grafts were used on the repaired urethras. The expression of common growth factors, such as VEGF and EGF staining, on the 21st day were evaluated, which is an appropriate time for wound healing and neo-vascular growth. Although it has been reported in previous studies that VEGF levels were increased after fat grafting [9], decreased density was found in the VEGF staining in the vascular endothelium. This could be explained by the day that the tissues were harvested, as this was the time that the VEGF levels started to decrease. Additionally, since the inflammatory process has already finished and growth factors have been exhausted in the tissue by the 21st day, the level of growth factors was low in fat grafted tissues. On the other hand, it is suggested that autologous fat grafts might cause decreased growth factor levels, which is contrary to the literature data. Therefore, future studies examining growth factor and other
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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Figure 3 Immunohistochemical examination of the vascular endothelial samples shows that the brown stained areas are positive (arrows) for 3a: vascular-endothelial growth factor (VEGF), 3b: VEGF-receptor (VEGF-R), 3c: endothelial-growth factor receptor (EGF) [left: control group, right: experimental group] (200, diaminobenzidine). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
inflammatory marker levels at different days of inflammation may clarify this issue.
Conclusion In conclusion, density of VEGF and EGFR staining in the EG was decreased at the late stages of healing. Judging from the immunohistochemical results of the present study, FG has an effect on the GF levels and is related to angiogenesis after urethral repair. The present study might inspire other researchers to study the dynamics of fat grafting on urethral tissue. It is difficult to have a firm conclusion about the role of fat grafting on urethral healing. Therefore, future studies are required in order to suggest whether fat grafts should to be
used as an alternative to other techniques in order to avoid complications.
Conflict of interest All authors accept that there are no financial or personal relationships with other people or organizations that could inappropriately influence (bias) their work. There is no conflict of interest.
Funding The study was supported by Kırıkkale University Scientific Council (KU-2011/47).
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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Ethical approval The experiments were performed after approval from the Local Ethical Committee.
Acknowledgements This study was presented at the 31st Congress of Turkish Pediatric Surgeons 2013, in Eskisehir, Turkey. It is supported by the Kırıkkale University Scientific Council (KU-2011/47).
References [1] Gardikis S, Giatromanolaki A, Ypsilantis P, Botaitis S, Perente S, Kambouri A, et al. Comparison of angiogenic activities after urethral reconstruction using free grafts in rabbits. Eur Urol 2005;47:417e21. [2] Muruganandham K, Ansarı MS, Dubey D, Mandhani A, Srivastava A, Kapoor R, et al. Urethrocutaneous fistula after hypospadias repair: outcome of three types of closure techniques. Pediatr Surg Int 2010;26:305e8. [3] Chung JW, Choi SH, Kim BS, Chung SK. Risk factors for the development of urethrocutaneous fistula after hypospadias repair: a retrospective study. Korean J Urol 2012;53:711e5. [4] Soyer T, Ayva S, Atasoy P, Aslan MK, Cakmak M. Comparison of growth factor levels in patients with normal and hypospadiac prepuce. Turk J Med Sci 2011;41:81e5. [5] Nazir Z, Masood R, Rehman R. Sensory innervation of normal and hypospadiac prepuce: possible implications in hypospadiology. Pediatr Surg Int 2004;20:623e7. [6] Yu ¨cel S, Gu ¨ntekin E, Kukul E, Karaguzel G, Ciftcioglu A, Melikoglu M, et al. Comparison of normal and hypospadiac preputial vascular anatomy. J Urol 2004;172:1973e6.
