口腔黏膜细胞与膀胱黏膜下脱细胞基质复合物构建组织工程化尿道的实验研究

目的 探讨以兔口腔黏膜细胞与同种异体膀胱黏膜下脱细胞基质(BAMG)复合物构建组织工程化尿道的可行性.方法 新西兰雄性兔24只,距尿道外口2.0 cm剥离尿道黏膜(2.0 cm×0.8 cm)后,随机分实验组和对照组,每组12只.切取实验组兔口腔黏膜组织分离细胞,在有灭活的3T3细胞培养皿上进行培养扩增,将培养获得的第2代口腔黏膜细胞种植于BAMG(2.2 cm×1.0 cm)上,植入实验组兔尿道缺损区域;对照组单纯采用无细胞植入的BAMG修复尿道.分别于术后1、2、6个月观察动物排尿情况,行尿道造影,8 F尿管插管确定有无狭窄;随后处死实验兔,取修复段尿道黏膜组织行组织学检查.结果 细胞培养获得的口腔黏膜细胞形态均一,生长良好;组织形态学、扫描电镜观察见口腔黏膜细胞与BAMG具有良好的相容性.实验组兔术后1、2、6个月伤口愈合良好、排尿通畅,无尿瘘发生,组织学和尿道造影检查显示带细胞修复的尿道形态完整、清晰宽敞,无狭窄发生;术后6个月植入的口腔黏膜细胞仍然存在,并明显扩增.对照组兔则出现排尿困难、尿道狭窄,光镜下发现黏膜及黏膜下存在严重的炎症反应.结论 兔口腔黏膜细胞与同种异体BAMG复合后,可成功用于尿道缺损的修复,构建组织工程化尿道.     

脱细胞基质在软骨组织工程中的应用

目的 综述脱细胞基质(acellular matrix,ACM)在软骨组织工程中的应用与研究进展. 方法 广泛查阅近年国内外相关文献,进行回顾与综合分析. 结果 许多研究者联合采用了渗透溶液法、冷冻干燥法和理化交联法等技术对软骨ACM支架进行了改进.软骨ACM支架用于构建组织工程软骨的实验结果 与ACM的特性密切相关. 结论 ACM在软骨组织工程领域有着广泛的应用前景,其特性的改进与完善非常重要.     

小肠黏膜下脱细胞基质修复前尿道狭窄的疗效分析

目的 探讨小肠黏膜下脱细胞基质(small intestinal submucosa,SIS)修复前尿道狭窄的可行性和有效性.方法 2009年6月至2010年8月采用4层SIS补片修复治疗尿道狭窄患者18例.患者年龄20～69岁,平均38岁;尿道狭窄段3.5～7.0 cm,平均4.6 cm;术前最大尿流率1.5～5.5 ml/s,平均3.8 ml/s.术中按需将SIS(长4.0～7.5 cm,宽 2.0 cm)植入尿道背侧缺损处,5-0可吸收线将SIS间断固定在阴茎海绵体上,SIS两侧与已剪开的狭窄段尿道作连续缝合,两端分别与尿道断端作间断吻合.结果 手术过程顺利,术后恢复好.随访6～18个月,平均10个月,患者未发生感染、排斥反应等并发症.17例排尿通畅,最大尿流率14.0～44.0 ml/s,平均25.4 ml/s.尿道造影显示尿道通畅;术后4、6周尿道镜检查示SIS移植物与周围组织分界清楚;术后14周尿道镜检查SIS已降解,修复段尿道与周围组织间限消失,黏膜光洁完整,管腔无明显狭窄;植入SIS部位活检显示黏膜表层为上皮细胞.1例尿道下裂术后患者术后5个月出现轻度尿道狭窄症状,行尿道扩张治疗.结论 利用SIS修复尿道狭窄具有创伤小、抗感染力强的特点,可作为组织工程尿道修复重建材料修复部分尿道狭窄患者.

Abstract：

Objective To investigate the feasibility of using small intestinal submucosa (SIS) graft for the repair of anterior urethral strictures. Methods From June 2009 to August 2010, 18 men (mean age, 38 yrs) with anterior urethral strictures underwent urethroplasty using a four-layer SIS as an onlay patch graft. SIS was used to augment the urethral caliber at the stricture site. The mean stricture length was 4.6 cm (range 3.5 to 7 cm). The pre-operative mean maximal flow rate was 3.8 ml/s (range 1.5 to 5.5 ml/s). The required SIS grafts (4 to 7.5 cm long and 2 cm wide) were positioned into the urethrotomy defect and were spread-fixed to the corpora cavernosa using 5-0 polyglactin interrupted sutures. Two apices of the graft were sutured to the proximal and distal apices of the urethrotomy with 5-0 polyglactin interrupted stitches. The margins of the opened urethra were sutured to the SIS patch with 5-0 polyglactin running sutures. Results The mean follow-up period was 10 mon. (range 6-18 mon.). No postoperative complication, such as infection or rejection related to the use of heterologous graft material was observed. Seventeen patients voided well postoperatively with the mean peak urine flow of 25.4 ml/s (14-44 ml/s). Cystoscopy revealed that at four weeks and six weeks, the SIS graft was well distinguishable from the normal surrounding tissue; and at 16 weeks, the urothelium was regenerated and the biomaterial was not distinguishable from the normal surrounding tissue. The squamosal epithelium was seen in the histological examination of the grafts. The remaining one patient with failed hypospadias developed a slight urethral narrowing at five months post-operatively and needed sound dilatations. Conclusions SIS matrix appears to be a safe and effective reconstructive material in selected urethral reconstructions.     

