Rapid Endothelialization Research Articles

Isolation and Transplantation of Autologous Circulating Endothelial Cells Into Denuded Vessels and Prosthetic Grafts

Blood-borne endothelial cells originating from adult bone marrow were reported previously. These cells have the properties of an endothelial progenitor cell (EPC) and can be mobilized by cytokines and recruited to sites of neovascularization, where they differentiate into mature endothelial cells. Current protocols for isolation of EPCs from peripheral blood rely on enrichment and selection of CD34+ mononuclear cells. In this report, we describe a streamlined method for the isolation and expansion of EPCs from peripheral blood and evaluate their therapeutic potential for autologous cell-based therapy of injured blood vessels and prosthetic grafts. A subset of unfractionated mononuclear cells exhibited the potential to differentiate in vitro into endothelial cells under selective growth conditions. The cells were efficiently transduced ex vivo by a retroviral vector expressing the LacZ reporter gene and could be expanded to yield sufficient numbers for therapeutic applications. Transplantation of these cells into balloon-injured carotid arteries and into bioprosthetic grafts in rabbits led to rapid endothelialization of the denuded vessels and graft segments, resulting in significant reduction in neointima deposition. We conclude that transplantation of EPCs may play a crucial role in reestablishing endothelial integrity in injured vessels, thereby inhibiting neointimal hyperplasia. These findings may have implications for novel and practical cell-based therapies for vascular disease.

Effect of biologically active coating on biocompatibility of Nitinol devices designed for the closure of intra-atrial communications

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n=10, each) and flat Nitinol bands (n=3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p<0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p<0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating>uncoated Nitinol>r-hirudin coating>heparin coating>basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

Autologous tissue-fragmented extracardiac conduit with rapid, stable endothelialization due to angiogenesis.

Autologous angiogenic cytokines are known to activated by mincing stimulation, and well regulated in vivo. We applied this tissue fragmentation technique to a low-pressure pulmonary extracardiac conduit to obtain rapid endothelialization and stable neointima formation due to angiogenesis. Subcutaneous adipose tissue was obtained, minced, suspended, and sieved through highly porous fabric vascular prosthesis by pressurized injection. The adipose tissue fragmented graft with an autologous fresh pericardial monocusp valve was implanted between the right ventricle and the pulmonary artery in 13 dogs. The same grafts without fragments were implanted in 8 dogs as controls. No anticoagulation therapy was given. Grafts were removed 6 to 1,128 days after implantation. In the developed grafts, angiogenesis occurred throughout the interstices of the graft wall from the adventitial side, and host cells proliferated and migrated. Endothelialization was completed throughout developed grafts at 2 weeks. The intima was still thin up to 1,128 days and free of degenerative changes. In control grafts, however, capillary infiltration was limited to perigraft tissue at 2 weeks and endothelialization was not completed by 3 months. Under the endothelial cell layer, laminal elastic fibers were formed through the developed graft wall by 4 months and still maintained at 1,128 days. The results demonstrated that adipose tissue fragmented extracardiac conduits induce rapid endothelialization and maintained thin intima with laminal elastic fibers. Long-term durability is expected based on results from using this technique in a low-pressure pulmonary system in dogs.

Histopathologic Evaluation of an Expanded Polytetrafluoroethylene-Nitinol Stent Endoprosthesis in Canine Iliofemoral Arteries

The authors assess a new ePTFE-nitinol stent for its long-term patency, healing, and properties of endothelialization. Adult greyhounds (n = 18) underwent bilateral iliofemoral placement of an endoprosthesis (Hemobahn) consisting of a nitinol stent lined with an ultrathin expanded polytetrafluoroethylene (ePTFE) material. Histologic and quantitative morphometric analyses were performed on devices explanted at 2 weeks and 1, 3, 6, and 12 months. The source of endothelialization was examined in four additional devices modified by sealing either the proximal and distal ends or the entire graft with poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP), a nonporous laminate to prevent potential transgraft endothelial cell migration. Device patency assessed with both intravascular ultrasound and histologic study showed minimal arterial stenosis, irrespective of implant duration. The neointima at less than 3 months consisted of fibrin and inflammatory infiltrate; at later time points, it was composed of mostly smooth muscle cells. Flow surfaces were more than 75% endothelialized by 3 months, which was nearly complete by 6 months. Modified endoprostheses entirely enveloped with FEP resulted in endothelialization of only the proximal and distal ends; the middle regions showed nonocclusive thrombi. Conversely, devices wrapped proximally and distally showed nearly complete endothelialization. This ePTFE-nitinol endoprosthesis demonstrated long-term patency at up to 1 year after implantation and showed early and nearly complete endothelialization by 6 months. The design promoted rapid endothelialization of flow surfaces, particularly in the midregion of the device possibly by transgraft migration.

