组织工程化血管研究进展

组织工程化血管技术为解决心外科血管移植物不足的难题提供了新思路,研究重点集中在种子细胞选择、支架材料改性、血管构建模式优化等方面,本文就组织工程化血管研究进展作一综述。     

组织工程化血管研究进展

组织工程化血管技术为解决心外科血管移植物不足的难题提供了新思路,研究重点集中在种子细胞选择、支架材料改性、血管构建模式优化等方面,本文就组织工程化血管研究进展作一综述。     

组织工程化血管研究进展

组织工程化血管技术为解决心外科血管移植物不足的难题提供了新思路,研究重点集中在种子细胞选择、支架材料改性、血管构建模式优化等方面,本文就组织工程化血管研究进展作一综述.     

骨髓间充质干细胞-聚乙醇酸支架复合体种植动物皮下构建组织工程化小口径血管(英文)

背景:课题组的前期工作已证实骨髓间充质干细胞可以诱导分化为血管内皮细胞和血管平滑肌细胞,并证实所诱导的细胞和胶原包埋的聚乙醇酸支架具有良好的组织相容性。目的:探讨利用动物皮下作为生物反应器构建小口径组织工程化血管的可行性。方法:骨髓间充质干细胞诱导分化为血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸支架表面,然后将细胞-支架复合体种植于动物皮下,构建小口径组织工程化血管。结果与结论:人工血管组织学观察见管壁结构清晰,其大体结构和天然血管相似,可承受26.6kPa的血管腔内压力不破裂。皮下培养8周免疫荧光观察Brdu标记的部分细胞核呈现明亮的黄绿色荧光。结果说明利用动物的皮下作为生物反应器,采用静态培养的方式构建小口径组织工程化血管是可行的。     

生物蛋白胶止血用于前列腺切除术

前列腺增生症是老年男性常见病。至今为止,手术仍是前列腺增生症最有效的治疗方法,防治术中、术后出血是前列腺切除术的关键步骤之一。我院自１９９８年１月～１１月对５０例前列腺增生症,行耻骨上前列腺剜出术,其中２６例缝合前列腺动脉结合腺窝喷洒生物蛋白胶止血方...     

生物蛋白胶外支架提高血管外膜腺病毒转染效率的实验研究

目的:将携带报告基因的腺病毒载体与生物蛋白胶混合,喷洒在移植静脉外膜,以观察生物蛋白胶是否能提高血管外膜基因转染效率。方法:健康纯种新西兰大耳白兔,随机分为两组单纯腺病毒对照组和生物蛋白胶腺病毒组,每组8只兔。建立兔颈外静脉颈总动脉旁路移植术模型。对照组移植静脉外周单独给予携带β-半乳糖苷酶报告基因的腺病毒载体,生物蛋白胶腺病毒组则给予生物蛋白胶腺病毒载体混合物。分别于术后第7天和第14天取出移植静脉,评价转染效率。结果:术后第7天,对照组腺病毒转染效率(2.1%±0.5%),而生物蛋白胶腺病毒组表现为高转染效率(13.2%±4.6%),两者比较有极显著差异;术后第14天,对照组偶见阳性转染细胞,而在生物蛋白胶腺病毒组虽然阳性转染细胞较术后第7天为少,但仍保持高转染率(6.3%±3.8%)。结论:生物蛋白胶作为一种新的药物缓释系统,能明显提高移植静脉外膜基因转染效率。     

医用生物蛋白胶用于腭裂整复术的疗效观察

目的 观察医用生物蛋白胶应用于腭裂整复术的疗效。方法 随机将68例患分为两组,其中38例使用生物胶填塞腭裂松弛切口患为观察组,30例使用碘仿纱条填塞腭裂松弛切口患为对照组。观察其术后体温、创口愈合、并发症等临床指标。结果 使用生物胶组术后体温恢复较快,术后72h体温较手术当天低0．48℃,差异有显性,P＜0．001;而使用碘仿纱条组术后72h体温较手术当天差异无显性,P＞0．05。使用生物胶组较使用碘仿纱条组创口愈合天数明显减少,经t检验有显性差异,P＜0．01。术后并发症发生则无明显差异。结论 腭裂整复术应用医用生物蛋白胶可以减少患术后反应,缩短创口愈合时间。     

