组织工程尿道中应用丝素蛋白支架的实验研究
发布时间：2018-08-11 08:57
【摘要】：长段前尿道狭窄通常需要应用替代材料来重建尿道。选择合适的尿道替代材料仍然是泌尿外科医生富有挑战的问题之一。目前，较多选用自体组织材料来修复重建狭窄的尿道，但是获取这些自体组织往往伴随着供区组织的多种并发症。组织工程技术为解决这些问题提供了一种可能。本实验采用新型的经拉伸处理后的静电纺丝素蛋白材料作为支架材料，构建组织工程尿道，修复重建比格犬缺损的尿道，从而评估丝素蛋白组织工程尿道在尿道修复重建中的应用效果。 第一部分拉伸处理后静电纺丝素蛋白支架材料制备及检测 目的：制备符合组织工程尿道修复重建要求的丝素蛋白支架材料，并对其结构及机械强度进行检测。 材料与方法：丝素蛋白来源于天然蚕茧。将天然蚕茧放置入碱性溶液中煮沸，充分洗去丝胶成分。将脱胶后的丝素溶液于LiBr水溶液中，使用去离子水连续透析，再浓缩到33%（质量分数）。获得的浓缩丝素蛋白水溶液转移入自制的静电纺丝装置进行制备。为了改善材料的机械强度，获得的丝素蛋白材料在90%的乙醇水溶液（质量体积）中进行单向拉伸处理。经过拉伸处理后的电纺丝素蛋白支架材料表面使用扫描电镜检测，并取5个区域，使用KS400图像分析系统进行分析表面孔隙大小，并检测其孔隙率，最后拉力试验检测其机械强度。 结果：我们制备获得的丝素蛋白支架材料多孔，三维结构。孔径约为40±4.96μm。纤维丝平滑而均匀，直径约为800-1200nm。材料经拉伸处理后，其断裂能及断裂强度分别为1.8MPa及93.2J/kg，缝合强度为1.4N，满足尿道修复重建要求。 结论：经制备、拉伸处理后的静电纺丝素蛋白支架材料为3D多孔结构,其空间结构及机械强度符合尿道重建修复需要。 第二部分丝素蛋白支架材料复合尿路上皮细胞修复重建犬尿道缺损 目的：评估拉伸处理后的静电纺丝素蛋白材料复合尿路上皮细胞在犬尿道重建中的应用效果。 材料与方法：制备新型的经拉伸处理的电纺丝素蛋白材料。使用9只比格犬作为实验动物，其中6只实验组，从实验组动物中获得，培养，扩增其尿路上皮细胞，并种植于材料上，培养一周，获得组织工程移植物。获得的组织工程移植物经过HE染色及扫描电镜检测。另外3只犬作为对照组，对9只实验动物都经过剥离尿道粘膜3×1cm2处理，实验组动物组应用组织工程移植物进行修复，3只对照组未使用任何材料进行修复。术后1,2及6个月后，行尿道造影检查，尿道修复部位组织切片HE染色，免疫组化染色。并使用KS400图像分析系统对上皮再生面积定量分析比较。 结果：尿路上皮细胞在丝素材料上生长良好，在材料表层形成多层结构，且细胞能渗透乳材料内部。在动物实验中，实验组中的比格犬未表现排尿困难，尿道造影示尿道管腔未显示明显狭窄，，组织学检测示上皮细胞逐渐生长，在术后6个月形成典型尿路上皮细胞结构。在对照组中，犬表现出排尿困难，尿道造影示尿道管腔狭窄，组织学显示上皮细胞生长缓慢，6个月后仅有1-2层上皮细胞覆盖在缺损处，并可见较严重炎性反应。 结论：拉伸处理后的丝素蛋白材料与尿路上皮细胞有良好的生物相容性。经证实复合尿路上皮细胞的丝素蛋白材料或可成为潜在的尿道重建材料。 第三部分丝素蛋白支架材料制备组织工程口腔粘膜修复重建犬尿道缺损 目的：评估丝素蛋白支架材料构建组织工程口腔黏膜在尿道修复重建中的应用效果。 材料与方法：我们分离、培养、鉴定自体口腔上皮细胞及成纤维细胞然后将种子细胞种植在丝素蛋白支架材料上，然后使用HE染色、免疫组化染色及扫描电镜检测。建立10只5cm尿道粘膜缺损动物模型，实验组5只使用组织工程口腔粘膜，对照组5只使用单纯的丝素蛋白支架材料，术后6个月应用尿道逆行造影、大体及组织学等检测。 结果：丝素蛋白支架与口腔上皮细胞及成纤维细胞表现良好的组织相容性。口腔上皮在支架材料上生长良好，形成多层上皮结构。实验组动物6个月后未见排尿困难，尿道逆行造影未见明显尿道狭窄，尿道组织学检测示分层上皮细胞形成。对照组动物在6个月随访期间发生排尿困难，尿道逆行造影显示尿道狭窄，组织学检测发现仅1-2层上皮细胞形成。 结论：以丝素蛋白材料作为支架的组织工程口腔粘膜有成为潜在的尿道重建材料的可能。
[Abstract]:Long-term anterior urethral stricture usually requires the use of alternative materials to reconstruct the urethra. Selection of suitable urethral substitutes remains a challenging issue for urologists. Tissue engineering provides a possibility to solve these problems. In this study, a novel stretched electrospun protein material was used as a scaffold material to construct tissue-engineered urethra to repair and reconstruct the defective urethra in Beagle dogs.
