人脐静脉细胞外基质构建组织工程尿道的实验研究
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摘要
研究背景及目的
对于尿道先天畸形、后天损伤或因其他疾病造成的尿道狭窄或缺损,大多数患者需要尿道成形重建手术治疗。现在尿道成形重建的手术方式多达300余种,但无论何种手术方式,尿瘘、尿道狭窄仍是术后较为常见的并发症,常需要再次、甚至是多次手术治疗,足以说明尿道重建的难度、复杂性及挑战性。为了解决尿瘘和尿道狭窄并最大程度的恢复阴茎尿道功能及外观,新的或改良的手术方式仍然不断涌现,但并无一种普遍适用的方法,关键在于尿道重建的材料。较常用的尿道重建材料有:①膀胱粘膜或口腔粘膜移植物,前者可供组织量大,后者可供组织有限,但两者均造成继发创伤,容易形成尿瘘和尿道狭窄。②阴茎包皮或皮肤游离移植再造尿道,由于缺乏血运,已少应用。③直接利用输尿管、动脉、静脉或阑尾组织进行替代等,取材不便,疗效也欠佳,一直未能成为主流的手术方法。④阴茎皮肤、阴囊纵隔皮肤或包皮瓣带蒂转移再造尿道,优点是血运良好,但可供组织有限,在包皮缺乏、重症尿道下裂以及多次尿道手术失败的患者常无可供利用的组织。
随着生命科学、材料科学以及相关学科的迅猛发展,诞生了一门新生交叉学科——组织工程学,它为泌尿系组织或器官的重建开辟了新的途径。在泌尿系组织工程的研究中,支架材料对再造工程化组织具有重要作用,细胞外基质作为支架材料越来越受到重视。
细胞外基质是一种特殊的天然生物材料,是生物体原型组织经过特殊脱细胞处理后,仅保留胶原蛋白、蛋白多糖、糖蛋白等低抗原性物质,能为组织、器官甚至整个机体提供力学支持和物理强度。研究表明,细胞外基质不仅为组织和机体提供力学支架和生物支架作用,还含有各种生物因子和细胞因子,对于调节细胞在材料上基因表达方式、细胞的增殖、黏附、分化、发育、扩散、移行、定居、凋亡等都有重要作用。在去除具有抗原性的细胞成分同时,保留其天然的细胞外基质和生物学特性,使其在体内基本达到无免疫原性,是这类材料能够应用于组织修复的理论基础。
很多学者己在组织工程尿道方面取得显著成果。脱细胞小肠粘膜基质,脱细胞膀胱粘膜基质,脱细胞的血管基质等被用于尿道重建研究,并取得一定效果,说明利用脱细胞组织基质再造尿道有其可行性。因此,我们在前人组织工程化尿道已有研究的基础上提出以人脐静脉细胞外基质重建修复尿道,观察其组织再生能力及能否恢复尿道的功能,以期寻找理想的尿道修复材料。
材料与方法
取健康孕妇剖宫产脐带,采用酶消化、去污剂和渗透溶液方法处理人脐静脉,制备人脐静脉细胞外基质。倒置显微镜和H-E染色观察细胞脱落情况。24只新西兰雄性白兔,随机分为A(n=12)、B(n=6)、C(n=6)组,A、B组切除前尿道2.0cm,A组用脐静脉细胞外基质修复尿道缺损;B组将其断端与周围组织直接缝合作为对照;C组切断前尿道2.0cm后将断端直接缝合作为另一对照组。三组受试对象均置8#硅胶管做支架及导尿。术后1w每只兔每天应用青霉素80万U肌注,术后10d拔除支架管。术后2w、4w、8w、16w行免疫组织学观察,术后16w行尿道膀胱造影及尿动力学检查。
结果
1.人脐静脉细胞外基质:大体观察无色透明,有一定的弹性及韧性,倒置显镜下观察呈网状结构,细胞外基质保持完好,细胞全部脱除。
2.实验组中有2只出现感染,经换药、抗生素应用等措施,感染控制,拔尿管后2只出现尿瘘;B组术后3只发生尿瘘,其余可见尿道闭塞,大量结缔组织生长,无正常上皮结构;C组恢复良好。
3.术后2w,A组移植段尿道有上皮细胞生长,伴有白细胞及巨噬细胞浸润,术后4w时移植段尿道已被上皮完全覆盖,无炎性细胞。术后8w时可见人脐静脉细胞外基质降解的微小碎片包裹,出现不规则紊乱的平滑肌细胞生长,术后16w时A组的上皮组织及基层下组织与C组无明显差别,平滑肌排列规则,血管数目进一步增多,炎症反应消失,未见脐静脉细胞外基质组织。
4.术后16w尿道膀胱造影A、C组见尿道完整、光滑,无尿液外渗、尿道憩室等形成,尿动力学检查示A、C组的膀胱容量、最大尿道压分别与术前比较,差异无统计学意义(P＞0.05),而B组不能置入测压管检测。
结论
1.酶消化、去污剂和渗透溶液联合处理人脐静脉是制备细胞外基质较为理想的方法。
2.人脐静脉细胞外基质可以修复2.0cm长的兔管状尿道缺损,再造尿道时仅有两个吻合口,术后能获得良好的粘膜覆盖,尿瘘及尿道狭窄发生的机率降低。
3人脐静脉细胞外基质替代尿道缺损,材料来源相对充足,手术简单,创伤小是一个良好的、值得进一步研究的可用于尿道重建的替代材料。
Background and Objectives
Urethral reconstruction is still the main therapy pathway for urethral stricture or defect caused by congenital malformation,acquired injury or other diseases.There are more than 300 operation methods for urethroplasty,but uerthral stricture and fistula are still common complications which explain the difficulty,complexity and challenge of urethroplasty.Patients often need to be operated again or for many times.Moreover, the repair for function and shape of penis is also difficult to urologists.Novel or improved methods continue to come forth to prevent urethral fistula and stricture in order to recover the function and shape of penis to the most extent.But no way is appropriate and generalized.The focus is on the stuff for urethral reconstruction.At present,biological materials for urethral reconstruction include:1 vesical or buccal mucosa grafts.The former can supply adequate amounts of tissue,but the later supply a limited amount.Both of them could bring secondary injuries,easily causing urethral fistula and stricture.2 prepuce of penis or skin free grafting.For lack of blood,this way has seldom been used now.3 ureter,artery,vein or appendix tissue.These ways are not the major methods at all times because of inconvenience to get them and bad treatment effect.4 Vascularized skin flaps from the prepuce,scrotum or penile shaft. These ways have the virtue of good blood circulation,but no adequate amounts of tissue.No tissue could be available for patients' lack of prepuce,with severe hypospadias,or urethral operation failure many times.
