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Healing characteristics of intraarterial stented grafts: Effect of intraluminal position on prosthetic graft healing
Healing characteristics of intraarterial stented grafts: Effect of intraluminal position on prosthetic graft healing Mark P. OmbreUaro, MD, Scott L. Stevens, MD, Kyle Kerstetter, DVM, Michael B. Freeman, MD, and Mitchell H. Goldman, MD, Knoxville, Tenn.
Background. The purpose of this study was to investigate the effect of complete intraluminal placement on prosthetic graft healing. Methods. Thirty dogs underwent infrarenal abdominal aorta polytetrafluoroethylene interposition (12) or intraluminal stented (18) grafting. Grafts were removed at 4 and 8 weeks. Length of endothelial ingrowth and intima to media height ratios (IMHRs) were calculated. Pevianastomotic endothelial (CD31+, factor VIII [FVIII+]), smooth muscle (actin+), macrophage (CD44+), and proliferating (PCNA +) cell content was determined. Results. In control grafts mean proximal and distal anastomotic endothelial cell ingrowth was 0.42 +- 0.06 and 0.47 +- 0.08 cm at 4 weeks and 1.10 +- 0.24 and 0.94 +- 0.17 cm at 8 weeks. In intraluminal grafts mean proximal and distal anastomotic endothelial cell ingrowth was 1.5 7 +- O.09 and 1.54 +- 0.12 cm at 4 weeks and 1.88 +-- 0.06 and 2.11 +_ 0.25 cm at 8 weeks. Endothelial ingrowth was greater in all stented grafts (p < 0.001). Mean proximal anastomosis IMHRs were 1.01 +- O.16 for 4-week and 1.42 + O.16 for 8-week control grafts and 0.59 +_ O.18 for 4-week and 0.50 +- 0.14 for 8-week stented grafts. Similar I M H R values were present at the distal anastomosis. Lower IMHRs were observed in stented grafts (p < 0.05). Content of C~44+, PCNA+, and FVIII+ cells were reduced both proximally and distally in 4-week stented grafts (p < 0.05). Distal content of C~31+ and actin+ cells was greater in 4-week stented grafts (p < O. 05). At 8 weeks C~44+ cell content decreased in controls (p < O.05). Conclusions. Intraluminal location enhances prosthetic graft reendothelialization and attenuates intimal thickening. (Surgery 1996;120:60-70.) From the Department of Surgery, Division of Vascular Surgery, University of Tennessee Medical Center, Knoxville, Tenn.
COMPLETE REPOPULATION OF A prosthetic graft surface with a functional endothelial cell layer has yet to be exhibited in h u m a n beings. Endothelial cells provide a n o n t h r o m b o g e n i c a n d antiinflammatory barrier between circulating proteins, coagulation factors, cellular elements, a n d the artificial biomaterial. 1' 2 In addition, they also play a regulatory role, interacting with smooth muscle cells, macrophages, platelets, a n d other vascular wall cellular constituents during acute and subacute vascular injury. Although it has b e e n suggested that reendothelialization would improve prosthetic graft patency, this has yet to be proved clinically.~-5
Supported by the Physicians'Medical Education and Research Foundation (PMERF), Knoxville,Tenn. Accepted for publication Oct. 6, 1995. Reprint requests: Mark Ornbrellaro, MD, Department of Surgery, Division of Vascular Surgery, Universityof Tennessee Medical CenterKnoxville, 1924 Alcoa Hwy, Knoxville,TN 37920. Copyright 9 1996 by Mosby-YearBook, Inc. 0039-6060/96/$5.00 + 0 11/56/71946 60
SURGERY
Refinements in endovascular techniques using stent and balloon catheter technology have lead to the evolution o f an intraluminal stented bypass graft. Whereas the healing characteristics of the various individual c o m p o n e n t s of stented grafts have b e e n described, those of the complete intraluminal device have yet to be defined. Intravascular stents, i m p o r t a n t adjuncts for the treatment o f postangioplasty intimal dissection a n d failures caused by elastic recoil, are characteristically resurfaced with endothelium. 6, 7 Because stents are placed in conjunction with angioplasty procedures, they are often associated with intimal hyperplasia. Intravascular stented grafts offer several theoretic advantages for enh a n c e d graft healing a n d r e d u c e d intimal hyperplasia including wide anastomoses, an in-line graft configuration, a n d direct contact with an endothelialized surface. Although the feasibility o f endovascular grafting has b e e n reported, the healing characteristics o f this type of graft a n d effect o f the intravascular location have not. 8-17 We have investigated the influence o f an intraluminal e n v i r o n m e n t on prosthetic graft healing a n d the cellu-
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lar composition of the perianastomotic intimal region by using intraarterial stented grafts.
METHOD Thirty adult male and female h o u n d dogs, weighing between 17 and 32 kg (average weight, 24.1 kg), were used for this study. Animals were cared for in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of LaboratoryAnimals" (National Institutes of Health publication no. 80-23, revised 1985). Each animal was premedicated with an intramuscular dose of acepromazine (0.022 m g / k g ) , atropine (0.044 m g / k g ) , and butorphanol (0.44 m g / k g ) , anesthetized with intravenous thiopental sodium (13 m g / k g ) , intubated, and maintained on a mixture of isofluorane (1.5 to 3.5 m i n i m u m alveolar concentration) and 100% oxygen (1 L / m i n ) via a semiclosed anesthesia circuit. Antibiotic prophylaxis with 500 mg intravenous cefazolin was administered 30 minutes before and 90 minutes after skin incision. Intravenous lactated Ringer's solution was given as required to maintain circulating blood volume. The control group consisted of 12 dogs who underwent standard interposition grafting of the infrarenal abdominal aorta. The aorta was exposed through a left lower quadrant incision and retroperitoneal approach from the renal arteries to the aortic trifurcation. A 2 cm segment of aorta was circumferentially dissected both proximally and distally, and the intervening central aortic segment was excluded with two ligatures of 0-0 polydioxanone sutures (Ethicon, Inc., Somerville, N.J.). Vascular clamps were applied, and the aorta was transected between each set of clamp and excluding ligature. Aortic continuity was restored with a 6 cm interposition graft of 10 m m diameter, thin-walled (30 p internodal distance with outer reinforcing wrap removed) polytetrafluoroethylene (PTFE, Gore-Tex; W. L. Gore & Associates, Flagstaff, Ariz.). Normal aortic diameters were previously measured in similar size hounds undergoing intraabdominal surgery. Diameters ranged from 9 to 12 mm, and on the basis of these findings a 10 m m diameter PTFE graft was chosen for this study. Anastomoses were constructed in an end-to-end fashion by using a continuous CV-6 PTFE suture under 2.5x loupe magnification. The experimental group consisted of 18 dogs who underwent intraluminal stented grafting of the infrarenal abdominal aorta. Stented grafts were constructed by securing the midportion of a 3.4 cm Palmaz balloon expandable stent (P 308; Johnson & J o h n s o n Interventional Systems, Warren, N.J.) to the proximal end of a 6 cm length of the previously described PTFE graft material by using a single CV-6 PTFE suture. In all cases 50% of the stent surface was overlapped by the PTFE graft. A pushing tube (14F outer diameter rigid plastic
OmbreUaro et al.
