组织工程技术构建阴茎及尿道海绵体的实验研究
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摘要
海绵体是男性阴茎和尿道的重要组成部分,男性外生殖器及尿道疾患往往伴有阴茎海绵体或尿道海绵体病变。以往多采用各种假体结合自体皮瓣重建阴茎和尿道,但海绵体组织缺失使上述手术难以兼顾形态学和功能学的效果。因此,如何重建海绵体一直是泌尿外科范畴内的一个难题。近年来,组织工程技术的发展为海绵体重建提供了可能。本研究探讨了组织工程技术重建海绵体的可行性,以期为阴茎和尿道的修复重建手术提供新的选择。全文共分三章:
第一章种子细胞的培养
1目的
探讨兔阴茎海绵体海绵体平滑肌细胞和内皮细胞的培养方法。
2材料和方法
应用胶原酶消化法和组织贴块法分离培养幼兔海绵体平滑肌细胞。差速贴壁法纯化海绵体平滑肌细胞。采用α-平滑肌肌动蛋白(α-SM-actin)和肌球蛋白重链(smMHC)免疫荧光鉴定海绵体平滑肌细胞,波形蛋白(Vimentin)衬染检测成纤维细胞。流式细胞仪检测海绵体细胞表达α-SM-actin的阳性率,比较不同培养条件下海绵体平滑肌细胞的纯度。应用0.02%的I型胶原酶消化分离幼兔海绵体内皮细胞,以EGM-2培养液进行传代培养。采用von Willebrand因子免疫荧光和透射电镜鉴定海绵体内皮细胞。通过流式细胞仪检测海绵体内皮细胞表达von Willebrand因子的阳性率。
3结果
体外培养的海绵体来源细胞在荧光显微镜下对α-SM-actin和smMHC仅有少量表达,随传代次数的增加,细胞表达α-SM-actin和smMHC的阳性率进一步降低。流式细胞仪检测酶消化法和贴块法原代培养的海绵体平滑肌细胞的纯度分别为16.91%、11.13%。经差速贴壁纯化后第1代海绵体平滑肌细胞的纯度分别提高到26.88%、21.98%。Vimentin免疫荧光检测到大量阳性表达。酶消化法原代培养的内皮细胞在接种后约12 h开始贴壁生长,7～10 d细胞可融合成铺路石状镶嵌排列,体外传代培养6～8代,细胞活力良好;免疫荧光可见内皮细胞胞浆中von Willebrand因子呈阳性反应;透射电镜下可见胞浆中有Weibel-Palade小体。经流式细胞仪检测海绵体血管内皮细胞的纯度达93.76%。
4结论
目前常规方法体外分离、培养的海绵体平滑肌细胞纯度较低,大量海绵体间质来源的成纤维细胞混杂生长,差速贴壁法可在一定范围内提高海绵体平滑肌细胞的纯度。采用0.02%的Ⅰ型胶原酶消化40～50 min,以EGM-2作为培养液可在体外分离培养得到较高纯度的海绵体内皮细胞。
第二章人脐带动脉平滑肌细胞复合海绵体脱细胞基质构建组织工程海绵体平滑肌的实验研究
1目的
探讨人脐动脉平滑肌与阴茎海绵体脱细胞基质复合在裸鼠体内构建海绵体平滑肌的可行性。
2方法
以1%的Triton-X100与0.1%NH3H2O制备海绵体脱细胞基质(ACCM)。异体肌肉内埋植实验评价ACCM的生物相容性。分离培养人脐动脉平滑肌细胞(HUASMCs),MTT法测定ACCM浸提液对HUASMCs增殖的影响。将3～5代的HUASMCs接种ACCM,共培养3 d、5 d和10 d后观察HUASMCs与ACCM复合情况。将细胞-ACCM体外复合10天后将复合物植入裸鼠背部皮下,术后10天,20天和40天分别回收移植物,进行组织学观察和器官浴槽实验评价其体内构建情况。
3结果
ACCM无细胞残留,异体肌肉内埋植实验证实ACCM生物相容性良好。浸提液实验显示体外培养第4 d和第6 d,浸提液组OD值明显高于对照组(p<0.05)。HUASMCs能渗入ACCM内部,并分化形成平滑肌束。自裸鼠背部皮下回收移植物,组织学观察发现随时间延长,逐渐形成结构良好的海绵体平滑肌和内皮。器官浴槽实验中,构建组织表现出与正常海绵体平滑肌类似的收缩功能;去氧肾上腺素和电刺激所诱导的最大收缩力分别为3.64±0.18 g和2.50±0.21 g.