Succinylated collagen crosslinked by thermal treatment for coating vascular prostheses.

Vascular prostheses coated with collagen carefully prepared to avoid contamination were tested to see if it could induce endothelial cell lining throughout the graft surface in a natural way. The collagen fibers were succinylated. Hydrogel produced with the succinylated collagen was used for the sealant to reduce the amount of solid substance. To avoid contamination and the side effects of chemical reagents, the collagen thermally crosslinked under sterile conditions. A suspension of the collagen fibers was enmeshed in the interstices of Dacron fibers of fabric prostheses, which were then thermally crosslinked at 130 degrees C for 20 h. The prostheses were porous when the collagen fiber network was dry. Under wet conditions, however, the water permeability of the grafts was reduced to 0.1 ml/min from the 1,250 ml/min of the original prostheses. Three weeks after implantation in the abdominal aortas of dogs, 81.2 +/- 11% of the luminal surface was macroscopically thrombus free, and 56 +/- 14% was endothelialized. More than 95% of the coated collagen had been absorbed. Numerous fibroblasts had migrated into the graft walls, and capillary blood vessels had infiltrated the inside of the graft walls without foreign body reaction. In the controls, thrombus free areas averaged 9.0 +/- 5%, and endothelialized areas averaged 5.2 +/- 4%. Many giant cells, plasma cells, and lymphocytes had migrated into the graft walls, but no fibroblasts. These results suggest that rapid endothelialization is possible when clean collagen is used.

Growth Regulation of Endothelial Cells by Tissue Fragments: Analysis on Mechanism of Rapid Endothelialization of Tissue‐Seeded Vascular Prosthesis in a Three‐Dimensional Culture System

Rapid endothelialization of the inner surface was reported in an autologous tissue-seeded vascular prosthesis. We applied a three-dimensional in vitro culture system to elucidate the precise mechanism of rapid endothelial coverage of a tissue-seeded vascular prosthesis. Human venous, omental, adipose and striated muscle tissue fragments were harvested from surgical specimens. They were embedded in collagen gel, and 2.0 x 10(5) bovine aortic endothelial cells (BAECs) were seeded on the gel. The number of BAECs was counted on Days 2 and 7. Growth rate of BAECs was facilitated on the collagen gel with omental and striated muscle tissue fragments (p < 0.05). Factor VIII-negative spindle cells migrated around tissue fragments, especially around the omental and striated muscle tissue fragments. Rapid endothelialization of a tissue-seeded vascular prosthesis may result from facilitation of EC proliferation by viable tissue fragments and migrated cells. These results confirm tissue fragments regulate EC growth, and are useful as bioengineering tools.

Enhanced Patency of Venous Dacron Grafts by Endothelial Cell Sodding

The effect of microvessel endothelial cell (EC) sodding on the patency of Dacron grafts interposed in canine inferior vena cava was studied. EC were harvested enzymatically from canine omentum and isolated by density gradient centrifugation. Preclotted, knitted Dacron grafts were sodded with > 10(6) EC/cm2 surface. The results demonstrate significant improvement in patency of sodded grafts placed in the inferior vena cava as compared with control grafts (p < 0.001 in grafts with a distal arteriovenous fistula and p < 0.05 in grafts without a distal fistula). The neointima of the sodded grafts were thinner and scanning electron microscopy confirmed the presence of a confluent layer of EC. In addition, the production of prostacyclin but not thromboxane A2 was significantly enhanced in the sodded grafts as compared with controls. We conclude that microvessel EC sodding of Dacron grafts significantly improves the patency rate and inhibits neointimal thickening of the prosthesis. The mechanism is unknown but may involve a more rapid endothelialization of the graft surface with the potential of producing more prostacyclin and less thromboxane A2.

Acceleration of neointima formation in vascular prostheses by transplantation of autologous venous tissue fragments: Application to small-diameter grafts

We have previously demonstrated rapid and complete endothelialization in synthetic fabric vascular prostheses that have been pretreated with autologous venous tissue fragments. However, significant thrombogenicity has been a major problem when this method has been applied to small-diameter grafts. By masking the positively charged collagen fibrils in the tissue fragments with negatively charged heparin, we were able to overcome this problem. A canine jugular vein was resected, minced into tissue fragments, and suspended. This mixture was sieved through the wall of a highly porous vascular prosthesis with a water porosity value of 4,000 ml/cm2 per minute by pressurized injection, which caused the tissue fragments to be trapped in the graft wall. Tissue-fragmented grafts (7 mm inside diameter, 5.7 cm long) were implanted into the thoracic aorta of 35 dogs. In addition, tissue-fragmented grafts of small diameter (4 mm inside diameter, 3.5 cm long) were pretreated with heparin and implanted into the carotid arteries of 16 dogs (32 grafts). Preclotted grafts without tissue fragmentation were implanted into the thoracic aorta (25 dogs) and carotid arteries (6 dogs, 12 grafts) as controls. Grafts were explanted from 1 to 495 days after implantation. New arterial wall formation was complete throughout the tissue-fragmented grafts within 2 weeks; however, in the control grafts, neointima formation was limited to the anastomotic sites even after 2 months. Twenty small-caliber tissue-fragmented grafts that were pretreated with heparin in the carotid position were patent, but all the control grafts were occluded within 1 week. These results demonstrate that neointima formation can be enhanced in synthetic fabric prostheses; furthermore, long-term patency of vascular grafts of small caliber is possible in dogs with this tissue-fragmentation technique.

