Abstract
BACKGROUND: At present, bladder acellular matrix grafts have been successfully used for substituting animal bladder and urinary canal, and for repairing hypospadia. However, reports on bladder acellular matrix grafts for substituting albuginea penis need to be investigated.

OBJECTIVE: Allogeneic bladder acellular grafts were used for substituting albuginea penis of rabbits, in order to observe repairing results.
DESIGN: A randomized controlled observation.

SETTING: West China Medical Laboratory Animal Center and West China Laboratory of Tissue Engineering of Sichuan University as well as Laboratory of Tissue Engineering of Guiyang Medical College.

MATERIALS: Fifty male healthy New Zealand Rabbits of grade 3, weighing 2.6–3.0 kg, without phimosis and penis dysplasia, and without presence of phallocampsis after normal saline being perfused, were provided by Huaxi Laboratory Animal Center of Sichuan University.

METHODS: This study was performed at the West China Laboratory Animal Center and West China Laboratory of Tissue Engineering of Sichuan University as well as Laboratory of Tissue Engineering of Guiyang Medical College between December 2005 and June 2007. Bladders were taken from 10 experimental rabbits for preparing bladder acellular matrix grafts. The other 40 New Zealand rabbits were randomly divided into the control group, and the bladder acellular matrix grafts group, with 20 in each. An area of 10 mm×5 mm of albuginea penis was resected from dorsum penis of each rabbit. Suture in situ of albuginea penis and bladder acellular matrix grafting were conducted in rabbits of the control group and bladder acellular matrix grafts group, respectively. In the 2nd, 6th, 12th and 24th weeks postoperatively, each rabbit was intracavernously perfused normal saline for inducing penile erection, separately, in order to observe phallocampsis. At above-mentioned each time point, experimental animals were sacrificed. Sample was taken from surgical region for haematoxylin-eosin (HE) staining and Masson trichrome staining, in order to observe the changes of tissue and structure of surgical region. Types Ⅰand Ⅲ collagen fiber areas were detected by Stirus red staining, and the expressions of inducible nitric oxide synthase(iNOS) and transforming growth factor-β1 (TGF-β1) were detected by immunohistochemical staining.

MAIN OUTCOME MEASURES: ①Phallocampsis status. ② Changes of tissue and structure of surgical region. ③iNOS and TGF-β1 expressions. ④TypeⅠand Ⅲ collagen fiber areas.

RESULTS: Forty experimental rabbits were involved in the penile surgery, two of them died from overdose anesthesia, two died from chordapsus, so the remaining thirty-six rabbits were involved in the final analysis. In the 6th week postoperatively, phallocampsis reached its highest level, and 2 rabbits in the control group and 1 rabbit in the bladder acellular matrix grafts group presented phallocampsis. In the 12th week, every rabbit presented phallocampsis. In the 24th week, 1 rabbit in the control group but none in the bladder acellular matrix grafts group presented phallocampsis. In the 2nd week, the structure of surgical regions of each rabbit was poorly clear, with remarkable inflammatory infiltration. In the bladder acellular matrix grafts group, grafting regions presented cells ingrowing the bladder acellular matrix grafts. Masson trichrome staining results showed that in the surgical region, tunica albuginea fibers were thin and poorly arranged. In the 6th week, tunica albuginea recovered its integrity, and bladder acellular matrix grafts could not be distinguished. No significant difference existed between two groups. In the 24th week, tunica albuginea was even and complete in the sugical region, and fibers restored their arrangement of circular muscle in inner layer and longitudinal muscle in outer layer, without difference from normal tunica albuginea. iNOS and TGF-β1 expressions were the strongest in the 2nd week, and they were found in the fibrocytes and vascular endothelial cells in the 6th week, but a little in the 12th and 24th weeks postoperatively. There were no remarkable differences in iNOS and TGF-β1 expressions between two groups at the same time point. In the 2nd week, typesⅠand Ⅲ collagen fibers co-existed with equivalent proportion. Then, typeⅠcollagen fibers were gradually increased, while type Ⅲ collagen fibers were on the contrary. In the 24th week, typeⅠcollagen fibers took the main place and type Ⅲ collagen fibers were unremarkable.

CONCLUSION: Bladder acellular matrix grafts have no remarkable inflammatory reactions and fibrosis in repairing tunica albuginea of New Zealand rabbits, so they are very ideal grafting materials for penile surgery.

INTRODUCTION

Quantitative analysis of experimental animals
Forty experimental rabbits were involved in the penile surgery, two of them died from overdose anesthesia, two died from chordapsus, so the other thirty-six rabbits were involved in the final analysis.

