利用两种不同上皮细胞体外构建组织工程尿道的比较研究
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摘要
目的比较利用两种不同上皮细胞的组织工程学技术培养片状细胞层体外构建尿道的方法,探索修复长段尿道狭窄的可行性。方法手术切取雄性新西兰兔2cm2左右的尿道黏膜和包皮,用酶分解获得上皮细胞后扩增培养成片状细胞层来构建尿道。培养出的片状细胞层裹在导尿管上继续培养进一步观察其完整性、张力以及强度。结果经过2~3周的扩增培养,两种上皮细胞都能形成完整的并具有一定张力的片状细胞层来构建尿道。病理学(HE染色)显示片状细胞层与天然的尿道黏膜和包皮上皮结构类似。给予一定的作用力与导尿管共培养1周后,片状细胞层仍具有完整性。而包皮来源的片状细胞层培养时间最短且具有更好的强度和厚度。结论通过组织工程学技术可以获得应用于临床的片状细胞层构建的尿道。包皮来源的细胞可能更加适合于长段尿道的修复。
Objective To compare two kinds of tissue-engineered multisource fullthickness mucosa sheets for using in urethral reconstruction in vitro,so as to verify the feasibility of long substitution urethroplasty grafting of urethral strictures. Methods After enzymatic treatment of a small(2cm2)BM biopsy from urethra buccal and foreskin of male New Zealand rabbit.Epithelial cells were obtained and cultured for urethral reconstruction.These cells were expanded and applied to sterilized deepidermized dermis to obtain a fullthickness mucosa sheet.The fullthickness mucosa sheets were assessed integrity,tension and strength histologically after mechanical stressing in vitro using catheterization. Results Pathological(HE staing)results revealed that histologically the two kings of fullthickness mucosa sheets were intact and tensional,closely resembled the native mucosa after culturing for 2or 3weeks.Despite subjecting the fullthickness mucosa sheets to mechanical stress and catheter,their integrity were maintained.And the foreskin derived cell sheet layer had better strength and thickness with shorter culture time. Conclusions Tissue-engineered fullthickness mucosa sheets for substitution urethroplasty were successfully cultured.Foreskin derived cell may more suitable for long substitution urethroplasty grafting.
Objective To compare two kinds of tissue-engineered multisource fullthickness mucosa sheets for using in urethral reconstruction in vitro,so as to verify the feasibility of long substitution urethroplasty grafting of urethral strictures. Methods After enzymatic treatment of a small(2cm2)BM biopsy from urethra buccal and foreskin of male New Zealand rabbit.Epithelial cells were obtained and cultured for urethral reconstruction.These cells were expanded and applied to sterilized deepidermized dermis to obtain a fullthickness mucosa sheet.The fullthickness mucosa sheets were assessed integrity,tension and strength histologically after mechanical stressing in vitro using catheterization. Results Pathological(HE staing)results revealed that histologically the two kings of fullthickness mucosa sheets were intact and tensional,closely resembled the native mucosa after culturing for 2or 3weeks.Despite subjecting the fullthickness mucosa sheets to mechanical stress and catheter,their integrity were maintained.And the foreskin derived cell sheet layer had better strength and thickness with shorter culture time. Conclusions Tissue-engineered fullthickness mucosa sheets for substitution urethroplasty were successfully cultured.Foreskin derived cell may more suitable for long substitution urethroplasty grafting.
引文
[1]Chapple C,Andrich D,Atala A,et al.SIU/ICUD consultation on urethral strictures:The management of anterior urethral stricture disease using substitution urethroplasty[J].Urology,2014,83(3Suppl):S31-S47.
[2]Palminteri E,Berdondini E,Fusco F,et al.Long-term results of small intestinal submucosa graft in bulbar urethral reconstruction[J].Urology,2012,79(3):695-701.
[3]Raya-Rivera A,Esquiliano DR,Yoo JJ,et al.Tissueengineered autologous urethras for patients who need reconstruction:an observational study[J].Lancet,2011,377(9772):1175-1182.
[4]Mangera A,Chapple CR.Tissue engineering in urethral reconstruction—an update[J].Asian J Androl,2013,15(1):89-92.
[5]Rheinwald JG,Green H.Serial cultivation of strains of human epidermal keratinocytes:the formation of keratinizing colonies from single cells[J].Cell,1975,6(3):331-343.
