膀胱脱细胞胶原基质复合血管内皮生长因子修复兔尿道缺损
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摘要
背景:采用膀胱无细胞基质作为游离移植物修复尿道缺损,诱导尿道再生取得一定的成果,但该方法仍未能从根本上解决移植物血管再生不足的问题。目的:观察复合血管内皮生长因子的异种膀胱脱细胞基质修复兔尿道缺损的效果。方法:将30只成年新西兰大白兔随机分为5组,每组6只,模型组、对照组、实验组均制作尿道缺损模型,随后对照组、实验组分别采用猪膀胱脱细胞基质、复合血管内皮生长因子的猪膀胱脱细胞基质进行修复,同时设立正常组、假手术组。术后4,12周分批处死动物,进行尿道造影、尿流率及免疫组织化学检测。结果与结论:(1)实验组术后4,12周的平均尿流率高于模型组、对照组(P <0.05),与正常组、假手术组比较无差异;(2)术后12周尿道造影显示,实验组尿路线条流畅;模型组、对照组尿路线条欠流畅;(3)苏木精-伊红染色显示,模型组胶原纤维细胞较多,纤维排列紊乱,有较多淋巴细胞浸润;实验组胶原基质基本降解,新生胶原纤维排列规律,其间血管较多,未见血管瘤等;对照组见团块状胶原沉积,视野内血管较少;(4)术后12周Masson染色显示,实验组胶原组织沉积明显少于对照组、模型组(P <0.05),接近正常组和假手术组(P> 0.05);(5)术后12周CD31染色显示,实验组新生血管数量明显多于对照组、模型组(P> 0.05),接近正常组和假手术组(P> 0.05);(6)术后12周a-SMA免疫组织化学染色显示,实验组再生肌肉含量明显高于对照组和模型组(P <0.05),接近正常组和假手术组(P> 0.05);(7)结果表明,复合血管内皮生长因子的异种膀胱脱细胞基质修复兔尿道缺损,可促进移植物局部血管新生,提高再生尿道肌肉含量,加速局部胶原降解,改善尿道再生微环境,促进尿道更好地再生。
BACKGROUND: The use of bladder acellular matrix as a free graft to repair urethral defects and induce urinary tract regeneration has achieved certain results, but this method cannot fundamentally solve insufficient angiogenesis of the graft.OBJECTIVE: To observe the effect of bladder acellular matrix(BAM) combined with vascular endothelial growth factor(VEGF) in the repair of rabbit urethral defects.METHODS: Thirty adult New Zealand rabbits were randomly allocated into five groups(n=6 per group), including healthy control group, sham operation group, model group, BAM group, and BAM combined with VEGF group(VEGF-BAM group). Animal models of urethral defect were made in the latter three groups. The animals were sacrificed at 4 and 12 weeks after surgery, respectively. The penis was immediately harvested for standardized passive flowmetry, urethral radiography and subsequently fixed for immunohistochemical staining for evaluation.RESULTS AND CONCLUSION:(1) The average urinary flow rate of the VEGF+BAM group was significantly higher than that of the model group and BAM group at 4 and 12 weeks after surgery(P < 0.05), while there was no difference from the healthy control group and sham operation group.(2) Urethral radiography at 12 weeks after surgery indicated that urethra continuity was repaired with no urethral fistula in the VEGF+BAM group, but not incompletely in the model and BAM groups.(3) Hematoxylin-eosin staining results indicated there were more collagen fibers and infiltrated lymphocytes in the model group, with the fiber arrangement being disordered; the collagen matrix in the VEGF+BAM group was basically degraded, new collagen fibers arranged regularly, and there were many blood vessels, but no hemangioma;and in the BAM group, massive collagen deposition and less blood vessels were observed.(4) Masson staining results indicated that less collagen deposition was observed in VEGF+BAM group compared with BAM group and model group at 12 weeks after surgery(P < 0.05),which was similar to the healthy control group and sham operation group(P > 0.05).(5) CD31 staining results indicated that the local density of new blood vessels in the VEGF+BAM group was significantly higher than that in the BAM and models groups at 12 weeks after surgery(P >0.05), which was also close to the value in the healthy control and sham operation groups(P > 0.05).(6) Regeneration of urethral smooth muscle in the VEGF+BAM group was significantly better than that in the BAM and model groups at 12 weeks after surgery(P < 0.05), as indicated by a-SMA immunohistochemical staining. Moreover, there was no significant difference among VEGF+BAM group, sham operation group and health control group. To conclude, BAM scaffolds combined with VEGF show a totally potential capacity in inducing the urethral reconstruction. The combined use of VEGF and BAM can stimulate angiogenesis, decrease the rate of collagen deposition, promote urethral smooth muscle regeneration, and improve the urethra regeneration microenvironment in urethral reconstruction.