利用脱细胞基质预构小口径组织工程血管的实验研究

目的 在犬腹腔内预置脱细胞基质后形成结缔组织管,观察将其作为血管替代物的力学特征及移植于自体股动脉后的组织结构变化.方法 在犬腹腔内埋置长8～12 cm用硅胶棒支撑的制备好的脱细胞基质,3周后将硅胶棒周围形成的管状物作为血管替代物移植于自体股动脉,同时对此管状物作力学、组织学检测及与颈动脉、股静脉的对比分析.在移植术后6个月观察血管通畅情况.光镜、透射及扫描电镜观察替代物的组织结构.结果 ①血管替代物的力学性能弱于正常动脉而强于正常静脉.②组织学观察:形成的管状物内腔有少量的间皮细胞黏附;弹性纤维、胶原纤维结构较完整;纤维网中充满成纤维母细胞.6个月后内皮细胞在替代物内壁覆盖连续,平滑肌细胞与对照组相近.③移植6个月后脱细胞基质组血管全部通畅.结论 ①用脱细胞基质预构的血管替代物力学性能符合血管移植、血运重建的需要.②所形成的血管替代物移植6个月后组织相容性良好,替代物管壁厚度及组织结构已接近正常血管.     

脱细胞基质在泌尿管路组织工程中的应用

脱细胞基质是经理化方法脱去组织中抗原成分并富含胶原的一种材料，在泌尿管路缺损的替代再生与扩大研究中，显示了诱导膀胱、输尿管与尿道各层成分再生的潜力。随着制备工艺的完善与分子机制的阐明，脱细胞基质将会成为一种“现货供应”的泌尿系统修复材料。     

脱细胞基质支架在尿道缺损修复中的应用及研究进展

随着组织工程学的发展,脱细胞基质材料越来越多地被应用于组织创伤及器官病变的修复中,其在泌尿外科中的应用也越来越广泛,在尿道狭窄、尿道损伤以及先天性尿道缺损中的应用也逐渐增加,且临床效果显著。本文通过复习以往文献及相关图书,归纳了尿道缺损疾病的类型、病因及目前临床上的相关治疗方法,并阐述了脱细胞基质材料的特点及其在尿道缺...     

脱细胞基质在组织工程气管移植中的研究进展

目的综述脱细胞基质在组织工程气管中的研究现状、进展及未来前景。方法广泛查阅脱细胞基质在组织工程气管研究中的相关文献,对不同脱细胞基质修复人和动物气管病损的研究现状进行综合分析。结果气管组织工程应用的脱细胞基质包括空肠、膀胱、主动脉和气管。结论脱细胞膀胱基质及空肠基质在修复气管小范围非环形病损中具有较好效果,脱细胞主动脉基质在长段气管病损中的应用还需进一步研究,脱细胞气管基质在长段气管病损中具有良好的应用前景。     

不同性状脂肪脱细胞基质的制备及研究进展

近年来,组织工程作为一种新兴的治疗手段在整形和修复重建中的应用越来越受到临床和基础研究的重视。然而,目前常见的支架存在可塑性低或生物相容性差等缺点,寻求一种理想的支架材料仍是目前研究的热点和难点。脂肪组织脱细胞外基质（Decellularized adipose tissue,DAT）是指脂肪组织通过一系列脱细胞处理后得到的无细胞提取物,可模拟天然脂肪细胞外基质的特性,具有良好的生物相容性及可调节性,有望作为新型的组织工程支架应用于整形和修复重建等领域。本文将从DAT的特点、不同性状脱细胞基质的制备方法及研究进展等方面进行综述。     

激光微孔脱细胞基质在尿道缺损修复中应用的体外实验研究

目的：研究不同脉宽的超脉冲CO2激光对尿道脱细胞基质的热损伤以及微孔脱细胞基质的生理降解和孔隙率。方法：对尿道脱细胞基质用不同脉宽的CO2激光器进行激光打孔,比较微孔洗涤前后孔径变化,评价热损伤率;并对制备的微孔基质生理降解和孔隙率比较;结果：60ms的超脉冲CO2激光对尿道脱细胞基质的热损伤轻,制备的微孔脱细胞基质生理降解性能和细胞生长增殖生长曲线趋势一致,孔隙率合适。结论：超脉冲CO2激光对尿道脱细胞基质进行精细的加工出合适性能的脱细胞基质。     