With the rapid development of life science,material science and relative disciplines,a new intersect science named tissue engineering arises.Tissue engineering has been proposed as a strategy for urethral defect.In the studies of urologic tissue engineering the support material is very important for tissue or organ's reconstruction.As a supporting stuff,extracellular matrix has been paid more and more attenation to nowadays.
ECM is a special natural biomaterial.It is a such low antigenicity substance as collagen protein,proteoglycans,or glucoprotein retained from organism via special decellularization,and can provide mechanical support and physical intensity for tissue,organ even the whole organism.Further study showed that ECM not only can provide mechanical support for organism,but also has profound influence on cell reproduction,adhere,differentiation,development,diffuseness,migration,ecesis anomaly,apoptosis etc.The theoretical basis of this material being used for tissue repair is reserving its natural extracellular matrix and biological characteristics, enabling it to no immunogenicity when antigenic cellular components are extracted.
Many researchers have acquired significant achievements in urethral tissue engineering.ECM such as SIS,BECM and VECM were used to reconstruct the urethra.These studies indicated that urethral reconstruction with ECM was feasible. Therefore,based on the study of the forward researchers,we will use ECM of umbilical vein to repair the urethra,and observe its tissue regeneration capacity so as to seek an ideal material for urethral reconstruction.
Materials and methods
Umbilical core was obtained from health woman of uterine-incision delivery. ECM of umbilical vein was prepared by use of hypotonic and hypertonic solutions, detergent and proteinase in a multistep process.Twenty-four male rabbits were divided into 3 groups:group A(n=12),group B(n=6),group C(n=6).In group A and B,a 2.0cm segment of urethra was resected.In group A,the resected urethra was replaced with ECM of human umbilical vein graft and in group B urethral defect was created as a control group.A 2.0cm segment of urethra was cut and then stitched up with 6-0 absorbable suture in group C.8~# silicone tubes were placed into the three groups of rabbits' urethras to play the role of support and urination.Every rabbit was injected with penicillin 8×10~6 u/d the first week after the operation.The silicone tube was removed in the tenth day after operation.Then the regenerative segment was studied with histological technique by hematoxylin-eosin straining and immunohistological straining after 2,4,8 and 16 weeks postoperatively.The retrograde urethrography and urodynamics were used to evaluate the function of the regenerative urethra at 16 weeks after operation.