BACKGROUND: The use of bladder acellular matrix as a free graft to repair urethral defects and induce urinary tract regeneration has achieved certain results, but this method cannot fundamentally solve insufficient angiogenesis of the graft.OBJECTIVE: To observe the effect of bladder acellular matrix(BAM) combined with vascular endothelial growth factor(VEGF) in the repair of rabbit urethral defects.METHODS: Thirty adult New Zealand rabbits were randomly allocated into five groups(n=6 per group), including healthy control group, sham operation group, model group, BAM group, and BAM combined with VEGF group(VEGF-BAM group). Animal models of urethral defect were made in the latter three groups. The animals were sacrificed at 4 and 12 weeks after surgery, respectively. The penis was immediately harvested for standardized passive flowmetry, urethral radiography and subsequently fixed for immunohistochemical staining for evaluation.RESULTS AND CONCLUSION:(1) The average urinary flow rate of the VEGF+BAM group was significantly higher than that of the model group and BAM group at 4 and 12 weeks after surgery(P < 0.05), while there was no difference from the healthy control group and sham operation group.(2) Urethral radiography at 12 weeks after surgery indicated that urethra continuity was repaired with no urethral fistula in the VEGF+BAM group, but not incompletely in the model and BAM groups.(3) Hematoxylin-eosin staining results indicated there were more collagen fibers and infiltrated lymphocytes in the model group, with the fiber arrangement being disordered; the collagen matrix in the VEGF+BAM group was basically degraded, new collagen fibers arranged regularly, and there were many blood vessels, but no hemangioma;and in the BAM group, massive collagen deposition and less blood vessels were observed.(4) Masson staining results indicated that less collagen deposition was observed in VEGF+BAM group compared with BAM group and model group at 12 weeks after surgery(P < 0.05),which was similar to the healthy control group and sham operation group(P > 0.05).(5) CD31 staining results indicated that the local density of new blood vessels in the VEGF+BAM group was significantly higher than that in the BAM and models groups at 12 weeks after surgery(P >0.05), which was also close to the value in the healthy control and sham operation groups(P > 0.05).(6) Regeneration of urethral smooth muscle in the VEGF+BAM group was significantly better than that in the BAM and model groups at 12 weeks after surgery(P < 0.05), as indicated by a-SMA immunohistochemical staining. Moreover, there was no significant difference among VEGF+BAM group, sham operation group and health control group. To conclude, BAM scaffolds combined with VEGF show a totally potential capacity in inducing the urethral reconstruction. The combined use of VEGF and BAM can stimulate angiogenesis, decrease the rate of collagen deposition, promote urethral smooth muscle regeneration, and improve the urethra regeneration microenvironment in urethral reconstruction.
引文
[1]陈恕柱,靳宏勇,刘毅东,等.舌黏膜与口腔黏膜在治疗多次尿道下裂手术失败患儿中的临床应用和比较[J].中华小儿外科杂志,2017,38(1):59-63.
[2]任晓敏,蒋跃庆,姚海军,等.后型尿道下裂二期膀胱黏膜半管状重建尿道术的临床研究[J].现代泌尿外科杂志,2011,16(6):502-504.
[3]Weiser AC,Franco I,Herz DB,et al.Single layered small intestinal submucosa in the repair of severe chordee and complicated hypospadias.J Urol.2003;(4 Pt.2):1593-1595;disussion 1595-0.
[4]李振武,张潍平,孙宁,等.国内医院尿道下裂治疗现状调查[J].中华小儿外科杂志,2016,37(6):453-458.
[5]肖元宏,刘贵麟,刘洲禄.Onlay岛状皮瓣尿道成形术治疗儿童尿道下裂适应证及并发症防治[J].解放军医学院学报,2016,37(11):1152-1154,1159.
[6]梁伟强,冀晨阳,张佳琦,等.尿道口周蒂肉膜瓣在阴茎中段型尿道下裂一期修复中的应用[J].中国美容整形外科杂志,2017,28(2):107-111.
