組織工程尿道中應(yīng)用絲素蛋白支架的實驗研究
發(fā)布時間：2018-08-11 08:57
【摘要】：長段前尿道狹窄通常需要應(yīng)用替代材料來重建尿道。選擇合適的尿道替代材料仍然是泌尿外科醫(yī)生富有挑戰(zhàn)的問題之一。目前，較多選用自體組織材料來修復(fù)重建狹窄的尿道，但是獲取這些自體組織往往伴隨著供區(qū)組織的多種并發(fā)癥。組織工程技術(shù)為解決這些問題提供了一種可能。本實驗采用新型的經(jīng)拉伸處理后的靜電紡絲素蛋白材料作為支架材料，構(gòu)建組織工程尿道，修復(fù)重建比格犬缺損的尿道，從而評估絲素蛋白組織工程尿道在尿道修復(fù)重建中的應(yīng)用效果。 第一部分拉伸處理后靜電紡絲素蛋白支架材料制備及檢測 目的：制備符合組織工程尿道修復(fù)重建要求的絲素蛋白支架材料，并對其結(jié)構(gòu)及機械強度進行檢測。 材料與方法：絲素蛋白來源于天然蠶繭。將天然蠶繭放置入堿性溶液中煮沸，充分洗去絲膠成分。將脫膠后的絲素溶液于LiBr水溶液中，使用去離子水連續(xù)透析，再濃縮到33%（質(zhì)量分?jǐn)?shù)）。獲得的濃縮絲素蛋白水溶液轉(zhuǎn)移入自制的靜電紡絲裝置進行制備。為了改善材料的機械強度，獲得的絲素蛋白材料在90%的乙醇水溶液（質(zhì)量體積）中進行單向拉伸處理。經(jīng)過拉伸處理后的電紡絲素蛋白支架材料表面使用掃描電鏡檢測，并取5個區(qū)域，使用KS400圖像分析系統(tǒng)進行分析表面孔隙大小，并檢測其孔隙率，最后拉力試驗檢測其機械強度。 結(jié)果：我們制備獲得的絲素蛋白支架材料多孔，三維結(jié)構(gòu)�？讖郊s為40±4.96μm。纖維絲平滑而均勻，直徑約為800-1200nm。材料經(jīng)拉伸處理后，其斷裂能及斷裂強度分別為1.8MPa及93.2J/kg，縫合強度為1.4N，滿足尿道修復(fù)重建要求。 結(jié)論：經(jīng)制備、拉伸處理后的靜電紡絲素蛋白支架材料為3D多孔結(jié)構(gòu),其空間結(jié)構(gòu)及機械強度符合尿道重建修復(fù)需要。 第二部分絲素蛋白支架材料復(fù)合尿路上皮細(xì)胞修復(fù)重建犬尿道缺損 目的：評估拉伸處理后的靜電紡絲素蛋白材料復(fù)合尿路上皮細(xì)胞在犬尿道重建中的應(yīng)用效果。 材料與方法：制備新型的經(jīng)拉伸處理的電紡絲素蛋白材料。使用9只比格犬作為實驗動物，其中6只實驗組，從實驗組動物中獲得，培養(yǎng)，擴增其尿路上皮細(xì)胞，并種植于材料上，培養(yǎng)一周，獲得組織工程移植物。獲得的組織工程移植物經(jīng)過HE染色及掃描電鏡檢測。另外3只犬作為對照組，對9只實驗動物都經(jīng)過剝離尿道粘膜3×1cm2處理，實驗組動物組應(yīng)用組織工程移植物進行修復(fù)，3只對照組未使用任何材料進行修復(fù)。術(shù)后1,2及6個月后，行尿道造影檢查，尿道修復(fù)部位組織切片HE染色，免疫組化染色。并使用KS400圖像分析系統(tǒng)對上皮再生面積定量分析比較。 結(jié)果：尿路上皮細(xì)胞在絲素材料上生長良好，在材料表層形成多層結(jié)構(gòu)，且細(xì)胞能滲透乳材料內(nèi)部。在動物實驗中，實驗組中的比格犬未表現(xiàn)排尿困難，尿道造影示尿道管腔未顯示明顯狹窄，，組織學(xué)檢測示上皮細(xì)胞逐漸生長，在術(shù)后6個月形成典型尿路上皮細(xì)胞結(jié)構(gòu)。在對照組中，犬表現(xiàn)出排尿困難，尿道造影示尿道管腔狹窄，組織學(xué)顯示上皮細(xì)胞生長緩慢，6個月后僅有1-2層上皮細(xì)胞覆蓋在缺損處，并可見較嚴(yán)重炎性反應(yīng)。 結(jié)論：拉伸處理后的絲素蛋白材料與尿路上皮細(xì)胞有良好的生物相容性。經(jīng)證實復(fù)合尿路上皮細(xì)胞的絲素蛋白材料或可成為潛在的尿道重建材料。 第三部分絲素蛋白支架材料制備組織工程口腔粘膜修復(fù)重建犬尿道缺損 目的：評估絲素蛋白支架材料構(gòu)建組織工程口腔黏膜在尿道修復(fù)重建中的應(yīng)用效果。 材料與方法：我們分離、培養(yǎng)、鑒定自體口腔上皮細(xì)胞及成纖維細(xì)胞然后將種子細(xì)胞種植在絲素蛋白支架材料上，然后使用HE染色、免疫組化染色及掃描電鏡檢測。建立10只5cm尿道粘膜缺損動物模型，實驗組5只使用組織工程口腔粘膜，對照組5只使用單純的絲素蛋白支架材料，術(shù)后6個月應(yīng)用尿道逆行造影、大體及組織學(xué)等檢測。 結(jié)果：絲素蛋白支架與口腔上皮細(xì)胞及成纖維細(xì)胞表現(xiàn)良好的組織相容性。口腔上皮在支架材料上生長良好，形成多層上皮結(jié)構(gòu)。實驗組動物6個月后未見排尿困難，尿道逆行造影未見明顯尿道狹窄，尿道組織學(xué)檢測示分層上皮細(xì)胞形成。對照組動物在6個月隨訪期間發(fā)生排尿困難，尿道逆行造影顯示尿道狹窄，組織學(xué)檢測發(fā)現(xiàn)僅1-2層上皮細(xì)胞形成。 結(jié)論：以絲素蛋白材料作為支架的組織工程口腔粘膜有成為潛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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R318.08
本文編號：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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R318.08
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