小口径异种血管作为冠状动脉旁路移植术桥材料的研究

目的 研制和评价一种新型小口径异种血管，为冠状动脉旁路移植术（CABG）提供新的移植血管来源。方法 将犬颈动脉采用酶-去污剂法进行脱细胞处理，脱细胞后随机分为两组，肝素结合组（n=24）：进一步进行肝素结合处理；肝素未结合组（n=24）：仅脱细胞作为对照。两组的脱细胞异种血管同时分别植入同一只兔左、右侧颈动脉，在3周、3个月和6个月时用B超观察移植血管的通畅情况；3个月和6个月时分别取材行组织学、电子显微镜及免疫组织化学检查。结果 犬颈动脉细胞被完全去除，肝素结合于血管管壁全层。脱细胞异种血管植入兔颈动脉后两组均无血管阻塞，而肝素结合组血栓形成少于肝素未结合组。移植后3个月两组中无血栓形成的血管管壁均出现大量平滑肌细胞，而内腔由内皮细胞覆盖。结论 酶-去污剂联合肝素处理的犬颈动脉，有可能作为CABG的一种新型小口径异种移植血管。     

犬骨髓CD34＋细胞种植人工血管置换后短期内皮化的实验研究

目的：探讨骨髓CD34＋细胞种植于常用膨体聚四氟乙烯（expanded polytera fluoroethylene，ePTFE）人工血管和涤纶人工血管后二者的内皮化程度。方法：选杂种犬8条，依支架覆膜不同分为ePTFE组和涤纶组，每组实验犬2条，对照犬2条。实验犬采自体骨髓，提取CD34＋细胞种植覆膜支架，对照犬采用单纯自体血预凝覆膜支架。将人工血管植入犬的下腔静脉和腹主动脉。在术后第10、30天观察植入的人工血管通畅情况，采用免疫组织化学方法鉴定内膜细胞来源，在光镜和电镜下观察人工血管新生内膜表面内皮化情况。结果：术后第10天实验组与对照组差异无统计学意义；术后第30天腔面新生内膜内皮细胞自人工血管吻合口向中间逐渐减少（P〈0．05）；而对照组内膜表面第10、30天均无内皮细胞覆盖。结论：经纯化的CD34＋细胞种植于ePTFE和涤纶人工血管，均获得理想的内皮化。     

脱细胞真皮基质作为Beagle犬骨髓基质细胞移植载体的实验研究

目的探讨脱细胞真皮基质（Acellular dermal matrix,ADM）作为Beagle犬骨髓基质细胞（Bone marrow stromalcells,BMSCs）移植载体的可行性。方法将BMSCs复合到ADM载体上,体外观察ADM与BMSCs的生物相容性：ADM—BMSCs复合物植入裸鼠皮下,以单纯ADM为对照组,分别于术后4周和8周进行大体标本观察及组织学分析。结果体外观察ADM与BMSCs的生物相容性良好。裸鼠皮下植入实验显示,实验组4周后ADM部分降解吸收,BMSCs生长良好,出现新生组织,部分形成类骨样结构;8周后,ADM大部分吸收,形成大量的新生组织和类骨样结构;对照组新生组织形成较少,ADM支架材料降解速度与实验组类似。结论ADM可作为Beagle犬BMSCs的移植载体。     

小口径脱细胞基质人工血管移植的研究

目的 探讨脱细胞基质( DCM)人工血管用于小口径血管移植的可行性.方法 40条雄性杂种犬随机分为DCM、膨体聚四氟乙烯(ePTFE)人工血管及自体颈外静脉3组行右颈总动脉置换术,彩超监测移植物通畅率.术后4、8周活体取材,标本行苏木素-伊红(HE)、免疫组织化学染色及扫描电镜检查.结果 3组移植物1周通畅率(75.0％、64.3％、100.0％)差异无统计学意义(P＞0.05);自体颈外静脉组4、8周通畅率( 100.0％、88.9％)优于DCM组(56.3％、26.7％)及ePTFE组(57.1％、23.1％,P＜0.05),后两组差异无统计学意义(P＞0.05).DCM人工血管4、8周血栓形成面积小于ePTFE人工血管,吻合口内膜内皮化程度高于后者.结论 小口径DCM人工血管在抑制血栓形成及加快内皮化方面优于ePTFE人工血管.     