[7]江志勇,李学德,何庆鑫,等.组织覆盖技术在Snodgrass术治疗尿道下裂的应用研究进展[J].中国性科学,2016,25(1):24-27.
[8]唐耘熳.尿道下裂术后尿道狭窄、阴茎头裂开及尿道憩室的认识及处理[J].临床小儿外科杂志,2017,16(3):212-214.
[9]王德娟,李科,黄文涛,等.游离舌黏膜在儿童再次尿道下裂成形术中的应用[J].新医学,2016,47(4):242-245.
[10]Sievert KD,Amend B,Stenzl A.Tissue engineering for the lower urinary tract:a review of a state of the art approach.Eur Urol.2007;52(6):1580-1589.
[11]Atala A.Recent developments in tissue engineering and regenerative medicine.Curr Opin Pediatr.2006;18(2):167-171.
[12]Svystonyuk DA,Mewhort HEM,Fedak PWM.Using Acellular Bioactive Extracellular Matrix Scaffolds to Enhance Endogenous Cardiac Repair.Front Cardiovasc Med.2018;5:35
[13]Udelsman B,Mathisen DJ,Ott HC.Bioprosthetics and repair of complex aerodigestive defects.Ann Cardiothorac Surg.2018;7(2):284-292.
[14]Emodi O,Ginini JG,van Aalst JA,et al.Cleft Palate Fistula Closure Utilizing Acellular Dermal Matrix.Plast Reconstr Surg Glob Open.2018;6(3):e1682.
[15]Griffith LG,Naughton G.Tissue engineering--current challenges and expanding opportunities.Science.2002;295(5557):1009-1014.
[16]Leslie B,Jesus LE,El-Hout Y,et al.Comparative histological and functional controlled analysis of tubularized incised plate urethroplasty with and without dorsal inlay graft:a preliminary experimental study in rabbits.J Urol.2011;186(4 Suppl):1631-1637.
[17]Kanematsu A,Yamamoto S,Ogawa O.Changing concepts of bladder regeneration.Int J Urol.2007;14(8):673-978.
[18]Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest.2002;109(3):337-346.
[19]Nieda M,Nicol A,Denning-Kendall P,et al.Endothdial cell precursors are normal components of human umbilical cord blood.Br J Haematol.1997;98(3):775-777.
[20]Tamaki T,Akatsuka A,Ando K,et al.Identification of myogenicendothelial progenitor cells in the interstitial spaces of skeletal muscle.J Cell Biol.2002;157(4):571-577.
[21]Dahms SE,Piechota HJ,Dahiya R,et al.Composition and biomechanical properties of the bladder acellular matrix graft:comparative analysis in rat,pig and human.Br J Urol.1998;82(3):411-419.
[22]De Filippo RE,Yoo JJ,Atala A.Urethral replacement using cell seeded tubularized collagen matrices.J Urol.2002;168(4-2):1789-1792.
[23]Shokeir A,Osman Y,El-Sherbiny M,et al.Comparison of Partial Urethral Replacement with Acellular Matrix versus Spontaneous Urethral Regeneration in a Canine Model.Eur Urol.2003;44(5):603-609.
[24]Atala A,Guzman L,Retik AB.A novel inert collagen matrix for hypospadias repair.J Urol.1999;162(3 Pt 2):1148-1151.
[25]Kanematsu A,Yamamoto S,Noguchi T,et al.Bladder regeneration by bladder acellular matrix combined with sustained release of exogenous growth factor.J Urol.2003;170(4 Pt 2):1633-1638.
[26]Kanematsu A,Yamamoto S,Ozeki M,et al.Collagenous matrices as release carriers of exogenous growth factors.Biomaterials.2004;25(18):4513-4520.
[27]Youssif M,Shiina H,Urakami S,et al.Effect of vascular endothelial growth factor Oil regeneration of blader acellular matrix graft:histologic and functional evaluation.Urology.2005;66(1):201-207.
[28]Wang JH,Xu YM,Fu Q,et al.Continued sustained release of VEGF by PLGA nanospheres modified BAMG stent for the anterior urethral reconstruction of rabbit.Asian Pac J Trop Med.2013;6(6):481-484.
[29]Xiong Q,Lin H,Hua X,et al.A nanomedicine approach to effectively inhibit contracture during bladder acellularmatrix allograft-induced bladder regeneration by sustained delivery of vascular endothelial growth factor.Tissue Eng Part A.2015;21(1-2):45-52.