利用复合生物支架材料构建全生物型小口径组织工程血管的研究

目的利用复合纤维蛋白胶生物支架材料和血管细胞成分体外构建具有完整中膜及内皮层的全生物型小口径组织工程血管。方法体外培养扩增猪主动脉血管内皮细胞及平滑细胞,将血管平滑肌细胞与猪纤维蛋白胶混合,均匀复合于犬颈总动脉脱细胞基质材料外表面,经培养后将血管内皮细胞悬液种植到基质材料的内表面,构建全生物型小口径组织工程血管。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外表面结构。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,血管的外表面可见分层排列的血管平滑肌细胞。结论利用纤维蛋白胶血管细胞混合液复合犬颈总动脉脱细胞基质可以构建出具有完整中膜及内膜层的全生物型小口径组织工程血管。     

组织工程化血管构架及构建环境相关研究与进展

目的:介绍组织工程血管的支架材料设计、种子细胞的选择及体外动态培养系统的应用现状及进展。资料来源:应用计算机检索PUBMED 1996/2007与组织工程化血管相关的文章,检索词“tissue engineering,vascular,scaffold,seed cell,bioreactor”,限定文献语种为“English”;同时检索万方数据库2000/2007相关文章,检索词“组织工程,血管,支架材料,种子细胞,生物反应器”,限定文献语种为中文。资料选择:共检索到相关文献200余条,选择与组织工程血管构建、组织工程血管支架材料及其生物相容性研究、种子细胞选择相关的文章,无论观察对象为人或动物、无论是否有对照组均纳入,排除综述和重复文献。资料提炼:根据以上标准共纳入30篇进行综述。其中12篇陈述了血管构架材料,12篇介绍种子细胞选择,8篇论述了构建环境对组织工程血管的影响。资料综合:①根据血管构架的来源和性能,可分为天然生物生材料和人工合成材料,因为2种材料均有其缺陷,目前已有研究将天然材料的重要氨基酸序列接在合成聚合物的表面,形成复合生物材料以克服两种材料各自的缺陷。②选取种子细胞的一种途径是从组织活检中直接捕获成熟的血管壁细胞,来源包括自体的非必须大血管壁细胞及微血管壁细胞;另一种途径就是通过构建具有自我更新、多向繁殖分化能力的干细胞工程来获得相应的种子细胞。③组织工程血管的培养环境应尽可能模仿生物体内血管生发的环境条件。结论:到目前为止,组织工程血管的研究主体仍处于实验阶段,现有的临床应用仅仅是少量的尝试性探索。只有从应力与生长关系出发,摸索出能够成功诱导分化成血管壁细胞的干细胞系,在体外完全模拟出新生血管的相同环境,并解决体内移植的组织相容性等问题,才能形成可生长性、可塑形性和高度顺应性的组织工程血管。     

动态反应器构建组织工程化血管的初步实验研究

目的 以自行设计的旋转细胞培养器和动态反应器比较静态和动态培养条件下细胞增殖的差异,以期为体外构建组织工程化血管提供依据.方法 将兔骨髓间充质干细胞种植在氧等离子体辐照10 min聚氨酯膜制成的管状支架上进行细胞培养,比较静态和动态种植、静态和动态培养细胞增殖率的差异.结果 自制的旋转培养器、动态反应器工作状态稳定;培养第7,14,28天动态培养组光密度值均高于静态培养组(P＜0.01);培养28 d甲苯胺蓝染色及扫描电子显微镜观察显示,动态培养组细胞增殖数较静态培养组多、生长更致密,且有顺培养液流动方向排列的趋势.结论 动态细胞的种植与培养优于静态.自制的旋转培养器、动态反应器可用于初步的动态培养实验,但仍有待改进.     

血管内皮细胞与聚羟基丁酯细胞相容性的实验研究

目的评价新型可降解生物材料聚羟基丁酯(PHB)与血管内皮细胞的细胞相容性。方法采用培养的牛血管内皮细胞接种在PHB膜片及胶原包埋的PHB膜片上,并与PGLA和空白对照作比较,用相差显微镜和扫描电镜观察细胞的粘附和生长情况,并对细胞增殖用MTT法进行测定。结果牛血管内皮细胞在四个实验组中粘附良好,生长符合其生长曲线,胶原包埋PHB较单纯PHB细胞更易粘附生长,较PGLA略差。结论牛血管内皮细胞与PHB细胞相容性好,其用胶原包埋可增加细胞亲和性。     

Both sides nanopatterned tubular collagen scaffolds as tissue‐engineered vascular grafts

Two major requirements for a tissue‐engineered vessel are the establishment of a continuous endothelium and adequate mechanical properties. In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co‐culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano‐scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the case in natural vessels. Both cell types were observed to proliferate and retain their phenotypes in the co‐culture. Copyright © 2010 John Wiley & Sons, Ltd.     