Part one preparation and detection of electrospun silk fibroin scaffold materials after stretching treatment
Objective: To prepare silk fibroin scaffolds for tissue engineering urethral reconstruction and to test their structure and mechanical strength.
Materials and Methods: Silk fibroin comes from natural silkworm cocoon. The natural silk cocoon is boiled in alkaline solution, and the sericin is washed out. The degummed silk fibroin solution is dialyzed in LiBr aqueous solution, and then concentrated to 33% (mass fraction). The concentrated silk fibroin protein solution is transferred to homemade electrospinning. In order to improve the mechanical strength of the material, the obtained silk fibroin material was stretched unilaterally in 90% ethanol aqueous solution (mass volume). The surface of the stretched silk fibroin scaffold material was examined by scanning electron microscope (SEM). Five regions were selected and analyzed by KS400 image analysis system. Gap size and porosity were measured, and tensile strength was tested to determine its mechanical strength.
Results: The silk fibroin scaffolds were porous and three-dimensional. The pore size was about 40
CONCLUSION: The electrospun protein scaffold prepared by stretching treatment has a 3D porous structure, and its spatial structure and mechanical strength meet the needs of urethral reconstruction and repair.
Second silk fibroin scaffolds combined with urothelial cells to reconstruct canine urethral defects
Objective: To evaluate the effect of tension-treated electrospun protein composite with urothelial cells on urethral reconstruction in dogs.
Materials and Methods: A novel electrospun protein material was prepared. Nine beagles were used as experimental animals, and six of them were used as experimental group. Urinary epithelial cells were obtained, cultured and amplified from the experimental group. The cells were planted on the material and cultured for one week to obtain tissue engineered grafts. The other three dogs were taken as control group, 9 experimental animals were treated with 3 Immunohistochemical staining and KS400 image analysis system were used for quantitative analysis of epithelial regeneration area.
Results: Urinary epithelial cells grew well on silk fibroin material, formed multi-layer structure on the surface of material and penetrated into the interior of milk material. In animal experiment, the beagles in the experimental group did not show dysuria, urethrography showed no obvious stricture in the urethral lumen, and histological examination showed epithelial cells grew gradually 6 months after operation. In the control group, the dogs showed dysuria, urethrography showed urethral lumen stenosis, histology showed that epithelial cells grew slowly, 6 months later only 1-2 layers of epithelial cells covered the defect, and there were serious inflammatory reactions.
Conclusion: The silk fibroin material after stretching has good biocompatibility with urothelial cells. It has been proved that the silk fibroin material combined with urothelial cells may be a potential material for urethral reconstruction.
Part III Repair and reconstruction of canine urethral defect with tissue-engineered oral mucosa made of silk fibroin scaffold material
Objective: To evaluate the effect of tissue-engineered oral mucosa constructed with silk fibroin scaffolds in urethral reconstruction.
Materials and Methods: We isolated, cultured and identified autologous oral epithelial cells and fibroblasts, then planted the seed cells on silk fibroin scaffolds, and then used HE staining, immunohistochemical staining and scanning electron microscopy to detect them. Five patients in the control group were treated with silk fibroin scaffolds. Retrograde urethrography, gross and histological examination were performed 6 months after operation.
Results: Silk fibroin scaffolds showed good histocompatibility with oral epithelial cells and fibroblasts. Oral epithelial cells grew well on the scaffolds and formed multi-layer epithelial structures. No dysuria, no obvious urethral stricture on retrograde urethrography were found in the experimental group after 6 months. In the control group, dysuria occurred during the follow-up period of 6 months. Retrograde urethrography showed urethral stricture. Histological examination revealed only 1-2 layers of epithelial cells.