人正常泌尿系上皮细胞系作为组织工程化尿道种子细胞的可行性研究

目的:研究人正常泌尿系上皮细胞系(SV-HUC-1)在传代20代之后的性质变化,探讨将其作为组织工程化尿道研究种子细胞的可行性.方法:对于SV-HUC-1传代20代的细胞进行形态观察,应用RT-PCR法和间接免疫荧光法检测细胞标志蛋白表达.应用Cell TrackerTMBlue CMF2HC,进行活细胞示踪研究.结果:对于第20代SV-HUC-1细胞,应用倒置相差显微镜观察生长到其形态正常.应用RT-PCR法和间接免疫荧光法均检测到第20代SV-HUC-1细胞中Vimentin和CK-18的mRNA表达.应用研究活细胞示踪研究,在第20代SV-HUC-1细胞中加入活细胞示踪剂Cell TrackerTMBlue CMF2HC后,发现SV-HUC-1细胞生长良好.结论:SV-HUC-1细胞能够正常生长传代20代,形态正常,能够表达泌尿系上皮细胞的标志蛋白,并能以活细胞的方式进行示踪和观察.SV-HUC-1细胞系是适合作为尿道组织工程实验用的种子细胞.     

人脂肪来源干细胞与膀胱脱细胞基质-丝素蛋白双层支架的生物相容性研究

目的观察人脂肪来源干细胞(Human adipose derived stem cells,h ASCs)在膀胱黏膜下脱细胞基质-丝素蛋白(Bladder acellular matrix graft-silk fibroin,BAMG-SF)双层支架材料中的生长情况,分析其生物相容性。方法取h ASCs,置于BAMG-SF浸提液中培养,CCK-8法检测其细胞活力,评价BAMG-SF支架的细胞毒性并绘制生长曲线。扫描电镜观察BAMG-SF双层支架材料的表面形貌。将h ASCs传代扩增后接种到BAMG-SF双层支架材料上,体外培养1周后,转至裸鼠皮下培养1周、2周,HE染色观察细胞在支架上的生长情况。HLA免疫荧光鉴定裸鼠皮下双层支架上细胞的种属来源。结果 h ASCs在BAMG-SF双层支架浸提液中可保持较高的增殖率,根据细胞相对增殖率与细胞毒性分级关系证实BAMG-SF双层支架浸提液无细胞毒性。由h ASCs在BAMG-SF浸提液和DMEM培养基中的生长曲线可知,BAMG-SF有利于h ASCs的生长。将h ASCs接种到BAMG-SF双层支架材料上,经过体外、内培养,h ASCs均能长入支架的空隙内,且体内培养比体外培养有更多的h ASCs细胞长入支架。HLA检测显示支架内细胞部分为h ASCs。结论新型BAMG-SF双层支架材料安全无毒,与h ASCs生物相容性好,可作为细胞载体应用于组织工程膀胱的研究。     

双层细菌纤维素基组织工程尿道支架的设计与初步构建

目的 设计并构筑具有模拟尿道组织微环境的三维微/纳支架材料。方法 通过对尿道脱细胞基质多尺度结构分析,设计仿尿道组织的双层细菌纤维素(Baterial cellulose,BC)基微/纳组织工程尿道支架。采用模板合成法,以明胶多孔支架为模板,在其上静态培养制备兼具微米多孔与纳米纤维的双层BC基微/纳支架材料。结果 双层BC基微/纳支架材料的致密层为细菌纤维素膜,疏松层为相互连通的多孔结构,平均孔径为(167±56)μm,同时在其微米级孔壁表面修饰着模拟天然细胞外基质(Extracellular matrix,ECM)结构的纳米纤维网络,纳米纤维直径为(20~40)nm。结论 采用模板合成法,成功构建出了兼具宏观双层结构、微米级多孔和纳米级纤维的双层BC微/纳组织工程尿道支架。     

Comparative Evaluation of the Book‐Type Acellular Bone Scaffold and Fibrocartilage Scaffold for Bone‐Tendon Healing

