Novel Vascular Graft Grown Within Recipient’s Own Peritoneal Cavity
Abstract—A method by which to overcome the clinical symptoms of atherosclerosis is the insertion of a graft to bypass an artery blocked or impeded by plaque. However, there may be insufficient autologous mammary artery for multiple or repeat bypass, saphenous vein may have varicose degenerative alterations that can lead to aneurysm in high-pressure sites, and small-caliber synthetic grafts are prone to thrombus induction and occlusion. Therefore, the aim of the present study was to develop an artificial blood conduit of any required length and diameter from the cells of the host for autologous transplantation. Silastic tubing, of variable length and diameter, was inserted into the peritoneal cavity of rats or rabbits. By 2 weeks, it had become covered by several layers of myofibroblasts, collagen matrix, and a single layer of mesothelium. The Silastic tubing was removed from the harvested implants, and the tube of living tissue was everted such that it now resembled a blood vessel with an inner lining of nonthrombotic mesothelial cells (the “intima”), with a “media” of smooth muscle–like cells (myofibroblasts), collagen, and elastin, and with an outer collagenous “adventitia.” The tube of tissue (10 to 20 mm long) was successfully grafted by end-to-end anastomoses into the severed carotid artery or abdominal aorta of the same animal in which they were grown. The transplant remained patent for at least 4 months and developed structures resembling elastic lamellae. The myofibroblasts gained a higher volume fraction of myofilaments and became responsive to contractile agonists, similar to the vessel into which they had been grafted. It is suggested that these nonthrombogenic tubes of living tissue, grown in the peritoneal cavity of the host, may be developed as autologous coronary artery bypass grafts or as arteriovenous access fistulae for hemodialysis patients.
Atherosclerosis is the principal cause of coronary occlusion, stroke, aortic aneurysm, and gangrene of the extremities and, as a single disease, is the major cause of mortality and morbidity in the United States, Europe, and other western nations. Atherosclerotic lesions are the result of an inflammatory response and damage of the arterial wall complicated by excessive lipid deposition.1 A method by which to overcome the clinical symptoms of atherosclerosis is the insertion of bypass grafts around an artery blocked or impeded by plaques. The most common vascular graft material in use today is saphenous vein or mammary artery from the patient him/herself (autografts). Both are flexible, viable, nonthrombogenic, and compatible. However, although the mammary artery seldom develops atherosclerosis, it may not always be the proper size or length, and saphenous vein may have varicose degenerative alterations that can lead to aneurysm formation when transplanted to a high-pressure arterial site. Furthermore, the nonthrombogenic surface of endothelial cells of saphenous veins is often damaged during graft preparation. Venous and arterial allografts have also been tried but have generally been abandoned clinically because they show a high incidence of rejection, deterioration, and complications. Similarly, the use of dialdehyde starch–tanned bovine xenografts has been generally abandoned because of a high incidence of aneurysm formation and poor resistance to infection.
For these reasons and because autologous grafts are not always available, synthetic vascular prostheses such as Dacron fabric grafts and expanded polytetrafluoroethylene have been developed. Although they perform reasonably satisfactorily in high-flow low-resistance conditions, these materials are not suitable for small-caliber arterial reconstructions because they are foreign bodies and because blood coagulation can occur on the luminal surface, resulting in occlusion. One innovation designed to improve their patency is the coating of the lumen of the graft with endothelial cells.2 Although flow through the graft is improved and thrombogenesis is reduced, graft failure due to occlusion by cell overgrowth can still occur. Gene therapy has been used to address this overgrowth, but retrovirally transduced cells on the graft are not able to withstand the stresses encountered by the flow of blood and are sheared off. Also, the procedure for obtaining endothelial cells from the patient is invasive, and the cells are hard to propagate in vitro. Attempts have been made to use skin granulation tissue as a vascular graft (the Sparks mandril prosthesis3 ). This process involves the placement of a plastic/glass rod under the skin. With time, cylindrical granulation tissue is formed, the rod is removed, and the tissue is tanned to remove all cellular components, leaving only a skeletonized collagen framework. However, findings of a low bursting strength led to the addition of external and internal synthetic grafts (such as Dacron) to the skeletonized tube, but these grafts were still found to have high rates of complication and low percentages of patency,4 and their usage is limited. Therefore, despite considerable experimental and clinical research, none of the biological and synthetic grafts produced thus far is an ideal substitute for an artery.