Increased platelet-derived growth factor production and intimal thickening during healing of Dacron grafts in a canine model

Purpose: Growth factor production by endothelial cells on grafts may play a role in the development of intimal hyperplasia and subsequent graft failure.Methods: To study the relationship between platelet-derived growth factor production and graft healing, 26 beagles underwent placement of 20 cm long, 6 mm internal diameter, knitted Dacron thoracoabdominal grafts, either seeded with autologous endothelial cells (n = 14) or unseeded controls (n = 12). The grafts and adjacent arteries were removed 4 or 20 weeks after implantation for measurement of platelet-derived growth factor production in organ culture, endothelial cell coverage, and intimal thickness.Results: Midgraft platelet-derived growth factor production by seeded graft segments increased from 41 ± 6 to 148 ± 27 pg/cm2/72 hr (p < 0.002) between 4 and 20 weeks. This was accompanied by a significant increase in inner-capsule thickness. Platelet-derived growth factor production by control graft segments also increased from 58 ± 21 to 163 ± 42 pg (p < 0.05) and was similar to that of seeded grafts despite more rapid endothelialization of seeded grafts. The increase in growth factor production by Dacron grafts was greater than that of the expanded polytetrafluoroethylene grafts studied previously despite similar endothelial cell coverage.Conclusion: This increase corresponded with the rapid appearance of smooth muscle cells in the pseudointima of Dacron grafts, suggesting that these cells may be responsible for the observed increase in platelet-derived growth factor production.

Rapid endothelialization of vascular prostheses by seeding autologous venous tissue fragments

A method was developed to obtain rapid endothelialization of a fabric vascular prosthesis by seeding autologous venous tissue fragments into its wall. In an animal study, complete endothelialization was observed in the entire inner surface of the prosthesis within 2 weeks after implantation. A piece of peripheral vein was minced with scissors and then stirred into saline to create a tissue suspension. This suspension was enmeshed into the wall of a highly porous fabric vascular prosthesis by repeated pressurized injections with a syringe. The prostheses (7 mm inside diameter and 5.7 cm in length), seeded with tissue fragments, were implanted into the descending thoracic aorta of 25 dogs, and they were removed from 1 hour to 2 months after implantation. Twenty-five prostheses, preclotted with fresh blood, were used as control prostheses. In the seeded graft, a thin fibrin layer covered the inner surface just after implantation, but countless numbers of endothelial cells migrated from the fragments and came up to the luminal surface like multiple "mushrooms" under the fibrin layer. Smooth muscle cells made multiple layers underneath the endothelial cell layer. The healing proceeded equally at every part. By this active migration and proliferation, the inner surface was completely healed within 2 weeks.

Rapid endothelialization of mieroporous vascular prostheses covered with meshed vascular tissue: a preliminary report

The aim of our study was to induce rapid endothelialization of vascular prostheses which replace malfunctioning blood vessels. We developed a technique of meshing a section of autologous arterial or venous tissue, wrapping it around a porous vascular prosthesis and fixing it with a running suture. This combination was used to replace a 1 cm segment of rat abdominal aorta. The implanted specimens were harvested after 8,16, 24 or 135 d and evaluated for cellular ingrowth and endothelial coverage with light and scanning electron microscopy. All grafts were patent when harvested. Cells of the meshed tissue were observed to migrate through the pores in the prosthetic wall to cover the luminal surface. The presence of meshed vascular tissue around the prosthesis resulted in complete endothelialization within 24 d and sometimes within 8 d after implantation. This endothelial layer is still present after 135 d. In control prostheses without meshed vascular tissue, complete endothelialization takes at least 42 d. We conclude that the application of meshed vascular tissue around porous vascular prostheses results in patent prostheses with a rapidly formed and long-term surviving endothelium layer.

New techniques of analyzing the healing process of artificial vascular grafts, transmural vascularization, and endothelialization.

Beside traditional techniques (light, immunofluorescence, transmission electron (TEM)- and scanning electron microscopy (SEM), a new more sensitive method for producing microcorrosion casts using a polyester-based resin has been developed. The SEM analyses of the microcorrosion casts of alloplastic vascular grafts was realized in an absolutely stable stage of polymerization. For the first time, a transprosthetic vascularization could be shown in great three-dimensional detail. The importance of a complete and rapid endothelialization of artificial vascular grafts is discussed.