M. Cakmak et al. [7] El-Galley R, Smith E, Cohen C, Petros JA, Woodard J, Galloway NT. Epidermal growth factor (EGF) and EGF receptor in hypospadias. Br J Urol 1997;79:116e9. [8] Pu LLQ. Towards more rationalized approach to autologous fat grafting. J Plast Reconstr Aesthetic Surg 2012;65:413e9. [9] Nishimura T, Hashimoto H, Nahanishi I, Furukawa M. Microvascular angiogenesis and apoptosis in the survival of free fat grafts. Laryngoscope 2000;110:1333e8. [10] Hai J, Li S, Lin Q, Pan QG, Gao F, Ding MX. Vascular endothelial growth factor expression and angiogenesis induced by chronic cerebral hypoperfusion in rat brain. Neurosurgery 2003;53: 963e72. [11] Springer A, Subramaniam R. Preliminary experience with the use of acellular collagen matrix in redo surgery for urethrocutaneous fistula. Urology 2012;80:1156e60. [12] Prestipino M, Bertozzi M, Nardi N, Appignani A. Outpatient department repair urethrocutaneous fistulae using n-butylcyanoacrylate (NBCA): a single-centre experience. BJU Int 2011;108:1514e7. [13] Kajbafzadeh A, Abolghasemi H, Eshghi P, Alizadeh F, Elmi A, Shafaattalab S, et al. Single-donor fibrin sealant for repair of urethrocutaneous fistulae following multiple hypospadias and epispadias repairs. J Pediatr Urol 2011;7:422e7. ¨ . Use of [14] Soyer T, Cakmak M, Aslan MK, S‚enyu ¨cel MF, Kisa U autologous platelet rich fibrin in urethrocutaneous fistula repair: preliminary report. Int Wound J 2013;10:345e7. [15] Sultan SM, Barr JS, Butala P, Davidson EH, Weinstein AL, Knobel D, et al. Fat grafting accelerates revascularization and decreases fibrosis following thermal injury. J Plast Reconstr Aesthet Surg 2012;65:219e27. [16] Kambouri K, Gardikis S, Giatromanolaki A, Efstathiou E, Pitiakoudis M, Ipsilantis P, et al. Comparison of angiogenic activity after urethral reconstruction using fat grafts and pedicle flap: an experimental study. Eur J Pediatr Surg 2006; 16:323e8.
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Journal of Pediatric Urology (2015) xx, 1.e1e1.e6
The effect of penile urethral fat graft application on urethral angiogenesis M. Cakmak a, I. Yazıcı b, O. Boybeyi c, S. Ayva d, M.K. Aslan c, M.F. Senyucel c, T. Soyer e a
Ankara University, Medical Faculty, Department of Pediatric Surgery, Ankara, Turkey
b
Kırıkkale University, Medical Faculty, Department of Reconstructive Surgery, Kırıkkale, Turkey
c Kırıkkale University, Medical Faculty, Department of Pediatric Surgery, Kırıkkale, Turkey
d
Baskent University, Medical Faculty, Department of Pathology, Ankara, Turkey
e
Hacettepe University, Medical Faculty, Department of Pediatric Surgery, Ankara, Turkey Correspondence to: M. Cakmak, Ankara University, Faculty of Medicine, Department of Pediatric Surgery, Ankara, Turkey, Tel.: þ90 532 3510811 [email protected] (M. Cakmak) [email protected] (I. Yazıcı) [email protected] (O. Boybeyi) [email protected] (S. Ayva) [email protected] (M.K. Aslan) [email protected] (M.F. Senyucel) [email protected] (T. Soyer) Keywords Hypospadias; Urethra; Fat graft; Angiogenesis Received 18 October 2014 Accepted 17 March 2015 Available online xxx
Summary Background Autologous fat grafts are rich in adipose-derived stem cells, providing optimal soft-tissue replacement and significant quantities of angiogenic growth factor. Although fat grafts (FG) are used in several clinical conditions, the use of FG in urethral repairs and the effects of FG to urethral repairs have not yet been reported. Objective An experimental study was performed to evaluate the effect of FG on urethral angiogenesis and tissue growth factor (GF) levels. Study design Sixteen Wistar albino, adult, male rats were allocated into two groups: the control group (CG) (n Z 8) and the experiment group (EG) (n Z 8). After anesthetization of all rats, 3-mm vertical incisions were made on the urethras, and then sutured with interrupted 5/0 vicryl sutures. The operations were performed under a stereo dissecting microscope under magnification (20). In the CG, no additional procedure was performed. In the EG after the same surgical procedure, 1 mm3 FG was removed from the inguinal region by sharp dissection with a knife. The grafts were trimmed to 1 1 mm dimensions on millimeter paper. The FGs were placed on the repaired urethras. The skin was then closed. Samples from urethral and penile skin were taken 21 days after surgery in both groups. Density and intensity of staining with vascular-endothelial GF (VEGF), VEGF-receptor, and endothelial-GF receptor