We describe here a “designer” artery, produced in the peritoneal cavity from cells of the same animal into which it is grafted. These arterial substitutes are composed of living myofibroblasts and the matrix they have produced, and they are antigenically acceptable and strong, respond to agonists and antagonists similar to blood vessels of the host, and are lined by living, nonthrombogenic, mesothelial (endothelium-like) cells.
Materials and Methods
Tubing Implantation and Harvest and Autologous Tissue Transplantation
Thirty male adult Wistar rats (250 to 350 g) and 20 New Zealand White rabbits (aged 3 to 4 months) were anesthetized with 2.5% halothane (Fluothane; Laser Animal Health, Queensland, Australia) (vol/vol O2). A small incision was made in the shaved abdominal wall, and four 10-mm (for rat) or four 20-mm (for rabbit) lengths of Silastic tubing with outer diameter of 3 mm (rat) and 5 mm (rabbit) were placed inside the peritoneal cavity of each animal.
Two weeks after insertion of the tubing, the rats were premedicated with atropine (0.25 mg/kg body wt administered intraperitoneally) and anesthetized with 1% halothane. The 4 implants were harvested from the peritoneal cavity of each rat, and any implants that had adhered to the wall or bowel were discarded. Of the free-floating implants, the one with the thickest capsule was selected for autologous transplantation, and the others were used for histology or Western analysis (see below). With the aid of an operating microscope, 2 vascular clamps were placed on the area above and below the transplant site in the abdominal aorta, which was then resected. The elastic recoil left a gap of 5 to 10 mm between the cut ends. The Silastic tubing was removed from the implant selected for autologous transplantation, and at the same time, the tissue capsule was gently everted such that the mesothelial layer now lined its inside surface (Figure 1⇓). The tube of living tissue was then trimmed and aligned with the gap ready for suturing. Two stay sutures (9-0 silk) were placed at each anastomosis to orient the graft and the artery and to facilitate the placing of other sutures. The mid anastomotic site was sutured with 10-0 (22-μm) Ethilon suture material and with round and nontraumatic needles (Ethicon, Inc). Suturing was first performed at the distal anastomosis, followed by the proximal anastomosis. A total of 8 interrupted sutures were placed at each end: one each was placed at the dorsal, ventral, medial, and lateral aspect of the anastomosis, and 4 more (Ethilon 9-0) interrupted sutures were then placed to fill the intervals between them. The use of stay sutures was important, because they prevent accidental suturing of the front and back wall of the same vessel. The grafts were not preclotted, nor were heparin or spasmolytics administered.
Similarly, 2 weeks after insertion of 4 pieces of Silastic tubing into their peritoneal cavities, 20 rabbits were preanesthetized with 1 mL Saffan (IV, Gloxovet) injected into their marginal ear veins, and continuous anesthesia was achieved with 2.5% halothane. The 4 grafts were harvested from each animal, the thickest free-floating capsule was set aside for autologous transplantation into the right carotid artery, and the others were fixed for histology. To expose the right carotid artery, a midline incision (approximately in line with the trachea) was made, the surrounding connective tissue was bluntly dissected, and the submandibular glands were clamped and retracted to one side. The transplantation procedure was similar to that in the rat, except a 10-mm segment of artery was removed. After elastic recoil, this left a gap of ≈20 mm between the cut ends into which the trimmed everted tube of tissue (with Silastic tubing discarded) was sutured.
In both rats and rabbits, after suturing was complete, the distal clamp was released to allow the graft to fill with blood under low pressure, and then the proximal clamp was released to allow blood flow under full arterial pressure through the graft. Light external pressure with Gelfoam (Upjohn, New South Wales, Australia) sealant was required at the anastomoses to control initial leakage. Hemostasis was achieved by ≈2 to 3 minutes after removal of the clamps, but the graft was continuously monitored for 10 to 14 minutes in case of secondary bleeding. Patency was determined by direct inspection. The wound was irrigated with saline solution and closed with Dexon (Davis & Geck) 4-0 sutures. The animals had free access to standard food and water. A graft was deemed successful at the time of operation if it was fully dilated and pulsating and if a femoral pulse was present. Unsuccessful grafts were limp and flaccid, with no detectable pulse.
The 30 rats were randomly divided into 5 groups (n=6 per group) and euthanized at 1, 1.5, 2, 3, and 4 months after transplantation. The 20 rabbits were divided randomly into 4 groups (n=5 per group) and euthanized at 1, 2, 3, and 4 months after transplantation.
