Ann Thorac Surg 2007;83:517-525
© 2007 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Novel Bioengineered Small Caliber Vascular Graft With Excellent One-Month Patency
Yosuke Ishii, MDa,
Russell T. Kronengold, PhDb,
Renu Virmani, MDc,
Elias A. Rivera, MHSc,
Scott M. Goldman, MSb,
Ericka J. Prechtel, MSb,
Richard B. Schuessler, PhDa,
Ralph J. Damiano, Jr, MDa,*
a Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri
b Kensey Nash Corp, Exton, Pennsylvania
c American Registry of Pathology, Washington, DC
Accepted for publication September 7, 2006.
* Address correspondence to Dr Damiano, Suite 3108, Queeny Tower, Barnes-Jewish Hospital Plaza, St. Louis, MO 63110 (Email: damianor{at}wustl.edu).
Presented at the Poster Session of the Forty-first Annual Meeting of The Society of Thoracic Surgeons, Tampa, FL, Jan 24–26, 2005.
| Dr Kronengold and Mr Goldman disclose that they have a financial relationship with Kensey Nash Corp.
|
 |
Abstract
|
|---|
BACKGROUND: A bioengineered microporous polycarbonate-siloxane polyurethane graft has been developed for coronary artery bypass grafting. Biological agents can be impregnated into its absorbable collagen and hyaluronan microstructure and stable macrostructure to promote patency. The objective of this study was to examine the biological performance and biomechanical characteristics of this graft.
METHODS: Heparin-sirolimus (HS) or heparin-sirolimus–vascular endothealial growth factor (HSV) grafts were manufactured for this study. Heparin (40 U) was embedded in the microstructure of the graft for early elution from the graft wall. Heparin (100 U) and sirolimus (450 µg) were incorporated into the macrostructure of the graft for late elution. Vascular endothelial growth factor was also embedded in the microstructure of the graft. Both grafts (3.6 mm internal diameter, 24 mm length) were implanted into the abdominal aortas of rabbits (n = 36) to compare with heparin-alone (H) grafts (n = 9). At 4 hours, 1 day, and 1, 2, and 4 weeks after surgery, the grafts were removed for histologic, immunohistochemical, and biomechanical evaluations.
RESULTS: The patency rate of all grafts was 100% at each time point. None of grafts had stenosis after surgery. Endothelial cells were observed at 4 weeks after surgery in the HS, HSV, and H grafts. Although there was no significant difference of neointima thickness among the HS, HSV, and H grafts (136 ± 75, 93 ± 64, and 125 ± 90 µm; p = 0.08), the H grafts did have more cellular infiltration in the graft than the HS or HSV grafts. There was neocapillary formation inside the graft wall at 4 weeks in all grafts. The graft macrostructure was unchanged based on biomechanical evaluation 4 weeks after surgery.
CONCLUSIONS: A unique drug-eluting graft had excellent patency at 1 month and may encourage luminal endothelialization without excessive intimal hyperplasia. Although vascular endothelial growth factor did not improve intimal formation, cell infiltration, or vascularization, sirolimus might inhibit cell proliferation. Further long-term study would need to evaluate the efficacy of impregnated sirolimus.
Coronary artery bypass grafting (CABG) is now performed routinely around the world. The autologous internal mammary arteries, radial artery, and saphenous vein are the most widely used conduits for coronary revascularization. These autologous grafts provide excellent mechanical stability and their intimae have a natural antithrombogenicity [1, 2]. However, they require surgical harvest procedures, are of variable quality and size, and are limited by availability in some cases. Saphenous vein conduits have the added disadvantage of relatively poor long-term patency [3]. There would be several significant advantages if an effective synthetic graft could be developed. It would have unlimited availability, and consistent quality and patency. Moreover, the biomechanical uniformity of a synthetic graft may allow for the development of effective anastomotic devices for minimally invasive surgery.
Unfortunately, satisfactory synthetic materials have not been developed in a size appropriate for CABG. Synthetic materials such as Dacron (C. R. Bard, Haverhill, Pennsylvania) or expanded polytetrafluoroethylene (ePTFE) have been used successfully in peripheral revascularization but failed in coronary revascularization [4]. Dacron grafts lead to thrombosis and neointimal thickening in low blood flow. The ePTFE grafts also fail owing to surface thrombogenicity for small vessels [5]. Endothelial cell seeded grafts might be more effective for anticoagulation compared with nonseeded grafts [6, 7]. However, the manufacturing process is complex, time consuming, and costly. Planned surgery is required for this graft, but CABG is usually performed emergently, without such lengthy planning time.