(EGFR) in the endothelial and mesenchymal cells of the penile urethral vessels were immunohistochemically evaluated. Data obtained from immunohistochemical evaluations were analyzed with SPSS 15.0. The P-values lower than 0.05 were considered as significant. Results Density of VEGF staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). Density of the EGFR staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05) (Table). Intensity of VEGF, VEGF-R and EGFR staining was not significantly different between the two groups. There were no significant differences between groups regarding to VEGFR staining and mesenchymal examination. Discussion Decreased density was found in the VEGF staining in the vascular endothelium. This could be explained by the day that the tissues were harvested or because autologous fat grafts might cause decreased growth factor levels, which is contrary to the literature data. Conclusion Fat grafting has an immunohistochemical effect on the growth factor levels that are related to angiogenesis after urethral repair. It is difficult to make a firm conclusion about the role of fat grafting on urethral healing. Therefore, future studies are needed to see if FG can be used as an alternative to other procedures in order to avoid complications.
Table The median values of density grades of VEGF, VEGF-R, and EGF staining in the vascular endothelium (interquartile ranges within brackets).
VEGF VEGF-R EGF
Control group
Experimental group
2.0 (1.75e2.0) a 1.50 (0e2.25) 2.0 (1.75e2.0) b
1.0 (0e1.0) a 0.50 (0e1.0) 1.0 (0e1.25) b
a, b, P < 0.05. vascular-endothelial growth factor: VEGF, VEGF-receptor: VEGF-R, endothelial-growth factor receptor: EGF. http://dx.doi.org/10.1016/j.jpurol.2015.03.014 1477-5131/ª 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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Introduction Reconstruction of the urethra is one of the most challenging issues in pediatric urology. It is mostly required to treat congenital urinary anomalies e mostly hypospadias e and less commonly in traumatic defects or urethral strictures [1]. A variety of surgical techniques for urethral repair, especially in hypospadias, have been introduced in order to get satisfying results after surgery. Besides some wellknown issues, including tension-free anastomosis, gentle tissue handling, using absorbable suture materials and covering with well-vascularized tissue, several surgical grafts or flaps have also been developed to minimize the complication rates [2,3]. Despite all of the advances in surgical techniques, complications after urethral repair are still problematic, indicating the presence of congenital or structural anomalies in the penile urethra and prepuce of hypospadiac children [4e7]. Autologous fat transfers have been widely used for improving the quality of underlying soft tissue, especially in plastic surgery [8]. These fat grafts (FG) can be injected into another tissue/organ as a free graft. They are rich in adipose-derived stem cells, providing optimal soft-tissue replacement. Fat grafts have also been reported to contain significant quantities of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), which have a possible role in wound healing [8,9]. Although these grafts are used in several clinical conditions, the use and effects of FG in urethral repair have not yet been reported. Therefore, an experimental study was performed to evaluate the effect of FG on urethral angiogenesis and tissue growth factor (GF) levels.
Materials and methods The experiments were performed after approval by the Local Ethical Committee and the study was supported by Kırıkkale University Scientific Research Council (KU-2011/ 47).