Free-floating implants from the rat and rabbit not used for autologous transplantation were processed for light microscopy, immunohistochemistry,5 and transmission electron microscopy.6 In addition, fresh implants from the rat (n=6) were used for organ bath experiments (see below). Total protein was extracted from another 6 rat implants and subjected to Western blot analysis7 using antibodies to cytoskeletal markers and contractile filaments. In both cases, rat aorta was used as a control. Antibodies used for Western blotting were α-smooth muscle actin (1-A4, mouse IgG2a, 42 kDa, Sigma Chemical Co), β-actin (AC-74, 44 kDa, Sigma), smooth muscle myosin heavy chain (mouse IgG1, hSM-V, 204 and 202 kDa, Sigma), vimentin (V9, mouse Ig G2a, 57 kDa, Sigma), and desmin (D33, mouse IgG1, 250 kDa, Dako), as well as collagen I (C-1926, Sigma), collagen IV (epitopes α1 and α2, Sigma), elastin (Sigma), and ED1 for macrophages (Dako). Densitometric analysis was performed on the bands by use of image analysis software (Mocha, Jandel Scientific), and values for each implant protein were expressed as a percentage of that expressed in the aorta. For light microscopy, sections were stained with hematoxylin and eosin and with both Weigert’s and Hart’s stains for elastin. For immunohistochemistry, sections were stained with antibodies to α-smooth muscle actin, smooth muscle myosin heavy chain, and von Willebrand factor (Serotec) by use of the biotin/streptavidin system. Negative controls were obtained by omitting the primary antibody or staining with an irrelevant monoclonal antibody of the same isotype. The volume fraction of myofilaments (Vvmyo) of cells of the “media” was determined as previously described.6
Rats and rabbits at 1, 2, 3, and 4 months after transplantation were euthanized with pentobarbital (Lethobarb, Provet Supplies) and perfusion-fixed. Wall thickness of the transplants was measured, and cell density was calculated by image analysis (modification of the method of Kleinert et al2 ). Sections were taken for light microscopy, electron microscopy, and immunohistochemistry of the tissue before transplantation. Transplants taken from the rat group killed at 1.5 months were taken for standard organ bath experiments to determine the response of the tissue to contracting and relaxing agents and were compared with 2-week implants and normal rat aorta.
All statistical analyses were performed by use of the statistical software package SIGMASTAT (Jandel Scientific).
Formation of a Granulation Tissue Tube in the Peritoneal Cavity
In both the rat and rabbit, an average of 2 of 4 pieces of Silastic tubing implanted in the peritoneal cavity remained free-floating and had become completely encased in a capsule of granulation tissue by 2 weeks (Figure 2a⇓). However, in 2 rats and 1 rabbit, all 4 harvested tubes had adhesions. These were removed, discarded, and replaced by fresh tubing. After 2 weeks, 3 of the 4 new implants in each of the 3 animals remained free-floating and by 2 weeks had developed a capsule of granulation tissue. The tissue was removed from the Silastic tubing of all free-floating implants by trimming one end and then pealing it back over the tubing, a procedure similar to taking off a sock (see Figure 1⇑). This caused little or no damage and, at the same time, everted the tube of living tissue. The Silastic tubing functioned only as an irritant and molding and was now discarded.
Light microscopy of the capsule from both the rat and rabbit showed that close to where the Silastic tubing had been was a layer of connective tissue covered by several layers of cell-rich granulation tissue (Figure 2b⇑). There was concentric layering of collagen bundles, and the spindle-shaped cells contained large amounts of synthetic organelles and abundant focal/dense bodies in myofilament bundles (Figure 2c⇑ and 2d⇑). Vvmyo in these cells of the rat tube was 35.7±1.6% compared with 63.7±5.7% (P<0.05) for smooth muscle cells in the aorta of the same animals (Table 1⇓). Macrophages, readily distinguished by their irregular shape and high vesicle content, were commonly seen. The inside lining of the everted tubes consisted of a single layer of cells that stained positively for von Willebrand Factor,8 and transmission electron microscopy confirmed their identification as mesothelial cells (Figure 2c⇑). The hollow tubes of living tissue thus resembled normal blood vessels, with an inner “endothelium” (mesothelium in this case), “media” of myofibroblasts, and a thin outer “adventitia” of connective tissue.
Western analysis showed that the rat granulation tissue tube contained a similar content of α-smooth muscle and desmin as the rat aorta, with less smooth muscle myosin heavy chain and much greater levels of β-actin and vimentin (Table 2⇓). The levels of collagen types I and IV were similar to levels in the aorta, but elastin levels were low.
