An experimental collagen-impregnated dacron graft: Potential for endothelial seeding

This study evaluates the potential for endothelial seeding of a collagen-impregnated Dacron graft with or without surface modifiers (fibronectin, heparin) to attach and retain these cells during flow. Human umbilical endothelial cells were harvested, cultured, labeled with Indium111-oxine and seeded onto 30 mm X 4 mm diameter grafts. Six graft surfaces were studied: 1) a collagen-impregnated Dacron graft, HemashieldR (C); 2) C + fibronectin (C + F); 3) C + heparin (C + H); 4) C + F + H; 5) HytrelR + F (Hyt + F); and 6) Hyt + F + H. Radioactive loss determined the percentage attachment and then percentage retention of labeled inoculum after a one-hour in vitro perfusion. Scanning electron and light microscopy demonstrated the endothelium on the graft surface following perfusion. Fibronectin-coated grafts had a significantly higher percentage attachment than those without fibronectin (ANOVA, P less than 0.05). However, the percentage retention following perfusion was similar for all Dacron grafts and statistically inferior to the HytrelR grafts studied (ANOVA, P less than 0.05). SEM evaluation of the C + F + H graft surface was qualitatively the most impressive Dacron surface for seeding, yet was inferior to the HytrelR graft. We conclude that fibronectin benefits the initial attachment of endothelium to collagen-coated Dacron rivaling the HytrelR surface. Fibronectin does not improve percentage retention of the HemashieldR surface during perfusion, therefore, some of its initial benefit is lost.     

Re-endothelialization of punctured ePTFE graft: an in vitro study under pulsed perfusion conditions.

BACKGROUND: When used as arteriovenous (AV) shunts for haemodialysis, small diameter expanded polytetrafluoroethylene (ePTFE) grafts have a high failure rate in vivo. Attempts to improve graft patency are various, and focus on either improvement of implantation techniques or graft tissue engineering. The tissue engineering approach attempts to reproduce in grafts the properties of pristine vasculature. As shown in previous experiments, it is possible to grow on ePTFE grafts under shear stress in vitro an autologous endothelial cell layer, which will withstand physiological stress under in vivo conditions of blood flow. The aim of this study was to investigate in an in vitro model the regenerative potency of a tissue-engineered prosthetic vascular graft after repeated cannulation with a haemodialysis cannula. METHODS: Pig endothelial cells were harvested from an external jugular vein. Following processing of the endothelial cells, seven ePTFE grafts were coated with an inner cell layer and were kept under pulsed perfusion. Each graft was then cannulated three times with a standard shunt needle. The endothelium was then left to regenerate for a maximum of 48 h. The grafts were stained with haematoxylin/eosin before histological study. RESULTS: All grafts were endothelialized over the puncture sites within 48 h. Histological analysis revealed a confluent endothelial cell lining at each puncture site. Cell morphology and cell pattern over puncture sites were not different from randomly picked locations over the graft lumen. CONCLUSION: Our results underline the potential of endothelial tissue engineering in vascular shunt surgery. Vascular bio-hybrids that have the properties of pristine vascular endothelium may be a key step forward in maintaining angio-access in patients who require haemodialysis.     

Improving endothelial cell retention for single stage seeding of prosthetic grafts: use of polymer sequences of arginine-glycine-aspartate.

OBJECTIVE: single stage seeding within the timeframe of a typical vascular operation has not been successful. One reason for this is poor cell adherence to the graft lumen once exposed to pulsatile blood flow. In this study we have carried out investigations with the use of two different fibronectin-based peptides, fibronectin-like engineered protein polymer (FEPP) which contains multiple copies of arginine-glycine-aspartate (RGD) and fibronectin adhesion promoting peptide (FAPP) to improve cell adherence. MATERIALS AND METHODS: FAPP and FEPP were coated onto native polyurethane and heparinised polyurethane grafts. The grafts were then seeded for either 1 or 2h with human umbilical vein endothelial cells (HUVEC). After the incubation period the cells were washed off and cell retention was calculated. Cell metabolism was measured using Alamar Blue, and confirmed with scanning electron microscopy (SEM). RESULTS: heparinised grafts coated with FEPP showed the best cell retention after both 1 and 2h seeding (80+/-4% vs 81+/-3%). This graft had no significant difference in cell retention after both times whilst all the other grafts had better cell retention after a 2h seeding. The Alamar blue and SEM results confirmed cell viability and function for all graft types. CONCLUSION: heparinised graft coated with FEPP allows significant cell retention after only 1h of seeding and shows promise for single stage seeding.     

