MAPCs/EPCs性组织工程化静脉瓣的在体研究
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摘要
研究背景和意义:原发性瓣膜功能不全、血栓形成完全再通后的瓣膜破坏以及先天性瓣膜缺乏等静脉疾患,静脉瓣移植常常是最后的选择。但是,自体静脉瓣移植除来源有限、损伤较大外,尚有瓣膜强度不足和带瓣静脉的管径往往难以符合要求等问题。近年来,心血管组织工程技术的发展为构建组织工程化静脉瓣膜提供了可能。研究证明,虽然每条深静脉的瓣膜有多对,但只要移植一对有功能的瓣膜就能明显改善血液逆流的问题,这就使得构建组织工程化静脉瓣更具有实用价值。因此应用细胞生物学与工程学原理开发出具有生物活性、无免疫原性的组织工程化静脉瓣是修复与功能重建深静脉功能不全的理想方案。
目前组织工程化静脉瓣的研究国外尚处于起步阶段,国内也仅我们研究所在进行该项研究。Pavcnik等采用金属架支撑的去细胞异体小肠黏膜下层(SIS)作为静脉瓣膜移植物经皮送至绵羊股静脉,1个月后可见到受体绵羊的内皮细胞、成纤维细胞等多种细胞成分黏附并进入到移植物内部及表面生长,移植物具有类似自体瓣膜的功能;但是由于没有种植细胞和进行再内皮化,虽然采用了肝素抗凝,部分移植物还是出现了出血、血栓、钙化等严重并发症。可见,未种植细胞的瓣膜支架材料体内种植不是瓣膜移植的理想选择。Teebken等应用组织工程学原理,采用同种异体去细胞绵羊带瓣静脉作为支架,在其上种植受体绵羊静脉壁来源的肌纤维母细胞和内皮细胞构建组织工程化静脉瓣,但是肌纤维母细胞没能长入支架内部,移植到受体绵羊股静脉后,虽然短期内多数移植瓣膜有功能,但12周时半数组织工程化静脉瓣失去功能,这可能与种植细胞的活性及寿命有关。
2008年,本研究所温昱博士进行了“犬MAPCs/EPCs性组织工程化静脉瓣应用基础”的研究工作,其研究结果显示,犬MAPCs/EPCs性组织工程化静脉瓣3个月时在颈外静脉内均能发挥静脉瓣生理功能,但和正常静脉瓣相比功能出现了一定程度上的减弱。原因可能是在种子细胞EPC在种植时只进行了顺血流方向种植和培养,而没有采用逆血流方向种植的缘故。另外,该研究将组织工程化静脉瓣移植到犬的颈外静脉仅进行了在体3个月的效用性研究,因颈外静脉和下肢静脉血液回流有很大的不同,所以在即使其在3个月时有一定的生理功能,也不能真正说明组织工程化静脉瓣在下肢长期生理功能问题。除此之外,本研究所前期还进行了自体MAPC和EPC细胞、异种脱细胞支架性组织工程化静脉瓣在体安全性评价研究,结果显示用自体骨髓来源的MAPC、EPC和同种异体脱细胞支架一起构建的组织工程化静脉瓣在受体动物体内没有免疫排斥反应,没有毒性,能与宿主很好的共存,说明用受体骨髓来源的MAPC和EPC种植在同种异体脱细胞支架材料上构建的组织工程化静脉瓣可安全应用于临床。
至今为止,组织工程化静脉瓣的研究已取得了不小的进展,但还有很多问题亟需解决。为此,本课题拟采用同种异体脱细胞绵羊静脉瓣作为支架,联合应用多点注射、加压灌注等技术,分批种植受体绵羊自体骨髓来源的成体多能祖细胞(Multipotent adult progenitor cells,MAPC)和内皮祖细胞(Endothelial progenitor cells,EPC),体外构建“绵羊MAPCs/EPCs性组织工程化静脉瓣”,并将其吻接于受体绵羊股静脉处,进行长时程(为期1年)在体观察,采用彩色多谱勒超声仪、小动物超声仪、数字减影成像仪进行效用性研究,不同时间段取材后作大体观察并运用HE染色、免疫组织化学染色、扫描电镜、透射电镜来检测种子细胞在支架材料上的分布、生长、分化演变情况,拟研制一种具有临床应用前景的组织工程化静脉瓣。
第一部分MAPCs/EPCs性组织工程化静脉瓣的构建
研究目的:通过多点注射和加压灌注的方法,将受体绵羊骨髓来源的MAPC和EPC,分批种植于绵羊同种异体脱细胞带瓣静脉支架上,体外培养构建组织工程化静脉瓣。
材料和方法:绵羊髂后上棘抽取骨髓,MAPC和EPC的原代培养、传代,分选CD45-的MAPC和CD133+的EPC,流式检测MAPC的SSEA-1、EPC的CD133和CD14;以GADPH为内参, RT-PCR检测MAPC的OCT-4、SM-MHC和EPC的KDR、VE-cadherin等标记分子;用免疫细胞荧光化学检测MAPC的CD13、SSEA-1、CD44、CD45、MHC-‖和EPC的CD133、CD14、VWF、CD31。0.5%Triton-100+0.05%NH4OH进行同种异体带瓣静脉支架脱细胞处理, DNase+RNase消化支架内酶, HE染色和van Gieson染色分别观察脱细胞情况、弹力纤维和胶原纤维的分布。分选扩增后的MAPC用Hochest标记、EPC用PKH26标记,应用多点注射和加压灌注的方法分批种植在脱细胞支架上,构建MAPCs/EPCs性组织工程化静脉瓣。体外培养不同时间的组织工程化静脉瓣行冰冻切片,荧光显微镜下观察种子细胞MAPC和EPC在支架材料上的生长和迁移。用上述方法将未标记的绵羊MAPC和EPC扩增后采用相同构建方法体外构建绵羊组织工程化静脉瓣。组织工程化静脉瓣、脱细胞支架、天然静脉,弹性回复实验检测三组血管的弹性断裂强度差异;扫描电镜检测组织工程化静脉瓣血管壁细胞与天然血管壁细胞分布差异。组织工程化静脉瓣行石蜡包埋、切片, HE染色和Desmin、α-actin、VWF、VEGF、CD133免疫组化分子检测,观察种子细胞在支架材料上的分布、分化及内皮化情况。
结果: CD45-的MAPC和CD133+的EPC细胞形态较均一,呈三角形或长梭形,折光性强,增殖旺盛,融合后均呈铺路石样排列。流式细胞仪检测显示:MAPC的SSEA-1阳性率为31.47%;EPC的CD14阳性率为50.26%、CD133阳性率为92.39%;细胞免疫荧光染色结果显示:MAPC为CD13+、SSEA-1+、CD44-、CD45-、CD133-、MHCⅡ-;EPC表达CD133、CD14,不表达CD45。RT-PCR结果显示:MAPC表达OCT-4、SM-MHC分子;EPC表达KDR、VE-cadherin分子。Hochest和PKH26能成功标记两种细胞,显微镜下能观察到MAPC和EPC分别发蓝光和红光,构建血管后冰冻切片荧光显微镜观察,可显示标记的种子细胞能成功种植在脱细胞支架上,并在血管内壁和瓣膜两侧生长形成细胞单层。构建血管与天然血管相比:在扫描电镜下内壁细胞分布相似;弹性断裂强度,构建血管和天然血管差异不明显,和脱细胞支架有显著性差异。HE染色显示脱细胞支架没有残留细胞,天然血管细胞排列整齐,组织工程化静脉瓣内壁和瓣膜两面有单层细胞。Van Gieson染色显示脱细胞支架、构建血管和天然血管弹力纤维和胶原纤维分布相似。组织工程化静脉瓣免疫组织化学染色显示:Desmin、α-actin、VWF、VEGF、CD133均呈阳性表达。
结论:以绵羊自体骨髓MAPC和EPC为种子细胞,以同种异体脱细胞带瓣静脉为支架材料,应用多点注射和加压灌注等方法,体外可以成功构建组织工程化静脉瓣。
第二部分MAPCs/EPCs性组织工程化静脉瓣的在体研究
研究目的:MAPCs/EPCs性组织工程化静脉瓣体内移植,研究其功能和体内演变规律,探讨自体组织干细胞、脱细胞支架性组织工程化静脉瓣临床应用的可能性。
材料和方法:雌性1 y龄绵羊12只,分3组,每组4只。将构建的含绵羊MAPCs/EPCs性组织工程化静脉瓣血管以端端吻合术吻接至受体绵羊右侧股静脉处作实验组。左侧移入同种异体脱细胞静脉瓣支架作对照组。术后3、6、12月时间点运用彩色多普勒超声仪、小动物超声以及数字减影血管造影(DSA)进行检测;并分别在3、6、12月时间点处死动物,取出移植物,大体观察后,4 %多聚甲醛固定,脱水、透明、石蜡包埋,制备石蜡切片,分别进行H-E染色及免疫组织化学染色,扫描电镜和透射电镜检测。
结果:绵羊MAPCs/EPCs性组织工程化静脉瓣植入体内后在体检测:彩色超声多普勒显示通畅率实验组(对照组)分别为3个月4/4(4/4),6个月4/4(1/4),12个月1/4(0/4);术后3个月DSA检查实验组未见返流,对照组可见明显返流;术后6个月小动物超声检查实验组可见瓣膜回声,但是瓣膜运动受限,对照组静脉壁增厚,瓣膜形态分辨不清。
大体、透射电镜、扫描电镜、组织学及免疫组织化学观察:术后3个月实验组未见血栓形成,静脉壁及瓣膜壁无明显增厚,形态与正常相似,内皮处及瓣膜上VE-cadherin、VWF和VEGFR阳性,中膜VE-cadherin、α-actin及Desmin阳性;对照组出现肉眼可见血栓,H-E染色为瓣膜处较大附壁血栓,内膜除表达VEGFR, VWF外,VE-cadherin、α-actin及Desmin也明显表达。
术后6个月实验组瓣膜处见附壁血栓,但静脉壁无明显增厚,瓣膜形态清晰可辨,内膜VEGFR, VWF阳性,中膜α-actin及Desmin阳性;对照组静脉壁极度增厚至阻塞管腔,瓣膜结构被破坏,瓣膜形态分辨不清,血栓明显,VEGFR, VWF外,VE-cadherin、α-actin及Desmin均不表达。
术后12个月时实验组瓣膜及静脉壁明显增厚,可见血栓,但瓣膜形态仍可辨,内膜VEGFR, VWF阳性,中膜VE-cadherin、α-actin及Desmin阳性;对照组瓣膜明显增厚似静脉壁,不能分清静脉壁三层结构,VEGFR, VWF外,VE-cadherin、α-actin及Desmin均不表达。。
结论:MAPC/EPC性组织工程化静脉瓣植入体内半年内具有一定瓣膜功能,但在一年时功能消失。可见在当前条件下构建的组织工程化静脉瓣在体内不能长时间发挥生理功能,需要进一步探索组织工程化静脉瓣的构建方法,改善组织工程化静脉瓣在体生理功能。
Background and significance:It may be critical to establish tissue-engineering bioprosthetic venous valves to treat chronic venous insufficiency. However, there are no satisfying tissue-engineering bioprosthetic venous valves for implying. To develop a graft bearing an immunologically tolerated tissue-engineered venous valve (TE graft) that will be incorporated into a native vessel, and restore normal valve function for the treatment of chronic venous insufficiency, a manufactured, tissue-engineered, nonimunogenic, nonanticoagulant venous valve that remains patent and competent over time is an attractive alternative to direct venous valves transplantation for the treatment of chronic venous insufficiency.