Granulation Tissue Tube as Autologous Vascular Graft (Artificial Artery)
The potential usefulness of the granulation tissue–derived tube as vascular graft material was demonstrated in both the rat and rabbit. The rabbit had a 10-mm segment of its right carotid artery removed and replaced with 20 mm of everted granulation tissue lined with mesothelial cells grown for 2 weeks in the peritoneal cavity of the same animal. The rat had its abdominal aorta cut, but no segment removed, and 10 mm of autologous granulation tissue inserted at the cut ends. After 1, 2, 3, and 4 months after implant, the presence or absence of a pulse through the transplant was evaluated, then the proximal site of the vessel was clamped, and the graft was perfusion-fixed followed by postdrop fixation.
One month after transplantation, grafts in all 6 rats of group 1 had a pulse and were patent. At 2 months, 4 of 6 grafts were patent, and at 3 and 4 months, 3 of 6 grafts were patent, giving an overall patency rate of 67%. None of the grafts had been preclotted, nor had heparin or spasmolytics been administered to the animals at the time of transplantation because we wished to test the antithrombotic benefits of the mesothelial lining. Nonpatent grafts had their lumens blocked by incorporated thrombi and by α-smooth muscle actin–stained cells. Signs of recanalization were sometimes seen. The patent rat grafts possessed a normal intact wall and a strong pulse. Mesothelium (or migrated endothelium), which stained for von Willebrand factor,8 constituted the inner lining. The average wall thickness of the graft increased from 0.18±0.02 mm (pretransplant) to 0.25±0.02 mm by 1 month, after which no further change was observed. No significant (P<0.1) increase in cell number was evident in the “media,” with the increase in graft thickness being due to the large amount of extracellular matrix, mainly collagen, that developed on the outer surface of the wall (Figure 3a⇓). This “adventitia” contained vasa vasorum as seen with antibodies to α-smooth muscle actin (Figure 3b⇓). Similar results were found for rabbit granulation tissue tubes grafted into the carotid artery, with an overall patency rate of 70% (14 of 20 at the different time points).
The cells within the wall of the grafts in both the rat and rabbit stained intensely for α-smooth muscle actin (Figure 3b⇑) and smooth muscle myosin (Figure 3c⇑). By 3 months, the Vvmyo of the cells in the rat transplant had increased to 58.7±1.4%, which was not significantly different from smooth muscle cells in the rat aorta near to the transplant site (Table 1⇑). Structures that resembled elastic lamellae and stained with both Hart’s and Weigert’s elastic stain began to appear in both rat and rabbit grafts by 1 month; at first they were found only near the lumen and then throughout the “media.”
To determine whether the graft responded to contractile and relaxing agents, organ bath experiments were carried out on grafts transplanted to the rat aorta for 6 weeks (n=6); these grafts were compared with rings of aorta from the same animal. First, 10−1 mol/L KCl was administered to determine whether the transplanted graft had any contractile activity. Rings from the aorta had an increase in contraction of 16 mN, whereas the transplanted graft contracted to 3 mN (Figure 4a⇓). Both contracted tissues relaxed in response to (10−5 mol/L) acetylcholine. Both tissues were then exposed to the contractile agonists 5-hydroxytryptamine (10−9 to 10−5 mol/L) and phenylephrine (10−9 to 10−4 mol/L). The transplant showed no contractile response to 5-hydroxytryptamine; however, it did respond to phenylephrine, with a contractile response of 1.5 mN at 10−5 mol/L compared with a response of 15.5 mN exhibited by the aorta at the same concentration (Figure 4b⇓). Before grafting into the host artery, the tubes of granulation at 2 weeks had no response to contractile or relaxing agents (n=6).
It has previously been observed that small foreign bodies (such as plastic disk, boiled liver, agar, gelatin, egg white, filter membranes, and boiled blood clot) introduced into the peritoneal cavity of the rat, rabbit, or mouse initiate an inflammatory response, with the resultant granulation tissue covered by a layer of mesothelium.9 10 11 We speculated that if a tube of tissue could be grown and then freed from the molding and everted, this process might be used to develop an autologous vascular substitute.
