内皮祖细胞复合膀胱无细胞基质的生物相容性

目的 应用内皮祖细胞复合膀胱无细胞基质构建组织工程学膀胱.方法 分离培养猪外周血内皮祖细胞,制备膀胱无细胞基质.体外检测细胞与膀胱无细胞基质的生物相容性.观察细胞与膀胱无细胞基质的体外复合.结果 成功分离培养猪外周血内皮祖细胞,其表面标志分别为CD133 25.1%、CD34 55.9%、flk-1 97.7%、CD31 82.0%、CDl44 95.4%.成功制备膀胱无细胞基质.生物相容性检测证明膀胱无细胞基质对细胞无明显的细胞毒性,细胞活力保持在90%以上.体外观察细胞可以复合于无细胞基质,并在其上增殖、生长.结论 可以应用内皮祖细胞作为种子细胞,膀胱无细胞基质作为支架材料构建组织工程学膀胱,可能成为提高体内组织工程膀胱血管化的一种新方法.     

碳化二亚胺交联犬颈总动脉脱细胞基质的组织相容性初步研究

目的应用碳化二亚胺交联同种异体的犬颈总动脉脱细胞基质，对其进行组织相容性评价。方法应用多步骤除垢剂-胰酶作用制备犬颈总动脉脱细胞基质，按脱细胞基质与碳化二亚胺质量比1：1和1：2制备不同交联程度的脱细胞基质。通过体外降解率，细胞毒性MTT实验和大鼠皮下埋置实验对交联效果和组织相容性进行评价。结果经碳化二亚胺交联的脱细胞基质形态上无明显变化，HE染色纤维排列规则无断裂。0．1％Ⅱ型胶原酶作用下，交联的脱细胞基质的降解率较单纯脱细胞基质显著降低，交联程度高的脱细胞基质较交联程度低的脱细胞基质降解率低。细胞毒性MTT结果显示碳化二亚胺交联的脱细胞基质细胞毒性分级为0-1级，无细胞毒性。大鼠皮下埋置实验显示交联的脱细胞基质抵抗组织酶降解的能力显著提高，同时免疫反应减轻。交联程度高，效果越明显。结论碳化二亚胺交联的脱细胞基质具有良好的组织相容性和抗组织酶解能力，作为血管移植物和组织工程化血管的支架材料有广阔的应用前景。     

碳化二亚胺交联并肝素化的血管脱细胞基质的组织相容性

目的应用碳化二亚胺交联同种异体犬颈总动脉脱细胞基质,并且利用其双功能交联特性将肝素共价结合于脱细胞基质上,评价其组织相容性。方法应用多步骤除垢剂-胰酶作用制备犬颈总动脉脱细胞基质,按脱细胞基质、碳化二亚胺和肝素的质量比1∶2∶1制备交联并肝素化的脱细胞基质,采用甲苯胺蓝染色法定性检测肝素结合;采用细胞毒性四甲基偶氮唑盐比色法(MTT)检测其细胞相容性;采用大鼠皮下埋植实验检测其体内组织相容性,并进行免疫组织化学和透射电镜检测。结果碳化二亚胺交联并肝素化的脱细胞基质的形态和强度无明显变化,甲苯胺蓝染色定性地显示了肝素与支架的结合;细胞毒性实验显示碳化二亚胺交联并肝素化的脱细胞基质浸提液提高了内皮细胞的增值率;大鼠皮下埋植实验显示,交联并肝素化的脱细胞基质有良好的组织相容性,并且促进成肌纤维细胞浸润。结论碳化二亚胺交联并肝素化的脱细胞基质具有良好的组织相容性,同时能够促进细胞的再分布过程。     

人羊膜脱细胞基质填充材料的生物相容性及动物有效性研究

目的探讨人羊膜脱细胞基质填充材料的生物相容性及动物有效性。方法通过生物学评价实验对羊膜脱细胞基质填充材料的刺激性、致敏性、细胞毒性等进行检测,并通过大鼠皮内植入实验检测材料的动物有效性(以上市产品双美胶原蛋白作为对照)。结果羊膜脱细胞基质填充材料无致敏性、无刺激性,且细胞毒性不大于1级;大鼠皮内植入有效性实验显示,羊膜脱细胞基质填充材料于植入后20周依然有肉眼可观察到的材料保留。双美胶原植入剂降解完全,肉眼和HE染色均未见材料。且两种材料在体内降解期间的炎症反应和免疫排斥反应均为轻度反应。结论人羊膜脱细胞基质填充材料具有良好的生物相容性,无明显炎症及免疫排斥反应。其在大鼠皮内耐降解性强,保留时间较长,可作为一种安全有效的填充注射产品应用于临床。     