Until now, few reports were found on this field and many challenges and questions remained to be solved. So, it is very significant to carry out this study of tissue-engineered venous valves. Pavcnik et al. used the mental rack with SIS as grafts of venous valves, and replanted the grafts to external jugular veins of sheeps. After one month, autogeneic EC and fibroblasts can immigrate into the grafts, so the grafts performed the functions of venous valves. But some grafts were accompanied severe complications of bleeding, thrombus, and calcification, because of without reendothelialization. Teebken et al. applied the theories of tissue engineering, used the allochthonous decellularized venous valves as scaffolds, and seeded myofibroblasts and EC to construct tissue engineered venous valves. But myofibroblasts can not grow into the walls of scaffolds. After being replanted, the grafts can not perform the long term function.
In 2008, in our Institute of Biomedical Engineering, Dr. Wen Yu carried out a study of "Canine MAPCs/EPCs Tissue Engineering Venous Valves applications basis" under the guidance of the Professor. Zhang. Her works were to implant seeding cells MAPC and EPC which were derived of the receptor canine bone marrow to the allogenic acellular venous scaffold material and construct tissue engineering venous valves in vitro. Then she transplanted TEVV into the external jugular vein of recipient canine and observed effective function for 3-month after operation in vivo. The results showed the canine TEVV could play it’s physiological functions after 3 months in the external jugular vein, but compared to the normal venous valve function, there was a certain degree of weakening. The reasons may be due to the seeding cells EPC which were implanted only along the direction of blood flow and training, but not against the direction of blood flow so that the back of valve could not be reendothelialized. In addition, her research was to implanted TEVV into canine's external jugular vein, blood flow are different between external jugular vein and low limb vein. Besides, 3 months for effectiveness research in vivo do not meet international standard which were accept for tissue engineering products pre-clinical effective studies in vivo. However, TEVV can play some physiological functions in 3 months in vivo, which can not suggest that TEVV can play long-term physical function in lower limb. In addition, the Institute of Professor.Zhang also conducted TEVV safety evaluation studies in vivo, the results showed that TEVV had no immune rejection response with the recipient animals, had no cellular toxic too, TEVV had good biocompatibility with the host animal. All these results suggested the TEVV constructed by implanted bone marrow-derived MAPC and the EPC into the allogenic acellular scaffolds were safe to recipient animal.
Up to now, TEVV research has made great progress, but there are many problems need to be solved. Facing all above problems, this subject tried to use sheep allogeneic acellular vein graft as scaffold material, then combined multi-point injection and adding pressure perfusion technique, respectively implanted adult multipotent progenitor cells (MAPC) and endothelial progenitor cells (EPC) into scaffold material, at last construct Tissue Engineering venous valve in vitro, which we called "sheep MAPC / EPC TEVV "; Next, we anastomosed TEVV to the recipient sheep femoral vein using end to end method, and carried on long time function observation in vivo. We respectively applied color Doppler ultrasound, small animal ultrasound, digital subtraction imaging instrument to investigate the effectiveness of TEVV, at last, in different time point, all the transplanted TEVV and accelular scaffolds were harvested, we used HE staining, immunohistochemistry, scanning electron microscopy, transmission electron microscopy to detect the seeding cells distribution, growth, differentiation and evolution situation in the scaffold material. Our purpose was to research successfully a kind of TEVV which can be applied for long time in vivo so that TEVV can be used to treat chronic venous insufficiency in clinic.
Part I. Tissue-Engineered Vein Valve Construction in Vitro
Objective: Implanted MAPC and EPC derived of sheep bone marrow into acellular venous scaffold containing venous valve and used multi-point injection and adding pressure perfusion method to construct tissue engineering venous valve. Our purpose is to provide a basic experimental evidence for tissue engineering venous valve’s long-term research in vivo.