61
tube attached to a hemostatic valve) and the stent graft complex were both loaded on a 5.8F balloon catheter (10 m m x 4 cm, Meditech; Boston Scientific, Watertown, Mass.) and placed into a 14F delivery sheath (Check-Flo II; Cook, Bloomington, Ind.) (Fig. 1). Arterial access was obtained via a left c o m m o n femoral artery cutdown. Fluoroangiography was routinely performed to delineate the location of the renal arteries and aortic trifurcation. A guide wire was passed, and tile stented graft delivery system was advanced over the wire into the infrarenal abdominal aorta. Stented grafts were deployed by ejecting the graft from the sheath by use of the pushing tube and balloon expansion of the stent. Straightening and expansion of the graft body were achieved by means of additional balloon inflations and gende traction of a partially inflated balloon inside the graft. No stents were used to secure the distal aspect of the graft. All grafts were placed without heparinization. Completion aortogram confirmed proper intraluminal graft placement and no evidence of perigraft leakage. The left c o m m o n femoral artery was ligated after stented graft deployment. Postoperative graft patency was monitored by means of daily femoral pulse palpation in both the control and experimental animals. Antiplatelet agents and anticoagulants were not administered. Animals in each group were killed at 4 and 8 weeks after grafting. At the time of death, each animal was reanesthetized and given an intravenous dose of heparin (3000 units) and Evans blue (30 m g / k g ) . The aorta and graft were exposed through a midline incision, and aortography was performed through a proximal aortic needle puncture. Lumbar and inferior mesenteric artery orifices covered by the intraluminal graft were absent on angiogram with no evidence of perigraft leakage. Both the interposition and intraluminal grafts remained relatively well matched to the native aortic size. When compared with normal aortic segments above and below intraluminally placed stented grafts, no external evidence of aortic distention related to graft deployment was noted. The infrarenal aorta was isolated, and perfusion cannulas were inserted retrograde through both c o m m o n iliac arteries to the aortic trifurcation. The aorta was flushed with normal saline solution and fixed for 5 minutes with a solution of 2% paraformaldehyde and 0.5% gluteraldehyde at 100 m m H g pressure after a lethal dose of intravenous pentobarbital sodium (1 m g / 4 . 5 kg Beuthanasia D; ScheringPlough, Kenilworth, N.J.). The aortic specimens were removed, o p e n e d longitudinally, photographed enface, and divided into two (longitudinal) segments (Fig. 2). Gross examination showed no evidence of h e m a t o m a between the aorta and stented intraluminal grafts in either early or late specimens. One segment was fixed in 2.5% gluteralde-
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> >
/A It
b
Fig. 1. a, Intraluminal stented graft; b, diagram of intraluminal stent graft delivery device.
hyde for 24 hours and processed for electron microscopy, and the other was fixed in zinc formalin (Z-fix; Anatech Ltd, Battle Creek, Mich.) and processed for light microscopy and immunohistochemical studies. Representative sections of the proximal and distal anastomotic and mid-graft regions were dehydrated, critical point dried, mounted by using colloidal graphite, grounded with silver paint, and vacuum coated with gold palladium for scanning electron microscopy. The zinc formalin-fixed tissue was bisected longitudinally, and one strip was embedded in paraffin and the other in methyl methacrylate plastic. One set of specimens in each type of embedding medium was required so that cell epitopes would be treated uniformly when processed for immunohistochemistry. Standard immunohistochemical staining techniques are typically performed on paraffin-embedded tissue. One potential problem, however, is that grafts containing metallic stents cannot be cut or processed in this fashion. Before stented graft specimens were embedded in paraffin, stents were carefully dissected from the graft material under magnification. Immunohistochemical staining for endothelial cell surface antigens CD-31 (Dako Corp., Carpinteria, Calif.) and yon Willibrand
factor VIII (BioGenex, San Ramon, Calif.), smooth muscle cell ed-actin (Dako Corp.), macrophage surface antigen CD44 (PharMingen, San Diego, Calif.), and proliferating cell nuclear antigen (PCNA) (Dako Corp.) was performed. To assure antibody staining was specific to the particular cell type of interest, all antibodies were tested with both positive and negative control tissues. Immunoantibodies were visualized by using an alkaline phosphatase chromagen developing kit (BioGenex). The purpose of embedding stented grafts in methylmethacrylate plastic was to preserve the in situ relationships of the artery, graft, and stent materials. Whereas grafts embedded in methylmethacrylate can be cut and processed without the need for stent removal, immunohistochemical staining of cellular antigens from soft tissue embedded in methylmethacrylate has yet to be described. Immunohistochemical staining techniques on methylmethacrylate embedded tissue are being performed in our laboratory, but additional testing is required for standardization. In this study methylmethacrylate specimens were used to confirm histologic architecture, whereas immunohistochemical comparisons between control and experimental grafts were made by using paraffin-embedded tissue samples only.