Results
1 The ECM of human umbilical vein is achromatic color and has elastic behavior. Morphological analyses showed that the cells were removed effectively from umbilical veins,and the elastic fibers and collagen fibers were preserved.
2 In group A,2 rabbits had infection which was controled after treatments,and urinary fistula occurred on two of them after the tube was removed.In group B,3 rabbits had urinary fistula,and in others urethral strictures and fibrous connective tissue were observed without normal epithelial structure.ln group C,all rabbits successfully recovered.
3 In group A,epithelial cells grew from each side in ECM with infiltration of inflammatory cells 2 weeks after operation.However,these cells completely disappeared 4 weeks after operation and at the same time the luminal surface of matrix was completely covered by urothelium.At 8 weeks after operation, minimal extracellular matrix of human umbilical veins graft was observed in the host tissue,and irregular mussy smooth muscle cells were confirmed.At 16 weeks postoperatively,the amount of regenerative tissue was equivalent to the urethral tissue of group C with regular smooth muscle cells,increased vessels and no inflammatory reaction,and ECM of human umbilical veins disappeared in group A.
4 The urethrography showed wide smooth urethra without urine exosmosis and urethral diverticulum in group A and C,meanwhile urodynamic evaluation didn't demonstrate significant statistical difference(P＞0.05) in the bladder volume and the maximum urethral pressure before and after operation between the two groups. Piezometer can't be placed in group B for detecting.
Conclusions
1 Hypotonic and hypertonic solutions,detergent and proteinase are ideal methods to prepare ECM from umbilical veins.
2 ECM of umbilical vein can repair 2.0 cm long tubiform urethral defect. There are only two anastomosises when using it to reconstruct urethra.Good regeneration of mucosa occurred and the rate of urethral fistula and stricture is low after operation.
3 Urethral reconstruction with ECM of umbilical vein permitted adequate materials, simple operation and few traumas.It is a good material to reconstruct urethra and worthy of further study.
对于尿道先天畸形、后天损伤或因其他疾病造成的尿道狭窄或缺损,大多数患者需要尿道成形重建手术治疗。现在尿道成形重建的手术方式多达300余种,但无论何种手术方式,尿瘘、尿道狭窄仍是术后较为常见的并发症,常需要再次、甚至是多次手术治疗,足以说明尿道重建的难度、复杂性及挑战性。为了解决尿瘘和尿道狭窄并最大程度的恢复阴茎尿道功能及外观,新的或改良的手术方式仍然不断涌现,但并无一种普遍适用的方法,关键在于尿道重建的材料。较常用的尿道重建材料有:①膀胱粘膜或口腔粘膜移植物,前者可供组织量大,后者可供组织有限,但两者均造成继发创伤,容易形成尿瘘和尿道狭窄。②阴茎包皮或皮肤游离移植再造尿道,由于缺乏血运,已少应用。③直接利用输尿管、动脉、静脉或阑尾组织进行替代等,取材不便,疗效也欠佳,一直未能成为主流的手术方法。④阴茎皮肤、阴囊纵隔皮肤或包皮瓣带蒂转移再造尿道,优点是血运良好,但可供组织有限,在包皮缺乏、重症尿道下裂以及多次尿道手术失败的患者常无可供利用的组织。
随着生命科学、材料科学以及相关学科的迅猛发展,诞生了一门新生交叉学科——组织工程学,它为泌尿系组织或器官的重建开辟了新的途径。在泌尿系组织工程的研究中,支架材料对再造工程化组织具有重要作用,细胞外基质作为支架材料越来越受到重视。