Materials and Methods: MAPC and EPC primary culture was done, they were from bone marrow of sheep posterior superior iliac spine, MAPC and EPC passage cells were selected by CD45- and CD133+ using magnetic bead separation method respectively; After cell sorting, MAPC and EPC were detected SSEA-1, CD13 and CD34, CD133 by FLC respectively; Using GADPH as control, MAPC’s markers OCT-4, SSEA-1 and EPC’s markers KDR, VE-cadherin, eNOS were detected by RT-PCR respectively ; MAPC for CD13, SSEA-1, CD44, CD45, MHC-‖and EPC for CD133, CD34, VWF were detected respectively by using immune cell fluorescent chemical methods. Acellular venous scaffolds containing venous valve were gained by utilizing 0.5% Triton-100 +0.05% NH4OH, then digested with DNase + RNase enzymes; Cells, elastic fibers and collagen fibers inside acellular scaffolds were observed using HE staining and van Gieson staining; MAPC were marked with Hochest and EPC were marked with PKH26 after separation and amplification; Used multi-point injection and adding pressure perfusion method to implant MAPC and EPC into acellular scaffold to construct tissue engineering venous valve in vitro, then observed MAPC’s and EPC’s distribution and migration inside scaffold by fluorescence microscope after frozen section; The same method was used to implant not marked sheep bone marrow drived MAPC and EPC into scaffold to construct tissue engineering venous valve. The differences of the cells distribution in vascular wall were observed among constructed tissue engineering venous valve , acellular scaffold, natural veins by using scan electron microscopy; The differences of blood vessels elastic fracture intensity among constructed tissue engineering venous valve , acellular scaffold, natural veins were detected by elastic recovery test. Seeding cells distribution in constructed tissue engineering venous valve were detected by HE staining; Expression for Desmin,α-actin of SMC and VWF, VEGFR, CD133 of EPC were detected using immunohistochemical fluorescence method in constructed tissue engineering venous valve .
Results: Sorted CD45- MAPC and CD133+ EPC morphology uniform, triangular or long spindle-shaped, two kinds of cells have strong proliferative ability, cells spread like cobblestone after confluence. After cells were sorted, through flow cytometry analysis showed: about positive rate, MAPC’s SSEA-1was 31.47%, CD13 was 21.19%, EPC’s CD34, CD133 was 57.69%, 53.63% respectively; immunofluorescence staining showed: MAPC express CD13, SSEA-1, but not express CD44, CD45, MHC-Ⅱ; EPC express CD133, CD34, VEGFR. RT-PCR results showed: MAPC express OCT-4, SSEA-1; EPC express KDR, VE-cadherin and eNOS. MAPC and EPC present blue and red through fluorescence microscope after being marked with Hochest and PKH26 respectively, after using marked MAPC and EPC to construct tissue engineering venous valve, through fluorescence microscopy after the frozen section showed the marked seed cells grew in acellular scaffolds and on both sides of valves leaflet and formatted cell monolayer. Compared with natural blood vessels, constructed tissue engineering venous valves have follow results: 1)Cells’distribution in blood vessel wall is similar through scanning electron microscope; 2)About elastic fracture strength, there was a little difference between constructed tissue engineering venous valve and natural blood vessels, but it was significant difference between constructed tissue engineering venous valve and acellular scaffold; 3)HE staining showed no cells remained in acellular scaffold, cells arranged regularly in natural blood vessel, there were cells monolayer on both sides of constructed tissue engineering venous valve and inner surface of blood vessel wall; 4)Van Gieson staining showed the distribution of elastic fibers and collagen fibers was similar among acellular scaffold, constructed blood vessels containing tissue engineering venous valve and natural blood vessels; 5) immunohistochemical staining showed Desmin,α-actin, VWF, VEGF, CD133 were positive on tissue engineering venous valve.
Conclusion: MAPC and EPC can be successfully cultured from sheep bone marrow; combination application of MAPC and EPC, using multi-point injection and adding pressure perfusion method to implant seeding cells into the acellular scaffold, then in vitro constructed tissue engineering venous valve which is similar to natural blood vessel wall structure; implanted seeding cells can spread successfully monolayer and endothelializate on both sides of constructed tissue engineering venous valve and inner surface of blood vessel wall; All these results provide experimental foundation for tissue engineering venous valve’s long-term research in vivo the next.
Part II. In vivo investigation of MAPCs/EPCs tissue engineered venous valves
Objective: To investigate physiological function and it’s lasting time of TEVV by sheep MAPCs/EPCs TEVV long time (one year) effective observation in vivo and to provide a reliable experimental basis and theoretical basis for the application of TEVV on treating deep venous insufficiency in clinic in the future.
Materials and Methods: 12 female sheep (age 1 y), divided into 3 groups, n = 4/group. The sheep TEVV were anastomosised to the right femoral vein of recipient sheep as the experimental group. The acellular scaffold were anastomosised to the left femoral vein of recipient sheep as the control group. In 3, 6,1 2 months after operation, respectively using color Doppler ultrasound, small animal ultrasound and digital subtraction angiography (DSA) to detect the function of TEVV; At last, the sheep were sacrificed and TEVV were taken out in 3,6,12, respectively, after general observation, the TEVV were fixed by 4% paraformaldehyde, and were embedded in paraffin, then paraffin sections were detected by using HE and immunohistochemical staining, scanning electron microscopy and transmission electron microscopy.
Results: Efftective investigation results were followed: color Doppler ultrasound examination showed that postoperative patency rate of experimental group (control group) were 4/4 (4/4) in 3 months, 4 / 4 (1 / 4) in 6 months, 1 / 4 (0 / 4) in 12 months; In 3 months after operation, DSA detection showed there no reflux in the experimental group and significant reflux in control group; In 6 months after operation, small animal ultrasound showed there was valves resonance in experimental group, but valve’s movement is limited, the control group vascular wall was thicker than experimental group and valve’s shape was incomplete. In general, transmission electron microscopy, scanning electron microscopy, histological and immunohistochemical detection showed: the experimental group in 3 months after operation, no thrombosis, no obvious being thicken on vascular wall and surface of valves, were similar to the shape of normal valves, on the surface of valves and endomembrane, VWF and VEGFR were positive; VE-cadherin,α-actin and Desmin were positive on the tunica media. However, the control group had visible thrombus, HE staining showed that there were larger thrombus on the valves, the expression of VEGFR, VWF,α-actin, VE-cadherin and Desmin were evident; For the experimental group, in 6 months after operation, there was light thrombus near the valve, but no obvious being thicken in the vascular wall, valve morphology could be clearly recognized, VEGFR and VWF were positive expression in the endomembrane,α-actin and Desmin were positive too in the tunica media; For the experimental group, in 12 months after operation, venous valves and vascular wall were significantly thicker, there were visible thrombus, but valve morphology could still be identified. VEGFR, VWF of endomembrane andα-actin, VE-cadherin and Desmin of tunica media were expressed positively; For the control group, venous valve was thickened to be similar to the vascular wall and it’s three-layer structure could not be distinguished, in 3 months after operation,α-actin, VE-cadherin and Desmin were positive in valves and endomembrane, and in 6,12 months after operation, valve’s wall were extremely thickened that lumen were blocked by thrombosis, valve’s structure was destroyed and valve’s morphology could not be recognized clearly, thrombosis were obvious.α-actin, VE-cadherin, Desmin, VEGFR, VWF were not expressed.
Conclusion: Sheep MAPCs/EPCs TEVV had valve function in six months after operation in vivo, but it’s function was disappear in a year after operation. All above test results suggested that constructed TEVV in the current condition could not make up their function long time in vivo, the construction method needed to be further improved so that TEVV physiological function in vivo can be increased.