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63
Fig. 2. Freshly excised 8-week control (A) and intraluminal stented (B) graft after administration of Evans blue dye. Black arrows delineate PTFE-artery interface. White arrows delineate interface between reendothelialized and unhealed surfaces. Enhanced reendothelializafion is apparent in intraluminal stented graft. To quantify endothelial cell ingrowth, specimen photographs (including scale markers) were traced by using a digitized drawing board and stored on an Apple IIci computer (Apple Computer Inc., Cupertino, Calif.). Graft area, anastomotic width, and area of endothelial cell resurfacing were measured by using Evans blue stain exclusion and IP Lab Spectrum 2.5 image analysis software (Signal Analytics Corp., Vienna, Va.). To control for differences in specimen diameter, area of endothelial cell ingrowth was divided by vessel width and reported as average length of cell ingrowth for each anastomosis. Intimal height for the proximal and distal perianastomotic (artery-graft interface) regions of each control and stented graft was calculated by averaging two measurements from the point of maximal intimal thickness overlying the PTFE graft in each region. To control for variation in intimal thickness related to specimen sectioning in different tangential planes, intireal height was related to medial height in each section. Medial thickness was measured on the arterial side of each anastomosis in a uniform region adjacent to the PTFE-artery interface. The ratio of maximum intimal to medial thickness (IMHR) was used to quantify intimal
hyperplasia in each graft perianastomotic region. All measurements were performed with the IP Lab Spectrum program on digital images of histologic specimens at 250x magnification acquired by a videomicroscope. Lesion composition was determined by counting the number of CD31 and factor VIII (endothelial), CD44 (macrophage), ~-actin (smooth muscle), and PCNA (proliferative) positive staining cells in three different high-power fields at 1000x magnification. Statistical analysis was performed in consultation with the statistics department at the University of Tennessee by using the JMP 3.0 statistical program (SAS Institute, Cary, N.C.). Mean value comparisons between control and experimental groups were performed with a two-tailed Student's t test and Wilcoxon rank sums test. A p value of <0.05 was considered significant. Numeric values are reported as a mean value _+ the standard error (SEM). RESULTS Three animals in the experimental group died within 48 hours of stented graft placement. Two deaths were attributable to technical complications including an aortic dissection with perforation during delivery system
64
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Fig. 3. Mean length of endothelial cell ingrowth at proximal (A) a n d distal (B) anastomoses. Endothelial cell ingrowth is significantly greater in stent grafts, compared with controls, in each anastomotic region at both 4 a n d 8 weeks (p < 0.001). Error bars denote standard error of mean. passage a n d bilateral r e n a l artery occlusions f r o m inaccurate graft p l a c e m e n t . O n e a n i m a l was f o u n d d e a d o f a n u n d e t e r m i n e d cause in its cage 12 h o u r s after operation. All r e m a i n i n g grafts in the c o n t r o l a n d experim e n t a l g r o u p s were p a t e n t at the time o f d e a t h Perianastomotic endothelial cell ingrowth. Average l e n g t h of e n d o t h e l i a l cell ingrowth was calculated for the p r o x i m a l a n d distal anastomoses in each g r o u p (Fig. 3). I n 4-week c o n t r o l grafts (n = 6) e n d o t h e l i a l cell ingrowth was 0.42 +- 0.06 c m at the p r o x i m a l anastomosis a n d 0.47 +_ 0.08 c m at the distal anastomosis. I n 4-week i n t r a l u m i n a l grafts (n = 7) e n d o t h e l i a l cell ingrowth was 1.57 _+ 0.09 c m at the p r o x i m a l anastomosis a n d 1.54 -+ 0.12 c m at the distal anastomosis. At 8 weeks endothelial cell ingrowth i n c r e a s e d b o t h proximally a n d distally to 1.10 _+ 0.24 c m a n d 0.94 -+ 0.17 cm, respectively, in c o n t r o l grafts ( n = 6 ) ) . E n d o t h e l i a l cell in-
growth in 8-week i n t r a l u m i n a l grafts ( n = 8 ) were 1.88 + 0.06 c m for the p r o x i m a l a n d 2.11 + 0.25 c m for the distal a n a s t o m o t i c regions. At each o f 4 a n d 8 weeks a significant increase in e n d o t h e l i a l cell i n g r o w t h was e v i d e n t in s t e n t e d grafts at b o t h anastomotic sites ( p < 0 . 0 0 1 ) . W i t h i n each c o n t r o l a n d e x p e r i m e n t a l g r o u p n o difference in e n d o t h e l i a l cell ingrowth was identified b e t w e e n p r o x i m a l a n d distal anastamotic regions. I n all regions o f Evans b l u e dye exclusion, e n d o thelial cells were c o n f i r m e d with s c a n n i n g electron microscopy (Figs. 4 a n d 5). Characterization o f i n t i m a l thickness. I M H R was u s e d to quantify the i n t i m a l hyperplastic r e s p o n s e to each type o f graft (Fig. 6). I n 4-week c o n t r o l grafts the I M H R w a s 1 . 0 1 -+ 0.16 at the p r o x i m a l anastomoses a n d 0.90 - 0.12 at the distal anastomosis. I n 4-week intralum i n a l grafts the I M H R was 0.59 -+ 0.18 at the p r o x i m a l
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65
Fig. 4. Scanning electron micrograph of 4-week control graft at proximal anastomosis. A, Endothelial cells along proximal graft surface; original magnification • B, Transition zone shows advancing edge of endothelial cells (arrows); original magnification • C, Unhealed central graft surface. Fibrin deposition and lack of endothelial cells are apparent along PTFE surface; original magnification x480.
Fig. 5. Scanning electron micrographs of 4-week intraluminal stented graft at proximal anastomosis. Images are from same relative position along stent arm with increasing magnification. Reendothelialized surface is shown over both the stent and PTFE graft materials, a, Original magnification x45; b, original magnification x90; C, original magnification •
and 0.47 _+ 0.13 at the distal anastomosis. At 8 weeks control IMHRs were 1.42 + 0.16 for the proximal and 0.84 + 0.12 for the distal anastomoses, respectively. IMHRs in 8-week intraluminal grafts were 0.50 _+ 0.14 for proximal and 0.42 -+ 0.1 for distal anastomotic regions. The intimal hyperplastic response at the proximal anastomosis was lower in stented grafts when compared with controls. At 8 weeks the difference achieved statistical significance (p < 0.001). At the distal anastomosis intireal thickness was significantly less in intraluminal stented grafts at both 4 and 8 weeks (p < 0.05). Lesion composition. Immunohistochemical stains for specific cell surface and intracellular antigens were used to characterize intimal lesion evolution and composition and to quantitate the various cellular constituents along control and stented grafts. Factor VIII and the more specific CD31 antigens were used to identify
endothelial cells (Fig. 7). Smooth muscle cells and macrophages were identified by using antibodies against ~l-actin and CD44, respectively (Fig. 8). Proliferating cells were quantified by using antibodies against PCNA. Mean counts for each cell type are reported for proximal and distal anastomotic sites (Table). The quantity of endothelial cells as identified by CD31 staining was greater in stented grafts, when cornpared with controls, at the 4-week distal and both 8-week anastomotic sites (p< 0.001). Factor VIII positive ce.ll content was significantly higher in control grafts at all sites and time intervals except at the 8-week distal anastomosis (p < 0.02). At the proximal anastomosis no difference in smooth muscle cell content was noted between control and experimental grafts at any time. At the distal anastomosis smooth muscle cell content was greater in stented grafts at both time intervals (p < 0.01).
