Long-term biologic fate of neoarteries regenerated in microporous, compliant, biodegradable, small-caliber vascular grafts in rats

Microporous, compliant, biodegradable vascular grafts prepared from a mixture of polyurethane [( PU], 95% weight) and poly-L-lactide [( PLLA] 5% weight) can function as temporary scaffolds for the regeneration of the arterial wall of small-caliber arteries. The purpose of this study was to determine the long-term biologic fate of these neoarteries that were regenerated in PU/PLLA vascular grafts. The PU/PLLA vascular grafts (1.5 mm internal diameter [ID]) were implanted into the abdominal aortas of rats (N = 8) and were evaluated 1 year after implantation by means of macroscopic inspection, light microscopy, and electron microscopy. All implants were patent; three implants were normally shaped, two were slightly dilated (+/- 10% of the original ID), and three implants were aneurysmal. Arterial pulsations were reduced but still visible in the normally shaped implants and absent in the other implants. In all implants, the neointima was complete. The neomedia varied among the implants: In the normally shaped implants, smooth muscle cells were predominantly circularly arranged as in normal arterial tissue; in the other implants, smooth muscle cells were predominantly longitudinally arranged. The neoadventitia showed a completely fragmented graft lattice, which was organized by fibrohistiocytic tissue. These results suggest that the pattern of arrangement of smooth muscle cells in the neomedia determines the ultimate biologic fate of neoarteries regenerated in microporous, compliant, biodegradable vascular grafts. Only those neoarteries with predominantly circularly arranged smooth muscle cells in the neomedia were able to function normally as an arterial substitute for a 1-year period after implantation into the rat abdominal aorta.     

Arterial wall regeneration in small-caliber vascular grafts in rats. Neoendothelial healing and prostacyclin production

Clinically available synthetic graft materials frequently fail when used as a small-caliber arterial substitute. Therefore, we developed a new type of graft material, prepared from a mixture of polyurethane and poly-L-lactic acid, to be used as a scaffold for the regeneration of the arterial wall. In this study microporous, compliant, biodegradable polyurethane/poly-L-lactic acid grafts (n = 16) and polytetrafluoroethylene grafts (n = 16) were implanted in the rat abdominal aorta and evaluated 3, 6, and 12 weeks after implantation. First, we evaluated the extent of neoendothelial healing (n = 8) by means of light microscopy and scanning electron microscopy. Next, we studied the ability of the neoendothelial cells to produce prostacyclin (n = 8) by means of bioassay for prostacyclin and radioimmunoassay for its stable hydrolysis product, 6-oxo-prostaglandin F1 alpha. There were no significant differences between the two graft types in the amount of prostacyclin production per unit graft area covered with neoendothelium, and this amount was the same as for normal endothelium. However, the polytetrafluoroethylene grafts showed incomplete neoendothelial healing, even after 12 weeks of implantation, in contrast to the polyurethane/poly-L-lactic acid grafts. The better healing characteristics of the polyurethane/poly-L-lactic acid grafts ensured the fast development of a complete neoarterial wall, possessing strength, compliance, and thromboresistance equivalent to normal arterial wall tissue. These results demonstrate that arterial wall tissue regeneration in polyurethane/poly-L-lactic acid grafts may open new perspectives in the field of arterial reconstructive surgery.     

Microporous, complaint, biodegradable vascular grafts for the regeneration of the arterial wall in rat abdominal aorta

Microporous, complaint, biodegradable vascular grafts prepared from mixtures of polyurethane (PU) and poly-L-lactic acid (PLLA) can function as temporary scaffolds for the regeneration of the arterial wall in small-caliber arteries. This study was undertaken to determine the most suitable composition for PU/PLLA vascular grafts to ensure an optimal regeneration. Four types of PU/PLLA vascular grafts differing in percent weight of the PU/PLLA mixture, molecular weight of PLLA, and pore size were implanted into the abdominal aorta of rats (n = 32). Six weeks after implantation two implants of each graft type were evaluated by means of scanning electron microscopy and six implants were evaluated by means of light microscopy. In two types of the PU/PLLA vascular grafts, both of which were prepared from a 95%/5% weight PU/PLLA mixture with PLLA of molecular weight 500,000 but which had a different pore size, there was (I) absence of aneurysm formation and maintenance of arterial implant pulsations, (II) regeneration of a complete antithrombogenic neointima, (III) regeneration of a neomedia of comparable thickness to the media of normal rat abdominal aorta with the regeneration of elastic laminae almost throughout its thickness, and (IV) regeneration of a sufficiently supporting neoadventitia. These results demonstrate that a 95%/5% weight PU/PLLA mixture with PLLA of molecular weight 500,000 is the most suitable composition for PU/PLLA vascular grafts to ensure an optimal regeneration of a neoarterial wall that is of sufficient strength, compliance, and thromboresistance to function as a small-caliber arterial substitute. Pore size of these PU/PLLA grafts does not affect regeneration.     