Conclusion: It is possible to use silk fibroin as a scaffold for tissue engineering of oral mucosa.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08
本文编号：2176513
[Abstract]:Long-term anterior urethral stricture usually requires the use of alternative materials to reconstruct the urethra. Selection of suitable urethral substitutes remains a challenging issue for urologists. Tissue engineering provides a possibility to solve these problems. In this study, a novel stretched electrospun protein material was used as a scaffold material to construct tissue-engineered urethra to repair and reconstruct the defective urethra in Beagle dogs.
Part one preparation and detection of electrospun silk fibroin scaffold materials after stretching treatment
Objective: To prepare silk fibroin scaffolds for tissue engineering urethral reconstruction and to test their structure and mechanical strength.
Materials and Methods: Silk fibroin comes from natural silkworm cocoon. The natural silk cocoon is boiled in alkaline solution, and the sericin is washed out. The degummed silk fibroin solution is dialyzed in LiBr aqueous solution, and then concentrated to 33% (mass fraction). The concentrated silk fibroin protein solution is transferred to homemade electrospinning. In order to improve the mechanical strength of the material, the obtained silk fibroin material was stretched unilaterally in 90% ethanol aqueous solution (mass volume). The surface of the stretched silk fibroin scaffold material was examined by scanning electron microscope (SEM). Five regions were selected and analyzed by KS400 image analysis system. Gap size and porosity were measured, and tensile strength was tested to determine its mechanical strength.
Results: The silk fibroin scaffolds were porous and three-dimensional. The pore size was about 40
CONCLUSION: The electrospun protein scaffold prepared by stretching treatment has a 3D porous structure, and its spatial structure and mechanical strength meet the needs of urethral reconstruction and repair.
Second silk fibroin scaffolds combined with urothelial cells to reconstruct canine urethral defects
Objective: To evaluate the effect of tension-treated electrospun protein composite with urothelial cells on urethral reconstruction in dogs.
Materials and Methods: A novel electrospun protein material was prepared. Nine beagles were used as experimental animals, and six of them were used as experimental group. Urinary epithelial cells were obtained, cultured and amplified from the experimental group. The cells were planted on the material and cultured for one week to obtain tissue engineered grafts. The other three dogs were taken as control group, 9 experimental animals were treated with 3 Immunohistochemical staining and KS400 image analysis system were used for quantitative analysis of epithelial regeneration area.
Results: Urinary epithelial cells grew well on silk fibroin material, formed multi-layer structure on the surface of material and penetrated into the interior of milk material. In animal experiment, the beagles in the experimental group did not show dysuria, urethrography showed no obvious stricture in the urethral lumen, and histological examination showed epithelial cells grew gradually 6 months after operation. In the control group, the dogs showed dysuria, urethrography showed urethral lumen stenosis, histology showed that epithelial cells grew slowly, 6 months later only 1-2 layers of epithelial cells covered the defect, and there were serious inflammatory reactions.
Conclusion: The silk fibroin material after stretching has good biocompatibility with urothelial cells. It has been proved that the silk fibroin material combined with urothelial cells may be a potential material for urethral reconstruction.
Part III Repair and reconstruction of canine urethral defect with tissue-engineered oral mucosa made of silk fibroin scaffold material
Objective: To evaluate the effect of tissue-engineered oral mucosa constructed with silk fibroin scaffolds in urethral reconstruction.
Materials and Methods: We isolated, cultured and identified autologous oral epithelial cells and fibroblasts, then planted the seed cells on silk fibroin scaffolds, and then used HE staining, immunohistochemical staining and scanning electron microscopy to detect them. Five patients in the control group were treated with silk fibroin scaffolds. Retrograde urethrography, gross and histological examination were performed 6 months after operation.
Results: Silk fibroin scaffolds showed good histocompatibility with oral epithelial cells and fibroblasts. Oral epithelial cells grew well on the scaffolds and formed multi-layer epithelial structures. No dysuria, no obvious urethral stricture on retrograde urethrography were found in the experimental group after 6 months. In the control group, dysuria occurred during the follow-up period of 6 months. Retrograde urethrography showed urethral stricture. Histological examination revealed only 1-2 layers of epithelial cells.
Conclusion: It is possible to use silk fibroin as a scaffold for tissue engineering of oral mucosa.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08
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