脱细胞肝素处理犬颈动脉的异种移植研究

目的：研究用去污剂-酶消化联合肝素结合处理法制备小口径异种移植血管.方法：犬颈动脉管壁经脱细胞后部分再行肝素结合处理,样本经组织学及电镜观察脱细胞效果,机械性能研究观察脱细胞对管壁弹性和强度的影响,甲苯胺蓝染色观察肝素结合效果和结合程度.将结合与未结合肝素的脱细胞血管同时植入16只兔双侧颈动脉,术后21 d B超观察移植血管的通畅情况.结果：犬颈动脉血管壁细胞被完全去除,细胞外基质保存完好,机械性能无明显破坏,肝素结合于管壁全层,植入兔颈动脉后两侧均无血管阻塞,而肝素结合组血栓形成少于未结合组.结论：去污剂-酶消化法联合肝素结合处理可以作为制备脱细胞小口径异种移植血管的新方法.     

四肢小口径组织工程血管的实验研究

目的研究以小肠黏膜下层组织（small—calibu tissue，SIS）为血管支架，在体内血流条件下培养小口径组织工程血管的可行性。方法自犬隐动脉分离出血管种子细胞，与Ⅰ型胶原蛋白凝胶均匀混合，种植于SIS膜表面，包绕3mm聚乙烯棒制成3层管状支架，为实验组；制成单层无细胞管状支架，为对照组。两种支架作为血管移植物，分别植入15只犬两侧股动脉缺损处进行桥接吻合。术后进行彩超、组织学评价血管的形成过程。结果术后12周，14个3层血管支架保持通畅，通畅率为93．3％，有明显的血管生物结构形成。单层血管支架5个出现部分狭窄，只有3个完全通畅，通畅率为52．6％。结论SIS膜制成的3层血管支架能直接修复血管缺损，并能在体内环境中培形成生物化的小口径组织工程血管。     

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。     

骨髓间充质干细胞与去细胞肌肉生物支架体外相容性研究

[目的]研究骨髓间充质干细胞与去细胞肌肉生物支架体外相容性,进一步探讨联合二者移植修复脊髓损伤可能性.[方法]采用改良化学方法制备去细胞肌肉生物支架并复合冷灭菌方法灭菌,密度梯度离心法体外分离、贴壁法培养BMSCs,注射法接种第3代细胞于支架.[结果]H-E常规染色观察支架内部呈平行走行,Masson染色示支架内平行结构主要为胶原纤维,几乎没有肌纤维.接种BMSCs于支架并孵育14 d后,Hoechest 33342荧光标记计数显示大量存活细胞,扫描电镜可见细胞贴附于支架内表面生长.[结论]去细胞肌肉生物支架具有规则的内部结构是细胞移植理想载体.BMSCs能与去细胞肌肉生物支架体外相容并存活至少2周,为进一步体内联合移植修复脊髓损伤提供实验支持.     