冻干法保存脱细胞组织工程血管支架的实验研究

目的：探讨冷冻干燥技术长期保存脱细胞组织工程血管支架材料的可行性。方法：将制备的脱细胞组织工程血管支架预冷后置入-70℃、6.67×10-4kPa的冷冻干燥机内6h作冷冻干燥处理。通过对冷冻干燥处理前后脱细胞血管支架材料的组织,超微结构观察、生物力学评估、细胞毒性测定以及动物体内的组织相容性检测,研究冷冻干燥处理对脱细胞血管支架材料生物相容性和生物力学特性的影响。结果：冷冻干燥处理对于脱细胞血管材料的生物力学及生物化学特性没有明显影响,处理后的材料依然具有良好的体内、外生物相容性。结论：冷冻干燥处理作为脱细胞组织工程血管支架材料的长期储存方法是可行的。     

骨骼肌无细胞基质的制备及其生物相容性研究

目的细胞外基质是脂肪组织工程材料的研究热点之一。通过探讨骨骼肌无细胞基质的制作方法及生物相容性,为其在脂肪组织工程中的应用奠定基础。方法取健康成年小香猪新鲜骨骼肌组织,横切成厚2～3 mm的组织块,采用低渗-去垢剂法脱细胞处理。处理后采用HE染色、Masson三色染色、免疫组织化学染色及扫描电镜检测骨骼肌无细胞基质是否有细胞成分残留,并观察其基本结构;应用MTT法检测骨骼肌无细胞基质细胞毒性。取乳腺癌患者自愿捐赠脂肪组织,分离培养人脂肪干细胞(human adipose-derived stem cells,hADSCs),从形态学、流式细胞学和成脂、成骨分化能力方面进行鉴定。将骨骼肌无细胞基质与第3代hADSCs共培养,于培养后第1、3、5、7天通过细胞活性检测材料上细胞黏附、扩散和增殖情况,了解其与细胞之间的相互作用。结果 HE、Masson、免疫组织化学染色及扫描电镜观察显示骨骼肌无细胞基质肌纤维去除完全,无细胞核残留,基质结构保留完整;大量连接成网状的胶原纤维呈多孔隙样结构,规则排列。MTT检测示骨骼肌无细胞基质细胞毒性为1级,细胞相容性好。细胞活性检测示hADSCs在骨骼肌无细胞基质上能很好地伸展,且能与周围基质黏附,进入基质内部并相互交织。结论经脱细胞处理的骨骼肌无细胞基质具有良好生物相容性,可能作为脂肪组织工程的支架材料。     

鹿茸软骨组织脱细胞基质材料的制备及生物相容性研究

目的探讨鹿茸软骨制备脱细胞基质材料的可行性以及生物相容性,为软骨修复重建探索新材料。方法取梅花鹿鹿茸生长中心间充质层,进行由DNA酶、RNA酶、抑肽酶等介导的脱细胞处理;行组织学和DNA含量检测,评价脱细胞效果。取第2代鹿生茸区骨膜(antlerogenic periosteum,AP)细胞,行荧光干细胞标记明确其干细胞特性后,用PKH26荧光标记并与制备的间充质层脱细胞基质进行复合培养;7 d后取材行HE染色观察以及荧光显微镜下观察PKH26标记的AP细胞在基质表面生长情况。以上观测均以未复合AP细胞的脱细胞基质作为对照。将复合培养7 d的样本移植至裸鼠一侧腹股沟(实验组),取空白培养样本移植于另一侧(对照组)。于移植后7、21 d取材行HE染色,同时对组织进行冰冻切片并在荧光显微镜下观察PKH26标记成功的AP细胞在脱细胞基质表面及内部的生长情况,评价含AP细胞的脱细胞基质在裸鼠体内的组织相容性。结果HE和DAPI染色显示脱细胞处理后材料中无细胞残留,DNA含量为(19.367±5.254)ng/mg,较脱细胞处理前的(3 805.500±519.119)ng/mg显著降低(t=12.630,P=0.000),提示成功制备间充质层脱细胞基质。AP细胞与间充质层脱细胞基质复合培养7 d后,AP细胞主要黏附于材料表面,部分进入脱细胞基质内部。植入裸鼠体内后,随观察时间延长,接种AP细胞可以在脱细胞基质材料中增殖并逐渐进入材料内部,并诱导血管生成。结论实验成功制备鹿茸软骨脱细胞基质,该基质材料在离体和活体情况下适于种子细胞(AP细胞)的黏附和增殖,并具有刺激血管生成的功能,为其用于软骨组织修复提供理论依据。     