Amorphous nanosilica particles evoke vascular relaxation through PI3K/Akt/eNOS signaling

There have been several reported studies on the distribution and/or toxicity of nanosilica particles. However, the influence of these particles on blood vessels through which they are distributed is poorly understood. Hence, we investigated the effects of nano‐ and micromaterials on blood vessel shrinkage and relaxation. Nanosilica particles with diameters of 70 nm (nSP70) were used as the nanomaterial, and particles of 300 and 1000 nm (nSP300 and mSP1000, respectively) were used as micromaterials. A rat thoracic aorta was used as the test blood vessel. The nano‐ and micromaterials had no effect on vessel shrinkage. Of the nano‐ and micromaterials tested, only nSP70 strongly evoked vascular relaxation. Vascular relaxation evoked by nSP70 was almost completely inhibited by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin. In addition, the selective nitric oxide synthesis inhibitor NG‐nitro‐l ‐arginine methyl ester, which inhibits endothelial nitric oxide synthase (eNOS) downstream of PI3K signaling, inhibited vascular relaxation evoked by nSP70. In an analysis using bovine aortic endothelial cells (bAECs), nSP70 phosphorylated protein kinase B (AKT) and eNOS acted downstream of PI3K signaling. PI3K inhibition by wortmannin reduced AKT and eNOS phosphorylation. These results demonstrated that 70‐nm amorphous nanosilica particles evoked vascular relaxation through PI3K/Akt/eNOS signaling. Moreover, it was suggested that nanomaterials, in general, control or disrupt vascular function by activating a known signal cascade.     

Omental grafting: a cell‐based therapy for blood vessel repair

Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin β4 (Tβ4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tβ4‐induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tβ4‐stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tβ4 promoted proliferation, migration and trans‐differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell‐based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm‐setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescent imaging of endothelial cells in bioengineered blood vessels: the impact of crosslinking of the scaffold

Fluorescent imaging is a useful tool to monitor and evaluate bioengineered tissues and organs. However, autofluorescence emitted from the scaffold can be comparable or even overwhelm signals generated by fluorescently labelled cells and biomarkers. Using standard fluorescent microscopy techniques, a simple and easy‐to‐measure signal to noise ratio metric was developed, which can facilitate the selection of fluorescent biomarkers and the respective biomaterials for tissue engineering studies. Endothelial cells (MS1) expressing green‐fluorescent protein and red fluorescent protein (mKate) were seeded on poly(epsilon‐caprolactone)–collagen hybrid scaffolds that were prepared by crosslinking with glutaraldehyde, genipin and ethyl(dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide. All scaffolds had comparable mechanical properties, which could meet the requirements for vascular graft applications. ethyl(dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide crosslinked scaffolds had a high signal to noise ratio value because of its low autofluorescence in green and red channels. Genipin crosslinked scaffolds had a high signal to noise ratio only in the green channel, while glutaraldehyde crosslinked scaffolds had a low signal to noise ratio in both green and red channels. The signal to noise ratio was independent of the exposure time. The data show that although similar in their mechanical properties and ability to support cell growth, scaffolds crosslinked with different agents have significant differences in causing autofluorescence of the scaffolds. This result indicates that scaffold's preparation method may have a significant impact on direct imaging of fluorescently labelled cells on scaffolds used for tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

局部枸橼酸钠抗凝-连续性血液净化治疗中血液透析管路采血代替血管采血的可行性研究

目的探讨局部枸橼酸钠抗凝-连续性血液净化(RCA-CBP)治疗过程中血透管路采血代替血管采血行电解质及血气监测的可行性,以减少危重症患者CBP过程中频繁采血带来的创伤和痛苦。方法选择2016年1月—2019年2月急诊重症监护室接受RCA-CBP治疗的89例患者为研究对象,采用自身对照法,经患者动脉和血液透析管路同时采集血标本,比较2处血标本电解质、血气检测结果的差异。结果采集血标本共400例,电解质及血气分析结果显示,2组血钙、血钠、血氯、血糖、pH值、乳酸、HCO 3-及碱剩余(BE)差异无统计学意义(P>0.05)。管路采血组的血钾和CO 2分压(PCO 2)高于动脉采血组[(3.91±0.34)mmol/L vs.(3.56±0.40)mmol/L,P<0.01;(44.95±9.40)mm Hg vs.(36.85±9.34)mm Hg,P<0.01]。结论RCA-CBP治疗时,正确的血液透析管路采血可以代替血管采血进行血钙、血钠、血钾、HCO 3-等指标的监测,但需注意动静脉血钾的差异。     