目前组织工程化静脉瓣的研究国外尚处于起步阶段,国内也仅我们研究所在进行该项研究。Pavcnik等采用金属架支撑的去细胞异体小肠黏膜下层(SIS)作为静脉瓣膜移植物经皮送至绵羊股静脉,1个月后可见到受体绵羊的内皮细胞、成纤维细胞等多种细胞成分黏附并进入到移植物内部及表面生长,移植物具有类似自体瓣膜的功能;但是由于没有种植细胞和进行再内皮化,虽然采用了肝素抗凝,部分移植物还是出现了出血、血栓、钙化等严重并发症。可见,未种植细胞的瓣膜支架材料体内种植不是瓣膜移植的理想选择。Teebken等应用组织工程学原理,采用同种异体去细胞绵羊带瓣静脉作为支架,在其上种植受体绵羊静脉壁来源的肌纤维母细胞和内皮细胞构建组织工程化静脉瓣,但是肌纤维母细胞没能长入支架内部,移植到受体绵羊股静脉后,虽然短期内多数移植瓣膜有功能,但12周时半数组织工程化静脉瓣失去功能,这可能与种植细胞的活性及寿命有关。
2008年,本研究所温昱博士进行了“犬MAPCs/EPCs性组织工程化静脉瓣应用基础”的研究工作,其研究结果显示,犬MAPCs/EPCs性组织工程化静脉瓣3个月时在颈外静脉内均能发挥静脉瓣生理功能,但和正常静脉瓣相比功能出现了一定程度上的减弱。原因可能是在种子细胞EPC在种植时只进行了顺血流方向种植和培养,而没有采用逆血流方向种植的缘故。另外,该研究将组织工程化静脉瓣移植到犬的颈外静脉仅进行了在体3个月的效用性研究,因颈外静脉和下肢静脉血液回流有很大的不同,所以在即使其在3个月时有一定的生理功能,也不能真正说明组织工程化静脉瓣在下肢长期生理功能问题。除此之外,本研究所前期还进行了自体MAPC和EPC细胞、异种脱细胞支架性组织工程化静脉瓣在体安全性评价研究,结果显示用自体骨髓来源的MAPC、EPC和同种异体脱细胞支架一起构建的组织工程化静脉瓣在受体动物体内没有免疫排斥反应,没有毒性,能与宿主很好的共存,说明用受体骨髓来源的MAPC和EPC种植在同种异体脱细胞支架材料上构建的组织工程化静脉瓣可安全应用于临床。
至今为止,组织工程化静脉瓣的研究已取得了不小的进展,但还有很多问题亟需解决。为此,本课题拟采用同种异体脱细胞绵羊静脉瓣作为支架,联合应用多点注射、加压灌注等技术,分批种植受体绵羊自体骨髓来源的成体多能祖细胞(Multipotent adult progenitor cells,MAPC)和内皮祖细胞(Endothelial progenitor cells,EPC),体外构建“绵羊MAPCs/EPCs性组织工程化静脉瓣”,并将其吻接于受体绵羊股静脉处,进行长时程(为期1年)在体观察,采用彩色多谱勒超声仪、小动物超声仪、数字减影成像仪进行效用性研究,不同时间段取材后作大体观察并运用HE染色、免疫组织化学染色、扫描电镜、透射电镜来检测种子细胞在支架材料上的分布、生长、分化演变情况,拟研制一种具有临床应用前景的组织工程化静脉瓣。
第一部分MAPCs/EPCs性组织工程化静脉瓣的构建
研究目的:通过多点注射和加压灌注的方法,将受体绵羊骨髓来源的MAPC和EPC,分批种植于绵羊同种异体脱细胞带瓣静脉支架上,体外培养构建组织工程化静脉瓣。
材料和方法:绵羊髂后上棘抽取骨髓,MAPC和EPC的原代培养、传代,分选CD45-的MAPC和CD133+的EPC,流式检测MAPC的SSEA-1、EPC的CD133和CD14;以GADPH为内参, RT-PCR检测MAPC的OCT-4、SM-MHC和EPC的KDR、VE-cadherin等标记分子;用免疫细胞荧光化学检测MAPC的CD13、SSEA-1、CD44、CD45、MHC-‖和EPC的CD133、CD14、VWF、CD31。0.5%Triton-100+0.05%NH4OH进行同种异体带瓣静脉支架脱细胞处理, DNase+RNase消化支架内酶, HE染色和van Gieson染色分别观察脱细胞情况、弹力纤维和胶原纤维的分布。分选扩增后的MAPC用Hochest标记、EPC用PKH26标记,应用多点注射和加压灌注的方法分批种植在脱细胞支架上,构建MAPCs/EPCs性组织工程化静脉瓣。体外培养不同时间的组织工程化静脉瓣行冰冻切片,荧光显微镜下观察种子细胞MAPC和EPC在支架材料上的生长和迁移。用上述方法将未标记的绵羊MAPC和EPC扩增后采用相同构建方法体外构建绵羊组织工程化静脉瓣。组织工程化静脉瓣、脱细胞支架、天然静脉,弹性回复实验检测三组血管的弹性断裂强度差异;扫描电镜检测组织工程化静脉瓣血管壁细胞与天然血管壁细胞分布差异。组织工程化静脉瓣行石蜡包埋、切片, HE染色和Desmin、α-actin、VWF、VEGF、CD133免疫组化分子检测,观察种子细胞在支架材料上的分布、分化及内皮化情况。
结果: CD45-的MAPC和CD133+的EPC细胞形态较均一,呈三角形或长梭形,折光性强,增殖旺盛,融合后均呈铺路石样排列。流式细胞仪检测显示:MAPC的SSEA-1阳性率为31.47%;EPC的CD14阳性率为50.26%、CD133阳性率为92.39%;细胞免疫荧光染色结果显示:MAPC为CD13+、SSEA-1+、CD44-、CD45-、CD133-、MHCⅡ-;EPC表达CD133、CD14,不表达CD45。RT-PCR结果显示:MAPC表达OCT-4、SM-MHC分子;EPC表达KDR、VE-cadherin分子。Hochest和PKH26能成功标记两种细胞,显微镜下能观察到MAPC和EPC分别发蓝光和红光,构建血管后冰冻切片荧光显微镜观察,可显示标记的种子细胞能成功种植在脱细胞支架上,并在血管内壁和瓣膜两侧生长形成细胞单层。构建血管与天然血管相比:在扫描电镜下内壁细胞分布相似;弹性断裂强度,构建血管和天然血管差异不明显,和脱细胞支架有显著性差异。HE染色显示脱细胞支架没有残留细胞,天然血管细胞排列整齐,组织工程化静脉瓣内壁和瓣膜两面有单层细胞。Van Gieson染色显示脱细胞支架、构建血管和天然血管弹力纤维和胶原纤维分布相似。组织工程化静脉瓣免疫组织化学染色显示:Desmin、α-actin、VWF、VEGF、CD133均呈阳性表达。
结论:以绵羊自体骨髓MAPC和EPC为种子细胞,以同种异体脱细胞带瓣静脉为支架材料,应用多点注射和加压灌注等方法,体外可以成功构建组织工程化静脉瓣。
第二部分MAPCs/EPCs性组织工程化静脉瓣的在体研究
研究目的:MAPCs/EPCs性组织工程化静脉瓣体内移植,研究其功能和体内演变规律,探讨自体组织干细胞、脱细胞支架性组织工程化静脉瓣临床应用的可能性。
材料和方法:雌性1 y龄绵羊12只,分3组,每组4只。将构建的含绵羊MAPCs/EPCs性组织工程化静脉瓣血管以端端吻合术吻接至受体绵羊右侧股静脉处作实验组。左侧移入同种异体脱细胞静脉瓣支架作对照组。术后3、6、12月时间点运用彩色多普勒超声仪、小动物超声以及数字减影血管造影(DSA)进行检测;并分别在3、6、12月时间点处死动物,取出移植物,大体观察后,4 %多聚甲醛固定,脱水、透明、石蜡包埋,制备石蜡切片,分别进行H-E染色及免疫组织化学染色,扫描电镜和透射电镜检测。
结果:绵羊MAPCs/EPCs性组织工程化静脉瓣植入体内后在体检测:彩色超声多普勒显示通畅率实验组(对照组)分别为3个月4/4(4/4),6个月4/4(1/4),12个月1/4(0/4);术后3个月DSA检查实验组未见返流,对照组可见明显返流;术后6个月小动物超声检查实验组可见瓣膜回声,但是瓣膜运动受限,对照组静脉壁增厚,瓣膜形态分辨不清。
大体、透射电镜、扫描电镜、组织学及免疫组织化学观察:术后3个月实验组未见血栓形成,静脉壁及瓣膜壁无明显增厚,形态与正常相似,内皮处及瓣膜上VE-cadherin、VWF和VEGFR阳性,中膜VE-cadherin、α-actin及Desmin阳性;对照组出现肉眼可见血栓,H-E染色为瓣膜处较大附壁血栓,内膜除表达VEGFR, VWF外,VE-cadherin、α-actin及Desmin也明显表达。
术后6个月实验组瓣膜处见附壁血栓,但静脉壁无明显增厚,瓣膜形态清晰可辨,内膜VEGFR, VWF阳性,中膜α-actin及Desmin阳性;对照组静脉壁极度增厚至阻塞管腔,瓣膜结构被破坏,瓣膜形态分辨不清,血栓明显,VEGFR, VWF外,VE-cadherin、α-actin及Desmin均不表达。
术后12个月时实验组瓣膜及静脉壁明显增厚,可见血栓,但瓣膜形态仍可辨,内膜VEGFR, VWF阳性,中膜VE-cadherin、α-actin及Desmin阳性;对照组瓣膜明显增厚似静脉壁,不能分清静脉壁三层结构,VEGFR, VWF外,VE-cadherin、α-actin及Desmin均不表达。。
结论:MAPC/EPC性组织工程化静脉瓣植入体内半年内具有一定瓣膜功能,但在一年时功能消失。可见在当前条件下构建的组织工程化静脉瓣在体内不能长时间发挥生理功能,需要进一步探索组织工程化静脉瓣的构建方法,改善组织工程化静脉瓣在体生理功能。
Background and significance:It may be critical to establish tissue-engineering bioprosthetic venous valves to treat chronic venous insufficiency. However, there are no satisfying tissue-engineering bioprosthetic venous valves for implying. To develop a graft bearing an immunologically tolerated tissue-engineered venous valve (TE graft) that will be incorporated into a native vessel, and restore normal valve function for the treatment of chronic venous insufficiency, a manufactured, tissue-engineered, nonimunogenic, nonanticoagulant venous valve that remains patent and competent over time is an attractive alternative to direct venous valves transplantation for the treatment of chronic venous insufficiency.
