组织工程化膀胱壁复层结构的体外构建

目的 探讨膀胱缺损组织工程修复方法.方法 机械法去除膀胱黏膜层、肌层和浆膜层,仅保留膀胱黏膜下层.经SDS、Trixon-100和三蒸水处理去除细胞结构,制备获得膀胱脱细胞基质移植物(BAMG)作为支架材料.取成年猪膀胱壁,机械法分离膀胱肌层和黏膜层.以酶消化法分别获取膀胱尿路上皮细胞和平滑肌细胞.常规细胞传代和扩增后,将膀胱平滑肌细胞和尿路上皮细胞分别接种在BAMG浆膜面和黏膜面.细胞-材料复合物体外培养4周并行组织学鉴定.结果 HE和Masson染色鉴定发现,BAMG以胶原成分为主,内部未见明显细胞残留.免疫荧光方法鉴定结果显示,膀胱尿路上皮细胞uroplakin Ⅱ阳性,膀胱平滑肌细胞α-平滑肌肌动蛋白阳性.体外培养4周后,BAMG浆膜面和黏膜面分别形成连续的复层结构尿路上皮层和膀胱平滑肌层,免疫组化显示平滑肌层α-平滑肌肌动蛋白表达阳性,尿路上皮层广谱角蛋白表达阳性.结论 以膀胱脱细胞材料作为支架,尿路上皮细胞和膀胱平滑肌细胞作为种子细胞,能够在体外构建形成包含膀胱黏膜层及平滑肌层的复层膀胱壁结构.细胞-材料复合物的体外构建将为体内膀胱缺损的组织工程修复提供实验依据.     

聚乳酸/聚羟基乙酸共聚物网管支架体外预血管化及体内植入的实验研究

目的研究预血管化对含网管的多孔可降解聚乳酸/聚羟基乙酸共聚物(polylacticl/glycolicacidcopolymer,PLGA)支架血管化的影响。方法将含网管的多孔可降解PLGA支架由小鼠微血管内皮细胞预血管化,分为体外预血管化组(A组)和单纯支架组(B组)。将两组支架植入12只雌性NIH小鼠肠系膜间,分别于术后2、4周取材,行组织学及免疫组织化学观察血管化情况,并应用图像分析软件计算支架血管化面积。结果支架体外预血管化后,其网管内可见微血管内皮细胞均匀贴壁。支架植入体内2周,切片血管化面积A组为2260.91±242.35μm2与B组823.64±81.29μm2比较,差异有统计学意义(P<0.01);4周时A组为17284.36±72.67μm2与B组17041.14±81.51μm2比较,差异无统计学意义(P>0.05)。植入2周支架切片肌动蛋白阳性染色面积A组为565.22±60.58μm2与B组205.91±16.25μm2比较,差异有统计学意义(P<0.01);4周时A组为4321.09±19.82μm2与B组4260.28±27.17μm2比较,差异无统计学意义(P>0.05)。结论含网管的多孔可降解PLGA支架是一种良好的简易血管化支架模型;预血管化可以明显增强支架植入早期的血管化。     

许旺细胞在聚羟基乙酸纤维上三维定向培养

目的 为制备组织工程化的神经桥接物。方法 将体外扩增的许旺细胞（ＳＣｓ）接种在纵向排列的、可吸收材料ＰＧＡ纤维支架上，并对这种立体培养的细胞进行光镜、电镜和免疫组化观察，结果 发现许旺细胞可与聚羟基乙酸（ＰＧＡ）纤维良好吸附。并在其上边分裂边迁移从而形成了类似Ｂｕｅｎｇｎｅｒ带的纵向排列的细胞链，且其间含有一定的间隙，同时细胞大量分泌的层粘蛋白（ＬＮ）沉积在纤维表面形成了纵向的、类似变性神经的结构     