A novel automated cell‐seeding device for tissue engineering of tubular scaffolds: design and functional validation

Obtaining an efficient, uniform and reproducible cell seeding of porous tubular scaffolds constitutes a major challenge for the successful development of tissue‐engineered vascular grafts. In this study, a novel automated cell‐seeding device utilizing direct cell deposition, patterning techniques and scaffold rotation was designed to improve the cell viability, uniformity and seeding efficiency of tubular constructs. Quantification methods and imaging techniques were used to evaluate these parameters on the luminal and abluminal sides of fibrous polymer scaffolds. With the automated seeding method, a high cell‐seeding efficiency (~89%), viability (~85%) and uniformity (~85–92%) were achieved for both aortic smooth muscle cells (AoSMCs) and aortic endothelial cells (AoECs). The duration of the seeding process was < 8 min. Initial cell density, cell suspension in matrix‐containing media, duration of seeding process and scaffold rotation were found to affect the seeding efficiency. After few days of culture, a uniform longitudinal and circumferential cell distribution was achieved without affecting cell viability. Both cell types were viable and spread along the fibres after 28 h and 6 days of static incubation. This new automated cell‐seeding method for tubular scaffolds is efficient, reliable and meets all the requirements for clinical applicability. Copyright © 2011 National Research Council Canada and John Wiley & Sons, Ltd.     

以胶原凝胶为支架原位构建可注射型工程化肝

背景：为了解决肝移植供体的不足，最具潜力的肝组织工程技术悄然兴起，为肝组织的修复与功能重建开辟了新的前景。目的：探索一种以新生大鼠肝细胞为种子细胞，以胶原凝胶为支架材料，可原位注射的工程化肝组织构建方法。设计、时间及地点：以动物为观察对象的实验方法探索，于2007—03／2008—02在解放军总医院普通外科研究所完成。材料：成年雌性SD大鼠及出生24h以内的雄性新生SD大鼠。方法：以胰酶消化法获取新生大鼠肝细胞，以PKH26标记后与液态胶原复合，采用注射方法植入大鼠肝脏。主要观察指标：于肝细胞／胶原凝胶复合物植入当天及植入后第3，7天序贯取样、切片，分别行苏木精-伊红染色和自蛋白免疫组织化学染色后对“类肝组织”进行形态学观察，并观察植入细胞的示踪荧光。结果：①以胶原为基质构建的工程化肝组织能方便地植入到肝脏。②荧光显微镜观察到移植区域主要由PKH26阳性细胞组成。③苏木精伊红染色显示，移植区域肝细胞在胶原凝胶中可存活、生长。④免疫组织化学染色证实植入肝细胞能够表达自蛋白。结论：以新生大鼠肝细胞，胶原凝胶为基础构建可原位注射的工程化肝组织是可行的，这种方法对于发展以组织工程为基础的原位肝脏重建具有良好的应用前景。     

断指再植术后应用脉搏血氧饱和度仪早期发现血管危象的研究

[目的]早期发现血管危象的发生,提高再植指成活率。[方法]将病人分为A组和B组,A组56例76指,于断指再植术后1d~5d内应用脉搏血氧饱和度仪监测再植指和对侧相应健指的经皮血氧饱和度(SpO2);B组为30例48指断指再植术后发生血管危象的病人,于危象时立即监测再植指和对侧相应健指的SpO2变化。[结果]A组再植指与健指SpO2变化无统计学意义(P>0.05),而B组再植指与健指SpO2差异有统计学意义(P<0.01)。[结论]应用脉搏血氧饱和度仪对再植指进行SpO2监测,能及时发现血管危象,并有助于分析血管危象的类型。     

动脉人工血管转流术后患者康复依从性的调查

目的了解动脉人工血管转流术后患者的康复依从性。方法采用自制的电话随访记录表,对102例动脉人工血管转流术后出院的患者进行6个月的电话随访。结果患者出院后1～3个月存在不同比例的伤口和患肢的问题,患者出院后4～6个月服药依从性下降(P0.01),出院后3～6个月健康行为与自我保健依从性下降(P0.05),出院6个月复诊依从性下降(P0.01)。结论患者出院3～6个月康复依从性有明显差异,为今后实施更加有效的院内健康教育及出院患者的后续健康干预提供了依据。