A synthetic small caliber graft should be resistant to thrombosis and biocompatible, resembling a native artery. The graft should have excellent biomechanical stability, and be able to withstand the long-term hemodynamic stress of the arterial circulation. Suturability and handling are also important factors in minimizing operative time and risk.
Our group has developed a microporous polycarbonate-siloxane polyurethane graft that incorporates soluble collagen and hyaluronic acid into the pores. Biological agents can be impregnated into the stable macrostructure and absorbable microstructure of the graft, creating a two-tiered drug-release system to promote patency. The objective of this study was to examine the biological performance and biomechanical characteristics of this new graft.
|
Material and Methods
|
|---|
Graft Materials
Test samples consisted of a small-caliber vascular graft with an internal diameter of 3.6 mm and a length of 24 mm. A porous tube consisting of polycarbonate-siloxane polyurethane was manufactured using proprietary PTM foaming technology (Kensey Nash Corporation, Exton, Pennsylvania). This process created a tube with a large distribution of interconnected pores, averaging approximately 25 µm in diameter (Fig 1). The "macrostructure" of this graft was designed to provide biomechanical properties comparable to native arteries, and allow for cellular in-growth while producing minimal tissue inflammatory response. The macrostructure also functioned as a vehicle for delivery of biological agents, and proved amenable to easy handling and suturing. Incorporated into the pores of the polyurethane macrostructure was a "microstructure" consisting of hyaluronan (LifeCore Biomedical, Chaska, Minnesota) and bovine hide-derived acid-soluble collagen (Kensey Nash Corporation). The microstructure was intended to create a cell-friendly environment to encourage cell attachment and proliferation, prevent leakage through the porous macrostructure, and provide a secondary vehicle for the delivery of biological agents. Biological agents can be incorporated into the vascular grafts during manufacturing with a goal of preventing short- and long-term thrombosis as well as chronic smooth muscle cell hyperplasia.
View larger version (54K):
[in this window]
[in a new window]
|
Fig 1. The synthetic small caliber graft with a bioengineered microporous matrix that did not contain cells and required no preclotting.
|
|
In this study, three types of graft were manufactured: heparin-alone (H) grafts; heparin and sirolimus (HS) grafts; and heparin, sirolimus and vascular endothelial growth factor (VEGF [HSV]) grafts. All grafts were impregnated with 40 U heparin (Celsus Laboratories, Cincinnati, Ohio) in the microstructure for early elution to prevent acute graft thrombosis and 100 U heparin in the macrostructure for prevention of late thrombosis. The HS graft was impregnated with 450 µg sirolimus in the macrostructure for prolonged elution to discourage late thrombosis and inhibit intimal hyperplasia. The HSV grafts also incorporated 450 µg sirolimus in the macrostructure, and 650 ng VEGF (VEGF165; Peprotech, Rocky Hill, New Jersey) within the microstructure to encourage endothelialization of the graft. All grafts were terminally sterilized by exposure to gamma radiation at a minimum dose of 25 kGy. The drug delivery profile of heparin in the graft was assessed in vitro by exposing the grafts to phosphate buffer solution at 37°C and 100 revolutions per minute (rpm) in an incubator-shaker over 40 days. Heparin was quantified using a dimethylmethylene blue assay (DMMB) to determine eluted concentration in solution spectrophotometrically at a wavelength of 525 nm.
Surgical Procedure
All animals received humane care in compliance with the "Principles of Laboratory Animal Care," formulated by the National Society for Medical Research, and the "Guide for the Care and Use of Laboratory Animals," prepared by the National Academy of Science and published by the National Institutes of Health (NIH Publication 86-23, revised 1985). In addition, the Animal Studies Committee of the Washington University School of Medicine approved this study protocol.
Forty-five New Zealand White rabbits weighing between 3 and 4 kg were used randomly in this study. All animals were anesthetized with ketamine (70 mg/kg) intramuscularly, intubated with a 3-mm cuffed endotracheal tube, and mechanically ventilated with a pressure-controlled ventilator. An adequate level of anesthesia was maintained by inhaled isoflurane (1% to 3%). A limb-lead electrocardiogram was monitored. A central ear artery catheter was inserted to monitor systemic arterial pressure continuously. Arterial blood samples were drawn every 30 minutes to determine arterial oxygen tension, acid-base balance, and electrolyte levels. Ringer’s lactate solution was infused continuously, and sodium bicarbonate, potassium chloride, and calcium chloride were supplemented to maintain pH and electrolytes within normal values. Enrofloxacin (5 mg/kg) was administrated preoperatively to reduce the risk of infection.