Smooth muscle cell seeding in biodegradable grafts in rats: a new method to enhance the process of arterial wall regeneration

In this study a smooth muscle cell seeding technique was developed and evaluated in biodegradable vascular grafts in rats. Cultured smooth muscle cells (3.5 to 5 X 10(6) in 0.1 ml whole blood) were seeded by means of a one-step preclotting technique in microporous, compliant, biodegradable vascular grafts (internal diameter 1.3 mm, length 1 cm; n = 16). Four seeded grafts as well as four nonseeded preclotted control grafts were histologically evaluated immediately after seeding and on preclotting; the other seeded grafts (n = 12) as well as nonseeded preclotted control grafts (n = 12) were implanted into the abdominal aorta of rats and were evaluated after 2 hours (n = 4), 2 days (n = 4), and 1 week (n = 4) with regard to the process of arterial wall regeneration. All grafts, except one seeded graft harvested at 1 week, were patent. All nonseeded control grafts showed no (at 2 hours and at 2 days) or limited (at 1 week) smooth muscle cell ingrowth. Of the 11 patent seeded grafts three also showed poor or limited neomedia development, probably because of improper preclotting. However, eight of the 11 patent seeded grafts showed fast and uniform noemedia development (clearly discernible at 2 days). Moreover, in two patent 1-week seeded grafts there were neomedia comparable in thickness and architecture (circularly arranged smooth muscle cells) to that of normal rat media, and a neointima had already lined 30% to 40% of the lumen. These results clearly demonstrate that smooth muscle cell seeding in biodegradable grafts is feasible and indicate that this seeding can enhance the process of arterial wall regeneration in these grafts.     

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.     

Hyaluronic acid biodegradable material for reconstruction of vascular wall: a preliminary study in rats

The objective of this preliminary study was to develop a reabsorbable vascular patch that did not require in vitro cell or biochemical preconditioning for vascular wall repair. Patches were composed only of hyaluronic acid (HA). Twenty male Wistar rats weighing 250-350 g were used. The abdominal aorta was exposed and isolated. A rectangular breach (1 mm × 5 mm) was made on vessel wall and arterial defect was repaired with HA made patch. Performance was assessed at 1, 2, 4, 8, and 16 weeks after surgery by histology and immunohistochemistry. Extracellular matrix components were evaluated by molecular biological methods. After 16 weeks, the biomaterial was almost completely degraded and replaced by a neoartery wall composed of endothelial cells, smooth muscle cells, collagen, and elastin fibers organized in layers. In conclusion, HA patches provide a provisional three-dimensional support to interact with cells for the control of their function, guiding the spatially and temporally multicellular processes of artery regeneration.     

Administration of granulocyte colony-stimulating factor enhances endothelialization and microvessel formation in small-caliber synthetic vascular grafts

OBJECTIVE: The purpose of this study was to determine whether systemic administration of granulocyte colony-stimulating factor (G-CSF) would promote endothelialization for small-caliber Dacron vascular grafts. METHODS: We implanted 4-mm preclotted Dacron grafts in both carotids of 12 dogs. For a fair comparison, all dogs had a comparable platelet aggregation profile with platelet aggregation scores less than 30. Five dogs served as controls, and the others were given 7-day subcutaneous injections of G-CSF (10 microg/kg per day), starting on the seventh postoperative day. The effect of G-CSF was evaluated by white blood cell count, which showed a 3.7-fold (+/- 2.7-fold) increase at the end of treatment. Grafts were harvested at 4 weeks. All G-CSF grafts were patent, and one control occluded. Endothelial-like cell coverage averaged 80.8% on G-CSF grafts, but only 35.6% for control grafts (P <.0004). With the exclusion of the anastomotic pannus healing factor, the difference in endothelial-like cell coverage was even greater (68.5% vs 9.8%; P <.0001). Immunocytochemical staining and electron microscopy studies demonstrated endothelial cells. Light microscopy also showed that there were more microvessels on and in the G-CSF grafts than in the control grafts. This study suggests that G-CSF can enhance early endothelialization of small-caliber vascular grafts. Further studies to determine the proper dosage and timing are needed before clinical application can be recommended.     

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.     

Experimental studies on application of small-caliber vascular prosthesis produced by polyurethane.