4结论
HUASMCs作为种子细胞与ACCM复合,可构建出具有一定形态和功能的组织工程海绵体平滑肌。
第三章骨髓基质细胞复合PGA重建兔尿道海绵体的实验研究
1目的
探讨兔自体骨髓基质细胞(BMSCs)与PGA体外复合的可行性,为尿道海绵体修复提供实验基础。
2方法
制备1×1cm的非编织状PGA膜片。采用密度梯度离心法分离培养BMSCs。5ug/ml CM-Dil标记BMSCs。将BMSCs以30×106 /ml与PGA体外复合培养2周。采用扫描电镜及荧光显微镜观察BMSCs与PGA的复合情况。建立新西兰大白兔尿道腹侧1 cm全层尿道缺损动物模型,将PGA膜片植入尿道缺损区域,修补尿道缺损。术后1个月、2个月、3个月进行尿道造影、大体观察和组织学观察评价尿道重建情况。
3结果
CM-Dil标记BMSCs阳性率达为90%以上。BMSCs与PGA复合后,BMSCs均匀密布于PGA纤维表面及PGA纤维形成的间隙中。PGA修复新西兰兔全层尿道缺损,6只动物除1只术后1个月发生尿瘘外,其余动物均排尿通畅。组织学检查发现尿道黏膜再生良好,但尿道海绵体区域主要以纤维瘢痕增生为主,未见明显尿道海绵体再生。
4结论
BMSCs与PGA在体外具有良好相容性。PGA能够修复新西兰大白兔1 cm尿道黏膜的缺损,但缺乏尿道海绵体再生。
CHAPTER ONE THE CULTURE OF SEEDING CELLS
1 Objective
To investigate the methods of isolating, culturing and evaluating the rabbit corpus cavernosal smooth muscle cells and endothelial cells.
2 Methods
The explant cell culture and digestion cell culture methods were used to isolate corporal smooth muscle cells from young rabbit cavernosal tissue. Velocity sedimentation were used to enrich the cells. Usingα-smooth muscle actin, myosin and vimentin monoclone mouse antibodies as markers, the cells purity in cultured cells was analysed by immunofluorescence and flow cytometry. The enzymatic digestion methods were used to isolate corpus cavernosal endothelial cells from young rabbit cavernosal tissue. The cells were suspended in endothelial basal medium(EBM-2) supplemented with EGM-2 BulletKit for culturing, Using endothelial specific von Willebrand Factor(vWF) as a marker, the 2nd passage cells was identified by immunofluorescence and transmission electron microscope. Flow cytometry was performed to analyses cells purity.
3 Results
Usingα-smooth muscle actin as the cell marker, the positive rate of corpus cavernosal smooth muscle cells was 16.91% in the digestion cell culture and 11.13% in the explant cell culture. After velocity sedimentation procedures, the cells purity was increased to 26.88% in the digestion cell culture and 21.98% in the explant cell culture. Immunofluorescence confirmed contaminating fibroblasts by vimentin labeling. The corpus cavernosal endothelial cells were cultured in vitro and passaged for six to eight generations. Immunofluorescence confirmed the characteristic of endothelial cells by vWF labeling. Weibel-Palade body (W-P body) was observed by transmission electron microscope. The purity of 2nd passage cells was 93.76%.
4 Conclusion
It was difficult to exclude fibroblasts contamination in corporal smooth muscle cells cultured in vitro. Velocity sedimentation procedures could only achieve slight purify of corporal smooth muscle cells. Rabbit corpus cavernosal endothelial cells can be isolated and cultured successfully in vitro by our method.