After a midline abdominal incision, the intestines were displaced to the right side and covered with moistened gauze. The infrarenal aorta was carefully dissected from the surrounding tissue. The lumbar arterial branches were spared to avoid spinal cord ischemia. Intravenous heparin (200 U/kg) was administered. The abdominal aorta was clamped by microapproximator clamps between the lumbar branches and transected. The H, HS, or HSV grafts were anastomosed to the aorta in an end-to-end fashion with a continuous 7-0 polypropylene suture. The total anastomosis time was less than 30 minutes (22 ± 3) in every animal. Blood flow was measured with an ultrasonic flow probe (Transonic System, Ithaca, New York) proximally and distally. The abdominal incision was closed. The animals received analgesia (buprenorphine 0.3 to 0.5 mg/kg) and antibiotic (enrofloxacin 5 mg/kg) treatments subcutaneously twice daily for 2 days postoperatively. Postoperative antiplatelet therapy (aspirin 15 mg/kg) was administered daily.
At 4 hours, 1 day, and 1, 2, and 4 weeks after surgery (n = 3, 3, 3, 4, and 5, respectively, for HS and HSV grafts), the animals were anesthetized again with intramuscular ketamine (70 mg/kg). The abdominal incision was reopened. The surgical site was examined for adhesions, fibrosis, postoperative bleeding or hematoma, and aortovenous fistula. The blood flow at the proximal and distal anastomoses was measured with an ultrasonic flow probe. The animal was euthanized, and the graft was carefully removed for biomechanical characterization, and histologic and immunohistochemical evaluations. The animals implanted with H graft were euthanized at 4 weeks after surgery (n = 9) to compare data with the HS and HSV grafts.
Biomechanical Characterization
Graft compliance test
The graft was removed from the aorta, leaving approximately 1 cm intact aorta proximally and distally. Both ends of the graft were tied securely to a customized fixture designed to hold the sample at a set length. A pressure gauge (Digimano 1000; Netech, Hicksville, NY) was inserted in-line at the downstream end of the graft, while a calibrated repeat pipettor (Repeater Plus; Eppendorf, Westbury, NY) was placed in-line in the upstream end of the graft. Static, internal, and volumetric compliances were determined by increasing fluid volume incrementally and recording pressure. Percent radial compliance was calculated using the formula: % Compliance = (R – R0)/
P x 100, where R = graft radius, R0 = initial graft radius, and p = pressure changes. Internal radius was calculated from the volume with the assumption that the length remained constant. To obtain arterial data for comparison, porcine carotid arteries were obtained from a local butcher on the day of sacrifice and excess tissue was dissected free. Compliance testing on three normal porcine carotid arteries was performed within 24 hours of animal sacrifice.
Graft tensile strength test
After grafts were removed, two 5-mm segments were cut from the midportion of the graft for tensile strength testing. Graft thickness, length, and outer diameter of each segment were carefully measured. Two dowel pins were inserted within each 5-mm sample and secured with custom fixtures to a Chatillon test stand (Model TCD200; Chatillon, Largo, FL) and 2-pound load cell (Model DFGS 2; Chatillon). The pins were then pulled apart at a rate of 50 mm/min. Maximum force was recorded and ultimate tensile strength (UTS) was calculated as: UTS = Max Load/(2 x thickness x length). To obtain arterial data for comparison, tensile strengths of the thoracic arteries obtained from the euthanized rabbits (n = 18) were measured.
Histologic and Immunohistochemical Evaluations
Graft patency, neointima formation, endothelialization of the graft, and tissue ingrowth and angiogenesis in the graft wall were examined histologically and immunohistochemically. The explanted graft was fixed in 10% buffered formalin. Longitudinal and transverse sections from the proximal and distal anastomoses and through the center of the graft were obtained for tissue processing. All sections were dehydrated in a graded series of ethanol and embedded in paraffin. The paraffin blocks were cut at 4 to 6 µm, and the sections were mounted on a charged glass slide and stained with hematoxylin-eosin, and movat pentachrome.