M. Cakmak et al. receptor, endothelial-GF receptor (EGFR) in the endothelial and mesenchymal cells of the penile urethral vessels were immunohistochemically evaluated. Data obtained from the immunohistochemical evaluations were analyzed with SPSS 15.0. The distribution between groups was analyzed with the KruskaleWallis test and the difference between two groups was evaluated with the ManneWhitney U test. The P-values lower than 0.05 were considered as significant.
Operation and postoperative care The rats were placed in the supine position. Their genitalia were prepared with poviodine-iodine. The operations were performed under stereo dissecting microscope (Amscope, China) magnification (20). A 23-gauge angiocath was used to catheterize the urethra. The penile skin was incised circumferentially at the level of the mucosa and hairy skin junction on the penis. In order to reach the urethra it was then degloved up to the glans. A 3-mm length vertical incision was performed on the urethra and then sutured with interrupted 5/0 vicryl sutures (Fig. 1). In the experimental group, the FG was then harvested from the inguinal fat pad. The inguinal regions of the animals were shaved and prepared with poviodine-iodine. The fat grafts were taken by sharp dissection with a knife. The grafts were prepared on cotton gauze before implanting onto the urethras. The excessive connective tissue was cleaned and grafts were trimmed to 1 1 mm dimensions on millimeter paper in order to standardize them (Fig. 2). The FGs were placed onto the repaired regions of the urethras. The skin was closed with interrupted 5/0 silk sutures. The repaired regions on the urethras were marked with non-absorbable sutures in all animals. All animals urinated well after the procedures. They were sacrificed on 21st postoperative day for sampling. The penises were harvested and fixed in 10% formalin.
Immunohistochemical evaluation Animals and experimental design Sixteen Wistar albino, male, adult rats, weighing between 250 and 300 g, were used in the study. They were housed in standard cages under the same environmental conditions. The rats were kept at 22 C room temperature and 12h day/night cycle. They were provided with tap water and standard food ad libitum. The rats were randomly divided into two groups: control group and experimental group. They were anesthetized with intraperitoneal ketamine hydrochloride (50 mg/kg, Ketalar, Eczacıbası, Istanbul, Turkey). In all animals, after anesthetization, 3-mm vertical incisions were performed on the urethra, and then they were sutured with interrupted 5/0 vicryl sutures. In the control group (CG) (n Z 8), no additional procedures were done. In the experiment group (EG) (n Z 8), 1 mm3 FG was taken from the inguinal region and placed onto the repaired urethra. In both groups, samples from the urethra and penile skin were taken 21 days after surgery. Density and intensity of staining with vascular-endothelial GF (VEGF), VEGF-
From paraffin embedded blocks, 4e6 mm sections were obtained. Sections were immunohistochemically stained using an automatic staining machine (Bond System, Leica Microsystems GmbH, Wetzlar, Germany) with a biotin-free Bond Polymer Define Detection System (Leica Microsystems GmbH, catalog no. DS9800, Germany). Vascular endothelial growth factor antibody (VEGF-Abcam; 1/50), vascular endothelial growth factor receptor-1 antibody (VEGFR1-Abcam; 1/50) and endothelial growth factor receptor antibody (EGFR, Abcam; 1/80) were used as primary antibodies. Ethylenediaminetetraacetic acid (EDTA) was used as the epitope retrieval solution for VEGFR-1 and EGFR antibodies. Citrate buffered solution (pH Z 6) was used as the antigen retrieval solution for VEGF antibodies. 3,30 -Diaminobenzidine (DAB) was used as the chromogen. Slides were washed between all steps in phosphate-buffered saline with a pH of 7.4. Placental tissues were used as a positive control for VEGF and VEGFR-1 and breast carcinoma sections were used for EGFR. Phosphate-buffered
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Figure 1 Urethral repair in the control group. A: degloving to reach the urethra; B and C: 3-mm length vertical incision; D: suturing with interrupted 5/0 vicryl sutures.