Gross observation of samples
Bladder acellular matrix grafts presented white semi-transparent membrane. In the 2nd week postoperatively, grafting regions in the two groups had obvious scars. In the 6th and 12th weeks, the scars gradually became small, and the structures of tunica albuginea and spongy body were clear. In the 24th week, tunica albuginea of surgical region was complete with smooth and even boundary, which could not be separated from normal tunica albuginea. There was no marked difference between two groups at each time point.

Observation of tissue structure of surgical regions
In the 2nd week postoperatively, the structure of surgical regions of rabbits in two groups were poorly clear, with remarkable inflammatory infiltration. In the bladder acellular matrix grafts group, grafting regions presented cells ingrowing the bladder acellular matrix grafts (Figure 1a). Masson trichrome staining results showed that in the surgical region, tunica albuginea fibers were thin and poorly arranged (Figure 1b). In the 6th week postoperatively, tunica albuginea recovered its integrity, and bladder acellular matrix grafts could not be distinguished. No significant difference existed between two groups. In the 24th week, tunica albuginea was even and complete in the surgical region, and fibers restored their arrangement of circular muscle in inner layer and longitudinal muscle in outer layer, without difference from normal tunica albuginea (Figure 2).

Detection of typesⅠand Ⅲ collagen fibers
In the 2nd week postoperatively, typesⅠand Ⅲ collagen fibers co-existed with equivalent proportion (Figure 3a,b). Then, typeⅠcollagen fibers were gradually increased, while type Ⅲ collagen fibers were on the contrary. In the 24th week, typeⅠcollagen fibers took the main place and type Ⅲ collagen fibers were unremarkable (Figure 3c,d).
Immunohistochemical observation
iNOS and TGF-β1 expressions were the strongest in the 2nd week postoperatively, and they were found in the fibrocytes and vascular endothelial cells in the 6th week postoperatively, but a little in the 12th and 24th weeks (Figure 4 c, d). There were no remarkable differences in iNOS and TGF-β1 expressions between two groups at the same time point.

Observation of phallocampsis
In the 6th week postoperatively, phallocampsis reached its highest level, and 2 rabbits in the control group and 1 rabbit in the bladder acellular matrix grafts group presented phallocampsis. In the 12th week, every rabbit presented phallocampsis. In the 24th week, 1 rabbit in the control group but none in the bladder acellular matrix grafts group presented phallocampsis.