利用聚羟基乙酸构建组织工程皮肤的实验研究

目的 以聚羟基乙酸(PGA)为培养支架，用组织工程方法在体外构建双层、有活性皮肤。方法应用酶消化法分别获取人成纤维细胞(FB)和表皮角质形成细胞(KC)，体外扩增培养。接种HF与PGA上1周后，在复合物表面接种KC，共培养1周将复合物置于气-液界面继续培养。于不同时间段取材。行组织学、超微结构及免疫组织化学检测。结果接种表皮角质形成细胞在“FB-PGA”复合物表面1周后可见2～3层连续的HK在复合物上生长良好；4周时PGA已部分降解，表皮分层分化较完善，抗involucrin染色阳性，透射电镜观察可见表皮细胞分层分化良好。细胞的相邻面有桥粒连接。结论 采用组织工程技术可以在体外构建双层、有活性的人工皮肤；组织工程皮肤具有和正常皮肤相似的组织学特征及生化成分。     

包埋后的聚羟基乙酸与软骨细胞体外培养实验研究

目的:通过卵磷脂和多聚赖氨酸分别和共同包埋以聚乳酸固定成形的聚羟基乙酸支架与软骨细胞体外培养,来观察其亲水性和对细胞吸附力的改变以及对细胞功能的影响。方法:将软骨细胞种于上述支架体外培养,通过倒置显微镜及扫描电镜观察支架的亲水性、对细胞的吸附力及基质产生情况。结果:支架以卵磷脂包埋后细胞悬液易浸入到支架内;以多聚赖氨酸包埋后细胞易吸附在支架纤维表面;以卵磷脂和多聚赖氨酸共同包埋后细胞均匀分布于支架纤维之间及吸附在支架纤维表面,且基质产生旺盛。结论:卵磷脂具有增强支架亲水性作用;而多聚赖氨酸除增加支架对细胞的吸附力外,还具有促进细胞功能的作用。以卵磷脂和多聚赖氨酸共同包埋支架可能是组织工程技术中较理想的支架之一。     

聚乳酸-聚羟基乙酸神经导管和骨髓间充质干细胞构建组织工程神经

[目的]用聚乳酸-聚羟基乙酸共聚物(poly lactide-co-glycolide acid,PLGA)为原料制备新型神经导管,同时对兔骨髓间充质干细胞与PLGA构建组织工程神经的可行性进行观察.[方法]以聚乳酸-聚羟基乙酸共聚物和壳寡糖为原料,采用静电纺丝工艺制备中空的PLGA神经导管.通过扫描电镜观察材料的微观结构,排水法测定材料的孔隙率.将兔的骨髓间充质干细胞(BMSCs)分离培养后与导管共培养,扫描电镜观察细胞在材料上的生长粘附情况,MTT方法检测细胞在材料上的增殖活性.[结果]PLGA神经导管管壁具有疏松多孔结构,管壁纤维直径为18 μm左右,孔隙率为85.4%±1.6%,MTT结果显示导管无细胞毒性.兔骨髓间充质干细胞在导管表面生长良好.[结论]PLGA神经导管具有良好的孔隙率,生物相容性好,是构建组织工程神经的良好材料.     

聚磷酸钙纤维作为肌腱组织工程支架材料的体外实验研究

目的　探讨编织的辫状聚磷酸钙纤维 (CPPF)支架 ,在体外物理性能及与肌腱细胞的吸附情况。　方法　 CPPF直径 1 5μm,编织成横截面积为 3mm× 2 mm的辫状支架 ;体外测试其拉伸强度、孔隙率及吸水率。将体外培养 SD大鼠原代趾屈肌腱细胞 ,以 5× 1 0 6/ ml浓度与 CPPF辫状支架或 型胶原涂层的 CPPF辫状支架进行体外混合培养 ,组织学观察材料与细胞的吸附情况。　结果　编织的 CPPF辫的平均拉伸强度为 (1 2 2 .80± 1 7.34) N;平均吸水率为61 .56%± 1 4 .57% ;平均孔隙率为 50 .2 9%± 8.1 6%。肌腱细胞与 CPPF支架纤维的吸附较差 ,与 型胶原涂层后的CPPF支架纤维吸附良好。　结论　CPPF有可能成为一种较理想的构建肌腱组织工程复合型支架的新型材料     