Indeed, it was shown that tubes of granulation tissue can be grown within an animal’s own peritoneal cavity by using moldings of Silastic tubing of different lengths and diameters. These tubes of tissue possess a lining of mesothelial cells and a living contractile wall composed of myofibroblasts with tensile strength provided by collagen type I. The mesothelial layer is extremely important because it possesses fibrinolytic and anticoagulant activity,12 and synthetic grafts seeded with mesothelium are known to have a high patency rate similar to grafts seeded with endothelium.13
We also showed that the artificial artery can be used successfully as an autologous arterial transplant and remain patent for at least 4 months, with constituent cells becoming more smooth muscle—like, with a Vvmyo similar to smooth muscle cells of the adjacent arterial wall. By 6 weeks after transplantation, the cells have begun to respond to the contractile agents phenylephrine and KCl and to relax in response to acetylcholine. However, we have not yet determined whether all the cells in the transplant come from the original myofibroblasts or whether some are derived from the transmural ingrowth of smooth muscle cells or “fallout” hemopoietic cells, as has been reported for vascular and synthetic grafts.14 Also, it is not known whether the mesothelium is sloughed off the grafts and replaced by local endothelium after their transplantation to high-pressure (rat abdominal aorta and rabbit carotid) arterial sites. The source of the lining cells will be determined in future experiments by staining sections with endothelium-specific antibodies (ED31) throughout the length of the transplants over time. Because the patency of the transplants reported in the present study was high, even though they were not preclotted nor had heparin or spasmolytics been administered to the animals, the source of the lining cells was not considered essential information for this initial study.
After the present study was complete, a report15 appeared in Science (April) describing the in vitro development of vascular grafts from cells isolated from biopsied pig carotid artery or bovine aorta grown for 8 weeks on pulsed or nonpulsed biodegradable polymer matrices. The engineered grafts responded to contractile agonists at a magnitude of ≈5% of control rabbit abdominal aorta. One pig had a pulsed xenograft transplanted into the right saphenous artery; 3 others had autologous grafts (1 pulsed and 2 nonpulsed) into the same site. The pulsed grafts remained patent for ≈4 weeks, whereas the nonpulsed grafts thrombosed after 3 weeks. There are several advantages of our grafts compared with those described above: our grafts are developed entirely in vivo over only 2 weeks by the donor/host themselves; they need no artificial mesh as part of the wall; and they develop elastic lamellae and have a demonstrated patency for at least 4 months. Their grafts before transplantation had contractile responses of ≈5% of control artery, whereas our tissue at 6 weeks after transplantation had a 10% to 20% response.
Although our results are preliminary and more extensive experiments are required before such grafts can be used in humans, we believe that this new type of graft material may open new perspectives in the field of arterial reconstructive surgery. These grafts may be developed to replace or bypass pieces of diseased artery for coronary artery bypass, thus avoiding the removal of saphenous vein (which is often varicose in the elderly) or mammary artery for this purpose. They may also be useful as readily replaceable arteriovenous access fistulae for hemodialysis patients. The grafts are biocompatible, have a nonthrombogenic surface, develop elastic fibers, exhibit a pulse, and contract and relax in response to exogenous agents, making them superior to synthetic grafts. Because they are grown inside the subject’s own body, there is no tissue rejection and limited graft complication. By altering the diameter and/or length of tubing used as a molding, grafts of different sizes can be produced. The procedure also allows for multiple or repeat grafting, because several tubes of tissue can be grown in the peritoneal cavity at the same or a later time.
This study was supported in part by a grant from the National Health and Medical Research Council of Australia and by a postgraduate award (to Dr Efendy) from The University of Queensland. This study is the subject of Australian Provisional Patent Application No. PP5422/98, filed August 21, 1998, with extra data added December 22, 1998 (PP7859/98). We thank Dr Lindsay Brown for his expertise with the organ bath experiments and Anita Thomas, Steve Stamatiou, Nathalie Worth, and Nicole Smith for technical assistance.
- Received May 3, 1999.
- Accepted September 8, 1999.
- © 1999 American Heart Association, Inc.
Rolfe BE, Campbell JH, Smith NJ, Cheong MW, Campbell GR. T-lymphocytes affect smooth muscle cell biology. Arterioscler Thromb. 1995;15:1204–1210.
Manderson JA, Campbell GR. Venous response to endothelial denudation. J Pathol. 1986;18:77–87.
Mosse PRL, Campbell GR, Ryan GB. A comparison of the avascular capsule surrounding free floating intraperitoneal blood clots in mice and rabbits. J Pathol. 1985;17:401–407.
Verhagen HJ, Heijnen-Snyder GJ, Pronk A, Vroom TM, van-Vroonhoven TJ, Eikelboom BL, Sixma JJ, de Groot PG. Thrombomodulin activity on mesothelial cells: perspectives for mesothelial cells as an alternative for endothelial cells for cell seeding on vascular grafts. Br J Haematol. 1996;95:542–549.
Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, Langer R. Functional arteries grown in vitro. Science. 1999;284:489–493.