新型多孔PLGA/HA复合支架体外细胞相容性的实验研究

目的研究新型多孔聚乳酸乙醇酸／羟基磷灰石（PLGA/HA）支架材料的体外细胞相容性。方法采用贴壁法对兔骨髓基质细胞（BMSCs）进行体外矿化诱导培养，扩增后与实验A组（含5％HA的PLGA／HA），实验B组（含10％HA的PLGA／HA）及对照C组（仅含PLGA）分别进行体外复合培养；并通过定性及定量法检测BMSCs在材料表面的粘附能力、增殖活力，验证细胞材料复合体的成骨活性。比较分析各组之间的差异。结果兔BMSCs在每组材料的表面均能生长，经体外诱导后在支架材料的表面形成钙结节，A、B组细胞的粘附及增殖能力均强于C组（P〈0．05），A、B组之间无差异。结论兔BMSCs与新型多孔PLGA／HA支架材料有良好的相容性。     

利用脂肪干细胞构建组织工程软骨修复兔膝关节软骨缺损

目的探讨以脂肪于细胞（ADSCs）复合脱细胞软骨基质支架构建组织工程软骨修复兔膝关节软骨缺损的效果。方法以人关节软骨脱细胞基质为支架，复合经诱导的兔ADSCs，体外分别经静态培养和生物反应器培养，构建组织工程软骨。对膝全厚关节缺损进行修复，并与单支架组、空白对照组比较，其中空白对照组12个关节，脱细胞软骨支架组16个关节，静态培养细胞支架组24个关节，生物反应器培养细胞支架组8个关节。分别于术后3、6个月对修复关节进行大体、组织学及免疫组化观察。结果实际完成观察的关节数为44个，其中空白对照组9个，脱细胞软骨支架组11个，静态培养细胞支架组18个，生物反应器培养细胞支架组6个。空白对照组全为纤维组织或纤维软骨样修复；单支架组5个关节为未成熟透明软骨，无成熟透明软骨形成；静态培养细胞支架组83．3％为透明软骨，其中3个关节为成熟透明软骨，12个关节为未成熟透明软骨；生物反应器培养细胞支架组100％为透明软骨，其中2个为成熟透明软骨，4个为未成熟透明软骨。Wakitani评分各组差异有统计学意义（P〈0．05）。结论ADSCs复合脱细胞软骨基质支架能良好地修复兔膝关节全厚软骨缺损，应用生物反应器技术有助于构建组织工程软骨，促进软骨缺损的修复。     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

Development and Validation of Small-diameter Vascular Tissue From a Decellularized Scaffold Coated With Heparin and Vascular Endothelial Growth Factor

To overcome shortcomings of current small‐diameter vascular prostheses, we developed a novel allogenic vascular graft from a decellularized scaffold modified through heparin immobilization and vascular endothelial growth factor (VEGF) coating. The VEGF coating and release profiles were assayed by enzyme‐linked immunosorbent assay, the biological activity of modified surface was validated by human umbilical vein endothelial cells seeding and proliferation for 10 days in vitro. In vivo, we implanted either a modified or a nonmodified scaffold as bilateral carotid allogenic graft in canines (n = 15). The morphological examination of decellularized scaffolds showed complete removal of cellular components while the extracellular matrix structure remained intact. After modification, the scaffolds possessed local sustained release of VEGF up to 20 days, on which the cells cultured showed significantly higher proliferation rate throughout the time after incubation compared with the cells cultured on nonmodified scaffolds (P < 0.0001). After 6 months of implantation, the luminal surfaces of modified scaffolds exhibited complete endothelium regeneration, however, only a few disorderly cells and thrombosis overlay the luminal surfaces of nonmodified scaffolds. Specifically, the modified scaffolds exhibited significantly smaller hyperplastic neointima area compared with the nonmodified, not only at midportion (0.56 ± 0.07 vs. 2.04 ± 0.12 mm², P < 0.0001), but also at anastomotic sites (1.76 ± 0.12 vs. 3.67 ± 0.20 mm², P < 0.0001). Moreover, modified scaffolds had a significantly higher patency rate than the nonmodified after 6 months of implantation (14/15 vs. 7/15, P = 0.005). Overall, this modified decellularized scaffold provides a promising direction for fabrication of small‐diameter vascular grafts.     

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.     

Decellularized vein as a potential scaffold for vascular tissue engineering

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.     