Until now, few reports were found on this field and many challenges and questions remained to be solved. So, it is very significant to carry out this study of tissue-engineered venous valves. Pavcnik et al. used the mental rack with SIS as grafts of venous valves, and replanted the grafts to external jugular veins of sheeps. After one month, autogeneic EC and fibroblasts can immigrate into the grafts, so the grafts performed the functions of venous valves. But some grafts were accompanied severe complications of bleeding, thrombus, and calcification, because of without reendothelialization. Teebken et al. applied the theories of tissue engineering, used the allochthonous decellularized venous valves as scaffolds, and seeded myofibroblasts and EC to construct tissue engineered venous valves. But myofibroblasts can not grow into the walls of scaffolds. After being replanted, the grafts can not perform the long term function.
In 2008, in our Institute of Biomedical Engineering, Dr. Wen Yu carried out a study of "Canine MAPCs/EPCs Tissue Engineering Venous Valves applications basis" under the guidance of the Professor. Zhang. Her works were to implant seeding cells MAPC and EPC which were derived of the receptor canine bone marrow to the allogenic acellular venous scaffold material and construct tissue engineering venous valves in vitro. Then she transplanted TEVV into the external jugular vein of recipient canine and observed effective function for 3-month after operation in vivo. The results showed the canine TEVV could play it’s physiological functions after 3 months in the external jugular vein, but compared to the normal venous valve function, there was a certain degree of weakening. The reasons may be due to the seeding cells EPC which were implanted only along the direction of blood flow and training, but not against the direction of blood flow so that the back of valve could not be reendothelialized. In addition, her research was to implanted TEVV into canine's external jugular vein, blood flow are different between external jugular vein and low limb vein. Besides, 3 months for effectiveness research in vivo do not meet international standard which were accept for tissue engineering products pre-clinical effective studies in vivo. However, TEVV can play some physiological functions in 3 months in vivo, which can not suggest that TEVV can play long-term physical function in lower limb. In addition, the Institute of Professor.Zhang also conducted TEVV safety evaluation studies in vivo, the results showed that TEVV had no immune rejection response with the recipient animals, had no cellular toxic too, TEVV had good biocompatibility with the host animal. All these results suggested the TEVV constructed by implanted bone marrow-derived MAPC and the EPC into the allogenic acellular scaffolds were safe to recipient animal.
Up to now, TEVV research has made great progress, but there are many problems need to be solved. Facing all above problems, this subject tried to use sheep allogeneic acellular vein graft as scaffold material, then combined multi-point injection and adding pressure perfusion technique, respectively implanted adult multipotent progenitor cells (MAPC) and endothelial progenitor cells (EPC) into scaffold material, at last construct Tissue Engineering venous valve in vitro, which we called "sheep MAPC / EPC TEVV "; Next, we anastomosed TEVV to the recipient sheep femoral vein using end to end method, and carried on long time function observation in vivo. We respectively applied color Doppler ultrasound, small animal ultrasound, digital subtraction imaging instrument to investigate the effectiveness of TEVV, at last, in different time point, all the transplanted TEVV and accelular scaffolds were harvested, we used HE staining, immunohistochemistry, scanning electron microscopy, transmission electron microscopy to detect the seeding cells distribution, growth, differentiation and evolution situation in the scaffold material. Our purpose was to research successfully a kind of TEVV which can be applied for long time in vivo so that TEVV can be used to treat chronic venous insufficiency in clinic.
Part I. Tissue-Engineered Vein Valve Construction in Vitro
Objective: Implanted MAPC and EPC derived of sheep bone marrow into acellular venous scaffold containing venous valve and used multi-point injection and adding pressure perfusion method to construct tissue engineering venous valve. Our purpose is to provide a basic experimental evidence for tissue engineering venous valve’s long-term research in vivo.
Materials and Methods: MAPC and EPC primary culture was done, they were from bone marrow of sheep posterior superior iliac spine, MAPC and EPC passage cells were selected by CD45- and CD133+ using magnetic bead separation method respectively; After cell sorting, MAPC and EPC were detected SSEA-1, CD13 and CD34, CD133 by FLC respectively; Using GADPH as control, MAPC’s markers OCT-4, SSEA-1 and EPC’s markers KDR, VE-cadherin, eNOS were detected by RT-PCR respectively ; MAPC for CD13, SSEA-1, CD44, CD45, MHC-‖and EPC for CD133, CD34, VWF were detected respectively by using immune cell fluorescent chemical methods. Acellular venous scaffolds containing venous valve were gained by utilizing 0.5% Triton-100 +0.05% NH4OH, then digested with DNase + RNase enzymes; Cells, elastic fibers and collagen fibers inside acellular scaffolds were observed using HE staining and van Gieson staining; MAPC were marked with Hochest and EPC were marked with PKH26 after separation and amplification; Used multi-point injection and adding pressure perfusion method to implant MAPC and EPC into acellular scaffold to construct tissue engineering venous valve in vitro, then observed MAPC’s and EPC’s distribution and migration inside scaffold by fluorescence microscope after frozen section; The same method was used to implant not marked sheep bone marrow drived MAPC and EPC into scaffold to construct tissue engineering venous valve. The differences of the cells distribution in vascular wall were observed among constructed tissue engineering venous valve , acellular scaffold, natural veins by using scan electron microscopy; The differences of blood vessels elastic fracture intensity among constructed tissue engineering venous valve , acellular scaffold, natural veins were detected by elastic recovery test. Seeding cells distribution in constructed tissue engineering venous valve were detected by HE staining; Expression for Desmin,α-actin of SMC and VWF, VEGFR, CD133 of EPC were detected using immunohistochemical fluorescence method in constructed tissue engineering venous valve .
Results: Sorted CD45- MAPC and CD133+ EPC morphology uniform, triangular or long spindle-shaped, two kinds of cells have strong proliferative ability, cells spread like cobblestone after confluence. After cells were sorted, through flow cytometry analysis showed: about positive rate, MAPC’s SSEA-1was 31.47%, CD13 was 21.19%, EPC’s CD34, CD133 was 57.69%, 53.63% respectively; immunofluorescence staining showed: MAPC express CD13, SSEA-1, but not express CD44, CD45, MHC-Ⅱ; EPC express CD133, CD34, VEGFR. RT-PCR results showed: MAPC express OCT-4, SSEA-1; EPC express KDR, VE-cadherin and eNOS. MAPC and EPC present blue and red through fluorescence microscope after being marked with Hochest and PKH26 respectively, after using marked MAPC and EPC to construct tissue engineering venous valve, through fluorescence microscopy after the frozen section showed the marked seed cells grew in acellular scaffolds and on both sides of valves leaflet and formatted cell monolayer. Compared with natural blood vessels, constructed tissue engineering venous valves have follow results: 1)Cells’distribution in blood vessel wall is similar through scanning electron microscope; 2)About elastic fracture strength, there was a little difference between constructed tissue engineering venous valve and natural blood vessels, but it was significant difference between constructed tissue engineering venous valve and acellular scaffold; 3)HE staining showed no cells remained in acellular scaffold, cells arranged regularly in natural blood vessel, there were cells monolayer on both sides of constructed tissue engineering venous valve and inner surface of blood vessel wall; 4)Van Gieson staining showed the distribution of elastic fibers and collagen fibers was similar among acellular scaffold, constructed blood vessels containing tissue engineering venous valve and natural blood vessels; 5) immunohistochemical staining showed Desmin,α-actin, VWF, VEGF, CD133 were positive on tissue engineering venous valve.