Figure 2 Fat grafting procedure. A: the 1 1 mm fat graft preparation on millimeter paper; B: fat graft placement onto the repaired region of the urethra; C: fat graft stabilization.
saline (PBS) was performed instead of using a primary antibody as a negative control. Brown staining was considered as positive. During immunohistochemical evaluation, growth factor expressions in the penile tissues were examined in the subepithelium. Density and intensity of staining with VEGF, VEGF-R, EGFR in endothelial and mesenchymal cells of the penile urethral vessels were evaluated semi-quantitatively, as described by Hai et al. [10]. Grading was as follows: Intensity Grade 1: Mild (+) Grade 2: Moderate (++) Grade 3: High (+++) Density Degree 0: No staining Degree 1: 1e10% of the field stained Degree 2: 11e50% of the field stained
Degree 3: 51e75% of the field stained Degree 4: 76e100% of the field stained
Results In the harvested samples, all grafts survived. The immunohistochemical examination was performed at the previously marked grafting region with non-absorbable sutures. Density and intensity of VEGF, VEGF-R and EGFR staining were evaluated separately in the vascular endothelial and mesenchymal tissue samples. The results are shown in Table 1. The density of the VEGF staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). The density of EGFR staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). There were no significant between-group differences regarding the density of VEGF-R staining. The intensity of the VEGF, VEGF-R and
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Table 1 The median values of density and intensity grades of VEGF, VEGF-R, and EGF staining (interquartile ranges within brackets). Density in vascular endothelium Control Group VEGF 2 VEGF-R 1.50 EGF 2 Experimental Group VEGF 1 VEGF-R 0.50 EGF 1
Intensity in vascular endothelium
(1.75e2) a (0e2.25) (1.75e2) b
1 (1e2) 1 (0e2) 1 (1e1)
(0e1) a (0e1) (0e1.25) b
1 (0e1.25) 0.50 (0e1) 1 (0e1)
Density in mesenchymal tissue
Intensity in mesenchymal tissue
1 (1e1.25) 1.50 (0.75e2) 1 (0.75e1.25) 0.50 (0e1) 1 (1e2) 0.50 (0e1)
1 (1e1) 1 (0.75e1) 1 (0.75e1) 0.50 (0e1) 1 (1e1) 0.50 (0e1)
a, b: P < 0.05. vascular-endothelial growth factor: VEGF, VEGF-receptor: VEGF-R, endothelial-growth factor receptor: EGF.
EGFR staining was not significantly different between the groups. In the mesenchymal tissue examinations, there were no significant differences between the groups regarding both density and intensity of VEGF, VEGF-R and EGFR staining (Fig. 3).
Discussion Several surgical techniques have been used for successful urethral reconstruction. Although it is accepted that a wellvascularized flap is the best technique, this is not always possible due to lack of tissue around the urethra [1]. Therefore, free grafts are commonly required from several donor sites. The donor tissues used for urethral repair include: free penile or preputial skin, hairless skin, bladder mucosa, buccal mucosa, tunica vaginalis, peritoneum, intestinal mucosa and deep fascia [1e3]. There are also some reports using other possible choices, such as: acellular collagen matrix [11], cyanoacrylates [12], fibrin sealant [13], and platelet-rich fibrin [14]. However, it is unclear as to which one of these is ideal. The advances in urethral reconstruction techniques remain insufficient enough to decrease the complications, which are usually urethrocutaneous fistulas [3]. It is not clear as to why fistulas do or do not develop in some people. Some reports have revealed that a local deficiency of growth factor, such as vascular endothelial growth factor (VEGF) and endothelial growth factor (EGF), may play a role besides surgical techniques [3,4,7]. Local or systemic hormone therapy has controversial results in decreasing complication rates [3]. Soyer et al. used platelet-rich fibrin, which is platelet concentrate containing several growth factors that have role in wound healing and angiogenesis [14]. They suggested that it could be a successful alternative in urethral repair [14]. However, free fat grafts have not been used in urethral repair so far. Autologous fat is commonly used for soft tissue replacement in several conditions associated with soft tissue defects [8]. From literature searches, it is believed that it has not yet been used in urethral repairs. The advantages of using autologous tissue are that it causes less inflammation and allergic reactions. Additionally, fat grafts are rich in adipose-derived stem cells and angiogenic growth