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.     

内皮化小口径人工血管的研究

目的 探讨成人大隐静脉内皮细胞(HSVECs)种植到人工血管内表面的可行性.方法 酶消化法获取成人大隐静脉内皮细胞,在体外扩增培养13～15 d,将扩增培养的内皮细胞种植于纤维蛋白胶预衬的聚四氟乙烯(ePTFE)人工血管内表面,继续体外培养9～12 d.在不同的时间点,分别剪取部分内皮化的人工血管,行荧光显微镜和扫描电镜检查.结果 内皮细胞种植于人工血管后,扫描电镜下可见细胞在血管表面黏附、生长、增殖.平均孵育12 d后,人工血管腔面见一层均匀的基质,其表面有内皮细胞单层,内皮细胞排列紧密,呈梭形.结论 成人大隐静脉内皮细胞可以种植到人工血管,在体外增殖形成内皮细胞单层,达到内皮化的效果.     

Scanning electron microscope study of human veins and aorta-coronary artery vein grafts.

Scanning electron microscope study of two vein grafts which were removed after 50 months of implantation as aorta-coronary artery vein grafts showed considerable subendothelial thickening, which consisted of relatively cell-poor fibrous layer. In one of the grafts the endothelial surface was intact; the second graft showed focal loss of endothelial cells. Sections of two fresh human veins, studied prior to their insertion as aorta-coronary artery vein grafts, have shown that they retained an intact endothelial layer. A third fresh vein, however, showed focal damage with missing endothelial cells and adherent fibrin.     

In vitro endothelialization of small-caliber vascular grafts

To establish the conditions for achieving immediate and complete endothelial cell coverage of the luminal surfaces of small-caliber (internal diameter:4 mm) vascular grafts in vitro, the attachment and spread of endothelial cells cultured from human umbilical veins to expanded polytetrafluoroethylene (PTFE) and knitted Dacron grafts was studied. Cell number was quantified by fluorescent measurements of deoxyribonucleic acid. The completeness of cell coverage and cell junction formation were assessed by means of both scanning and transmission electron microscopy. Cell attachment was compared after expanded PTFE or knitted Dacron grafts were precoated with gelatin, laminin, fibronectin, fibrin, or collagen, singly or in combination. Saturation cell attachment of 3.5 +/- 0.7 X 10(5) cm-2 was completed within 15 minutes when (1) type I collagen was used to form the substrate matrix, (2) human umbilical vein endothelial cells were suspended in phosphate-buffered saline solution supplemented with calcium and magnesium, and (3) the suspended cell number was greater than or equal to 5 X 10(5). In contrast, attachment to untreated or laminin-treated surfaces was 1.3 +/- 0.33 X 10(5) cells cm-2 and attachment to fibrin- or fibronectin-treated surfaces was 2.8 +/- 0.47 and 2.4 +/- 1.1 cells X 10(5) cm-2, respectively. However, to produce a confluent flow surface, the attached cells required several hours in culture medium to spread completely. Maintenance of confluent cell coverage on the graft surface for 3 days in vitro was achieved by means of continuous perfusion with oxygenated tissue culture medium RPMI-1640-HEPES supplemented with 20% fetal bovine serum. We conclude that optimum immediate confluent endothelial coverage of small-caliber vascular grafts requires a high concentration of cells, attachment to collagen-precoated grafts, and several hours of incubation in complete culture medium.     

Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness

Objective: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. Methods: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm² shear stress and then for an additional 3 days with 1 or 25 dyne/cm² shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with β-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. Results: The pretreatment with 25 dyne/cm²—but not with 0 or 1 dyne/cm²—shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that β-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained α–smooth muscle actin were present. The thickness of this neointima averaged 41 ± 12 μm and 60 ± 23 μm in endothelial cell–seeded grafts that were pretreated with 25 dyne/cm² shear stress and 1 dyne/cm² shear stress, respectively, and 158 ± 46 μm in grafts that were not seeded with endothelial cells. Conclusion: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness. (J Vasc Surg 1999;29:157-67.)     

Leukocyte depletion enhances cultured endothelial retention on vascular prostheses

Approximately 90% of endothelial cells that are seeded or cultured onto vascular prostheses are lost from the flow surface within 24 hours of implantation. To determine the contribution of leukocytes to endothelial cell loss, 111In-labeled, cultured canine jugular venous endothelial cells were grown to confluence on fibronectin-coated polyester elastomer tubes measuring 4 mm inner diameter and 30 mm in length. Autogenous cell-lined tubes were implanted as bilateral carotid replacement grafts in six dogs made leukopenic by cyclophosphamide. Similar unilateral grafts were placed in 12 control dogs. Grafts were removed and perfusion-fixed from six control animals after 2 hours of in vivo arterial perfusion and from the other six animals after 6 hours of perfusion. One graft was removed and perfusion-fixed from each leukopenic animal after 2 hours of implantation and the other after 6 hours. Attachment of endothelial cells to the grafts was measured by indium-labeling technique. Retention of endothelium on grafts removed after 2 hours was measured by planimetric counting with scanning electron microscopy and on those removed after 6 hours by radioisotope quantification. Endothelial cell retention after 2 hours was 37.6% +/- 27.0% in control dogs and 97.0% +/- 3.4% in leukopenic animals (p less than 0.0007). After 6 hours retention was 35.9% +/- 23.2% in control animals and 86.5% +/- 6.0% in leukopenic animals (p less than 0.0009). Leukocyte surface activity was present in less than 1% of the leukopenic dogs compared with 8.5% of the other in vivo midgrafts after 2 hours. These results suggest that leukocytes play a significant role in the loss of seeded endothelium from vascular prostheses.     

Delayed exposure to pulsatile shear stress improves retention of human saphenous vein endothelial cells on seeded ePTFE grafts

Since significant loss of endothelial cells (ECs) from the surface of a seeded prosthetic graft occurs after implantation, improved cell retention following exposure to flow should increase the likelihood of long-term success with this technology. An in vitro pulsatile flow circuit was developed to study the effects of two variables on cell retention: cell density at the time of seeding and postseeding incubation time. Fibronectin-coated ePTFE grafts (4 mm x 5 cm) were seeded with human saphenous vein ECs at two densities, confluent (1 x 10(5) cells/cm2) or subconfluent (2 x 10(4)), and incubated in vitro for varying time intervals (90 min, 1, 3, or 7 days). Test grafts were exposed to 90 min of pulsatile flow in an in vitro flow circuit, then fixed, and stained, and in situ cell counts (cells/cm2) were determined for nine representative fields per graft. Paired control grafts were treated identically but were not exposed to flow. Cell retention was calculated using the formula: % retention = cells/cm2 perfused graft divided by cells/cm2 control graft. Grafts exposed to flow 90 min after seeding demonstrated significantly lower cell retention when compared to later time points. When cells were seeded at confluent density, maximal retention (92 +/- 3%) occurred 24 hr after seeding. Prolonged culture of cells seeded on ePTFE grafts at confluent density resulted in increased cell loss. In contrast, on grafts seeded at subconfluent density, retention improved as cells grew to confluence (16 +/- 4.5% initially to 82 +/- 7% at 7 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

双层血管细胞种植提高人工血管内皮细胞粘附性

目的　采用血管内皮细胞 (EC)和平滑肌细胞 (SMC)双层种植方法 ,提高人工血管腔面EC的保存率。　方法　聚四氟乙烯 (PTFE)人工血管经纤维连结蛋白预衬处理 ,管腔面先后种植SMC和EC ,将受试的人工血管接入脉冲式体外灌注装置灌注 1h ,计算比较灌注前后受试标本SMC和EC密度。　结果　灌注 1h后 ,单纯EC种植组 61%细胞脱落 ,单纯SMC种植组 3 6%细胞脱落 ,而双层细胞种植组仅有 2 7%EC脱落 ,双层细胞种植组细胞保存率明显高于单纯EC种植组 (P <0 0 1)。低流速预灌注未能改善EC的保存率。　结论　在EC和人工血管壁之间种植SMC可提高EC的保存率     

Optimal endothelialisation of a new compliant poly(carbonate-urea)urethane vascular graft with effect of physiological shear stress.