Silyl-heparin adsorption improves the in vivo thromboresistance of carbon-coated polytetrafluoroethylene vascular grafts

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE) remains the most commonly utilized synthetic graft material for infrainguinal arterial reconstruction. However, patency rates of ePTFE bypass grafts are inferior to those observed with autogenous vein grafts. Modification of the luminal surface of ePTFE grafts such as coating with carbon or heparin, may prevent early graft failures and improve overall patency rates. We now report our results with a silyl-heparin adsorbed carbon-coated ePTFE graft. METHODS: Silyl-heparin was adsorbed onto carbon-coated ePTFE vascular grafts (Bard Peripheral Vascular, Tempe, Arizona), which were then evaluated for patency and platelet deposition acutely (2 hours after implantation) and 7 days after graft implantation in mongrel dogs. Dogs underwent bilateral aortoiliac grafting where one heparin adsorbed carbon-coated graft and one carbon-coated graft (control) were placed on either (alternating) side. Platelet deposition was determined by injection of autologous (111)Indium radiolabeled platelets followed by a 2-hour circulation period prior to explantation of grafts. Heparin activity of the silyl-heparin grafts (at preimplantation and explantation) was determined using an antithrombin-III based thrombin binding assay. RESULTS: Graft patency was 100% for both heparin coated (5 of 5) grafts and control (5 of 5) grafts in the acute group of dogs. In the 7-day group, patency was 87.5% for heparin coated (7 of 8) grafts and 50% for control (4 of 8) grafts (P = 0.28, Fisher's exact test). Radiolabeled platelet studies revealed a significantly lower deposition of platelets on heparin coated grafts compared with control grafts in the acute group (17.3 +/- 13.5 versus 35.2 +/- 17.9 counts per minute, per cm(2) per million platelets, mean +/- SEM; n = 5, P <0.05, paired Student t test). In the 7-day group of dogs with bilaterally patent grafts (4 of 8), a trend toward a lower deposition of platelets on heparin coated grafts compared with control grafts was observed (1.55 +/- 0.409 versus 2.14 +/- 1.13 counts per minute, per cm(2) per million platelets, mean +/- SEM; n = 4, P = 0.52, paired Student t test). Eight percent of the preimplantation heparin activity remained on the explanted silyl-heparin grafts after 2 hours and only 2% after 7 days. CONCLUSIONS: Silyl-heparin adsorption onto carbon-coated ePTFE vascular grafts resulted in improved acute thromboresistance in a canine bilateral aortoiliac model. Ongoing laboratory efforts are aimed at improving the silyl-heparin retention on vascular grafts. This graft may prove to be useful in the clinical setting.     

Improvements in GORE-TEX vascular graft performance by Carmeda BioActive surface heparin immobilization.

OBJECTIVES: a performance improvement in small-diameter bypass grafts remains a clinical objective. The purpose of the present investigation was to evaluate the potential of enhancing the thromboresistance of ePTFE grafts using a bioactive heparinized graft luminal surface in a canine model. MATERIAL AND METHODS: this study investigated the utility of heparin immobilization onto expanded polytetrafluoroethylene using Carmeda BioActive Surface technology (CBAS-ePTFE) as a means of improving vascular graft thromboresistance. Graft luminal surfaces were covered uniformly with the stably bound, end-point immobilized heparin. RESULTS: acute canine (5 greyhounds) interposition experiments comparing CBAS-ePTFE grafts to control ePTFE grafts showed that CBAS-ePTFE grafts remained patent and had significantly greater thrombus-free luminal surface (p<0.05). In a chronic canine (16 greyhounds) interposition experiment, significantly improved patency (p<0.05) was observed with CBAS-ePTFE grafts compared to controls. Long-term in vivo heparin bioactivity was demonstrated on CBAS-ePTFE grafts explanted between 1 and 12 weeks. On all CBAS-ePTFE grafts, heparin activity levels ranged from 15-25pmol/cm(2) and did not differ significantly (p>0.05). DISCUSSION: these results support the conclusion that a stable, CBAS-ePTFE surface provides improved thromboresistance and improved patency in canine interposition models. Maintenance of heparin catalytic activity on the graft surface in vivo likely contributes to this outcome and holds promise for the utility of this graft surface for clinical applications.     