DISCUSSION

Hypospadia, congenital penile dysplasia and other penile lesions lead to chordee and dyspareunia. Phallocampsis is corrected by enlarging or reconstructing tunica albuginea in the diseased region. The grafting materials include autologous tissues (such as dermis, perididymis, vagina femoris, and great saphenous vein)[1-4], xenogenic tissues (such as human and animal cardial sacs [5]) and artificial synthetic materials. The sample harvesting of autologous grafting material will cause the injury of another region, and grafting material will present scar shrinkage with time going, accordingly, phallocampsis reoccurs. Both corpse tissues and artificial materials have the disadvantages of remarkable scar shrinkage, induration and so on. With the development of tissue engineering, some investigators are in attempt to reconstruct tunica-albuginea penis with acellular extracellular matrix, for example, submucous layer of porcine small intestine.
Bladder acellular matrix grafts are a kind of acellular biomaterials with abundant collagens. By observation under the electron microscope, we found that its surface presented space-like structure. In comparison with artificial polymer, bladder acellular matrix grafts have some cytokines, which are beneficial to cell growing, and provide the environment and fiber arrangement for cell growth and differentiation as well as tissue formation. Bladder acellular matrix grafts have been successfully used to substitute animal bladder and urinary canal, and to repair hypospadia[6-8]. Reports on bladder acellular matrix grafts for penile surgery have not been found at home and abroad.
Piechota et al [8] transplanted bladder acellular matrix grafts into hamster, rabbits and dogs, and they did not found immunological rejections. So they thought that bladder acellular matrix grafts are extracellular matrix without immunogenicity. In this study, we found that in the 2nd week, both groups presented slight scar formation and many inflammatory cells. Then, scar was gradually subsided. In the 24th week, the structure of tunica albuginea in the surgical region could not be differentiated from that of normal tunica albuginea. The expressions of iNOS and TGF-β1 were the strongest in the 2nd week, and were unconspicuous in the later time, demonstrating that the repair process of bladder acellular matrix grafts is similar to that of autologous tissue in reconstructing tunica albuginea. No noticeable inflammatory reactions and fibrosis were found in the repair of albuginea penis with bladder acellular matrix grafts, indicating that good histocompatibility exists.
It is also found in this study that in the process of repair of albuginea penis, the changes of fibrous components in two groups are very similar: Fibrous components are composed of types Ⅰand Ⅲ collagen fibers with comparable proportion in the early stage. With time going, tunica albuginea began to proliferate and mold, fine type Ⅲ collagen fibers are gradually replaced by bulky type Ⅰcollagen fibers. Fibrous arrangement direction is also in step with function, presenting circular muscle in inner layer and longitudinal muscle in outer layer. The characteristics of fibrous composition and arrangement might be suitable for bearable pressure of tunica albuginea under the status of penile erection.
Study results showed that 10 days after bladder acellular matrix grafts were used for substituting rat bladders, there were cells ingrew the grafts, 20 weeks after grafting, mucosa and bladder detrusor muscle were the same as normal wall of urinary bladder, and vascularization and growing nerves were found [6]. Urakanmi et al [9] used bladder acellular matrix grafts in the rat bladder enlargement. They found that bladder acellular matrix grafts had been completely replaced by normal bladder tissue by histological detection in the 8th week. In this study, bladder acellular matrix grafts with inflammatory cells and fibroblasts could be found in partial samples in the 2nd week, and bladder acellular matrix grafts were replaced by fibrous tissue. It demonstrates that bladder acellular matrix grafts used for penile surgery are basically degraded within 12 weeks. Bladder acellular matrix grafts mainly play a cytoskeletal role. Tunica albuginea formed in the operated region might be induced by peripheral cell creeping, proliferation and differentiation. This is in accordance with reports on reconstruction of bladder.
Our previous study demonstrated that under the electron microscope, bladder acellular matrix grafts presented reticular fibrous arrangement, could bear multi-directional stress, with the largest stress of（8.87±1.51）MPa, and the largest strain of 38.60±34.83. We thought that the structural characteristics of bladder acellular matrix grafts in constructing albuginea penis might have the following advantages: ①providing a very good three-dimensional scaffold for cellular proliferation and growth; ②reticular structure could bear multi-directional stress, stress is multiaxial when spongy body is in the status of erection, and without grafting and disruption in the test. Studies on large animal models and large size of sample should be needed.
Through perfusing water into spongy body, we found that with time going, the occurrence of phallocampsis was gradually decreased. There was no noticeable difference in occurrence of phallocampsis between the experimental group and the control group. In the initial stage, granulation tissue ingrows the spongy body and forms a very big fibrous scar, leading insufficient filling of spongy body, in addition, tunica albuginea scar limits the local extension of tunica albuginea, as well as tunica albuginea extends asymmetrically in the status of penile erection, all these cause phallocampsis. With the absorption of tunica albuginea and scar in the spongy body, tunica albuginea and spongy body recover their compliance, and then occurrence of phallocampsis is decreased.
There were no studies on bladder acellular matrix grafts for reconstructing tunica albuginea. By making a comparison with control group, we did not find noticeable immunological rejections and local fibrosis in the stimulated tissue. Reticular structure has formed a better scaffold for cell growth and can also bear multiaxial stress under the status of erection of tunica albuginea. We think that bladder acellular matrix grafts are a kind of promising grafting materials for penile surgery.

REFERENCES

1 Devine CJJr, Horton CE. Use of dermal graft to correct chordee. J Urol 1975;113(1):56-58
2 Perlmutter AD，Montgomery BT，Steinhardt GF. Tunica vaginalis free graft for the correction of chordee. J Urol 1985;134(2):311-313
3 Thomas R，Palomar JM，Evans BB，et al. Correction of intrinsic penile chordee with a ventral penile graft of fascia lata. J Urol 1988;140(1): 191-193
4 Caesar RE, Caldamone AA. The use of free grafts for correcting penile chordee. J Urol 2000;164(5):1691-1693
5 Chun JL, McGregor A, Krishnan R, et al. A comparison of dermal andcadaveric pericardial grafts in the modified Horton–Devine procedure for Peyronie’s disease. J Urol 2001;166(1):185-188
6 Probst M, Dahiya R,Carrier S, et al. Reproduction of functional smooth muscle tissue and partial bladder replacement.Br J Urol 1997; 79(4):505-515
7 Dahms SE, Piechota HJ, Dahiya R, et al. Composition and biomechanical properties of the bladder acellular matrix graft: comparative analyBAMG in rat， pig and human. Br J Urol 1998;82(3): 411-419
8 Piechota HJ, Dahms SE, Probst M, et al. Functional rat bladder regeneration through xenotransplantation of the bladder acellular matrix graft. Br J Urol 1998;81(4):548-559
9 Urakami S, Shiina H, Enokida H, et al. Functional improvement in spinal cord injury-induced neurogenic bladder by bladder augmentation using bladder acellular matrix graft in the rat. World J Urol 2007;25(2): 207-213