Immunohistochemical studies were performed for Ram-11, von Willebrand factor (vWF), and 
-actin. Dewaxed paraffin sections were treated with 0.3% hydrogen peroxide for 20 minutes to inactivate endogenous peroxidases. The sections were then immersed in protein-free block (Dako, Carpinteria, California) for 10 minutes to block non-specific binding of primary antibodies. Sections were incubated for 1 hour at room temperature with primary antibodies against human smooth muscle -actin (clone HHF35, dilution 1:20; Enzo, Farmingdale, New York), the macrophage marker Ram11 (dilution 1:200; Dako), and a purified polyclonal antibody to vWF (dilution 1:2000; Strategic Biosolutions, Newark, Deleware).
The labeling of primary antibodies was achieved with an anti-mouse biotinylated link antibody from a peroxidase-based kit (LSAB; Dako). Positive staining (rose reaction product) was visualized using a 3-amino-9-ethylcarbazole substrate-chromogen system; the sections were counterstained with Gill’s hematoxylin.
Luminal surface fibrin/platelet aggregation, endothelialization, and cellular infiltration of the grafts were graded from grade 0 to 4. The definition of the grade is shown in Table 1. Histological index was calculated by dividing the sum of the grade by the number of sample grafts (grade/n) at the anastomotic sites and center of the grafts.
Statistical Analysis
All continuous values were expressed as mean ± 1 SD. The continuous variables were compared by an unpaired t test. Multigroup data were compared using the analysis of variance mode. Post hoc multiple comparisons were made using Fisher’s least significant difference technique. A value of p less than 0.05 was considered statistically significant.
|
Results
|
|---|
All rabbits in groups H, HS, and HSV survived after surgery. Paraplegia was not observed in any animals during the postoperative period in all groups. The percentage of heparin eluted from the grafts based on the in vitro study is shown in Figure 2. Sixty percent of heparin eluted within 1 day after soaking in the bath. Almost 80% of heparin eluted from the grafts within first the 10 days after soaking. Heparin continued eluting for at least 40 days.
Rheological Data and Graft Morphology
There were no significant differences in blood flow at the proximal and distal anastomoses after the initial implantation and postoperatively in all groups. Blood flow at the proximal and distal anastomoses was 42 ± 21 and 44 ± 18 mL/min in group H (p = 0.9), 37 ± 18 and 32 ± 18 mL/min in group HS (p = 0.4), and 43 ± 24 and 40 ± 22 mL/min in group HSV (p = 0.7) after initial implantation. Blood flow at the proximal and distal anastomoses before euthanasia was 39 ± 11 and 36 ± 16 mL/min in group H (p = 0.6), 36 ± 15 and 31 ± 13 mL/min in group HS (p = 0.4), and 50 ± 21 and 42 ± 17 mL/min in group HSV (p = 0.3) at each postoperative time point.
The patency rate of all grafts was 100% (45 of 45) at each time point. Although the grafts were covered by an adhesion capsule, there was no stenosis observed histologically at the proximal or distal anastomoses in any groups (Fig 3). There were no apparent changes in the graft, including dilatation, dehiscence, or aneurysm formation in any of the grafts during the 4-week postoperative period. None of the grafts was collapsed owing to surrounding tissue. There was no bleeding from the grafts, and no arteriovenous fistulae formation.
View larger version (58K):
[in this window]
[in a new window]
|
Fig 3. Explanted heparin-sirolimus (HS) (top) and heparin-sirolimus vascular endothelial growth factor (HSV) (bottom) grafts. There was no stenosis at the anastomotic sites and center of the grafts.
|
|
Biomechanical Characterization
Graft compliance
The graft compliance data are shown in Figure 4A. The H graft compliance between 80 and 120 mm Hg was 7.4% ± 2.4% and 8.5% ± 1.6% preoperatively and postoperatively, respectively (p = 0.55). The preoperative and postoperative compliance of the HS grafts was 3.6% ± 1.1% and 5.9% ± 2.7%, respectively (p = 0.23). In the HSV grafts, the preoperative and postoperative compliance was 6.1% ± 2.2% and 11.0% ± 2.2%, respectively (p = 0.02). The postoperative graft compliance was significantly higher than the preoperative compliance in the HSV grafts. Although the preoperative compliance was not different among the H, HS, and HSV grafts (p = 0.13), there was a statistical significance in the postoperative compliance among the three grafts (p = 0.001). Fresh porcine carotid artery compliance was 9.4% ± 2.2%. There was no significant difference between the porcine carotid arterial compliance and the postoperative graft compliance of the H, HS, and HSV grafts (p = 0.6, 0.1, and 0.4, respectively).