factors [8,9]. They cause more-rapid revascularization in the grafted region [15,16]. Despite widespread usage of fat grafts, the long-term viability of transferred fat remains uncertain. Several studies have revealed that volume loss of grafted fat tissue is inevitable, despite apparently increased vascularization [9]. On the other hand, some studies have revealed that fibrosis is less commonly seen in fat grafted tissues [15]. However, the effect of fat graft on urethral repair is also unknown. The success of urethral reconstruction depends on the viability of the engrafted tissue, with excellent wound healing preventing recurrence of complications. A graft’s survival strictly depends on revascularization. Therefore, examination of angiogenesis, which is an important indication of wound healing, can be a predictor for engrafted tissue viability [15,16]. Vascular endothelial growth factor (VEGF) and endothelial growth factor (EGF) are some of the growth factors that have a role in wound healing. The vascular endothelial growth factor, angiogenic peptide, enhances angiogenesis and endothelial cell growth [4,9]. The VEGF is released in response to tissue hypoxia after grafting and causes revascularization, which is increased by the seventh day of wound healing [9]. It was reported in a study that VEGF release decreases after the 30th day of wound healing, but neo-vessels continue to survive [9]. Endothelial growth factor also plays an important role in cellular proliferation and wound healing [4]. In the present study, autologous fat grafts were used on the repaired urethras. The expression of common growth factors, such as VEGF and EGF staining, on the 21st day were evaluated, which is an appropriate time for wound healing and neo-vascular growth. Although it has been reported in previous studies that VEGF levels were increased after fat grafting [9], decreased density was found in the VEGF staining in the vascular endothelium. This could be explained by the day that the tissues were harvested, as this was the time that the VEGF levels started to decrease. Additionally, since the inflammatory process has already finished and growth factors have been exhausted in the tissue by the 21st day, the level of growth factors was low in fat grafted tissues. On the other hand, it is suggested that autologous fat grafts might cause decreased growth factor levels, which is contrary to the literature data. Therefore, future studies examining growth factor and other
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Figure 3 Immunohistochemical examination of the vascular endothelial samples shows that the brown stained areas are positive (arrows) for 3a: vascular-endothelial growth factor (VEGF), 3b: VEGF-receptor (VEGF-R), 3c: endothelial-growth factor receptor (EGF) [left: control group, right: experimental group] (200, diaminobenzidine). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
inflammatory marker levels at different days of inflammation may clarify this issue.
Conclusion In conclusion, density of VEGF and EGFR staining in the EG was decreased at the late stages of healing. Judging from the immunohistochemical results of the present study, FG has an effect on the GF levels and is related to angiogenesis after urethral repair. The present study might inspire other researchers to study the dynamics of fat grafting on urethral tissue. It is difficult to have a firm conclusion about the role of fat grafting on urethral healing. Therefore, future studies are required in order to suggest whether fat grafts should to be
used as an alternative to other techniques in order to avoid complications.
Conflict of interest All authors accept that there are no financial or personal relationships with other people or organizations that could inappropriately influence (bias) their work. There is no conflict of interest.
Funding The study was supported by Kırıkkale University Scientific Council (KU-2011/47).
Please cite this article in press as: Cakmak M, et al., The effect of penile urethral fat graft application on urethral angiogenesis, Journal of Pediatric Urology (2015), http://dx.doi.org/10.1016/j.jpurol.2015.03.014
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Ethical approval The experiments were performed after approval from the Local Ethical Committee.
Acknowledgements This study was presented at the 31st Congress of Turkish Pediatric Surgeons 2013, in Eskisehir, Turkey. It is supported by the Kırıkkale University Scientific Council (KU-2011/47).
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