膀胱无细胞基质移植物重建兔阴茎白膜的动态观察**☆

孙 发1,杨宇如2,魏 强2,卢一平2,李 虹2,韩 平2,宋 超2,石家齐1, 谷 江1
1贵阳医学院附属医院泌尿外科，贵州省贵阳市 550004；2四川大学华西医院泌尿外科，四川省成都市 610041
孙 发☆，男，1969年生，贵州省六盘水市人，汉族，博士，副主任医师，硕士生导师，主要从事男性泌尿系统组织工程的研究。
贵州省科学技术基金（黔科合J字[2007]2106号）*；贵州省省长基金（黔省专合（2007）70号）*
摘要
背景：目前膀胱无细胞基质移植物已成功用于替代动物膀胱、尿道和修复尿道下裂，但膀胱无细胞基质移植物重建阴茎白膜有待观察。
目的: 采用同种异体膀胱无细胞移植物替代兔阴茎白膜，观察重建修复效果。
设计：随机对照观察。
单位：四川大学华西医学实验动物中心、华西组织工程实验室及贵阳医学院组织工程实验室。
材料：选用50只雄性健康封闭新西兰兔，动物级别3级，体质量为2.6~3.0 kg,均无包茎及阴茎发育不良，注射生理盐水后无阴茎弯曲，由四川大学华西实验动物中心提供。
方法：实验于2005-12/2007-06在四川大学华西实验动物中心、四川大学组织工程实验室及贵阳医学院组织工程实验室完成。①取10只实验兔膀胱制备膀胱无细胞基质移植物。随机数字表法将40只新西兰兔分为对照组和膀胱无细胞基质移植物组，分别在阴茎背侧切除白膜10 mm×5 mm造成缺损，分别采用白膜原位缝合及膀胱无细胞基质移植物修复，每组20只。②分别于术后2，6，12及24周对2组动物进行阴茎海绵体内快速注射生理盐水诱导阴茎勃起，观察阴茎弯曲情况；于上述时间点分别处死实验兔，于术区取材进行苏木精-伊红、Masson染色观察修复部位组织结构变化；Stirus 染色检测检测Ⅰ型和Ⅲ型胶原纤维阳性面积，免疫组化染色检测炎性标志因子诱导型一氧化氮合酶和促纤维化因子转化生长因子β1的表达。
主要观察指标：①阴茎弯曲情况。②修复部位组织结构变化。③炎性标志因子诱导型一氧化氮合酶和促纤维化因子转化生长因子β1的表达。④Ⅰ型和Ⅲ型胶原纤维阳性面积。
结果：纳入重建阴茎白膜术实验兔40只，2只死于麻醉药物过量，2只死于急性肠炎，其余36只均进入结果分析。①术后6周时弯曲发生率最高，对照组2例，膀胱无细胞基质移植物组1例；术后12周对照组及膀胱无细胞基质移植物组各1例发生弯曲；术后24周对照组1例发生弯曲，膀胱无细胞基质移植物组无弯曲发生。②修复部位组织结构变化：术后2周两组修复部位结构欠清，炎性细胞浸润明显，膀胱无细胞基质移植物组移植部位可见膀胱无细胞基质移植物内细胞生长，Masson染色显示术区白膜纤维纤细，排列欠规律。术后6周时白膜恢复完整性，膀胱无细胞基质移植物不能辨别，两组间无明显差异。术后24周移植部位白膜完整均匀，纤维恢复内环外纵排列，和正常白膜不能区别。③两组术后诱导型一氧化氮合酶及转化生长因子β1，2周表达最强，术后6周只有少数纤维细胞和血管内皮细胞有表达，术后12，24周仅有极少的血管内皮细胞表达诱导型一氧化氮合酶和转化生长因子β1，同时间点2组诱导型一氧化氮合酶和转化生长因子β1没有差别。④术后2周两组Ⅰ型和Ⅲ型胶原纤维共同存在，比例相当。以后Ⅰ型胶原纤维逐渐增加，而Ⅲ型胶原纤维逐渐减少，术后24周以Ⅰ型胶原纤维为主，Ⅲ型胶原纤维已不明显。
结论：膀胱无细胞基质移植物修复新西兰兔阴茎白膜无明显的炎症反应和纤维化，是较为理想的阴茎白膜修复材料。
关键词：阴茎；细胞外基质；移植；兔
中图分类号: R617 文献标识码: A 文章编号: 1673-8225(2008)05-00983-05
孙发,杨宇如,魏强,卢一平,李虹,韩平,宋超,石家齐,谷江.膀胱无细胞基质移植物重建兔阴茎白膜的动态观察[J].中国组织工程研究与临床康复，2008，12(5):983-987
[www.zglckf.com/zglckf/ejournal/upfiles/08-5/5k-983(ps).pdf]
（Edited by Zhang J/Song LP/Wang L）