细胞外基质是一种特殊的天然生物材料,是生物体原型组织经过特殊脱细胞处理后,仅保留胶原蛋白、蛋白多糖、糖蛋白等低抗原性物质,能为组织、器官甚至整个机体提供力学支持和物理强度。研究表明,细胞外基质不仅为组织和机体提供力学支架和生物支架作用,还含有各种生物因子和细胞因子,对于调节细胞在材料上基因表达方式、细胞的增殖、黏附、分化、发育、扩散、移行、定居、凋亡等都有重要作用。在去除具有抗原性的细胞成分同时,保留其天然的细胞外基质和生物学特性,使其在体内基本达到无免疫原性,是这类材料能够应用于组织修复的理论基础。
很多学者己在组织工程尿道方面取得显著成果。脱细胞小肠粘膜基质,脱细胞膀胱粘膜基质,脱细胞的血管基质等被用于尿道重建研究,并取得一定效果,说明利用脱细胞组织基质再造尿道有其可行性。因此,我们在前人组织工程化尿道已有研究的基础上提出以人脐静脉细胞外基质重建修复尿道,观察其组织再生能力及能否恢复尿道的功能,以期寻找理想的尿道修复材料。
材料与方法
取健康孕妇剖宫产脐带,采用酶消化、去污剂和渗透溶液方法处理人脐静脉,制备人脐静脉细胞外基质。倒置显微镜和H-E染色观察细胞脱落情况。24只新西兰雄性白兔,随机分为A(n=12)、B(n=6)、C(n=6)组,A、B组切除前尿道2.0cm,A组用脐静脉细胞外基质修复尿道缺损;B组将其断端与周围组织直接缝合作为对照;C组切断前尿道2.0cm后将断端直接缝合作为另一对照组。三组受试对象均置8#硅胶管做支架及导尿。术后1w每只兔每天应用青霉素80万U肌注,术后10d拔除支架管。术后2w、4w、8w、16w行免疫组织学观察,术后16w行尿道膀胱造影及尿动力学检查。
结果
1.人脐静脉细胞外基质:大体观察无色透明,有一定的弹性及韧性,倒置显镜下观察呈网状结构,细胞外基质保持完好,细胞全部脱除。
2.实验组中有2只出现感染,经换药、抗生素应用等措施,感染控制,拔尿管后2只出现尿瘘;B组术后3只发生尿瘘,其余可见尿道闭塞,大量结缔组织生长,无正常上皮结构;C组恢复良好。
3.术后2w,A组移植段尿道有上皮细胞生长,伴有白细胞及巨噬细胞浸润,术后4w时移植段尿道已被上皮完全覆盖,无炎性细胞。术后8w时可见人脐静脉细胞外基质降解的微小碎片包裹,出现不规则紊乱的平滑肌细胞生长,术后16w时A组的上皮组织及基层下组织与C组无明显差别,平滑肌排列规则,血管数目进一步增多,炎症反应消失,未见脐静脉细胞外基质组织。
4.术后16w尿道膀胱造影A、C组见尿道完整、光滑,无尿液外渗、尿道憩室等形成,尿动力学检查示A、C组的膀胱容量、最大尿道压分别与术前比较,差异无统计学意义(P＞0.05),而B组不能置入测压管检测。
结论
1.酶消化、去污剂和渗透溶液联合处理人脐静脉是制备细胞外基质较为理想的方法。
2.人脐静脉细胞外基质可以修复2.0cm长的兔管状尿道缺损,再造尿道时仅有两个吻合口,术后能获得良好的粘膜覆盖,尿瘘及尿道狭窄发生的机率降低。
3人脐静脉细胞外基质替代尿道缺损,材料来源相对充足,手术简单,创伤小是一个良好的、值得进一步研究的可用于尿道重建的替代材料。
Background and Objectives
Urethral reconstruction is still the main therapy pathway for urethral stricture or defect caused by congenital malformation,acquired injury or other diseases.There are more than 300 operation methods for urethroplasty,but uerthral stricture and fistula are still common complications which explain the difficulty,complexity and challenge of urethroplasty.Patients often need to be operated again or for many times.Moreover, the repair for function and shape of penis is also difficult to urologists.Novel or improved methods continue to come forth to prevent urethral fistula and stricture in order to recover the function and shape of penis to the most extent.But no way is appropriate and generalized.The focus is on the stuff for urethral reconstruction.At present,biological materials for urethral reconstruction include:1 vesical or buccal mucosa grafts.The former can supply adequate amounts of tissue,but the later supply a limited amount.Both of them could bring secondary injuries,easily causing urethral fistula and stricture.2 prepuce of penis or skin free grafting.For lack of blood,this way has seldom been used now.3 ureter,artery,vein or appendix tissue.These ways are not the major methods at all times because of inconvenience to get them and bad treatment effect.4 Vascularized skin flaps from the prepuce,scrotum or penile shaft. These ways have the virtue of good blood circulation,but no adequate amounts of tissue.No tissue could be available for patients' lack of prepuce,with severe hypospadias,or urethral operation failure many times.