[30]Jiang X,Xiong Q,Xu G,et al.VEGF-Loaded Nanoparticle-Modified BAMAs Enhance Angiogenesis and Inhibit Graft Shrinkage in Tissue-Engineered Bladder.Ann Biomed Eng.2015;43(10):2577-2586.
[31]Roelofs LA,Oosterwijk E,Kortmann BB,et al.Bladder Regeneration Using a Smart Acellular Collagen Scaffold with Growth Factors VEGF,FGF2 and HB-EGF.Tissue Eng Part A.2016;22(1-2):83-92.
[32]Wang L,Shi Q,Dai J,et al.vascularization promotes functional recovery in the transected spinal cord rats by implanted vascular endothelial growth factor-targeting collagenscaffold.J Orthop Res.2018;36(3):1024-1034.
[33]Ferrara N,Henzel WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.Biochem Biophys Res Commun.2012;425(3):540-547.
[34]Koch S,Yao CH,Grieb G,et al.Enhancing angiogenesis in collagen matrices by covalent incorporation of VEGF.J Mater Sci Mater Med.2006;17(8):735-741.
[35]Yao N,Gao M,Ren K,et al.PD806:a novel oral vascular disrupting agent shows antitumor and antivascular effects in vitro and in vivo.Anticancer Drugs.2015;26(2):148-159.
[36]Giatromanolaki A,Koukourakis MI,Theodossiou D,et al.Comparative evaluation of angiogenesis assessment with anti-factor-VIII and anti-CD31 immunostaining in non-small cell lung cancer.Clin Cancer Res.1997;3(12 Pt 1):2485-2492.
[2]任晓敏,蒋跃庆,姚海军,等.后型尿道下裂二期膀胱黏膜半管状重建尿道术的临床研究[J].现代泌尿外科杂志,2011,16(6):502-504.
[3]Weiser AC,Franco I,Herz DB,et al.Single layered small intestinal submucosa in the repair of severe chordee and complicated hypospadias.J Urol.2003;(4 Pt.2):1593-1595;disussion 1595-0.
[4]李振武,张潍平,孙宁,等.国内医院尿道下裂治疗现状调查[J].中华小儿外科杂志,2016,37(6):453-458.
[5]肖元宏,刘贵麟,刘洲禄.Onlay岛状皮瓣尿道成形术治疗儿童尿道下裂适应证及并发症防治[J].解放军医学院学报,2016,37(11):1152-1154,1159.
[6]梁伟强,冀晨阳,张佳琦,等.尿道口周蒂肉膜瓣在阴茎中段型尿道下裂一期修复中的应用[J].中国美容整形外科杂志,2017,28(2):107-111.
[7]江志勇,李学德,何庆鑫,等.组织覆盖技术在Snodgrass术治疗尿道下裂的应用研究进展[J].中国性科学,2016,25(1):24-27.
[8]唐耘熳.尿道下裂术后尿道狭窄、阴茎头裂开及尿道憩室的认识及处理[J].临床小儿外科杂志,2017,16(3):212-214.
[9]王德娟,李科,黄文涛,等.游离舌黏膜在儿童再次尿道下裂成形术中的应用[J].新医学,2016,47(4):242-245.
[10]Sievert KD,Amend B,Stenzl A.Tissue engineering for the lower urinary tract:a review of a state of the art approach.Eur Urol.2007;52(6):1580-1589.
[11]Atala A.Recent developments in tissue engineering and regenerative medicine.Curr Opin Pediatr.2006;18(2):167-171.
[12]Svystonyuk DA,Mewhort HEM,Fedak PWM.Using Acellular Bioactive Extracellular Matrix Scaffolds to Enhance Endogenous Cardiac Repair.Front Cardiovasc Med.2018;5:35
[13]Udelsman B,Mathisen DJ,Ott HC.Bioprosthetics and repair of complex aerodigestive defects.Ann Cardiothorac Surg.2018;7(2):284-292.
[14]Emodi O,Ginini JG,van Aalst JA,et al.Cleft Palate Fistula Closure Utilizing Acellular Dermal Matrix.Plast Reconstr Surg Glob Open.2018;6(3):e1682.
[15]Griffith LG,Naughton G.Tissue engineering--current challenges and expanding opportunities.Science.2002;295(5557):1009-1014.