Bone‐tendon (B‐T) healing is a clinical challenge due to its limited regeneration capability. Fibrocartilage regeneration and bone formation at the healing site are two critical factors for B‐T healing. Promoting fibrocartilage regeneration and bone formation by tissue‐engineering may be a promising treatment strategy. In this study, we innovatively fabricated two kinds of acellular scaffolds from bone or fibrocartilage tissues, namely the book‐type the acellular bone scaffold (BABS) and the book‐type acellular fibrocartilage scaffold (BAFS). Histologically, the two scaffolds well preserved the native extracellular matrix (ECM) structure without cellular components. In vitro studies showed BABS is superior in osteogenic inducibility, while BAFS has good chondrogenic inducibility. To comparatively investigate the efficacy on B‐T healing, the BABS or BAFS were, respectively, implanted into a rabbit partial patellectomy model. Macroscopically, a regenerated bone‐tendon insertion (BTI) was bridging the residual patella and patellar‐tendon with no signs of infection and osteoarthritis. Radiologically, more new bone was formed at the healing interface in the BABS group as compared with the BAFS or control (CTL) groups (p < 0.05). Histologically, at postoperative week 16, histological scores were significantly better for regenerated fibrocartilage in the BAFS group or BABS group compared with the CTL group, but the BAFS group showed a significantly larger score than the BABS groups (p < 0.05). Biomechanical evaluation indicated a higher failure load and stiffness were shown in the BAFS group than those in the BABS or CTL groups at week 16 (p < 0.05). This study indicated that the BAFS is a more promising scaffold for B‐T healing in comparison with the BABS. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1709–1722, 2019     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的：研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架。方法：应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物。采用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性;采用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态。结果：网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性。尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞,长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架贴附紧密,适度伸展并有基质分泌。结论：网状尿道支架适合尿道移行上皮细胞黏附生长．可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道。     

舌黏膜补片治疗复杂性尿道狭窄的疗效观察

目的：探讨使用舌黏膜补片尿道成形治疗复杂性尿道狭窄的有效性。方法：2006年12月～2009年1月应用舌黏膜补片法治疗复杂性前尿道狭窄患者36例。结果：术后随访3～25个月，平均13．6个月。2例术后出现尿瘘，经再次手术修补后好转；另2例术后出现尿道狭窄，经尿道扩张后治愈。余者术后排尿通畅，平均最大尿流率达23．8ml／s。结论：舌黏膜具有取材方便、取材部位并发症少等优点，是一种治疗前尿道狭窄较好的替代物。     

Use of Homologous Acellular Dermal Matrix for Abdominal Wall Reconstruction in Rats

Homologous acellular dermal matrix graft (HADMG) has been used for the reconstructions of bowel, bladder, or urethra, but its suitability in the reconstruction of abdominal wall has not been tested. Therefore an experimental study was performed to evaluate the use of HADMG for the reconstruction of abdominal wall defects in weanling rats. Thirty weanling Wistar rats were used. A patch of abdominal wall 20 × 20 mm in dimension was removed. The defects were reconstructed with HADMGs that were derived from rat skin and prepared through a detergent enzymatic method. The reconstructed abdominal walls were evaluated as hernia rate and graft take ratio, excised and prepared for histological examination at 21 (n = 10), 40 (n = 10), and 90 (n = 10) days postoperation. The healing of repaired abdominal walls was uneventful. Histological evaluation demonstrated the migration of fibroblasts and neovascularization within the HADMG. Hernia in four rats were developed at 90 days. Neither significant wound contraction nor inflammation was seen at 21, 40, and 90 days after surgery in wounds receiving HADMGs. Thus, the use of a HADMG for reconstructing the abdominal wall in weanling rats has not given rise to any complications. HADMG has progressively remodeled into fibrous tissue. It appears to represent an important alternative substitute for the reconstruction of abdominal wall.     

Feasibility of Autologous Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Matrix Scaffold for Cartilage Tissue Engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell–derived ECM (aBMSC‐dECM) membrane was collected and fabricated into a three‐dimensional porous scaffold via cross‐linking and freeze‐drying techniques. Articular chondrocytes were seeded into the aBMSC‐dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC‐dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC‐dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC‐dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC‐dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.     

脱细胞真皮基质医用组织补片在鼻唇沟充填中的应用

目的 探索脱细胞真皮基质医用组织补片在鼻唇沟充填术中的应用,并对各种材料充填效果进行初步评价.方法 自2005年3月至2006年6月,经口内入路、松解、剥离鼻唇沟皮下粘连,应用脱细胞真皮基质医用组织补片充填于鼻唇沟凹陷皮下,所有病例随访3个月～6个月.结果 本组23例病人除1例因放置不平取出重放,另1例因放置腔隙出血、青紫,清创后重新放置,其余21例均取得满意效果.结论 鼻唇沟皱纹祛除术,除中面部切开拉紧皮肤筋膜效果肯定外,其他的充填材料虽然有效,但效果不是很满意或容易复发.脱细胞真皮基质医用组织补片用鼻唇沟充填术不仅有良好的组织相容性,手术操作简单、损伤小、易成活等特点,充填效果满意,并且在鼻唇沟参与动态表情方面不会随面部运动显现充填物轮廓,是一种理想的鼻唇沟充填的新型生物材料.