OBJECTIVE: to define the optimal seeding conditions of a new stress free poly(carbonate-urea)urethane (CPU) graft with compliance similar to that of human artery with honeycomb structure engineered during the manufacturing process to enhance adhesion and growth of endothelial cells. METHODS:(111)Indium-oxine radiolabeled human umbilical vein endothelial cells (HUVEC) were seeded onto CPU grafts at (a) concentrations from 2-24x10(5)cells/cm(2)and (b) incubated for 0.5, 1, 2, 4 and 6 h. Following incubation, graft segments were subjected to three washing/gamma counting procedures and scanning electron microscopy (SEM). Cell viability was measured using a modified Alamar blue(TM)assay. To test physiological retention a pulsatile flow phantom was used to subject optimally seeded (16x10(5), 4 h) CPU grafts to arterial shear stress for 6 h with real time acquisition of scintigraphic images of seeded grafts using a nuclear medicine gamma camera system. RESULTS: the seeding efficiency of 54+/-13% post three washes was achieved using 16x10(5)cells/cm(2). Similarly in SEM micrographs a seeding density of 16x10(5)cells/cm(2)resulted in a confluent monolayer. Seeded CPU segments incubated for 4 h exhibited significantly higher resistance to wash-off than segments incubated for 30 min (p <0.05). Exposure of seeded grafts to pulsatile shear stress resulted in some cell loss with 67+/-3% of cells adherent following 6 h of perfusion with ongoing metabolic activity. Thus, optimal conditions were 16x10(5)cells/cm(2)at 4 h. CONCLUSIONS: the optimal seeding conditions have been defined for "tissue-engineered" vascular graft which allow complete endothelialisation and high cell-to-substrate strength that resists hydrodynamic stress.     

Optimal seeding conditions for human endothelial cells

Anin vitro model of endothelial cell seeding has been developed to individually evaluate the steps required for seeding arterial prostheses. Human saphenous vein endothelial cells are radiolabeled with tritiated thymidine and seeded onto 4 mm polytetrafluorethylene grafts. Grafts are then placed into a perfusion circuit for determination of cellular retention. Using this model, the following variables were studied: (1) graft coating (fibronectin versus serum versus plasma); (2) time of incubation of cells with graft (0, 20, 90 minutes); (3) density of the initial seeding solution (4×10³-6×10⁵ cells/cm²). The data suggest that incubation of a graft with plasma provides an adhesive surface that is as effective as fibronectin for enhancing cell retention. With this particular model, seeding densities between 1 and 2 × 10⁵ cells/cm² produce a confluent monolayer with optimal utilization of cells. A shorter 20 minute incubation period resulted in the retention of only half of the seeded cells, while postperfusion attachment increased significantly with a 90 minute incubation period. Data derived from this system can be used to construct a protocol that may be useful for clinicalin vivo seeding trials.     

脱细胞牛颈静脉血管支架的内皮化研究

目的 探讨在脱细胞牛颈静脉血管支架上进行内皮细胞再种植的可行性.方法 将人骨髓间充质干细胞(BMDCs)诱导分化为内皮种子细胞后种植在脱细胞牛颈静脉血管支架上,分为动态培养和静态培养2组.培养7 d后,对标本进行病理和扫描电镜观察.结果 静态培养的血管支架表面形成连续的单细胞层.动态培养后血管支架表面的细胞仍有50%残留,沿流场方向排列.结论 人骨髓间充质干细胞诱导分化的内皮种子细胞在脱细胞牛颈静脉血管支架上可以黏附生长.     