It has been suggested that a microporous structure enhances fast and complete endothelialization. For long-term patency, antithrombogenicity and microporous structure are very important factors. In this paper, we have developed a new technique to give a micro-porous structure to small-caliber vascular prosthesis produced by polyurethane which has favorable antithrombogenecity. A mixed solution (tetrahydrofuran: dimethylformamide = 1:1) containing 13 wt% of segmented polyurethane and a variable amount of calcium carbonate (mean particle size of 8 mm in diameter) was dip-coated on a glass mandril of 3 mm and 6 mm in diameter and placed into distilled water for 24 hours. After the glass mandrill was removed, this polyurethane tube was placed into 1 mmol hydrochloric acid for 1 hour, and a microporous polyurethane vascular prosthesis of 20 mm in length was completed. These prostheses of 3 mm and 6 mm in diameter were implanted into the femoral and the carotid arteries, and the abdominal aorta of the dogs, respectively. Patency was recognized by arteriography and Duplex scanning and the removed grafts were inspected macro- and microscopically. Greater hydraulic permeability of this graft was obtained with an increase in the quantity of calcium carbonate mixed with polyurethane. In elasticity, this graft was more similar to the canine jugular vein than the polytetrafluoroethylene graft. Patency was observed 8 weeks after implantation on the arteriogram, and neointima was observed microscopically on the smooth and lustrous lumen. The new polyurethane vascular prosthesis we developed might provide a potential prosthesis for small-caliber vascular reconstruction.     

Endothelial cell seeding of small-caliber synthetic grafts in the baboon

Endothelial cell (EC) seeding has been proposed as a method to improve the performance of small-caliber synthetic vascular prostheses. Seeding experiments to date have all been carried out in the dog. This study investigates EC seeding of small-caliber Dacron carotid interposition grafts compared with contralateral control grafts in the baboon. Surface thrombogenicity was assessed at 24 hours, 2 weeks, and 4 weeks after implantation with indium 111-labeled autologous platelets. Morphologic and immunohistochemical techniques were used to assess the identity and homogeneity of the EC inoculum before seeding and to identify cell types on the harvested grafts. There was no significant difference in patency rates between seeded and control grafts at 5 weeks. Platelet accumulation on seeded grafts was significantly less (p less than 0.05) than on paired controls at 2 and 4 weeks after implantation. The luminal lining of seeded grafts had more cellular ingrowth, less adherent thrombus, and more surface cells with the morphologic and histochemical characteristics of EC than did the lining of controls. EC seeding reduces the platelet reactivity and accelerates EC coverage of small-caliber grafts in the baboon.     

Advances in the development of grafts for bypass of small-caliber arteries

The standard for coronary bypass is now clearly the autogenous internal mammary artery (IMA). Complete revascularization of the left ventricle can be attained in most cases using only the IMA's with as many sequential anastomoses as necessary. This advance has freed the saphenous vein (SV) for use below the knee joint.Properly preclotted, noncrimped, externally-supported Dacron^® grafts with an external velour surface merit consideration as the standard for axillofemoral bypass. The limitation of this graft is its dependence on a precise technique of preclotting to transform the Dacron^® framework into a biosynthetic complex of dethrombinated fibrin and Dacron^®.The autogenous SV is clearly the standard for below-knee bypass. However, for above-knee femoropopliteal bypass, the results obtained with polytetrafluoroethylene, umbilical vein, and externally-supported, noncrimped, knitted Dacron^® prostheses are sufficiently competitive with the results obtained with the SV to make their use an acceptable option in the above-knee location. The saphenous vein is, thus, saved for use in the more demanding below-knee sites such as a femoropopliteal, femorotibial, or femoroperoneal bypass.

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Resumen Indudablemente el estándar actual para la derivación (bypass) coronaria es la arteria mamaria interna del propio paciente. En la mayoría de los casos se puede lograr la revascularización completa del ventrículo izquierdo utilizando sólo la arteria mamaria interna con tantas anastomosis secuenciales como sean necesarias. Este reconocido avance libera a la vena safena y hace posible su utilization para derivaciones arteriales por debajo de la rodilla.Los Injertos EXC (USCI^® Sauvage EXS Vascular Prosthesis, Bard Cardiosurgery Division, Billerica, Massachusetts, U.S.A.) son prótesis vasculares porosas de Dacrón^® con soporte externo constituídos por una superficie exterior de valour y una estructura fibrosa que debe ser convertida, antes de su implantación, en un complejo biosintético de Dacrón^® y fibrina destrombinada capaz de proveer una superficie de flujo hipotrombogénica y una pared impermeable. Debidamente precoagulado, el Injerto EXC merece consideración para derivaciones axilofemorales. Sus limitaciones se derivan de su dependencia de una meticulosa técnica de precoagulación para convertir la estructura de Dacrón^® en el complejo biosintético de fibrina destrombinada y Dacrón^®.La vena safena del propio patiente es el estándar indudable para derivación (bypass) arterial por debajo de la rodilla. Sin embargo, para derivaciones femoropoplfteas por encima de la rodilla los resultados obtenidos con politetrafluoroetileno, con vena umbilical, y con prótesis de Dacrón^® no corrugado y con soporte externo, son suficientemente competitivos con los obtenidos con la vena safena para hacer de su uso una opción alterna aceptable para la región superior a la rodilla. Con ello se puede salvar la vena safena para su posible utilization en derivaciones más exigentes, por debajo de la rodilla, tales como las derivaciones femoropoplíteas, femorotibiales, o femoroperoneas.