Conclusion: MAPC and EPC can be successfully cultured from sheep bone marrow; combination application of MAPC and EPC, using multi-point injection and adding pressure perfusion method to implant seeding cells into the acellular scaffold, then in vitro constructed tissue engineering venous valve which is similar to natural blood vessel wall structure; implanted seeding cells can spread successfully monolayer and endothelializate on both sides of constructed tissue engineering venous valve and inner surface of blood vessel wall; All these results provide experimental foundation for tissue engineering venous valve’s long-term research in vivo the next.
Part II. In vivo investigation of MAPCs/EPCs tissue engineered venous valves
Objective: To investigate physiological function and it’s lasting time of TEVV by sheep MAPCs/EPCs TEVV long time (one year) effective observation in vivo and to provide a reliable experimental basis and theoretical basis for the application of TEVV on treating deep venous insufficiency in clinic in the future.
Materials and Methods: 12 female sheep (age 1 y), divided into 3 groups, n = 4/group. The sheep TEVV were anastomosised to the right femoral vein of recipient sheep as the experimental group. The acellular scaffold were anastomosised to the left femoral vein of recipient sheep as the control group. In 3, 6,1 2 months after operation, respectively using color Doppler ultrasound, small animal ultrasound and digital subtraction angiography (DSA) to detect the function of TEVV; At last, the sheep were sacrificed and TEVV were taken out in 3,6,12, respectively, after general observation, the TEVV were fixed by 4% paraformaldehyde, and were embedded in paraffin, then paraffin sections were detected by using HE and immunohistochemical staining, scanning electron microscopy and transmission electron microscopy.
Results: Efftective investigation results were followed: color Doppler ultrasound examination showed that postoperative patency rate of experimental group (control group) were 4/4 (4/4) in 3 months, 4 / 4 (1 / 4) in 6 months, 1 / 4 (0 / 4) in 12 months; In 3 months after operation, DSA detection showed there no reflux in the experimental group and significant reflux in control group; In 6 months after operation, small animal ultrasound showed there was valves resonance in experimental group, but valve’s movement is limited, the control group vascular wall was thicker than experimental group and valve’s shape was incomplete. In general, transmission electron microscopy, scanning electron microscopy, histological and immunohistochemical detection showed: the experimental group in 3 months after operation, no thrombosis, no obvious being thicken on vascular wall and surface of valves, were similar to the shape of normal valves, on the surface of valves and endomembrane, VWF and VEGFR were positive; VE-cadherin,α-actin and Desmin were positive on the tunica media. However, the control group had visible thrombus, HE staining showed that there were larger thrombus on the valves, the expression of VEGFR, VWF,α-actin, VE-cadherin and Desmin were evident; For the experimental group, in 6 months after operation, there was light thrombus near the valve, but no obvious being thicken in the vascular wall, valve morphology could be clearly recognized, VEGFR and VWF were positive expression in the endomembrane,α-actin and Desmin were positive too in the tunica media; For the experimental group, in 12 months after operation, venous valves and vascular wall were significantly thicker, there were visible thrombus, but valve morphology could still be identified. VEGFR, VWF of endomembrane andα-actin, VE-cadherin and Desmin of tunica media were expressed positively; For the control group, venous valve was thickened to be similar to the vascular wall and it’s three-layer structure could not be distinguished, in 3 months after operation,α-actin, VE-cadherin and Desmin were positive in valves and endomembrane, and in 6,12 months after operation, valve’s wall were extremely thickened that lumen were blocked by thrombosis, valve’s structure was destroyed and valve’s morphology could not be recognized clearly, thrombosis were obvious.α-actin, VE-cadherin, Desmin, VEGFR, VWF were not expressed.
Conclusion: Sheep MAPCs/EPCs TEVV had valve function in six months after operation in vivo, but it’s function was disappear in a year after operation. All above test results suggested that constructed TEVV in the current condition could not make up their function long time in vivo, the construction method needed to be further improved so that TEVV physiological function in vivo can be increased.
引文
[1] Pavcnik D,Barry T,Hans A,et a1. Percutaneous bioprosthetic venous valve:A long—term study in sheep[J].J VaseSurg,2002,35(3):598—603.
[2] Pavcnik D,Kaufman J,Uchida B,et a1.Second generation pereutaneous bioprosthetic valve:A short—term study in sheep[J].J Vase Surg,2004,40(6):1223—1227.
[3] de Borst G J,Teijink J A,Patterson M,et a1.A pereutaneous approach to deep venous valve insufficiency with a new selfexpanding venous frame valve[J].J Endovasc Ther,2003,10(2):341～349.
[4] Abilez O,Benharash P,M ehrotra M ,et a1.A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts[J].J SurgRes,2006,132(2):170-178.
[5] Pavcnik D, Barry T, Hans A, et al. Percutaneous bioprosthetic venous valve: A long-term study in sheep. J Vasc Surg, 2002, 35(3):598-603.
[6] Teebken OE, Puschmann C, Aper T, et al. Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. Eur J Vasc Endovasc Surg, 2003, 25(4):305-312.
[7]沈曼茹,袁建明,张永珍,熊绍虎,党瑞山,张传森.细胞外基质材料的安全性能.中国组织工程研究与临床康复,2010,14(38):7145-7148.
[8] HSU SH, Tsai IJ, Lin DJ, et al. The effect of dynamic culture conditions on endothelial cell seeding and retention on small diameter polyurethane vascular grafts. Med Eng Phys, 2005, 27(3):267-272.
[1] Pavcnik D, Barry T, Hans A, et al. Percutaneous bioprosthetic venous valve: A long-term study in sheep. J Vasc Surg, 2002, 35(3):598-603.
[2] Teebken OE, Puschmann C, Aper T, Haverich A, Mertsching H. Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. Eur J Vasc Endovasc Surg 2003; 25: 305-12.
[3] April B, Nima E, Masayuki OFU, Molly NH, Laura L, Jiang YH. Multipotent adult progenitor cell isolation and culture procedures. Experimental Hematology 2006; 34: 1596–1601.
[4] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275(5302):964-967.
[5] Reyes M,Lund T,Lenvik T, et al. Purification and ex vivo expamion of postnatal human marrow mesodermal cells.Blood,200l,98(12):2615—2625.
[6] Jiang YH,Balkrishna N,Jahagirdar,et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,2O02,418(1):41-49.
[7] Reyes M, Verfailie CM.Charaeterization of multilptent adult progenitor cells,a subpopulation of mensechymal stem cells.Ann.N.Y.Acad. Sci,2001,938(2): 231-235.
[8] Friedenstein AJ,Corskaja U,Kalugina NN,et al.Precursors for fibroblasts in differentpopulations of hematopoietic cells as detected by the in vitro colonyassay method . Exp.Hematol,1974,2(1):83-92.
[9] Hristov M,Erl W,Weber PC.Endothelial progenitor cells:mobilization,diferentiation,and homing[J].Arterioscler Thromb Vasc Biol,2OO3,23(7):l185-l189.
[10] Heeschen C,Aicher A,Lehmann R,el a1. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization.Blo d,2003,102(4):1340-1346.
[11] Dignat-George F.Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry.Cytometry B Clin Cytom,2006,70B(2):104-105.
[12] Lydia B,Fabrizio I ,Astrid P.M orphological and phenotypical characte rization of human endothelial progenitor cells in an early stage of differentiation.FEBS Lett,2005,579(12):2731-2736.
[13] Hristov M, Erl W, Weber PC. Endothelial progenitor cells: isolation and characterization.Trends Cardiovasc Med. 2003;13 (5):201-206.