CHAPTER TWO CONSTRUCTION OF CORPUS CAVERNOSUM SMOOTH MUSCLE USING UMBILICAL ARTERY SMOOTH MUSCLE CELLS RESEEDED ON ACELLULAR CORPORAL COLLAGEN MATRICES
1 Objective
To investigate the feasibility of constructing tissue engineered corpus cavernosum smooth muscle.
2 Methods
Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting foreign body reaction. HUASMCs were isolated from human umbilical arteries through explant techniques, and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30×106 cells/ml. After that, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyze the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues.
3 Results
The decellularization process successfully extracted all cellular components and maintained their original collagen fibers. The foreign body reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro. Histologic analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64±0.18 g/100 mg and 2.50±0.21 g/100 mg.
4 Conclusion
Our study demonstrates that HUASMCs can be seeded on
3-dimensional ACCM scaffolds and will develop a tissue similar to that of the native corpus cavernosum smooth muscle.
CHAPTER THREE FEASIBILITY OF CONSTRUCTING TISSUE-ENGINEERED CORPUS CAVERNOUS URETHRA WITH RABBIT BONE MARROW STROMAL CELLS
1 Objective
To investigate the feasibility to construct urethra with PGA and the compatibility of PGA with BMSCs.
2 Methods
BMSCs were harvested, and their phenotype was identified. The cells were cultured onto PGA after amplification. BMSCs were labeled with CM-Dil. Histochemistry and scanning electron microscopy were performed to analyze the morphological characteristics of the engineered tissues. The PGA was implanted into the cavernous urethra of six rabbits after partial resection of 1×1 cm full-thickness urethra. Catheter examination and retrograde urethrography were used to evaluate results 1, 2 and 3 months after the operation, and then all dogs were sacrificed for macroscopical and histological examination.
3 Results PGA could be seeded with BMSCs in vitro, and BMSCs had the potential of attachment and proliferation on the three-dimensional PGA scaffolds. All dogs survived the procedure.1 of 6 rabbits developed a fistula formation. The other 5 rabbits voided spontaneously without difficulties. Retrograde urethrography revealed no sign of stricture. Histological examination showed the urethral walls were covered by squamous epithelium with slight keratinization.