With the rapid development of life science,material science and relative disciplines,a new intersect science named tissue engineering arises.Tissue engineering has been proposed as a strategy for urethral defect.In the studies of urologic tissue engineering the support material is very important for tissue or organ's reconstruction.As a supporting stuff,extracellular matrix has been paid more and more attenation to nowadays.
ECM is a special natural biomaterial.It is a such low antigenicity substance as collagen protein,proteoglycans,or glucoprotein retained from organism via special decellularization,and can provide mechanical support and physical intensity for tissue,organ even the whole organism.Further study showed that ECM not only can provide mechanical support for organism,but also has profound influence on cell reproduction,adhere,differentiation,development,diffuseness,migration,ecesis anomaly,apoptosis etc.The theoretical basis of this material being used for tissue repair is reserving its natural extracellular matrix and biological characteristics, enabling it to no immunogenicity when antigenic cellular components are extracted.
Many researchers have acquired significant achievements in urethral tissue engineering.ECM such as SIS,BECM and VECM were used to reconstruct the urethra.These studies indicated that urethral reconstruction with ECM was feasible. Therefore,based on the study of the forward researchers,we will use ECM of umbilical vein to repair the urethra,and observe its tissue regeneration capacity so as to seek an ideal material for urethral reconstruction.
Materials and methods
Umbilical core was obtained from health woman of uterine-incision delivery. ECM of umbilical vein was prepared by use of hypotonic and hypertonic solutions, detergent and proteinase in a multistep process.Twenty-four male rabbits were divided into 3 groups:group A(n=12),group B(n=6),group C(n=6).In group A and B,a 2.0cm segment of urethra was resected.In group A,the resected urethra was replaced with ECM of human umbilical vein graft and in group B urethral defect was created as a control group.A 2.0cm segment of urethra was cut and then stitched up with 6-0 absorbable suture in group C.8~# silicone tubes were placed into the three groups of rabbits' urethras to play the role of support and urination.Every rabbit was injected with penicillin 8×10~6 u/d the first week after the operation.The silicone tube was removed in the tenth day after operation.Then the regenerative segment was studied with histological technique by hematoxylin-eosin straining and immunohistological straining after 2,4,8 and 16 weeks postoperatively.The retrograde urethrography and urodynamics were used to evaluate the function of the regenerative urethra at 16 weeks after operation.
Results
1 The ECM of human umbilical vein is achromatic color and has elastic behavior. Morphological analyses showed that the cells were removed effectively from umbilical veins,and the elastic fibers and collagen fibers were preserved.
2 In group A,2 rabbits had infection which was controled after treatments,and urinary fistula occurred on two of them after the tube was removed.In group B,3 rabbits had urinary fistula,and in others urethral strictures and fibrous connective tissue were observed without normal epithelial structure.ln group C,all rabbits successfully recovered.
3 In group A,epithelial cells grew from each side in ECM with infiltration of inflammatory cells 2 weeks after operation.However,these cells completely disappeared 4 weeks after operation and at the same time the luminal surface of matrix was completely covered by urothelium.At 8 weeks after operation, minimal extracellular matrix of human umbilical veins graft was observed in the host tissue,and irregular mussy smooth muscle cells were confirmed.At 16 weeks postoperatively,the amount of regenerative tissue was equivalent to the urethral tissue of group C with regular smooth muscle cells,increased vessels and no inflammatory reaction,and ECM of human umbilical veins disappeared in group A.
4 The urethrography showed wide smooth urethra without urine exosmosis and urethral diverticulum in group A and C,meanwhile urodynamic evaluation didn't demonstrate significant statistical difference(P＞0.05) in the bladder volume and the maximum urethral pressure before and after operation between the two groups. Piezometer can't be placed in group B for detecting.
Conclusions
1 Hypotonic and hypertonic solutions,detergent and proteinase are ideal methods to prepare ECM from umbilical veins.
2 ECM of umbilical vein can repair 2.0 cm long tubiform urethral defect. There are only two anastomosises when using it to reconstruct urethra.Good regeneration of mucosa occurred and the rate of urethral fistula and stricture is low after operation.
3 Urethral reconstruction with ECM of umbilical vein permitted adequate materials, simple operation and few traumas.It is a good material to reconstruct urethra and worthy of further study.
引文
1.龚春雨,黄云.尿道下裂病因学的实验动物研究进展.West China Medical Journal,2003,18(2):268-71.
2.Woolf AS,Winyard PJ.Molecular Mechanisms of human Embryogenesis:Developmental Pathogenesis of Renal Tract Malformations.Pediatric and Developmental Pathology,2002,5(2):108-29.
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