[14] Medina J, Arroyo AG, Sánchez-Madrid F, Moreno-Otero R. Angiogenesis in chronic inflammatory liver disease.Hepatology. 2004;39 (5):1185-1195.
[15]崔磊,于海莹,尹烁,等.体外诱导大鼠骨髓内皮祖细胞成内皮细胞的实验研究[J].中华创伤骨科杂志,2004,6(1O):ll54-1159.
[16] Li W, Liu WY, Yi DH, et al. Histological/biological characterization of decellularized bovine jugular vein. Asian Cardiovasc Thorac Ann. 2007;15(2):91-96.
[17] Kim HI, Takai M, Ishihara K. Bioabsorbable material-containing phosphorylcholine group-rich surfaces for temporary scaffolding of the vessel wall. Tissue Eng Part C Methods. 2009;15(2):125-133.
[18] Amir G, Miller L, Shachar M,et al.Evaluation of a peritoneal-generated cardiac patch in a rat model of heterotopic heart transplantation. Cell Transplant. 2009;18(3):275-282.
[19] Gerontas S, Farid SS, Hoare M. Windows of operation for bioreactor design for the controlled formation of tissue-engineered arteries. Biotechnol Prog. 2009;25(3):842-853.
[20] Sun M, Zhou P, Pan LF,et al. Enhanced cell affinity of the silk fibroin- modified PHBHHx material. J Mater Sci Mater Med. 2009;20(8):1743-1751.
[21] Sodian R , Hcemtrup SP , Speding JS , et a1 . Evaluation of biodegradable , three dimensionalmatricesfortissue engineering af heart valve.ASAIO J.2000;46(3):107-110.
[22] Chenite A,Chaput C.Wang D.et al.NoveI injectable neutraI solutions of ch~osan f0rm biod egradable gels in situ. Biomaterials. 2000;21(21):2155-2161.
[23] Hench LL,Polak JM.Third-generation biomedical materials.Science.2002:295(5557):10-14.
[24] Liu B,Jiang ZL,Zhang Y,et al.The vessel organ culture system under stress in vitro:a new model for vascular biomechanical experiment.Yiyong Shengwu Lixue.2001;16(4):225-230.
[25] Cebotari S,Mertsching H,Kallenbach K,et al.Construction of autologous human heart valves based on an aeellular allograft.
[26] Gerontas S, Farid SS, Hoare M. Windows of operation for bioreactor design for the controlled formation of tissue-engineered arteries. Biotechnol Prog. 2009;25(3):842-853.
[27] Sun M, Zhou P, Pan LF,et al. Enhanced cell affinity of the silk fibroin- modified PHBHHx material. J Mater Sci Mater Med. 2009;20(8):1743-1751.
[1] Pereira MM, Jones JR, Hench LL.Bioactive glass and hybrid scaffolds prepared by solgel method for bone tissue engineering. Advances in Applied Ceramics, 2005, 104: 35-42
[2] Butler CE, Yannas IV, Compton CC, Correia CA, Orgill DP, Comparison of cultured and uncultured
[3] Teebken OE, Puschmann C, Breitenbach I, Rohde B, Burgwitz K, Haverich A.Preclinical development of tissue-engineered vein valves and venous substitutesusing re-endothelialised human vein matrix. Eur J Vasc Endovasc Surg. 2009 Jan;37(1):92-102.
[4] Teebken OE, Puschmann C, Aper T, Haverich A, Mertsching H. Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. Eur J Vasc Endovasc Surg.2003 Apr;25(4):305-312.
[5] Pavcnik D,Barry T,Hans A,et a1. Percutaneous bioprosthetic venous valve:A long—term study in sheep[J].J VaseSurg,2002,35(3):598—603.
[6] Pavcnik D,Kaufman J,Uchida B,et a1.Second generation pereutaneous bioprosthetic valve:A short—term study in sheep[J].J Vase Surg,2004,40(6):1223—1227.
[7] de Borst G J,Teijink J A,Patterson M,et a1.A pereutaneous approach to deep venous valve insufficiency with a new selfexpanding venous frame valve[J].J Endovasc Ther,2003,10(2):341～349.
[8] Abilez O,Benharash P,M ehrotra M ,et a1.A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts[J].J SurgRes,2006,132(2):170-178.
[9]周永昌,郭万学。超声医学[M]。第3版。北京:科学技术文献出版社,1998. 7342735.
[10] M asuda EM , Kister RL. P ro spective comparison of duplex scanning and descending venography in the assessment of ve2nous insufficiency. Am J Surg 1992, 164: 254.
[11]徐秋华,燕山,等。下肢静脉溃疡的血流动力学检测[J ]。中国超声医学杂志,1999 ,15 (4) :287.
[12] Van Bemmelen PS ,Bedford G,Bench KW,et al. Quantitative seg2 mental evaluation of venous valvular reflux with oluplex vltrasound scannig[J ] . J Vasc Sarg ,1989 ,10 :4252431.
[13]袁建明,管松晖,党瑞山,杨向群,沈曼茹,张传森.绵羊骨髓来源内皮祖细胞的培养与鉴定.中国组织工程研究与康复, 2010,14(36): 6662-6666.
[14] Wen yu, Li bin, Dang ruishan, et al. Characterization and differentiation of endothelial progenitor cells from canine bone marrow. Chinese Journal of Anatomy. 2008, 31(3):317-321.
[15]李琼,曹谊林. bFGF对BMSC体外软骨构建的影响及机制[博士学位论文].上海,国家组织工程研发中心,2011.
[16] Hoffman-Kim D, Maish MS, Krueger PM, Lukoff H, Bert A, Hong T, Hopkins RA. Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves. Tissue Eng. 2005;11(1-2): 288-301.
[17]温昱,李彬,党瑞山,等.犬骨髓成体多能祖细胞体外培养、鉴定及向平滑肌样细胞的诱导分化.第二军医大学学报. 2007,28(6):593-597.
[18] Mutuberria R, Satijn S, Huijbers A, et al. Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries. J Immunol Methods. 2004;287(1-2):31-47.
[19]刘太华,张炎,姜宗来,等.组织T程血管脱细胞支架主要组织相容性复合体I抗原性的免疫组化观察[J].第二军医大学学报,2004,25(1):72—74.
[20] Cebotari S,Mertsching H,Kallenbach K,et a1.Construction of autologous human heart valves based on an aeellular allograft matrix[J].Circulation,2002,106(12 Suppl 1):163-168.
[2] Pavcnik D,Kaufman J,Uchida B,et a1.Second generation pereutaneous bioprosthetic valve:A short—term study in sheep[J].J Vase Surg,2004,40(6):1223—1227.
[3] de Borst G J,Teijink J A,Patterson M,et a1.A pereutaneous approach to deep venous valve insufficiency with a new selfexpanding venous frame valve[J].J Endovasc Ther,2003,10(2):341～349.
[4] Abilez O,Benharash P,M ehrotra M ,et a1.A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts[J].J SurgRes,2006,132(2):170-178.
[5] Pavcnik D, Barry T, Hans A, et al. Percutaneous bioprosthetic venous valve: A long-term study in sheep. J Vasc Surg, 2002, 35(3):598-603.
[6] Teebken OE, Puschmann C, Aper T, et al. Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. Eur J Vasc Endovasc Surg, 2003, 25(4):305-312.
[7]沈曼茹,袁建明,张永珍,熊绍虎,党瑞山,张传森.细胞外基质材料的安全性能.中国组织工程研究与临床康复,2010,14(38):7145-7148.
[8] HSU SH, Tsai IJ, Lin DJ, et al. The effect of dynamic culture conditions on endothelial cell seeding and retention on small diameter polyurethane vascular grafts. Med Eng Phys, 2005, 27(3):267-272.
[1] Pavcnik D, Barry T, Hans A, et al. Percutaneous bioprosthetic venous valve: A long-term study in sheep. J Vasc Surg, 2002, 35(3):598-603.
[2] Teebken OE, Puschmann C, Aper T, Haverich A, Mertsching H. Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. Eur J Vasc Endovasc Surg 2003; 25: 305-12.