4 Conclusion
PGA may be used as the scaffold for BMSCs growing . PGA can results in regeneration of urethral mucosa,but no regeneration of corpus spongiosum.
第一章种子细胞的培养
1目的
探讨兔阴茎海绵体海绵体平滑肌细胞和内皮细胞的培养方法。
2材料和方法
应用胶原酶消化法和组织贴块法分离培养幼兔海绵体平滑肌细胞。差速贴壁法纯化海绵体平滑肌细胞。采用α-平滑肌肌动蛋白(α-SM-actin)和肌球蛋白重链(smMHC)免疫荧光鉴定海绵体平滑肌细胞,波形蛋白(Vimentin)衬染检测成纤维细胞。流式细胞仪检测海绵体细胞表达α-SM-actin的阳性率,比较不同培养条件下海绵体平滑肌细胞的纯度。应用0.02%的I型胶原酶消化分离幼兔海绵体内皮细胞,以EGM-2培养液进行传代培养。采用von Willebrand因子免疫荧光和透射电镜鉴定海绵体内皮细胞。通过流式细胞仪检测海绵体内皮细胞表达von Willebrand因子的阳性率。
3结果
体外培养的海绵体来源细胞在荧光显微镜下对α-SM-actin和smMHC仅有少量表达,随传代次数的增加,细胞表达α-SM-actin和smMHC的阳性率进一步降低。流式细胞仪检测酶消化法和贴块法原代培养的海绵体平滑肌细胞的纯度分别为16.91%、11.13%。经差速贴壁纯化后第1代海绵体平滑肌细胞的纯度分别提高到26.88%、21.98%。Vimentin免疫荧光检测到大量阳性表达。酶消化法原代培养的内皮细胞在接种后约12 h开始贴壁生长,7～10 d细胞可融合成铺路石状镶嵌排列,体外传代培养6～8代,细胞活力良好;免疫荧光可见内皮细胞胞浆中von Willebrand因子呈阳性反应;透射电镜下可见胞浆中有Weibel-Palade小体。经流式细胞仪检测海绵体血管内皮细胞的纯度达93.76%。
4结论
目前常规方法体外分离、培养的海绵体平滑肌细胞纯度较低,大量海绵体间质来源的成纤维细胞混杂生长,差速贴壁法可在一定范围内提高海绵体平滑肌细胞的纯度。采用0.02%的Ⅰ型胶原酶消化40～50 min,以EGM-2作为培养液可在体外分离培养得到较高纯度的海绵体内皮细胞。
第二章人脐带动脉平滑肌细胞复合海绵体脱细胞基质构建组织工程海绵体平滑肌的实验研究
1目的
探讨人脐动脉平滑肌与阴茎海绵体脱细胞基质复合在裸鼠体内构建海绵体平滑肌的可行性。
2方法
以1%的Triton-X100与0.1%NH3H2O制备海绵体脱细胞基质(ACCM)。异体肌肉内埋植实验评价ACCM的生物相容性。分离培养人脐动脉平滑肌细胞(HUASMCs),MTT法测定ACCM浸提液对HUASMCs增殖的影响。将3～5代的HUASMCs接种ACCM,共培养3 d、5 d和10 d后观察HUASMCs与ACCM复合情况。将细胞-ACCM体外复合10天后将复合物植入裸鼠背部皮下,术后10天,20天和40天分别回收移植物,进行组织学观察和器官浴槽实验评价其体内构建情况。
3结果
ACCM无细胞残留,异体肌肉内埋植实验证实ACCM生物相容性良好。浸提液实验显示体外培养第4 d和第6 d,浸提液组OD值明显高于对照组(p<0.05)。HUASMCs能渗入ACCM内部,并分化形成平滑肌束。自裸鼠背部皮下回收移植物,组织学观察发现随时间延长,逐渐形成结构良好的海绵体平滑肌和内皮。器官浴槽实验中,构建组织表现出与正常海绵体平滑肌类似的收缩功能;去氧肾上腺素和电刺激所诱导的最大收缩力分别为3.64±0.18 g和2.50±0.21 g.
4结论
HUASMCs作为种子细胞与ACCM复合,可构建出具有一定形态和功能的组织工程海绵体平滑肌。
第三章骨髓基质细胞复合PGA重建兔尿道海绵体的实验研究
1目的
探讨兔自体骨髓基质细胞(BMSCs)与PGA体外复合的可行性,为尿道海绵体修复提供实验基础。
2方法
制备1×1cm的非编织状PGA膜片。采用密度梯度离心法分离培养BMSCs。5ug/ml CM-Dil标记BMSCs。将BMSCs以30×106 /ml与PGA体外复合培养2周。采用扫描电镜及荧光显微镜观察BMSCs与PGA的复合情况。建立新西兰大白兔尿道腹侧1 cm全层尿道缺损动物模型,将PGA膜片植入尿道缺损区域,修补尿道缺损。术后1个月、2个月、3个月进行尿道造影、大体观察和组织学观察评价尿道重建情况。
3结果
CM-Dil标记BMSCs阳性率达为90%以上。BMSCs与PGA复合后,BMSCs均匀密布于PGA纤维表面及PGA纤维形成的间隙中。PGA修复新西兰兔全层尿道缺损,6只动物除1只术后1个月发生尿瘘外,其余动物均排尿通畅。组织学检查发现尿道黏膜再生良好,但尿道海绵体区域主要以纤维瘢痕增生为主,未见明显尿道海绵体再生。
4结论
BMSCs与PGA在体外具有良好相容性。PGA能够修复新西兰大白兔1 cm尿道黏膜的缺损,但缺乏尿道海绵体再生。
CHAPTER ONE THE CULTURE OF SEEDING CELLS
1 Objective
To investigate the methods of isolating, culturing and evaluating the rabbit corpus cavernosal smooth muscle cells and endothelial cells.