小口径人工血管的研究进展

为提高小口径人工血管的远期通畅率，一方面需要寻找顺应性更好的材料；另一方面则是在现有人工血管内壁种植内皮细胞。这包括单期种植法，二期种植法以及自体静脉碎片快速种植法。用于种植的内皮细胞可来源于自体静脉、人脐静脉和皮下脂肪微血管。通过在人工血管内壁上衬附可吸收的细胞外黏附蛋白、细胞生长刺激因子，利用生物素与抗生物素蛋白的特异结合能力，或改变植入的内皮细胞的带电性，以及在体外对植入的内皮细胞进行流体切应力锻炼等方法，可以提高内皮细胞的黏附力。植入基因工程改造过的内皮细胞，也是提高小口径人工血管通畅率的一种新尝试。     

Comparative In vitro evaluation of two different preparations of small diameter polyurethane vascular grafts

Polyurethane (PU) frequently has been used to manufacture small diameter vascular grafts due to its good bicompatibility. In this study, sponge PU small diameter vascular grafts were fabricated from Pellethane 2103, as well as a self-synthesized PU, by utilization of a salt casting technique and by varying the salt/polymer ratios. Two types were made; one of them had a thin solid layer on the outer surface. The inner surface was identical and was coated with gelatin crosslinked by epoxide. Tensile properties, compliance, platelet activation, and endothelial cell attachment were evaluated in vitro. It was found that for the grafts with an outer nonporous coating, compliance could not be estimated from modulus due to anisotropy. The most compliance-matched grafts were made of self-synthesized polyurethane with a salt/polymer ratio 4 to 8 or Pellethane with a ratio 6 to 8 without the outer nonporous coating. The former had better elongation. Self-synthesized PU had lower platelet adhesion as well as more endothelial attachment than Pellethane. Porosity activated platelets strongly and reduced endothelial adhesion unless the surface was modified by the crosslinked gelatin layer. It was concluded that PU synthesized in our lab with a salt/polymer ratio of at least 4 and coated with epoxy crosslinked gelatin was a better substrate for preparation of small diameter vascular grafts.     

In vivo retention of endothelial cells adenovirally transduced with tissue-type plasminogen activator and seeded onto expanded polytetrafluoroethylene

PURPOSE: Seeding a prosthetic graft with genetically engineered vascular endothelial cells (ECs) has the potential to enhance the graft's antithrombotic properties. However, it has been reported that ECs transduced with tissue-type plasminogen activator (tPA) have very low levels of retention on grafts, probably because of increased proteolytic activity. We examined the retention of human tPA (htPA)-transduced ECs after the cells were seeded onto expanded polytetrafluoroethylene (ePTFE) grafts and implanted into dogs. We also examined the function of secreted htPA in this model.Methods and Results: Canine jugular venous ECs were transduced with adenoviral vectors encoding htPA (Adex1CAhtPA) and beta-galactosidase (Adex1CALacZ). There was a positive relationship between the percentage of X-gal ECs staining and the multiplicity of infection (MOI) of Adex1CALacZ. The level of htPA production in vitro increased with the increasing MOI of Adex1CAhtPA, but decreased gradually 4 days after infection. ECs coinfected with Adex1CAhtPA and Adex1CALacZ (htPAEC) or ECs infected with Adex1CALacZ alone (LacZEC) were seeded onto ePTFE grafts at densities equivalent to confluence to visualize retained ECs in an in vivo flow study. The grafts were implanted into canine carotid arteries and harvested after 5 hours of exposure to blood flow. The harvested grafts showed patchy defects in ECs, most of which were covered with mural thrombi. There was no significant difference in retention between htPAEC (29.3% +/- 8.7%) and LacZEC (19.5% +/- 3. 6%). There was a significant negative correlation between the in vivo EC retention on the grafts and the in vitro cellular passage level of ECs (P =.041; r = -.40). htPAEC produced 210.3 +/- 22.2 ng htPA antigen/10(6) cells per 6 hours in vitro and continued to secrete htPA on the harvested graft. CONCLUSIONS: We demonstrated that a large amount of functional htPA was produced by adenovirally modified canine ECs. The results of the in vivo study may suggest that overexpression of tPA has little effect on the short-term retention of early passage ECs seeded onto ePTFE grafts.     