View larger version (19K):
[in this window]
[in a new window]
|
Fig 4. (A) Graft compliance at preoperative (white) and postoperative phase (black). (B) Graft tensile strength at preoperative (white) and postoperative phase (black). (H = heparin alone; HS = heparin-sirolimus; HSV = heparin-sirolimus vascular endothelial growth factor.)
|
|
Graft tensile strength
The tensile strength of the H graft was 490 ± 77 and 690 ± 99 kPa preoperatively and postoperatively (p = 0.002; Fig 4B). The preoperative and postoperative HS graft tensile strength was 468 ± 75 and 354 ± 77 kPa respectively (p = 0.01). In the HSV grafts, the preoperative and postoperative graft tensile strength was 443 ± 86 and 515 ± 82 kPa respectively (p = 0.17). The HSV grafts had higher postoperative tensile strength than the HS grafts (p = 0.01). Although the preoperative tensile strength was not different among the H, HS, and HSV grafts (p = 0.50), there was a statistical significance in the postoperative tensile strength among the three grafts (p = 0.001). The tensile strength of the descending thoracic aorta was measured to be 1,436 ± 450 kPa, which was significantly higher than that of the H, HS, and HSV grafts (p < 0.001).
Histologic Evaluations
All the H, HS, and HSV grafts were patent and had no stenoses histologically during the 4-week follow-up. In the early postoperative period (4 hours and 1 day) in the HS and HSV grafts, all grafts were covered with luminal surface fibrin. The graft wall was predominantly infiltrated by red blood cells and aggregates of platelets with some leukocytes. At 1 week after implantation, a thin layer of luminal surface fibrin with platelet aggregation and inflammatory cells was observed in all grafts with minimal to mild cellular infiltration (grade 2) within the grafts and neutrophil cell infiltration in the walls of both graft groups. There were no significant histologic differences between the HS and HSV grafts after 1 week. Serial changes of graft luminal surface fibrin/platelet aggregation, endothelialization, and cellular infiltration are shown in Figure 5A and B.
View larger version (32K):
[in this window]
[in a new window]
|
Fig 5. Serial change of endothelialization (top) and cellular infiltration (bottom) at anastomotic sites and center of the grafts. (A) Percentage of grafts that had endothelialization or cellular infiltration. (B) Histologic index, which was calculated by dividing the sum of the grade by the number of sample grafts (grade/n). (Open bars = anastomosis site; hatched bars = center of graft.)
|
|
At 2 weeks, a layer of spindle-shaped, immature mesenchymal cells covered the anastomotic site from the native aorta in the HS grafts, with occasional patches of endothelial cells (grade 1). However, the HSV grafts showed no endothelialization in the anastomosis sites at 2 weeks. There was no endothelialization in the midportion of any HS or HSV grafts. Mild inflammatory cells infiltrated into the pores in one-third of all the grafts, along with spindle-shaped cells in both graft groups. Both HS and HSV graft macrostructures appeared intact at 2 weeks.
At 4 weeks after implantation, the HS and HSV grafts showed a thin neointima with elongated endothelial cells that extended smoothly into the adjacent graft from the anastomotic site. The presence of a functional layer of endothelial cells was confirmed using vWF. Sixty-seven percent of the HS grafts had endothelialization at the anastomotic sites and the graft midportion (Fig 6). However, the HSV grafts did not show any endothelialization on the graft midportion despite the presence of endothelial cells at the anastomosis sites in 67% of the grafts (Fig 7). There was no excessive intimal hyperplasia in any HS or HSV grafts. The average neointimal thickness at the region of anastomotic sites at 4 weeks was 136 ± 75 µm in the HS grafts and 93 ± 64 µm in the HSV grafts (p = 0.1). There were moderate neocapillary formations inside the graft wall in the HS and HSV grafts. The graft macrostructure appeared unchanged at 4 weeks in all grafts. Vascular endothelial growth factor did not improve intimal formation, cell infiltration, or vascularization when compared with sirolimus only.