2 Methods
The explant cell culture and digestion cell culture methods were used to isolate corporal smooth muscle cells from young rabbit cavernosal tissue. Velocity sedimentation were used to enrich the cells. Usingα-smooth muscle actin, myosin and vimentin monoclone mouse antibodies as markers, the cells purity in cultured cells was analysed by immunofluorescence and flow cytometry. The enzymatic digestion methods were used to isolate corpus cavernosal endothelial cells from young rabbit cavernosal tissue. The cells were suspended in endothelial basal medium(EBM-2) supplemented with EGM-2 BulletKit for culturing, Using endothelial specific von Willebrand Factor(vWF) as a marker, the 2nd passage cells was identified by immunofluorescence and transmission electron microscope. Flow cytometry was performed to analyses cells purity.
3 Results
Usingα-smooth muscle actin as the cell marker, the positive rate of corpus cavernosal smooth muscle cells was 16.91% in the digestion cell culture and 11.13% in the explant cell culture. After velocity sedimentation procedures, the cells purity was increased to 26.88% in the digestion cell culture and 21.98% in the explant cell culture. Immunofluorescence confirmed contaminating fibroblasts by vimentin labeling. The corpus cavernosal endothelial cells were cultured in vitro and passaged for six to eight generations. Immunofluorescence confirmed the characteristic of endothelial cells by vWF labeling. Weibel-Palade body (W-P body) was observed by transmission electron microscope. The purity of 2nd passage cells was 93.76%.
4 Conclusion
It was difficult to exclude fibroblasts contamination in corporal smooth muscle cells cultured in vitro. Velocity sedimentation procedures could only achieve slight purify of corporal smooth muscle cells. Rabbit corpus cavernosal endothelial cells can be isolated and cultured successfully in vitro by our method.
CHAPTER TWO CONSTRUCTION OF CORPUS CAVERNOSUM SMOOTH MUSCLE USING UMBILICAL ARTERY SMOOTH MUSCLE CELLS RESEEDED ON ACELLULAR CORPORAL COLLAGEN MATRICES
1 Objective
To investigate the feasibility of constructing tissue engineered corpus cavernosum smooth muscle.
2 Methods
Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting foreign body reaction. HUASMCs were isolated from human umbilical arteries through explant techniques, and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30×106 cells/ml. After that, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyze the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues.
3 Results
The decellularization process successfully extracted all cellular components and maintained their original collagen fibers. The foreign body reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro. Histologic analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64±0.18 g/100 mg and 2.50±0.21 g/100 mg.
4 Conclusion
Our study demonstrates that HUASMCs can be seeded on
3-dimensional ACCM scaffolds and will develop a tissue similar to that of the native corpus cavernosum smooth muscle.
CHAPTER THREE FEASIBILITY OF CONSTRUCTING TISSUE-ENGINEERED CORPUS CAVERNOUS URETHRA WITH RABBIT BONE MARROW STROMAL CELLS
1 Objective
To investigate the feasibility to construct urethra with PGA and the compatibility of PGA with BMSCs.
2 Methods
BMSCs were harvested, and their phenotype was identified. The cells were cultured onto PGA after amplification. BMSCs were labeled with CM-Dil. Histochemistry and scanning electron microscopy were performed to analyze the morphological characteristics of the engineered tissues. The PGA was implanted into the cavernous urethra of six rabbits after partial resection of 1×1 cm full-thickness urethra. Catheter examination and retrograde urethrography were used to evaluate results 1, 2 and 3 months after the operation, and then all dogs were sacrificed for macroscopical and histological examination.
3 Results PGA could be seeded with BMSCs in vitro, and BMSCs had the potential of attachment and proliferation on the three-dimensional PGA scaffolds. All dogs survived the procedure.1 of 6 rabbits developed a fistula formation. The other 5 rabbits voided spontaneously without difficulties. Retrograde urethrography revealed no sign of stricture. Histological examination showed the urethral walls were covered by squamous epithelium with slight keratinization.
4 Conclusion
PGA may be used as the scaffold for BMSCs growing . PGA can results in regeneration of urethral mucosa,but no regeneration of corpus spongiosum.
引文
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