66
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Surgery July 1996
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Fig. 6. Mean IMHRs at proximal (A) and distal (B) anastomoses. At 8 weeks intimal thickness was significantly lower in stented grafts at proximal anastomosis (p < 0.001). At 4 weeks this difference was not statistically significant, lnfimal thickness was significantly lower in stented graft group, when compared with controls, at both 4 and 8 weeks at distal anastomosis (p < 0.05). Error bars denote standard error of mean.
At 4weeks the macrophage content in each anastomotic region was lower in intravascular graft specimens when compared with controls (p < 0,0005). By 8 weeks macrophage content remained stable in stented grafts and a significant macrophage efflux occurred in control s. As a result of the decline in control macrophage content, macrophages were more prevalent in stented graft specimens at 8 weeks. At the distal anastomosis this difference in 8-week macrophage content achieved statistical significance (p < 0.003). The n u m b e r of actively proliferating cells (PCNA positive) were significantly less in stented grafts at each anastomosis at up to 8 weeks (p < 0.05).
DISCUSSION The inability of h u m a n beings to repave a prosthetic graft luminal surface with functional endothelial cells remains an unexplained p h e n o m e n o n . After implancation a perianastomotic intimal accumulation of smooth muscle cells, extracellular matrix, and endothelial cells occurs that typically extends I to 2 cm beyond the suture line onto the graft surface. 2 Beyond this region the graft is laminated with a pseudointima of platelets, red blood cells, and compacted fibrin and is unhealed. Furthermore, a variety of animal species exhibit a great propensity to heal prosthetic grafts, but the mechanisms responsible for these observed differ-
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Fig. 7. Composite p h o t o m i c r o g r a p h shows reendothelialization of proximal, mid-graft, and distal anastomotic sites along control and stented grafts at 8 weeks. Arrows denote cells along luminal surface staining positive for endothelial cell CD31 antigens. Mid-graft region is layered with endothelial cells in intraluminal grafts and fibrin and debris in controls. CD 31 antibodies with alkaline phosphatase chromagen; original magnification x400
T a b l e . P e r i a n a s t o m o t i c cell counts (cells p e r h i g h - p o w e r field; x l 0 0 0 m a g n i f i c a t i o n )
Antigen Endothelial cells Group
CD 31
A (4-wk control) (n = 15) PA 10 • 0.6 DA 8 • 0.5 B (4-wk stented graft) (n = 12) PA 10 • 0.6 DA 11 • 0.6* C (8-wk control) (n = 18) PA 8 + 0.6 DA 7 + 0.5 D (8-wk stented graft) (n : 24 PA 11 • 0.5]DA 10 • OAt
Macrophage
Factor VIII
SMC actin
11 _+ 0.5 11 _+ 0.6 7 _+ 0,7* 7 • 0.8* I2 +- 0.5
10• 7 -+ 0.5t 9-+0.6
CD44
PCNA
108 • 8 88 + 6
23 • 2 32 • 4
30 -+ 4.5 21 +- 2.5
129 • 9 108 • 7*
10 • 2* 9 • 2*
126 • 7 108•
12 -+ 1.5 7-+ 1
121 + 6 146•
9 • 1.5 10-+ 1t
8 • 5* 9 • 2.5* 20 -+ 4 11 • 7 _+ 3.5t 7_+2t
Data shown as mean -+ standard error.
PA, Proximal anastomosis; DA, distal anastomosis; SMC, smooth muscle cells. *p < 0.05 compared with group A. tP < 0.05 compared with group C.
e n c e s are u n k n o w n , z' 18 It is h y p o t h e s i z e d that an intact, i n t r a l u m i n a l e n d o t h e l i a l cell layer is i m p o r t a n t in p r e v e n t i n g graft failure. E n d o t h e l i a l cell d a m a g e o r dysfunction f r o m the bypass p r o c e d u r e itself o r indirectly t h r o u g h the effects o f graft material, shear stress, a n d c o m p l i a n c e m i s m a t c h o n h e m o d y n a m i c s can
c o n t r i b u t e to a m i c r o e n v i r o n m e n t o f c h r o n i c arterial injury a n d i n d u c e p e r i a n a s t o m o t i c intimal hyper-plasia. 1'1926 T h e m e t a b o l i c r e s p o n s e o f m o n o c y t e s , m a c r o p h a g e s , T lymphocytes, a n d s m o o t h m u s c l e cells to a c h r o n i c injury stimulus f u r t h e r p e r p e t u a t e s this process.20, 27
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Fig. 8. Composite photomicrograph shows smooth muscle cell (SMC) content of proximal, mid-graft, and distal anastomotic sites along control and stented grafts at 8 weeks. Mid-graft region appears covered with thin SMC layer in intraluminal grafts, whereas fibrin and debris are present in controls. SMC ix-1 actin antibodies with alkaline phosphatase chromagen; original magnification x400.