人舌黏膜上皮细胞体外构建组织工程尿道的初步研究

目的 探索人舌黏膜上皮细胞和脱细胞支架体外复合构建组织工程尿道的可行性.方法 前尿道狭窄患者10例,手术切取0.5 cm×0.8 cm大小舌黏膜组织,分离获得舌黏膜上皮细胞,AE1/AE3抗体行细胞免疫荧光鉴定.收集第3代上皮细胞,按1×107/ml密度分别接种于脱水BAMG支架、液体保存BAMG支架以及4层脱细胞基质(SIS)支架表面,体外培养7d后行HE及扫描电镜检测.结果 10例患者术后1个月未出现舌部相关并发症.14 d后原代舌黏膜上皮细胞融合达90％～95％并呈典型的“鹅卵石”状生长.第3代时增殖速度达顶峰,平均7d达到90％～95％融合,第4代开始细胞逐渐老化.HE及扫描电镜检测液体保存BAMG支架表面仅复合极少量细胞,脱水BAMG以及SIS支架表面可见明显多层上皮细胞覆盖,其中4层SIS支架内部可见局部细胞浸润性生长迹象 结论 人舌黏膜上皮细胞可以作为组织工程尿道上皮种子细胞来源之一,与脱水处理后的SIS和BAMG有很好的组织相容性和黏附能力,二者的有效复合可以构建适合尿道修复重建需要的组织工程替代材料.     

胎猪脱细胞主动脉作为小口径组织工程血管替代物的体内试验研究

目的:研究胎猪脱细胞主动脉(decellularized aorta of fetal pigs,DAFP)的生物相容性,确定其是否有作为支架材料用于小口径组织工程血管移植的潜力。方法:利用胰酶和核酸酶联合的脱细胞方法来制备胎猪主动脉脱细胞基质(DAFP),将其作为小口径组织工程血管的生物支架材料移植在成年犬单侧颈总动脉处,并监测其移植处的血流通畅情况,后期又通过组织学染色观察组织工程血管的组织学结构;扫描电镜观察血管的内表面结构;透射电镜观察其内表面的内皮细胞再生情况。结果:组织学染色结果表明构建的小口径组织工程血管具有完整的内膜层及中膜层结构;扫描电镜结果显示组织工程血管内表面覆盖着完整的内皮细胞层。结论:DAFP有作为小直径组织工程血管支架用于体内移植的潜力。     

软骨脱细胞基质复合脂肪源性干细胞构建软骨组织的实验研究

[目的] 探讨软骨脱细胞基质支架材料的制备,及其体外复合脂肪源性干细胞构建软骨组织的技术方法.[方法] 成年新西兰大白兔脂肪源性干细胞获得,培养,扩增.成年新西兰大白兔新鲜软骨,低温冻干12 h,后经曲拉通、DNA、RNA酶等处理制备成为软骨脱细胞基质支架材料,终浓度为2×107/L的脂肪干细胞种植于软骨脱细胞基质中于软骨细胞方向诱导培养基中培养2周,构建软骨组织.新鲜制备的软骨脱细胞基质及构建的软骨组织分别行组织学、免疫组织化学及透射电镜检测.[结果] 实验制备的软骨脱细胞基质支架材料内无细胞结构存在,仅残留空白软骨陷窝.具有合适的孔隙率和孔径大小;复合脂肪源性干细胞后细胞向材料内部迁移,粘附,生长良好.部分载体内细胞Ⅱ型胶原免疫组化染色阳性.[结论] 软骨脱细胞基质可作为支架材料应用于软骨组织工程,复合脂肪源性干细胞培养可成功构建软骨组织.     

Long-term study of male rabbit urethral mucosa reconstruction using epidermal cell

Aim： To investigate the transformation of characteristics of epidermal cells from foreskin which were used to reconstruct male rabbit anterior urethra in combination with acellular collagen matrices. Methods： In three rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and seeded （inoculated） onto a tubular acellular collagen matrix, acquired from allogeneic rabbit bladder submucosa. A urethral mucosal defect was created, and urethral reconstruction was performed with the tubular acellular collagen matrix seeded with epidermal cells. Results： On gross examination at 12 months following the procedure, the mucosa of the urethral grafts appeared lubricous and smooth. Urethrography showed that a wide urethral caliber had been maintained without any sign of strictures. Histological examination showed a transitional cell layer in the graft without evidence of a margin between the graft and the host tissue at 12 months postoperatively. Conclusion： Epidermal cells seeded onto acellular collagen matrices can be successfully used to reconstruct urethras that have defects and are transformed to transitional epithelial cells.     