Complete in vitro prosthesis endothelialization induced by artificial extracellular matrix.

This report presents our research on the conditions necessary to substain optimal in vitro prosthetic endothelialization using human endothelium cultures. Human vein endothelial cells were seeded at a concentration of 3 x 10(5)/cm2 in a gelatinized Dacron patch graft coated with a commercial collagen film, using a solution of fibrin glue. Endothelium adhesion, proliferation, and survival were measured by [3H]thymidine incorporation, after 7 days of incubation. Finally, the morphology of prosthetic endothelialization was analyzed by scanning electron microscopy. We observed that the Dacron patch grafts coated with collagen film were able to promote endothelialization better than the prostheses coated with highly concentrated collagen solution or gelatin. We therefore concluded that the collagen film that supports endothelial cell adhesion and proliferation uniformly covers the entire synthetic endoluminal surface of the Dacron graft, thus preventing endothelial cell alterations induced by direct contact with the synthetic prosthetic surface.     

Microvenous graft length determination for arterial repair

Varying lengths of femoral vein grafts from 36 New Zealand rabbits were used to bridge defects in the ipsilateral femoral artery in an attempt to estimate the correct length of vein graft needed to replace an arterial defect. Harvested venous segments contracted by 35% to 55% of their in vivo length. Short grafts subjected to linear tension had poor patency rates. Increasing the length improved patency. Long redundant grafts were initially tortuous, but lost their redundancy when studied long term. Silicone perfusion studies, however, revealed a persistence of luminal irregularity and tortuosity. Histologically, arterialized vein grafts thickened by accumulation of fibrous tissue in the medial layer, but otherwise maintained identifiable venous morphological characteristics. Scanning electron microscopic studies identified endothelial changes in vein grafts after arterialization. Based on these results, the ideal microvenous graft should be approximately 35% longer than the arterial defect to be bridged when measured in vivo to permit optimal balance between linear and radial deforming forces.     

A hybrid compliant vascular graft seeded with microvascular endothelial cells extracted from human omentum

We report the development of a hybrid vascular graft using an innovative compliant poly(carbonate-urea)urethane unlike any previous polyurethane MyoLink as a permanent scaffold. The engineered graft has a hierarchical arterial structure: a monolayer of oriented microvascular endothelial cells (MVECs) and 3-D matrix (human collagen Type 4/dermatan sulfate) bonded onto MyoLink. The grafts' clinical feasibility was evaluated by determining optimal MVEC seeding density, incubation time, viability, and adhesion of these cells when exposed to arterial shear stress. MVECs from human omentum were isolated by a new centrifugation protocol, radiolabeled and seeded onto hybrid graft 2 to 18 x 105 cells/cm(2) for 24 h at 37 degrees C and for 1 to 5 h at 6 x 105 cells/cm(2), washed 3 times, and gamma counted. Qualitative assessment of seeding density/incubation was also undertaken with scanning electron microscopy (SEM) and viability tested with a modified Alamar Blue assay. A pulsatile flow phantom was used to subject the hybrid graft (200 mm length, 5 mm internal diameter) seeded with radiolabeled MVECs (6 x 105 cells/cm(2) at 2 h) to arterial shear and dynamic scintigraphy images acquired in real time using a nuclear medicine gamma camera system during 14 h of perfusion (n = 6). The optimal seeding density was 6 x 105 cells/cm(2), and qualitative SEM confirmed this. Incubating cells for 2 h produced significantly greater cell attachment than was seen for 1 h incubation (p < 0.05), and there was no significant difference in adhesion between cells incubated over the 2 h. Exposure of grafts to acute shear stress resulted in significantly higher proportions of initial cells attached to hybrid grafts compared to native MyoLink grafts (67 +/- 3% versus 55 +/- 2%, p < 0.001). As shown here, tissue engineering of native Myolink grafts significantly reduces the seeding density and incubation time to produce a monolayer onto which cells adhere to better.