Whereas the internal graft environment may affect patency, the importance of the external graft environment and its potential role in graft healing are now being appreciated. Recent animal investigations have shown enhanced endothelial cell deposition and capillary ingrowth within PTFE arterial grafts wrapped with omentum. 2s Because endothelial cell ingrowth is characteristically limited to within 1 to 2 cm of the anastomotic regions of grafts placed in a nonendothelialized environment, placing these conduits in a milieu with a high concentration of endothelial cells may improve graft healing. Sources of endothelial cells for in situ graft resurfacing include the native vessel in contact with the ends of the graft, capillary ingrowth through the graft interstices, and circulating endothelial microemboli.1, 2, 29, a0 The relative contribution to graft resurfacing and clinical significance of each mechanism to graft reendothelialization remains undefined. The concept of intraluminal placement of an arterial bypass conduit is a recent development made possible by the introduction of stented grafts. Early animal investigations have described the technique, as applied to aortic aneurysm repair, and the presence of endothelial-like cells along the intraluminal surface, alluding to a healed graft. 8-n These devices make it possible to study the relationship between external graft environm e n t and prosthetic graft healing with regard to the specific effects of a completely intraarterial location.
In our study intraluminal prosthetic (stented) grafts were associated with a significant increase in graft surface reendothelialization and decrease in perianastc~ motic intimal hyperplastic response when compared with interposition control grafts. The presence of endothelial cells along most of the stented graft surface, including the central graft region, was shown by use of scanning electron microscopy and, unequivocally, by using antibodies against endothelial cell specific CD31 surface antigens. Although yon Willibrand factor VIII surface antigen is often used to identify endothelial cells, these epitopes are also present on fibroblasts and other cell types. For this reason we also tested for the presence of endothelial cells by using the more specific CD31 cell marker. Factor VIII data were included in this report because of its acceptance in the literature. The significant increase in factor VIII positive cell content (in relation to CD31 cell staining) in control grafts may be a manifestation of its lower specificity and reflect cross-reaction with other cell types. In stented grafts a significant increase in smooth muscle cell content was observed at the distal anastomosis, whereas IMHR remained unchanged. Differences in intimal thickness and smooth muscle cell content may be attributable to cell migration, as well as changes in extracellular matrix volume. An increase in cellular concentration in the presence of diminished or unchanged lesion thickness suggests a reduction of ex-
Surgery Volume 120, Number 1
tracellular m a t r i x v o l u m e has o c c u r r e d . R e d u c e d peria n a s t o m o t i c synthetic a n d m e t a b o l i c activity m a y be s u p p o r t e d by t h e persistently low m a c r o p h a g e c o n t e n t , in a d d i t i o n to the o b s e r v e d d e c r e a s e in actively proliferating cells, as d e t e r m i n e d by P C N A staining. A l t h o u g h a low, stable m a c r o p h a g e c o n t e n t is o b s e r v e d in stented grafts, a significant effiux o f m a c r o p h a g e s is i d e n t i f i e d in c o n t r o l grafts by 8 weeks. T h e cause a n d significance o f t h e shift in c o n t r o l m a c r o p h a g e c o n t e n t are u n c e r tain. O n the basis o f the i m m u n o h i s t o c h e m i c a l c h a n g e s in s m o o t h muscle, m a c r o p h a g e , a n d p r o l i f e r a t i n g cell c o n t e n t in this m o d e l , i n t r a l u m i n a l grafts a p p e a r to be associated with a q u i e s c e n t m e t a b o l i c state, suggestive o f c o m p l e t e healing. O n e c o n c e r n with any a n i m a l study is the applicability o f e x p e r i m e n t a l l y o b s e r v e d p h e n o m e n a to the clinical arena. A n i m a l s d o n o t typically d e v e l o p atherosclerosis, n o r are grafts p l a c e d in n o r m a l vessels. N o r m a l vessels w e r e p u r p o s e l y c h o s e n to assess the effect o f the e x t e r n a l graft e n v i r o n m e n t o n prosthesis healing. Alt h o u g h o p i n i o n s vary r e g a r d i n g the m o s t a p p r o p r i a t e m o d e l for studying p r o s t h e t i c graft healing, we conc l u d e d that a m o d e r a t e - s i z e d PTFE graft p l a c e d in a d o g for 4 to 8 weeks simulates the characteristic, n o n h e a l e d state f o u n d in h u m a n beings, z' 9-11,18 A s t a n d a r d porosity P T F E graft was u s e d because it is the m o s t clinically relevant. In this study i n t r a l u m i n a l location e n h a n c e s graft h e a l i n g a n d suggests that t h e role o f the e x t e r n a l graft e n v i r o n m e n t m a y be i m p o r t a n t . T h e m e c h a n i s m s r e s p o n s i b l e for e n h a n c e d e n d o t h e l i a l cell ingrowth, r e d u c e d intimal thickness, a n d t h e b e h a v i o r o f intral u m i n a l grafts in diseased vessels r e m a i n to b e determ i n e d . A l t h o u g h e n d o t h e l i a l d a m a g e occurs in r e g i o n s o f c o m p l e x a t h e r o s c l e r o t i c plaques, r e c e n t investigations suggest that m a n y vascular lesions o c c u r in the p r e s e n c e o f an intact e n d o t h e l i a l cell layer. E n d o t h e l i a l cell c o n t a c t with the i n t r a l u m i n a l graft m a y be a significant factor affecting graft healing. A l t h o u g h the longt e r m results o f i n t r a l u m i n a l s t e n t e d grafts in h u m a n beings is u n d e f i n e d , in a d o g m o d e l significant graft heali n g o c c u r s as the result o f an intravascular location. We thank W. L. Gore & Associates, Inc, for the PTFE graft material, Johnson & Johnson Interventional Systems for the Palmaz stents, and Boston Scientific Corp. for the balloon catheters. We also thank Dorcas Schaeffer, DVM, MS, and Sonia Doss for technical assistance with the animals,Jeni Scirrotta for specimen processing, Dr. Esteban Walker for statistical analysis, and Paride Ombrellaro for the illustrations.
REFERENCES
1. Clowes AW, Gown AM, Hanson SR, Reidy MA. Mechanisms of arterial g'raft failure: 1. Role of cellular proliferation in early healing of PTFE prostheses. Am J Pathol 1985;118:43-54. 2. Berger K, Sauvage LR, Rao AM, Wood SJ. Healing of arterial
OmbreUaro et al.