Healing and Long-Term Viability of Grafts in the Venae Cavae Reconstruction

The need for superior vena cava (SVC) or inferior vena cava (IVC) recon struction is not uncommon: lung cancer, mediastinal tumors, or retroperitoneal neoplasms are the most frequent indications for caval replacement. Since auto genous veins, which represent the most satisfactory venous substitute, have not been applicable to caval replacement, because of the lack of suitable length and caliber, many types of materials have been tested in the venous system. The present study was planned to compare the potential of expanded polytetrafluoroethylene (e-PTFE) and glutaraldehyde-tanned human umbilical vein (HUV) grafts as caval substitutes. Patch (4 x 5 cm) reconstruction of the SVC was carried out in 10 dogs, tubu lar (3 x 10 mm) reconstruction of the SVC in 5 dogs, and patch (1 x 2 cm) recon struction of the IVC in 8 dogs. In the patch reconstruction groups, HUV grafts were used in half of the cases and 30 μm pore sizes e-PTFE grafts in the other half, while in the tubular SVC replacement group, only e-PTFE grafts were employed. The grafts were removed fifteen to thirty days after IVC patch re construction, thirty to two hundred seventy days after SVC patch reconstruc tion, and thirty-three to forty-one months after SVC tubular replacement. In every instance, specimens were obtained for light microscopy (LM) and scan ning electron microscopy (SEM). HUV patches implanted in the SVC showed hemorrhagic foci at their central portion and marked constriction at the anastomotic regions. On the contrary, the inner surface of e-PTFE patch grafts was covered with a uniform and glis tening neointima. By LM, e-PTFE patches showed a smooth and regular lumi nal surface, while the inner surface of HUV patches was irregular, with hemorrhagic areas in the underlying layers. By SEM, e-PTFE patches revealed a more rapid and orderly endothelialization of their inner surface than HUV patches did. Tubular SVC reconstruction showed the good long-term viability of the e-PTFE neointima (as long as forty-one months after grafting). Only spotty areas revealed neointima alterations, such as fibrinoid degeneration, fo cal necrosis, or minor thrombotic foci. In the IVC patch reconstruction model, HUV grafts showed an exuberant fibrin deposition on their inner surface and delayed endothelialization patterns, as compared with e-PTFE grafts. Since rapid healing with rapid endothelialization is accepted as the most important factor providing the graft luminal surface with the best antithrombotic proper ties, the present study demonstrated that e-PTFE is a more suitable material than HUV for SVC and IVC reconstruction.

Balloon-expandable intracoronary stents in the adult dog.

We studied the acute and chronic biological reaction to balloon-expandable intracoronary stents in the adult dog. Twenty stainless steel stents were placed, by standard angioplasty techniques, into the left anterior descending, left main, or circumflex coronary arteries of 20 dogs. Angiography was performed at 1, 3, 6, and 12 months and animals were killed in groups of three at 1, 3, 8, and 32 weeks, for gross, light, and electronmicroscopic analysis. All dogs survived, all stents were patent, and there was no evidence of myocardial infarction, spasm, rupture, or aneurysm formation during follow-up (longest 18 months; average, 12 months). The stent was initially covered by a thin layer of thrombus that was replaced later by neointimal muscular proliferation that reached its maximal thickness by 8 weeks (p less than .01). This neointima gradually thinned as it became more sclerotic and less cellular. The stents were covered completely by immature endothelium by 1 week without loss of side branches. We conclude that balloon-expandable intraluminal stents can be safely placed percutaneously into normal canine coronary arteries. Because of rapid endothelialization high patency rates can be expected, thus offering promise for clinical applications in man.

Thromboresistant properties of hydrophilic gels

Hydrophilic gels were made using methyl methacrylate and N-vinylpyrrolidon. Simple designs of hydrophilic gels (films, tubes) for the in vitro studying of thromboresistant properties were manufactured. On the base of measurements of blood clotting time, concentration of prothrombin complex factors fibrinogen, heparin, thromboelastography, and thrombocyte adhesion onto hydrogel surface in blood contact; it was demonstrated that a hydrophilic gel consisting of 86 mol% of methyl methacrylate, 14 mol% of N-vinylpyrrolidon and 0.652 mol% of hydrolysed N-vinylpyrrolidon has optimum thromboresistant properties. Thromboresistant was studied in the dogs during 44 operations, in which both the aorta and peripheral vessels replaced by polypropylene prostheses or by Lavsan, modified by the addition of hydrophilic gels, were applied. It was shown that hydrophilic gels decrease thrombogenesis and promote rapid endothelialization and periprosthetic tissue vascularization.