[6]Goberdhan NJ,Dawson RA,Freedlander E,et al.A Calmodulin-like protein as an extracellular mitogen for the keratinocyte[J].Br J Dermatol,1993,129(6):678-688.
[7]Angermeier KW,Rourke KF,Dubey D,et al.SIU/ICUD consultation on urethral strictures:evaluation and followup[J].Urology,2014,83(3Suppl):S8-S17.
[8]Romagnoli G,De Luca M,Faranda F,et al.Treatment of posterior hypospadias by the autologous graft of cultured urethral epithelium[J].N Engl J Med,1990,323(8):527-530.
[9]杨嗣星,王雪芬,宋超,等.尿道细胞外基质修复尿道缺损的研究[J].中华实验外科杂志,2005,22(1):29-30.
[10]翟弘峰,李森恺,刘红,等.以L929细胞为滋养层的尿道黏膜上皮细胞体外培养[J].中国修复重建外科杂志,2003,17(5):414-417.
[11]梁健升,李宇洲,姚干,等.游离包皮内板移植术一期修复尿道下裂[J].实用医学杂志,2007,23(2):253-254.
[12]Hussein MM,Moursy E,Gamal W,et al.The use of penile skin graft versus penile skin flap in the repair of long bulbo-penile urethral stricture:a prospective randomized study[J].Urology,2011,77(5):1232-1237.
[13]De Filippo RE,Yoo JJ,Atala A.Urethral replacement using cell seeded tubularized collagen matrices[J].J Urol,2002,168(4Pt 2):1789-1793.
[14]Bhargava S,MacNeil S,Chapple CR.Tissue-engineered buccal mucosa-preliminary results in substitution urethroplasty[J].Eur Urol,2006,5(2Suppl):45.
[2]Palminteri E,Berdondini E,Fusco F,et al.Long-term results of small intestinal submucosa graft in bulbar urethral reconstruction[J].Urology,2012,79(3):695-701.
[3]Raya-Rivera A,Esquiliano DR,Yoo JJ,et al.Tissueengineered autologous urethras for patients who need reconstruction:an observational study[J].Lancet,2011,377(9772):1175-1182.
[4]Mangera A,Chapple CR.Tissue engineering in urethral reconstruction—an update[J].Asian J Androl,2013,15(1):89-92.
[5]Rheinwald JG,Green H.Serial cultivation of strains of human epidermal keratinocytes:the formation of keratinizing colonies from single cells[J].Cell,1975,6(3):331-343.
[6]Goberdhan NJ,Dawson RA,Freedlander E,et al.A Calmodulin-like protein as an extracellular mitogen for the keratinocyte[J].Br J Dermatol,1993,129(6):678-688.
[7]Angermeier KW,Rourke KF,Dubey D,et al.SIU/ICUD consultation on urethral strictures:evaluation and followup[J].Urology,2014,83(3Suppl):S8-S17.
[8]Romagnoli G,De Luca M,Faranda F,et al.Treatment of posterior hypospadias by the autologous graft of cultured urethral epithelium[J].N Engl J Med,1990,323(8):527-530.
[9]杨嗣星,王雪芬,宋超,等.尿道细胞外基质修复尿道缺损的研究[J].中华实验外科杂志,2005,22(1):29-30.
[10]翟弘峰,李森恺,刘红,等.以L929细胞为滋养层的尿道黏膜上皮细胞体外培养[J].中国修复重建外科杂志,2003,17(5):414-417.
[11]梁健升,李宇洲,姚干,等.游离包皮内板移植术一期修复尿道下裂[J].实用医学杂志,2007,23(2):253-254.
[12]Hussein MM,Moursy E,Gamal W,et al.The use of penile skin graft versus penile skin flap in the repair of long bulbo-penile urethral stricture:a prospective randomized study[J].Urology,2011,77(5):1232-1237.
[13]De Filippo RE,Yoo JJ,Atala A.Urethral replacement using cell seeded tubularized collagen matrices[J].J Urol,2002,168(4Pt 2):1789-1793.
[14]Bhargava S,MacNeil S,Chapple CR.Tissue-engineered buccal mucosa-preliminary results in substitution urethroplasty[J].Eur Urol,2006,5(2Suppl):45.