血管移植物置换犬颈总动脉的研究

目的 观察以猪血纤维蛋白/微孔聚氨酯弹性膜为管形支架内皮化构建的小口径血管移植物在体内血流动力条件下血管壁重塑过程.方法 用体外培养的小口径血管移植物置换6条犬双侧颈总动脉,于术后1 d、1周、2周、4周行影像学检查,并于术后5 d、2周、4周取材行组织学、免疫组织化学和扫描电镜检查以评价移植血管在体内重塑.结果 10根血管移植物中有8根仍保持通畅(通畅率80%);8根通畅的血管在术后至4周的不同时点取出发现其内表面菲薄、光滑,被覆盖一连续、鹅卵石样单层细胞,Ⅷ因子相关抗原抗体染色阳性.术后4周时新生动脉壁厚900μm,并于血管壁中层可见较多平滑肌细胞,而且最早于术后4周时在血管壁中层就可见弹力纤维.结论 猪血纤维蛋白/微孔聚氨酯弹性膜管形支架内皮化体外构建的小口径血管移植物在体内重塑后,可形成具有类似自体动脉壁结构.     

Novel synthetic selectively degradable vascular prostheses: a preliminary implantation study

BACKGROUND: Vascular grafts perform less well than autologous arterial or vein grafts. The purpose of this study was to evaluate the short-term performance of selectively biodegradable filament-wound vascular prostheses, comprising elastomeric poly(ether urethane) (Lycra) scaffolds and flexible, hydrophilic biodegradable coatings. MATERIALS AND METHODS: Two types of selectively biodegradable vascular grafts were manufactured, comprising a filament-wound Lycra scaffold, subsequently coated with a biodegradable poly(ethylene glycol)/poly(lactic acid) (PELA) block copolymer. The two types of grafts differed in both the overall porosity of the scaffold and the hydrophilicity of the biodegradable constituent. A 60-mm-long and 6-mm-diameter filament-wound and polytetrafluoroethylene (ePTFE) grafts were implanted as interposition prostheses, randomly, at the right- and left-side carotid arteries. RESULTS: Implantation studies proved the grafts to be patent and pulsatile for periods of up to 3 months. Increasing the scaffold porosity and enhancing the hydrophilicity of the biodegradable component improved both the transmural tissue ingrowth process and the vascularization of the prosthesis wall. Also, a well-adhered peripheral tissue and a thin, uniform intima and endothelial lining were obtained. All ePTFE graft controls, although patent, were rather stiff and nonpulsatile. A thick pseudointima, poorly attached to the prosthesis inner surface, was observed. The compliance of the wet grafts was significantly higher than in the dry state, stemming mainly from the water-plasticizing effect on the biodegradable component. The grafts explanted after a period of 6 weeks exhibited compliance only slightly lower than that of the wet grafts. After 12 weeks, however, the hoop compliance was 20% lower than that prior to implantation. At 100 mm Hg, for example, the original compliance of the wet graft was 2.5%/100 mm Hg decreasing to 2.0%/100 mm Hg after a 3-month implantation. The compliance reduction with implantation is attributed to the ingrowth of the perigraft tissue as revealed by the histological study. A compliance of 2.0%/100 mm Hg is slightly better than that of a standard PTFE graft with an original compliance of 1.6%/100 mm Hg. Yet it is still an order of magnitude smaller than that of a canine carotid artery. CONCLUSIONS: The improved mechanical properties and enhanced healing of the highly porous filament-wound Lycra scaffold graft coated with hydrophilic biodegradable PELA has the potential of being a highly effective small caliber prosthetic graft.