经胶原包埋、修饰的聚羟基乙酸与软骨细胞体外培养的实验研究

目的：探讨经胶原包埋、修饰后的聚羟基乙酸（poloyglycolic acid简称PGA）作为组织技术中细胞培养支架的可行性；方法：将用胶原包埋的PGA和没有包埋的PGA分别与软骨细胞共同置于二氧化碳培养箱中培养，观察二者的亲水性，对细胞的吸附能力和细胞分泌基质的能力进行比较；结果：以胶原包埋的PGA和没有包埋的PGA亲水性没有明显的差异，二者亲水性均不强，而前者对细胞的吸附能力和细胞在其上面生长、分泌基质的能力明显增强；结论：以胶原包埋、修饰的PGA作为组织工程技术中细胞生长的支架，具有很大的应用前景。     

新型聚β-羟基丁酯作为血管组织工程支架材料的实验研究

目的：探讨经改性处理后的可降解生物材料聚β-羟基丁酯(PHB)作为血管组织工程支架材料与血管平滑肌细胞的细胞相容性。方法：采用体外培养的免血管平滑肌细胞接种在经胶原包埋处理后的管型PHB支架材料上，用相差显微镜和扫描电镜观察细胞的粘附和生长情况，并行HE染色观察。结果：免血管平滑肌细胞在改性后的管型PHB支架材料上粘附生长良好，扫描电镜下可见细胞生长融合成片状，HE染色示细胞在PHB材料上生长良好。结论：改性后的聚β-羟基丁酯材料与兔血管平滑肌细胞有较好的生物相容性。     

组织工程学材料作为膀胱替代材料的研究和应用

细胞外基质（Ｅｘｔｒａｃｅｌｌｕｌａｒ Ｍａｔｒｉｘ,ＥＣＭ）作为膀胱替代材料是近年来的研究方向之一。目前常用的ＣＥＭ有三种：（１）人工合成ＥＣＭ替代物;（２）天然ＥＣＭ;（３）天然ＥＣＭ框架。其作为膀胱替代材料的研究及应用有着广阔的前景。     

Improvement of the Tissue-adhesive and Sealing Effect of Fibrin Sealant Using Polyglycolic Acid Felt

Although fibrin sealant (FS) has an advantage of high biocompatibility, its adhesive force and sealing effect have been generally considered to be inadequate. In the present study, a high adhesive force and sealing effect were obtained by first rubbing fibrinogen solution into the target tissue, attaching polyglycolic acid (PGA) felt to the treated area, and finally spraying it with FS. This method was compared with three conventional FS application methods and a method using fibrin glue-coated collagen fleece. The adhesive force resulting from the present method was 12 times higher than that for the sequential application method, 4.5 times higher than the spray method, 2.5 times higher than the rubbing and spray method, and 2.2 times higher than the use of fibrin glue-coated collagen fleece. The high adhesive force of FS with PGA felt seemed to be due the high fibrin content of the fibrin gel (FG). Light and electron microscopic observations suggested that the formation of FG in closer contact with the muscle fibers was a factor contributing to this superior adhesive force. Comparison of the sealing effect of the present method with other methods using various biomaterials in combination with FS showed that the sealing effect of FS with PGA felt was 1.4 times higher that of polyglactin 910, 1.8 times that of polytetrafluoroethylene, and 6.7 times that of oxidized regenerated cellulose.     