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Résumé Le conduit le plus utilisé pour le pontage aortocoronaire est indiscutablement l'artère mammaire interne autogène (AMI). On peut ainsi revasculariser complètement le ventricule gauche avec une seule AMI en réalisant autant d'anastomoses successives qu'il le faut. Cette technique permet de garder la veine saphène interne (VSI) pour les pontages distaux.Dans les pontages axillofémoraux, on préconise surtout les prothèses en Dacron^®, précoagulées, non plisées, à anneaux rigides, tricotées avec une surface externe en velours. Les limites de l'utilisation de cette prothèse dépendent de la précision de la précoagulation qui transforme les mailles du Dacron^® en un complexe biosynthétique de fibrine déthrombinée et de Dacron^®.La VSI est sans aucun doute le matériel de choix pour les pontages sous-géniculaires. Cependant pour les pontages fémoropoplités sus-géniculaires, les résultats obtenus avec les prothèses en polytétrafluoéthylène, veine ombilicale et les prothèses en Dacron^® annelées, tricotées, non plissées sont suffisamment compétitifs avec la VSI pour être prises en compte dans cette situation. On sauvegarde alors la VSI pour les pontages sous géniculaires fémoropoplité, fémorotibial ou fémoropéronier éventuels.

     

The effect of wall mechanical properties on patency of arterial grafts

Normal arteries have properties which match the low output impedance of the heart to the high peripheral impedance. These properties can be assessed in terms of compliance (% diameter change per unit pressure change) as well as by other haemodynamic parameters. Experiments were designed using vein, Dacron and expanded polytetrafluoroethylene (PTFE) in a low flow canine femoral artery bypass model. No graft group achieved perfect patency. At twelve weeks 80% of vein grafts, 30% of Dacron grafts, and 15% of PTFE grafts remained patent. The compliance of vein grafts was maintained despite marked thickening of the wall. Patency was correlated at a highly significant level with compliance. The studies demonstrate that the matching of the mechanical properties of grafts to host arteries is important in the design of successful synthetic arterial grafts.     

Use of small-caliber polytetrafluoroethylene (Gore-Tex) grafts in microsurgical training

We present the use of polytetrafluoroethylene (PTFE) graft material as a microsurgical training model that better simulates live vessel repairs. PTFE grafts have mechanical advantages over polyethylene or silicone tubing in that they better mimic the "feel" of an arterial vessel wall, thus allowing the student to perfect counterpressor maneuvers before attempting live vessel repairs. Saving the PTFE "repairs" for later comparison and study provides the student with positive feedback that is not possible with living models. This cost-effective model has helped to shorten our directed teaching program and has reduced the use of laboratory animals.     

Morphological studies of cellular responses to experimental arterial grafts

Cross-carotid microvascular bypass grafts 2-3 mm in diameter were implanted using microsurgical techniques for end-to-end anastomosis in four dogs. One autograft control and one of three denatured human umbilical artery xenografts (HUAG) were patent at 5 weeks. One of the other two denatured HUAGs had thrombosed at 1 week, and the other was occluded at 5 weeks. Host and graft vessel specimens were evaluated histologically as well as with transmission electron microscopy after sacrifice. Results indicate that failure of reconstitution of a true endothelial layer, presence of a subintimal myofibroblast population, increased collagen deposition of the muscularis, and occlusion of the adventitial and mural microcirculation were observed in both the early and late nonpatent vessels but not in the patent specimens. Evidence for myointimal cell proliferation was present in both patent and occluded grafts. A cohesive intimal layer was observed in both patent cases. Microvascular occlusion, due to an excess of endothelial cells, of new vasa vasorum in one case seems related to increased fibrosis, which could have resulted in graft stenosis. The surgical procedures and subsequent morphological analyses were adequate and sufficient for use in a long-term study of the possible causes of graft failure.