Urethral replacement using epidermal cell-seeded tubular acellular bladder collagen matrix

OBJECTIVES: To investigate the feasibility of replacing urinary epithelium cells with foreskin epidermal cells to reconstruct engineered anterior urethra with an acellular collagen matrix. MATERIALS AND METHODS: Acellular collagen matrices were generated from allogeneic rabbit bladder submucosa. In nine rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and labelled with 5-bromo2'-deoxy-uridine (BrdU) before seeding onto a tubular acellular collagen matrix (1.5x1 cm). In male rabbits, a urethral mucosal defect was created, and urethroplasty performed with a tubular acellular collagen matrix seeded with epidermal cells (nine rabbits) or with a matrix with no cell seeding (nine rabbits; control group). Urethrography was done at 1, 2 and 6 months after grafting. The urethral grafts were harvested and analysed grossly and histologically. RESULTS: In the control group, gross views and urethrography revealed stricture of repaired defects at the different sample times. In the experimental group, a wide urethral calibre was maintained with no sign of strictures. Histology in the control group showed a single layer of epithelium cells with disorganized muscle fibre bundles in the submucosa layer at 1 month after grafting, and a transitional cell layer surrounded by disorganized muscle fibre bundles at 2 and at 6 months. Grafts seeded with epidermal cells formed a single-layer structure by 1 month, and at 2 and 6 months there were several layers of epidermal cells with abundant vessels in the submucosa. There was an evident margin between graft epidermal cells and host epithelium at 6 months. The implanted cells expressed keratin, shown by staining with anti-pancytokeratins. Immunofluorescence for BrdU confirmed the presence of implanted epidermal cells at 1 month after grafting; there were fewer positive cells at the implantation site at 2 months. At 6 months, there were several layers of epidermal cells with no signs of BrdU staining. CONCLUSIONS: Urethral reconstruction was better with an acellular collagen matrix seeded with epidermal cells than with the acellular collagen matrix alone. Foreskin epidermal cells seem adequate in replacing urethral epithelium cells for urethral reconstruction.     

应用异体脱细胞尿道基质修复尿道缺损

目的探讨应用同种脱细胞尿道基质修复尿道缺损的可行性。方法将14只雄性新西兰兔分为两组，切除实验组长约1.0～1．5cm的尿道，用相应长度脱细胞尿道基质修复；对照组行假手术。术后行尿道造影并取尿道标本作病理检查。结果12只实验兔的脱细胞基质移植物没有移位。除2例狭窄、2例尿瘘外，其余满意效果。病理检测示，术后3周尿道管腔上皮化，6个月基质中平滑肌及血管再生明显。结论同种脱细胞尿道基质材料可以修复兔尿道部分缺损。     

异种膀胱无细胞基质替代尿道的研究

目的探讨异种膀胱无细胞基质(ACM)管状替代尿道的可行性。方法19只成年雄性新西兰白兔分成3组：A组3只，为假手术对照组；B组10只，切除一段1．0cm尿道；C组6只，切除一段3．5～4．0cm尿道，之后应用已经事先制备好的异种膀胱ACM制成相当长度的管状替代被切除的尿道。术后1、2、4、8、16周动态观察替代尿道的尿道上皮、平滑肌和血管的再生情况。结果所有实验动物在术后7d拔除尿管后都恢复了自主排尿，没有排斥、尿瘘、感染等并发症发生。组织学检查显示实验组术后2周尿道上皮再生良好，4周完全覆盖尿道内腔，术后8周平滑肌见于近吻合口处，平滑肌生长缓慢，观察期内未能覆盖全长尿道。尿道造影未见明显尿道狭窄和憩室。结论异种膀胱ACM是一种良好的尿道修复和替代的材料。     

脱细胞尿道及其海绵体基质制备的实验研究

目的 探索脱细胞尿道及其海绵体基质的制备方法。方法取健康壮年兔完整尿道及其海绵体组织，以Triton-X100与NH3H2O联合提取法进行脱细胞处理。标本做HE染色，组织学观察分析脱细胞效果。结果脱细胞处理11天后，成功获得脱细胞及其海绵体基质，所得基质外观良好。HE染色观察无细胞存在。弹力纤维排列规整，间隙较大，结构无破坏。结论利用Triton-X100与NH3H2O联合提取法可成功制备完整无细胞尿道及其海绵体基质，为尿道再造修复提供崭新思路。     

Tissue engineering of vascular grafts: human cell seeding of decellularised porcine matrix.