Bladder wall grafting in rats using salt-modified and collagen-coated polycaprolactone scaffolds: Preliminary report

AIM: A rat model was used for the evaluation of collagen-coated and salt-modified polycaprolactone (PCL) scaffolds for bladder grafting after hemicystectomy. METHODS: SD rats underwent partial cystectomy and cystoplasty with collagen-coated and salt-modified polycaprolactone scaffolds. The grafts of the regenerated bladder wall were harvested at different intervals and tissue regeneration was evaluated microscopically. Anatomic and functional characters were evaluated by cystography and urodynamics. RESULTS: At harvesting, after 1 and 2 months, we found good preservation of the bladder shape and volume in all 16 rats receiving PCL cystorrhaphy. No stone formation was observed. Good epithelialization and ingrowth of smooth muscle cells were seen after 2 months grafting. Collagen-coated PCL scaffolds showed considerable encrustation, which appeared to be absorbed and disappear with time. The cystographic and urodynamic examinations revealed intact contour and a well-accommodated bladder with reservoir volume and contractility. CONCLUSIONS: In the rat model, we have successfully demonstrated the applicability of collagen coated and salt-modified PCL in reconstruction of the partial cystectomized bladder.     

膀胱替代的组织工程策略

组织工程学足应用细胞生物学和生物材料学的原理,研究开发用于修复或改善人体病损组织或器官的结构、功能的生物活性替代物的一门新兴学科。近年来,组织工程学的蓬勃发展为膀胱替代提供了崭新的思路。尤其进入21世纪以来,随着干细胞研究的深入和新的生物材料不断出现,使应用组织工程技术再造的新膀胱从文验室走向临床成为可能。本文就组织工程膀胱替代的基础研究和临床应用的发展和现状进行了概述。     

肌腱和韧带组织工程支架材料研究进展

肌腱和韧带损伤的修复治疗方法包括自体移植、异体移植和合成假体替代物的植入.组织工程肌腱和韧带作为生物性替代治疗具有其独特的优点,支架材料作为绀织工程的重要部分对肌腱和韧带的成功构建起着重要的作用.本文就近年来用于肌腱和韧带组织工程的天然和人工合成的支架材料进行综述.     

Urinary Bladder Matrix Does Not Improve Tenogenesis in an In Vitro Equine Model

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1848–1859, 2019     

组织工程尿道粘膜的体外构建

目的:利用组织工程方法在体外构建形成尿道粘膜结构。方法:酶消化法获得猪膀胱尿路上皮细胞(UC),以免疫荧光和RT-PCR方法进行UC鉴定。制备膀胱脱细胞基质移植物(BAMG)作为支架材料,以苏木精-伊红染色、Masson染色、免疫组化和扫描电镜进行评价。经过体外培养和扩增后,将P3代UC接种在BAMG上。结果:BAMG支架上的UC经过体外培养1周后即可形成沿基膜排列的复层上皮结构。扫描电镜显示细胞-材料复合良好,免疫组化检测支架上的UC广谱角蛋白表达阳性。结论:运用组织工程方法能够在体外快速进行尿道粘膜上皮结构的初步构建,为进一步临床修复诸如尿道下裂、尿道狭窄等尿道缺损奠定技术基础。     

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     

PLLA细胞支架的制备方法及形态结构的研究

目的 以聚乳酸为基质材料,分别用三种不同的方法,制备了细胞支架,并比较了制备方法对支架孔结构的影响.方法 分别用溶剂浇铸/致孔剂溶出法、固-液相分离法、液-液相分离法,制得了PLLA的多孔支架,测定其孔隙率及平均孔径,并观察其微观结构.结果 溶液浇铸/致孔剂溶出法可以调节致孔剂的用量及颗粒大小,制得符合设计要求的孔隙率及孔径的细胞支架.固-液相分离法结合致孔剂的溶出,可以获得孔径大于200 μm并且提高孔与孔之间贯通的支架.液-液相分离法通过调节相分离条件,可以制得最大孔径在300 μm～400 μm的细胞支架,大大超过文献报道的只有120 μm孔径.结论 三种制备方法均可得到组织工程用的细胞支架,可以按需控制孔隙率及孔径,溶剂浇铸/致孔剂溶出法只限于制得板状支架,固-液相分离和液-液相分离则不受限制,并且孔径可以达到200 μm以上.