[16]Leslie B,Jesus LE,El-Hout Y,et al.Comparative histological and functional controlled analysis of tubularized incised plate urethroplasty with and without dorsal inlay graft:a preliminary experimental study in rabbits.J Urol.2011;186(4 Suppl):1631-1637.
[17]Kanematsu A,Yamamoto S,Ogawa O.Changing concepts of bladder regeneration.Int J Urol.2007;14(8):673-978.
[18]Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest.2002;109(3):337-346.
[19]Nieda M,Nicol A,Denning-Kendall P,et al.Endothdial cell precursors are normal components of human umbilical cord blood.Br J Haematol.1997;98(3):775-777.
[20]Tamaki T,Akatsuka A,Ando K,et al.Identification of myogenicendothelial progenitor cells in the interstitial spaces of skeletal muscle.J Cell Biol.2002;157(4):571-577.
[21]Dahms SE,Piechota HJ,Dahiya R,et al.Composition and biomechanical properties of the bladder acellular matrix graft:comparative analysis in rat,pig and human.Br J Urol.1998;82(3):411-419.
[22]De Filippo RE,Yoo JJ,Atala A.Urethral replacement using cell seeded tubularized collagen matrices.J Urol.2002;168(4-2):1789-1792.
[23]Shokeir A,Osman Y,El-Sherbiny M,et al.Comparison of Partial Urethral Replacement with Acellular Matrix versus Spontaneous Urethral Regeneration in a Canine Model.Eur Urol.2003;44(5):603-609.
[24]Atala A,Guzman L,Retik AB.A novel inert collagen matrix for hypospadias repair.J Urol.1999;162(3 Pt 2):1148-1151.
[25]Kanematsu A,Yamamoto S,Noguchi T,et al.Bladder regeneration by bladder acellular matrix combined with sustained release of exogenous growth factor.J Urol.2003;170(4 Pt 2):1633-1638.
[26]Kanematsu A,Yamamoto S,Ozeki M,et al.Collagenous matrices as release carriers of exogenous growth factors.Biomaterials.2004;25(18):4513-4520.
[27]Youssif M,Shiina H,Urakami S,et al.Effect of vascular endothelial growth factor Oil regeneration of blader acellular matrix graft:histologic and functional evaluation.Urology.2005;66(1):201-207.
[28]Wang JH,Xu YM,Fu Q,et al.Continued sustained release of VEGF by PLGA nanospheres modified BAMG stent for the anterior urethral reconstruction of rabbit.Asian Pac J Trop Med.2013;6(6):481-484.
[29]Xiong Q,Lin H,Hua X,et al.A nanomedicine approach to effectively inhibit contracture during bladder acellularmatrix allograft-induced bladder regeneration by sustained delivery of vascular endothelial growth factor.Tissue Eng Part A.2015;21(1-2):45-52.
[30]Jiang X,Xiong Q,Xu G,et al.VEGF-Loaded Nanoparticle-Modified BAMAs Enhance Angiogenesis and Inhibit Graft Shrinkage in Tissue-Engineered Bladder.Ann Biomed Eng.2015;43(10):2577-2586.
[31]Roelofs LA,Oosterwijk E,Kortmann BB,et al.Bladder Regeneration Using a Smart Acellular Collagen Scaffold with Growth Factors VEGF,FGF2 and HB-EGF.Tissue Eng Part A.2016;22(1-2):83-92.
[32]Wang L,Shi Q,Dai J,et al.vascularization promotes functional recovery in the transected spinal cord rats by implanted vascular endothelial growth factor-targeting collagenscaffold.J Orthop Res.2018;36(3):1024-1034.
[33]Ferrara N,Henzel WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.Biochem Biophys Res Commun.2012;425(3):540-547.
[34]Koch S,Yao CH,Grieb G,et al.Enhancing angiogenesis in collagen matrices by covalent incorporation of VEGF.J Mater Sci Mater Med.2006;17(8):735-741.
[35]Yao N,Gao M,Ren K,et al.PD806:a novel oral vascular disrupting agent shows antitumor and antivascular effects in vitro and in vivo.Anticancer Drugs.2015;26(2):148-159.
[36]Giatromanolaki A,Koukourakis MI,Theodossiou D,et al.Comparative evaluation of angiogenesis assessment with anti-factor-VIII and anti-CD31 immunostaining in non-small cell lung cancer.Clin Cancer Res.1997;3(12 Pt 1):2485-2492.
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