OBJECTIVES: To develop a biocompatible and mechanically stable vascular graft combining human cells and a xenogenic acellular matrix. DESIGN/MATERIALS: Decellularised matrix tubes were obtained by enzymatic cell extraction of native porcine aortas. Endothelial cells and myofibroblasts were isolated from human saphenous veins and grown in cell cultures. The inner surface of the tubes was seeded with endothelial cells or myofibroblasts and exposed to pulsatile flow. RESULTS: After cell extraction, the absence of cellular components, as well as the maintenance of matrix integrity, was demonstrated by means of light microscopy and scanning electron microscopy. Furthermore, the porcine matrix was successfully seeded with human endothelial cells, which grew to a monolayer under flow conditions. Stable biomechanical properties were achieved at physiological perfusion pressures in vitro. CONCLUSIONS: Cellular components can be extracted from native porcine blood vessels. Vascular grafts can be generated in vitro of animal acellular matrix and human cells.     

Comparison of partial urethral replacement with acellular matrix versus spontaneous urethral regeneration in a canine model

OBJECTIVE: To determine whether acellular matrix could be used for partial urethral replacement and to compare regeneration over acellular matrix versus normal spontaneous urethral regeneration. MATERIALS AND METHODS: The study included 21 male mongrel dogs in which a 3-cm segment including half of the urethral circumference was excised. In 13 dogs (study group), the defect was covered by acellular matrix of the same length and width obtained from female mongrel dogs and prepared to have complete cell lysis with keeping of the fiber framework. In 8 dogs (control group), the urethral defect was not covered by any urethral tissue. In both groups, an 8F feeding tube was kept inside the urethra for a mean duration of 2 weeks. In the study group, dogs were sacrificed at 1 week, 2 weeks, 3 weeks and then one dog every month for 10 months. In the control group, one dog was sacrificed every month for 8 months. RESULTS: All dogs survived the procedure. In the study group, 10 dogs underwent urethrogram; 8 were normal, 1 had diverticulum and 1 had relative narrowing. In the control group, 6 dogs underwent urethrogram; 5 were normal and 1 showed relative narrowing. Histopathological examination of the study group showed gradual regeneration over the acellular matrix with normal appearance at 20 weeks. In the control group, normal healing was observed at 2 months and thereafter. CONCLUSION: Regeneration of all components of the urethra can occur gradually over acellular matrix and is complete at 20 weeks. Regeneration of a urethral defect 3-cm long including half of the urethral lumen is possible with or without acellular matrix.     

Acellular matrix tube for canine urethral replacement: is it fact or fiction?

PURPOSE: We evaluated the results of acellular matrix used as a tube for replacement of a relatively long segment of the canine urethra. MATERIALS AND METHODS: The study included 9 female and 5 male mongrel dogs in which a 3 cm segment of the whole urethral circumference was excised and replaced by a tube of acellular matrix of the same length and width. The acellular matrix was obtained by excision of the whole urethra of donor female dogs that were sacrificed and not included in the study group. The retrieved urethra was treated to have complete cell lysis with maintenance of the fiber framework. In all dogs the urethra was stented for 4 weeks. Ascending urethrogram was done after stent removal every month until the dogs were sacrificed. The dogs were sacrificed at a rate of 1 weekly for 4 weeks and then monthly. If urine retention occurred, ascending urethrogram was performed and the dog was sacrificed. Before sacrifice the urethra was exposed and carefully examined. The whole specimen was then excised for histopathological examination. RESULTS: All dogs survived surgery and no postoperative complications were seen during urethral stenting. After stent removal all dogs had a urethral fistula and/or stricture, which increased in severity until urine retention occurred by the end of month 3. Exploration at 1, 2, 3 and 4 weeks showed an intact graft, although there was progressive shrinkage in length and urethral calibration also demonstrated progressive narrowing of the lumen. At 3 months there was marked shrinkage (length equaled 0.3 cm) and complete obliteration of the lumen. Histopathological examination showed progressive fibrosis, which was extensive by week 4. CONCLUSIONS: Acellular matrix tube is not able to replace a 3 cm segment of canine urethra.