View larger version (71K):
[in this window]
[in a new window]
|
Fig 6. Histology and immunohistochemical data of the heparin-sirolimus (HS) grafts at 4 weeks. Moderate cellular infiltration was observed in the graft wall in hematoxylin-eosin (H-E) stain. Normal-appearing endothelial cells were aligned with the blood flow (right: arrows). There was moderate neocapillary formation in the graft wall (left: arrows). Clumps of red blood cells were observed inside the neocapillaries.
|
|
View larger version (57K):
[in this window]
[in a new window]
|
Fig 7. Histology and immunohistochemical data of the heparin-sirolimus vascular endothelial growth factor grafts at 4 weeks. Moderate cellular infiltration was observed in the graft wall in hematoxylin-eosin (H-E) stain.
|
|
All H grafts at 4 weeks had minimal neointima thickening, surface thrombosis (fibrin/platelet), and endothelialization inside the graft. Graft endothelialization was observed at the anastomosis sites and center in 100% of the grafts. The neointimal thickness of the H grafts was 125 ± 90 µm. There was no significant difference of neointima thickness among the H, HS, and HSV grafts (p = 0.08). However, the H grafts had moderate-severe cellular infiltration (100%, grade 3 to 4) at the anastomosis sites and center of the graft, which was more than the HS or HSV grafts. Neocapillary formation was observed in the H grafts as in the HS and HSV graft.
|
Comment
|
|---|
Synthetic small-caliber grafts will require patency equivalent to that of autologous arterial vessels. Bioengineering innovations have enabled the seeding of endothelial and smooth muscle cells onto synthetic grafts [6–9]. Although a seeded graft worked well experimentally, graft preparation was time consuming and technically difficult. If a synthetic graft had good endothelialization without any seeding, it would be more practical to use than a seeded graft. In the present study, this novel bioengineered synthetic graft had a 100% patency rate in the H, HS, and HSV grafts at 1 month. It had good endothelialization 4 weeks after implantation.
Synthetic small caliber grafts have not been practical for CABG because of their poor patency rate. Graft thrombosis and intimal hyperplasia are the major cause of graft failure. Studies have shown that heparin-coated grafts prevent thrombogenicity compared with nonheparin-coated grafts [10, 11]. There are conflicting studies on the beneficial effect of heparin on intimal hyperplasia [10–14]. Animal and clinical studies have demonstrated that the local delivery of sirolimus inhibits the proliferation of smooth muscle cells and reduces neointimal thickening [15–18]. The graft used in the present study contained drugs within the graft wall. Heparin was embedded in the microstructure of the graft for early elution from the graft wall. Heparin and sirolimus were incorporated into the macrostructure of the graft for late elution from the graft wall. No thrombosis or excessive neointima hyperplasia was observed in the H, HS, and HSV grafts after 4 weeks. Although there was no significant difference in neointimal thickness among the H, HS, and HSV grafts, the H grafts had more cellular infiltration in the graft wall than the HS and HSV grafts. That may suggest that sirolimus helped to inhibit cell proliferation. However, the role of impregnated drugs in the long-term prevention of these complications remains unknown, and will require further chronic studies.
The HSV grafts did not improve intimal formation, cell infiltration, or vascularization compared with the HS grafts in this study. There could be two possible reasons for this. First, the high blood flow rates encountered in the aortic circulation might have caused VEGF to elute from the microstructure too quickly for adequate cellular uptake to occur, thus obviating any stimulation of endothelialization. The impregnated drug in the microstructure begins eluting following graft implantation. Because most drugs were eluted from the graft microstructure within the first 10 days (Fig 2), only a small amount of VEGF should have remained when cells started infiltrating into the micropores of the graft. In addition, the graft macrostructure itself supplies a scaffold for endothelialization and cellular infiltration. The HS grafts had good endothelialization and cellular infiltration without VEGF or any cell-seeding process. Therefore, the benefit of adding VEGF to the scaffold might be overshadowed by the advantages of the scaffold itself.
There are several hypotheses on the source of the endothelial cells found inside the graft [19]. First, endothelial cells may have migrated into the graft from the normal endothelium at both anastomotic sites. Second, the source for the endothelial cells may have been from the circulating blood. Third, the source may be transinterstitial ingrowth of either multipotent cells or endothelial cells from neocapillaries in the porous graft wall. Determining the source of the endothelialization is important in creating an efficacious graft that can be used clinically. If endothelial cells inside the graft come only from the anastomotic sites, the synthetic graft may have a limited graft length that reduces its clinical availability. This study demonstrated that the H, HS, and HSV grafts had neocapillary formation within 4 weeks after implantation. Early neocapillary formation should be beneficial for graft endothelialization.