69
prostheses in man: its incompleteness. Ann Surg 1972;175:11827. 3. Schmidt SP, Hunter TJ, Sharp WV, Malindzak GS, Evancho MM. Endothelial cell-seeded four-millimeter Dacron vascular grafts: effects of blood flow manipulation through the grafts.J Vasc Surg 1984;1:434-41. 4. Douville EC, Kempczinski RF, Bitinyi LK, Ramalanjaona GR. hnpact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft. J Vasc Surg 1987;5:544-50. 5. Herring M, Smith J, Dalsing M, et al. Endothelial seeding of polytetrafluoroethylene femoral popliteal bypasses: the failure of low-density seeding to improve patency. J Vasc Surg 1994;20: 650-5. 6. PalmazJC. Balloon-expandable intravascular stent. AJR 1988;150: 1263-9. 7. Katzen BT, Becker GJ. Intravascular stents. Surg Clin North Am 1992;72:941-57. 8. Balko A, Piasecki GJ, Shah DM, et al. Transfemoral placement of intraluminal polyurethane prosthesis for abdominal aortic aneurysm. J Surg Res 1986;40:305-9. 9. Lawrence DD Jr, Charnsangavej C, Wright KC, Gianturco C, Wallace S. Percutaneous endovascular graft: experimental evaluation. Radiology 1987;163:357-60. 10. Mirich D, Wright KC, Wallace S, et al. Percutaneously placed endovascular grafts for aortic aneurysms: feasibilitystudy. Radiolo~,y 1989;170:1033-7. 11. Laborde JC, Parodi JC, Clem MF, et al. Intraluminal bypass of abdominal aortic aneurysm: feasibility study. Radiology 1992; 184:185-90. 12. parodi JC, Palmaz JC, Barone HD. Transfemoral intraluminal graft implantation for abdominal aortic aneurysms. Ann Vasc Surg 1991;5:491-9. 13. Lazarus HM. Endovascular grafting for the treatment of abdominal aortic aneurysms. Surg Clin North Am 1992;72:959-68. 14. Chuter TAM, Green RM, Ouriel K, Fiore WM, DeWeese JA. Transfemoral endovascular aortic graft placement. J Vasc Surg 1993;18:185-97. 15. May J, White G, Waugh R, Yu W, Harris J. Transluminal placement of a prosthetic graft-stent device for treatment of subclavian artery aneurysm. J Vasc Surg 1993;18:1056-9. 16. Matin ML, Veith FJ, Panetta TF, et al. Transfemoral endoluminal stented graft repair of a popliteal aneurysm. J Vasc Surg 1994;19:754-7. l 7. Moore WS, Vescera CL. Repair of abdominal aortic aneurysm by transfemoral endovascular graft placement. Ann Surg 1994;220: 33141. 18. Sauvage LR, Berger KE, Wood SJ, et al. Interspecies healing of porous arterial prostheses: observations, 1960-1974. Arch Surg 1974;109:698-705. 19. Ojha M, Cobbold RSC,Johnston KW. Hemodynamics of a sideto-end proximal arterial anastomosis model. J Vasc Surg 1993;17: 646-55. 20. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med 1994;330:1431-8. 21. Galt SW, Zwolak RM, Wagner RJ, Gilbertson .[J. Differentia][ response of arteries and vein grafts to blood flow reduction.J Vasc Surg 1993;17:563-70. 22. Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and ath-. erosclerosis in the human carotid bifurcation: positive correlation between plaque location and low and oscillating shear stress. Arteriosclerosis 1985;5:293-302. 23. Zarins CK, Giddens DP, Bharadvaj BK, Sottiural VS, Mabron RF, Glagov S. Carotid bifurcation atherosclerosis: quantitative correlation of plaque localization with flow velocity profiles and wall shear stress. Circ Res 1983;53:502-14.
70 Ombrellaro et al.
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24. Kamiya A, Togawa T. Adaptive regadation of wall shear stress to flow change in the canine carotid artery. AmJ Physiol 1980;239: H14-21. 25. Sterpetti AV, Cucina A, D'Angelo LS, Cardillo B, Cavallaro A. Shear stress modulates the proliferation rate, protein synthesis, and rnitogenic activity of arterial smooth muscle cells. Surgery 1993;113:691-9. 26. Morinaga K, Okadome K, Kuroki M, et al. Effect of wall shear stress on intimal thickening of arterially transplanted autogenous veins in dogs. J Vasc Surg 1985;2:430-3. 27. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-9.
O
28. Sterpetti AV, Hunter WJ, Schultz RD, Farina C. Healing of high-porosity polytetrafluoroethylene arterial grafts is influenced by the nature of the surrounding tissue. Surgery 1992;111: 677-82. 29. Clowes AW, Kirkman TR, Reidy MA. Mechanisms of arterial graft healing: rapid transmural capillary ingrowth provides a source of endothelium and smooth muscle in porous PTFE prostheses. Am J Pathol 1986;123:220-30. 30. Shi Q, Wu MH, Hayashida N, Wechezak AR, Clowes AW, Sauvage LR. Proof of fallout endothelialization of impervious Dacron grafts in the aorta and inferior vena cava of the dog. J Vasc Surg 1994;20:546-57.
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Background. The purpose of this study was to investigate the effect of complete intraluminal placement on prosthetic graft healing. Methods. Thirty dogs underwent infrarenal abdominal aorta polytetrafluoroethylene interposition (12) or intraluminal stented (18) grafting. Grafts were removed at 4 and 8 weeks. Length of endothelial ingrowth and intima to media height ratios (IMHRs) were calculated. Pevianastomotic endothelial (CD31+, factor VIII [FVIII+]), smooth muscle (actin+), macrophage (CD44+), and proliferating (PCNA +) cell content was determined. Results. In control grafts mean proximal and distal anastomotic endothelial cell ingrowth was 0.42 +- 0.06 and 0.47 +- 0.08 cm at 4 weeks and 1.10 +- 0.24 and 0.94 +- 0.17 cm at 8 weeks. In intraluminal grafts mean proximal and distal anastomotic endothelial cell ingrowth was 1.5 7 +- O.09 and 1.54 +- 0.12 cm at 4 weeks and 1.88 +-- 0.06 and 2.11 +_ 0.25 cm at 8 weeks. Endothelial ingrowth was greater in all stented grafts (p < 0.001). Mean proximal anastomosis IMHRs were 1.01 +- O.16 for 4-week and 1.42 + O.16 for 8-week control grafts and 0.59 +_ O.18 for 4-week and 0.50 +- 0.14 for 8-week stented grafts. Similar I M H R values were present at the distal anastomosis. Lower IMHRs were observed in stented grafts (p < 0.05). Content of C~44+, PCNA+, and FVIII+ cells were reduced both proximally and distally in 4-week stented grafts (p < 0.05). Distal content of C~31+ and actin+ cells was greater in 4-week stented grafts (p < O. 05). At 8 weeks C~44+ cell content decreased in controls (p < O.05). Conclusions. Intraluminal location enhances prosthetic graft reendothelialization and attenuates intimal thickening. (Surgery 1996;120:60-70.) From the Department of Surgery, Division of Vascular Surgery, University of Tennessee Medical Center, Knoxville, Tenn.