[3] April B, Nima E, Masayuki OFU, Molly NH, Laura L, Jiang YH. Multipotent adult progenitor cell isolation and culture procedures. Experimental Hematology 2006; 34: 1596–1601.
[4] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275(5302):964-967.
[5] Reyes M,Lund T,Lenvik T, et al. Purification and ex vivo expamion of postnatal human marrow mesodermal cells.Blood,200l,98(12):2615—2625.
[6] Jiang YH,Balkrishna N,Jahagirdar,et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,2O02,418(1):41-49.
[7] Reyes M, Verfailie CM.Charaeterization of multilptent adult progenitor cells,a subpopulation of mensechymal stem cells.Ann.N.Y.Acad. Sci,2001,938(2): 231-235.
[8] Friedenstein AJ,Corskaja U,Kalugina NN,et al.Precursors for fibroblasts in differentpopulations of hematopoietic cells as detected by the in vitro colonyassay method . Exp.Hematol,1974,2(1):83-92.
[9] Hristov M,Erl W,Weber PC.Endothelial progenitor cells:mobilization,diferentiation,and homing[J].Arterioscler Thromb Vasc Biol,2OO3,23(7):l185-l189.
[10] Heeschen C,Aicher A,Lehmann R,el a1. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization.Blo d,2003,102(4):1340-1346.
[11] Dignat-George F.Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry.Cytometry B Clin Cytom,2006,70B(2):104-105.
[12] Lydia B,Fabrizio I ,Astrid P.M orphological and phenotypical characte rization of human endothelial progenitor cells in an early stage of differentiation.FEBS Lett,2005,579(12):2731-2736.
[13] Hristov M, Erl W, Weber PC. Endothelial progenitor cells: isolation and characterization.Trends Cardiovasc Med. 2003;13 (5):201-206.
[14] Medina J, Arroyo AG, Sánchez-Madrid F, Moreno-Otero R. Angiogenesis in chronic inflammatory liver disease.Hepatology. 2004;39 (5):1185-1195.
[15]崔磊,于海莹,尹烁,等.体外诱导大鼠骨髓内皮祖细胞成内皮细胞的实验研究[J].中华创伤骨科杂志,2004,6(1O):ll54-1159.
[16] Li W, Liu WY, Yi DH, et al. Histological/biological characterization of decellularized bovine jugular vein. Asian Cardiovasc Thorac Ann. 2007;15(2):91-96.
[17] Kim HI, Takai M, Ishihara K. Bioabsorbable material-containing phosphorylcholine group-rich surfaces for temporary scaffolding of the vessel wall. Tissue Eng Part C Methods. 2009;15(2):125-133.
[18] Amir G, Miller L, Shachar M,et al.Evaluation of a peritoneal-generated cardiac patch in a rat model of heterotopic heart transplantation. Cell Transplant. 2009;18(3):275-282.
[19] Gerontas S, Farid SS, Hoare M. Windows of operation for bioreactor design for the controlled formation of tissue-engineered arteries. Biotechnol Prog. 2009;25(3):842-853.
[20] Sun M, Zhou P, Pan LF,et al. Enhanced cell affinity of the silk fibroin- modified PHBHHx material. J Mater Sci Mater Med. 2009;20(8):1743-1751.
[21] Sodian R , Hcemtrup SP , Speding JS , et a1 . Evaluation of biodegradable , three dimensionalmatricesfortissue engineering af heart valve.ASAIO J.2000;46(3):107-110.
[22] Chenite A,Chaput C.Wang D.et al.NoveI injectable neutraI solutions of ch~osan f0rm biod egradable gels in situ. Biomaterials. 2000;21(21):2155-2161.
[23] Hench LL,Polak JM.Third-generation biomedical materials.Science.2002:295(5557):10-14.
[24] Liu B,Jiang ZL,Zhang Y,et al.The vessel organ culture system under stress in vitro:a new model for vascular biomechanical experiment.Yiyong Shengwu Lixue.2001;16(4):225-230.
[25] Cebotari S,Mertsching H,Kallenbach K,et al.Construction of autologous human heart valves based on an aeellular allograft.
[26] Gerontas S, Farid SS, Hoare M. Windows of operation for bioreactor design for the controlled formation of tissue-engineered arteries. Biotechnol Prog. 2009;25(3):842-853.
[27] Sun M, Zhou P, Pan LF,et al. Enhanced cell affinity of the silk fibroin- modified PHBHHx material. J Mater Sci Mater Med. 2009;20(8):1743-1751.
[1] Pereira MM, Jones JR, Hench LL.Bioactive glass and hybrid scaffolds prepared by solgel method for bone tissue engineering. Advances in Applied Ceramics, 2005, 104: 35-42
[2] Butler CE, Yannas IV, Compton CC, Correia CA, Orgill DP, Comparison of cultured and uncultured
[3] Teebken OE, Puschmann C, Breitenbach I, Rohde B, Burgwitz K, Haverich A.Preclinical development of tissue-engineered vein valves and venous substitutesusing re-endothelialised human vein matrix. Eur J Vasc Endovasc Surg. 2009 Jan;37(1):92-102.
[4] Teebken OE, Puschmann C, Aper T, Haverich A, Mertsching H. Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. Eur J Vasc Endovasc Surg.2003 Apr;25(4):305-312.
[5] Pavcnik D,Barry T,Hans A,et a1. Percutaneous bioprosthetic venous valve:A long—term study in sheep[J].J VaseSurg,2002,35(3):598—603.
[6] Pavcnik D,Kaufman J,Uchida B,et a1.Second generation pereutaneous bioprosthetic valve:A short—term study in sheep[J].J Vase Surg,2004,40(6):1223—1227.
[7] de Borst G J,Teijink J A,Patterson M,et a1.A pereutaneous approach to deep venous valve insufficiency with a new selfexpanding venous frame valve[J].J Endovasc Ther,2003,10(2):341～349.
[8] Abilez O,Benharash P,M ehrotra M ,et a1.A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts[J].J SurgRes,2006,132(2):170-178.
[9]周永昌,郭万学。超声医学[M]。第3版。北京:科学技术文献出版社,1998. 7342735.
[10] M asuda EM , Kister RL. P ro spective comparison of duplex scanning and descending venography in the assessment of ve2nous insufficiency. Am J Surg 1992, 164: 254.
[11]徐秋华,燕山,等。下肢静脉溃疡的血流动力学检测[J ]。中国超声医学杂志,1999 ,15 (4) :287.
[12] Van Bemmelen PS ,Bedford G,Bench KW,et al. Quantitative seg2 mental evaluation of venous valvular reflux with oluplex vltrasound scannig[J ] . J Vasc Sarg ,1989 ,10 :4252431.
[13]袁建明,管松晖,党瑞山,杨向群,沈曼茹,张传森.绵羊骨髓来源内皮祖细胞的培养与鉴定.中国组织工程研究与康复, 2010,14(36): 6662-6666.
[14] Wen yu, Li bin, Dang ruishan, et al. Characterization and differentiation of endothelial progenitor cells from canine bone marrow. Chinese Journal of Anatomy. 2008, 31(3):317-321.
[15]李琼,曹谊林. bFGF对BMSC体外软骨构建的影响及机制[博士学位论文].上海,国家组织工程研发中心,2011.
[16] Hoffman-Kim D, Maish MS, Krueger PM, Lukoff H, Bert A, Hong T, Hopkins RA. Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves. Tissue Eng. 2005;11(1-2): 288-301.
[17]温昱,李彬,党瑞山,等.犬骨髓成体多能祖细胞体外培养、鉴定及向平滑肌样细胞的诱导分化.第二军医大学学报. 2007,28(6):593-597.
[18] Mutuberria R, Satijn S, Huijbers A, et al. Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries. J Immunol Methods. 2004;287(1-2):31-47.
[19]刘太华,张炎,姜宗来,等.组织T程血管脱细胞支架主要组织相容性复合体I抗原性的免疫组化观察[J].第二军医大学学报,2004,25(1):72—74.
[20] Cebotari S,Mertsching H,Kallenbach K,et a1.Construction of autologous human heart valves based on an aeellular allograft matrix[J].Circulation,2002,106(12 Suppl 1):163-168.