Polyurethanes have been used extensively as biomaterials due to their desirable physical properties, such as high tensile strength and elasticity [20]. However, polyurethanes historically have had limited use in long-term implants owing to degradation. Polyester polyurethanes have been found to be susceptible to hydrolysis through the ester linkage, while polyether polyurethanes were shown to be vulnerable to oxidative cleavage in vivo [21]. The incorporation of polydimethylsiloxane and polycarbonate into the polymer backbone instead of polyether or polyester segments has been shown to improve the resistance of the material to both hydrolytic and oxidative degradation [22]. Polycarbonate polyurethanes have been found to be more stable than traditional polyurethanes against chronic biodegradation and stress cracking [23]. The polyurethane used in this study is a polycarbonate and polydimethylsiloxane polyurethane copolymer, which should enable it to resist degradation and function as a long-term implantable vascular graft.
Human saphenous vein and ePTFE graft compliance are known to be 2.0% and 1.0% at 80 to 120 mm Hg, respectively [24, 25]. The postoperative graft compliance of the H, HS, and HSV grafts was higher than that of human saphenous vein or ePTFE grafts, and not significantly different from that of the porcine carotid artery. Therefore, the graft compliance of each graft was acceptable for a synthetic graft. Although the graft compliance in this study was not significantly changed during the 4-week study period in the H and HS grafts, postoperative compliance of the HSV grafts increased significantly after surgery. The tensile strength of each graft was not consistent during the 4-week study period. This may have been caused by the degradation of the collagen/hyaluronan microstructure, the infiltration of cells into the graft, the normal variability of the materials, or the limited sample size. Histologic data showed the graft structure was still changing as cellular infiltration into the pores of the grafts was observed over the 4-week period while the microstructure degraded. The graft compliance and tensile strength will likely continue to change as the graft becomes completely incorporated with the host. The H grafts, which had much more cellular infiltration at 4 weeks than the HS or HSV graft, had higher tensile strength. Once the process of graft remodeling is completed, the conduit could reach a stable condition in which its mechanical properties will become equivalent over time. There was no visual or histological evidence to suggest that the macrostructure was degrading during the 4 weeks, so we conclude that the microstructure is primarily responsible for the fluctuations in the biomechanical stability of graft. A long-term study is currently being conducted to evaluate serial changes in the graft biomechanical stability.
Study Limitations
This study was a preliminary evaluation of a novel bioengineered synthetic small caliber graft. Since grafts were examined for histology and biomechanical properties at several different time points, the number of implanted grafts at each time point was small. However, data of each time point were similar and consistent. The H grafts were evaluated at only 4 weeks after surgery to be compared with the HS and HSV grafts. Histologic data of the H grafts revealed the effect of sirolimus on cellular infiltration. A longer term study is needed to evaluate the efficacy of the eluted drugs.
|
Acknowledgments
|
|---|
We acknowledge the excellent technical assistance of P. Diane Toeniskoetter, Kathryn L. Cook, Naomi R. Still, Geneva R. Baca, Dina Harilal, Timothy Ringeisen, Louis Sessa, James Hill, and William Maas. We also thank Dawn G. Schuessler for preparation of the manuscript. This research was supported by the NIST Advanced Technology program (Award no. 70NANB1H3032).