COMPLETE REPOPULATION OF A prosthetic graft surface with a functional endothelial cell layer has yet to be exhibited in h u m a n beings. Endothelial cells provide a n o n t h r o m b o g e n i c a n d antiinflammatory barrier between circulating proteins, coagulation factors, cellular elements, a n d the artificial biomaterial. 1' 2 In addition, they also play a regulatory role, interacting with smooth muscle cells, macrophages, platelets, a n d other vascular wall cellular constituents during acute and subacute vascular injury. Although it has b e e n suggested that reendothelialization would improve prosthetic graft patency, this has yet to be proved clinically.~-5
Supported by the Physicians'Medical Education and Research Foundation (PMERF), Knoxville,Tenn. Accepted for publication Oct. 6, 1995. Reprint requests: Mark Ornbrellaro, MD, Department of Surgery, Division of Vascular Surgery, Universityof Tennessee Medical CenterKnoxville, 1924 Alcoa Hwy, Knoxville,TN 37920. Copyright 9 1996 by Mosby-YearBook, Inc. 0039-6060/96/$5.00 + 0 11/56/71946 60
SURGERY
Refinements in endovascular techniques using stent and balloon catheter technology have lead to the evolution o f an intraluminal stented bypass graft. Whereas the healing characteristics of the various individual c o m p o n e n t s of stented grafts have b e e n described, those of the complete intraluminal device have yet to be defined. Intravascular stents, i m p o r t a n t adjuncts for the treatment o f postangioplasty intimal dissection a n d failures caused by elastic recoil, are characteristically resurfaced with endothelium. 6, 7 Because stents are placed in conjunction with angioplasty procedures, they are often associated with intimal hyperplasia. Intravascular stented grafts offer several theoretic advantages for enh a n c e d graft healing a n d r e d u c e d intimal hyperplasia including wide anastomoses, an in-line graft configuration, a n d direct contact with an endothelialized surface. Although the feasibility o f endovascular grafting has b e e n reported, the healing characteristics o f this type of graft a n d effect o f the intravascular location have not. 8-17 We have investigated the influence o f an intraluminal e n v i r o n m e n t on prosthetic graft healing a n d the cellu-
Surgery Volume 120, Number 1
lar composition of the perianastomotic intimal region by using intraarterial stented grafts.
METHOD Thirty adult male and female h o u n d dogs, weighing between 17 and 32 kg (average weight, 24.1 kg), were used for this study. Animals were cared for in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of LaboratoryAnimals" (National Institutes of Health publication no. 80-23, revised 1985). Each animal was premedicated with an intramuscular dose of acepromazine (0.022 m g / k g ) , atropine (0.044 m g / k g ) , and butorphanol (0.44 m g / k g ) , anesthetized with intravenous thiopental sodium (13 m g / k g ) , intubated, and maintained on a mixture of isofluorane (1.5 to 3.5 m i n i m u m alveolar concentration) and 100% oxygen (1 L / m i n ) via a semiclosed anesthesia circuit. Antibiotic prophylaxis with 500 mg intravenous cefazolin was administered 30 minutes before and 90 minutes after skin incision. Intravenous lactated Ringer's solution was given as required to maintain circulating blood volume. The control group consisted of 12 dogs who underwent standard interposition grafting of the infrarenal abdominal aorta. The aorta was exposed through a left lower quadrant incision and retroperitoneal approach from the renal arteries to the aortic trifurcation. A 2 cm segment of aorta was circumferentially dissected both proximally and distally, and the intervening central aortic segment was excluded with two ligatures of 0-0 polydioxanone sutures (Ethicon, Inc., Somerville, N.J.). Vascular clamps were applied, and the aorta was transected between each set of clamp and excluding ligature. Aortic continuity was restored with a 6 cm interposition graft of 10 m m diameter, thin-walled (30 p internodal distance with outer reinforcing wrap removed) polytetrafluoroethylene (PTFE, Gore-Tex; W. L. Gore & Associates, Flagstaff, Ariz.). Normal aortic diameters were previously measured in similar size hounds undergoing intraabdominal surgery. Diameters ranged from 9 to 12 mm, and on the basis of these findings a 10 m m diameter PTFE graft was chosen for this study. Anastomoses were constructed in an end-to-end fashion by using a continuous CV-6 PTFE suture under 2.5x loupe magnification. The experimental group consisted of 18 dogs who underwent intraluminal stented grafting of the infrarenal abdominal aorta. Stented grafts were constructed by securing the midportion of a 3.4 cm Palmaz balloon expandable stent (P 308; Johnson & J o h n s o n Interventional Systems, Warren, N.J.) to the proximal end of a 6 cm length of the previously described PTFE graft material by using a single CV-6 PTFE suture. In all cases 50% of the stent surface was overlapped by the PTFE graft. A pushing tube (14F outer diameter rigid plastic
OmbreUaro et al.