|
References
|
|---|
- Angelini GD, Newby AC. The future of saphenous vein as a coronary artery bypass conduit Eur Heart J 1989;10:273-280.[Abstract/Free Full Text]
- Cameron A, Davis KB, Green G, Schaff HV. Coronary bypass surgery with internal-thoracic-artery grafts—effects on survival over a 15-year period N Engl J Med 1996;334:216-219.[Abstract/Free Full Text]
- Loop FD, Lytle BW, Cosgrove DM, et al. Influence of the internal-mammary-artery graft on 10-year survival and other cardiac events N Engl J Med 1986;314:1-6.[Abstract]
- Jones DN, Rutherford RB, Ikezawa T, Nishikimi N, Ishibashi H, Whitehill TA. Factors affecting the patency of small-caliber prostheses: observations in a suitable canine model J Vasc Surg 1991;14:441-448.[Medline]
- Esquivel CO, Blaisdell FW. Why small caliber vascular grafts fail: a review of clinical and experimental experience and the significance of the interaction of blood at the interface J Surg Res 1986;41:1-15.[Medline]
- Pasic M, Muller-Glauser W, von Segesser LK, Lachat M, Mihaljevic T, Turina MI. Superior late patency of small-diameter Dacron grafts seeded with omental microvascular cells: an experimental study Ann Thorac Surg 1994;58:677-683.[Abstract]
- Laube HR, Duwe J, Rutsch W, Konertz W. Clinical experience with autologous endothelial cell-seeded polytetrafluoroethylene coronary artery bypass grafts J Thorac Cardiovasc Surg 2000;120:134-141.[Abstract/Free Full Text]
- Dunn PF, Newman KD, Jones M, et al. Seeding of vascular grafts with genetically modified endothelial cellsSecretion of recombinant TPA results in decreased seeded cell retention in vitro and in vivo. Circulation 1996;93:1439-1446.[Abstract/Free Full Text]
- Niklason LE, Gao J, Abbott WM, et al. Functional arteries grown in vitro Science 1999;284:489-493.[Abstract/Free Full Text]
- Walpoth BH, Rogulenko R, Tikhvinskaia E, et al. Improvement of patency rate in heparin-coated small synthetic vascular grafts Circulation 1998;98(19 Suppl):II319-II323.[Medline]
- Lin PH, Chen C, Bush RL, Yao Q, Lumsden AB, Hanson SR. Small-caliber heparin-coated ePTFE grafts reduce platelet deposition and neointimal hyperplasia in a baboon model J Vasc Surg 2004;39:1322-1328.[Medline]
- Kiesz RS, Buszman P, Martin JL, et al. Local delivery of enoxaparin to decrease restenosis after stenting: results of initial multicenter trialPolish-American Local Lovenox NIR Assessment study (the POLONIA study). Circulation 2001;103:26-31.[Abstract/Free Full Text]
- Welt FG, Woods TC, Edelman ER. Oral heparin prevents neointimal hyperplasia after arterial injury: inhibitory potential depends on type of vascular injury Circulation 2001;104:3121-3124.[Abstract/Free Full Text]
- Esquivel CO, Bjorck CG, Bergentz SE, et al. Reduced thrombogenic characteristics of expanded polytetrafluoroethylene and polyurethane arterial grafts after heparin bonding Surgery 1984;95:102-107.[Medline]
- Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells Circ Res 1995;76:412-417.[Abstract/Free Full Text]
- Burke SE, Lubbers NL, Chen YW, et al. Neointimal formation after balloon-induced vascular injury in Yucatan minipigs is reduced by oral rapamycin J Cardiovasc Pharmacol 1999;33:829-835.[Medline]
- Gallo R, Padurean A, Jayaraman T, et al. Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle Circulation 1999;99:2164-2170.[Abstract/Free Full Text]
- Morice MC, Serruys PW, Sousa JE, et al. RAVEL Study Group Randomized study with the sirolimus-coated Bx velocity balloon-expandable stent in the treatment of patients with de novo native coronary artery lesionsA randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 2002;346:1773-1780.[Abstract/Free Full Text]
- Greisler HP. Arterial regeneration over absorbable prostheses Arch Surg 1982;117:1425-1431.[Abstract/Free Full Text]
- Brothers TE, Stanley JC, Burkel WE, Graham LM. Small-caliber polyurethane and polytetrafluoroethylene grafts: a comparative study in a canine aortoiliac model J Biomed Mater Res 1990;24:761-771.[Medline]
- Schubert MA, Wiggins MJ, Schaefer MP, Hiltner A, Anderson JM. Oxidative biodegradation mechanisms of biaxially strained poly(etherurethane urea) elastomers J Biomed Mater Res 1995;29:337-347.[Medline]
- Hergenrother RW, Yu XH, Cooper SL. Blood-contacting properties of polydimethylsiloxane polyurea-urethanes Biomaterials 1994;15:635-640.[Medline]
- Eberhart A, Zhang Z, Guidoin R, et al. A new generation of polyurethane vascular prostheses: rara avis or ignis fatuus? J Biomed Mater Res 1999;48:546-558.[Medline]
- Sawyer P. Modern vascular graftsNew York: McGraw-Hill; 1987. pp. 326.
- Conklin BS, Richter ER, Kreutziger KL, Zhong DS, Chen C. Development and evaluation of a novel decellularized vascular xenograft Med Eng Phys 2002;24:173-183.[Medline]
Top
Abstract
Introduction