61
tube attached to a hemostatic valve) and the stent graft complex were both loaded on a 5.8F balloon catheter (10 m m x 4 cm, Meditech; Boston Scientific, Watertown, Mass.) and placed into a 14F delivery sheath (Check-Flo II; Cook, Bloomington, Ind.) (Fig. 1). Arterial access was obtained via a left c o m m o n femoral artery cutdown. Fluoroangiography was routinely performed to delineate the location of the renal arteries and aortic trifurcation. A guide wire was passed, and tile stented graft delivery system was advanced over the wire into the infrarenal abdominal aorta. Stented grafts were deployed by ejecting the graft from the sheath by use of the pushing tube and balloon expansion of the stent. Straightening and expansion of the graft body were achieved by means of additional balloon inflations and gende traction of a partially inflated balloon inside the graft. No stents were used to secure the distal aspect of the graft. All grafts were placed without heparinization. Completion aortogram confirmed proper intraluminal graft placement and no evidence of perigraft leakage. The left c o m m o n femoral artery was ligated after stented graft deployment. Postoperative graft patency was monitored by means of daily femoral pulse palpation in both the control and experimental animals. Antiplatelet agents and anticoagulants were not administered. Animals in each group were killed at 4 and 8 weeks after grafting. At the time of death, each animal was reanesthetized and given an intravenous dose of heparin (3000 units) and Evans blue (30 m g / k g ) . The aorta and graft were exposed through a midline incision, and aortography was performed through a proximal aortic needle puncture. Lumbar and inferior mesenteric artery orifices covered by the intraluminal graft were absent on angiogram with no evidence of perigraft leakage. Both the interposition and intraluminal grafts remained relatively well matched to the native aortic size. When compared with normal aortic segments above and below intraluminally placed stented grafts, no external evidence of aortic distention related to graft deployment was noted. The infrarenal aorta was isolated, and perfusion cannulas were inserted retrograde through both c o m m o n iliac arteries to the aortic trifurcation. The aorta was flushed with normal saline solution and fixed for 5 minutes with a solution of 2% paraformaldehyde and 0.5% gluteraldehyde at 100 m m H g pressure after a lethal dose of intravenous pentobarbital sodium (1 m g / 4 . 5 kg Beuthanasia D; ScheringPlough, Kenilworth, N.J.). The aortic specimens were removed, o p e n e d longitudinally, photographed enface, and divided into two (longitudinal) segments (Fig. 2). Gross examination showed no evidence of h e m a t o m a between the aorta and stented intraluminal grafts in either early or late specimens. One segment was fixed in 2.5% gluteralde-
62
Ombrellaro et al.
Surgery July 1996
> >
/A It
b
Fig. 1. a, Intraluminal stented graft; b, diagram of intraluminal stent graft delivery device.
hyde for 24 hours and processed for electron microscopy, and the other was fixed in zinc formalin (Z-fix; Anatech Ltd, Battle Creek, Mich.) and processed for light microscopy and immunohistochemical studies. Representative sections of the proximal and distal anastomotic and mid-graft regions were dehydrated, critical point dried, mounted by using colloidal graphite, grounded with silver paint, and vacuum coated with gold palladium for scanning electron microscopy. The zinc formalin-fixed tissue was bisected longitudinally, and one strip was embedded in paraffin and the other in methyl methacrylate plastic. One set of specimens in each type of embedding medium was required so that cell epitopes would be treated uniformly when processed for immunohistochemistry. Standard immunohistochemical staining techniques are typically performed on paraffin-embedded tissue. One potential problem, however, is that grafts containing metallic stents cannot be cut or processed in this fashion. Before stented graft specimens were embedded in paraffin, stents were carefully dissected from the graft material under magnification. Immunohistochemical staining for endothelial cell surface antigens CD-31 (Dako Corp., Carpinteria, Calif.) and yon Willibrand
factor VIII (BioGenex, San Ramon, Calif.), smooth muscle cell ed-actin (Dako Corp.), macrophage surface antigen CD44 (PharMingen, San Diego, Calif.), and proliferating cell nuclear antigen (PCNA) (Dako Corp.) was performed. To assure antibody staining was specific to the particular cell type of interest, all antibodies were tested with both positive and negative control tissues. Immunoantibodies were visualized by using an alkaline phosphatase chromagen developing kit (BioGenex). The purpose of embedding stented grafts in methylmethacrylate plastic was to preserve the in situ relationships of the artery, graft, and stent materials. Whereas grafts embedded in methylmethacrylate can be cut and processed without the need for stent removal, immunohistochemical staining of cellular antigens from soft tissue embedded in methylmethacrylate has yet to be described. Immunohistochemical staining techniques on methylmethacrylate embedded tissue are being performed in our laboratory, but additional testing is required for standardization. In this study methylmethacrylate specimens were used to confirm histologic architecture, whereas immunohistochemical comparisons between control and experimental grafts were made by using paraffin-embedded tissue samples only.
Surgery Volume 120, Number I
OmbreUaro et al.
63
Fig. 2. Freshly excised 8-week control (A) and intraluminal stented (B) graft after administration of Evans blue dye. Black arrows delineate PTFE-artery interface. White arrows delineate interface between reendothelialized and unhealed surfaces. Enhanced reendothelializafion is apparent in intraluminal stented graft. To quantify endothelial cell ingrowth, specimen photographs (including scale markers) were traced by using a digitized drawing board and stored on an Apple IIci computer (Apple Computer Inc., Cupertino, Calif.). Graft area, anastomotic width, and area of endothelial cell resurfacing were measured by using Evans blue stain exclusion and IP Lab Spectrum 2.5 image analysis software (Signal Analytics Corp., Vienna, Va.). To control for differences in specimen diameter, area of endothelial cell ingrowth was divided by vessel width and reported as average length of cell ingrowth for each anastomosis. Intimal height for the proximal and distal perianastomotic (artery-graft interface) regions of each control and stented graft was calculated by averaging two measurements from the point of maximal intimal thickness overlying the PTFE graft in each region. To control for variation in intimal thickness related to specimen sectioning in different tangential planes, intireal height was related to medial height in each section. Medial thickness was measured on the arterial side of each anastomosis in a uniform region adjacent to the PTFE-artery interface. The ratio of maximum intimal to medial thickness (IMHR) was used to quantify intimal
hyperplasia in each graft perianastomotic region. All measurements were performed with the IP Lab Spectrum program on digital images of histologic specimens at 250x magnification acquired by a videomicroscope. Lesion composition was determined by counting the number of CD31 and factor VIII (endothelial), CD44 (macrophage), ~-actin (smooth muscle), and PCNA (proliferative) positive staining cells in three different high-power fields at 1000x magnification. Statistical analysis was performed in consultation with the statistics department at the University of Tennessee by using the JMP 3.0 statistical program (SAS Institute, Cary, N.C.). Mean value comparisons between control and experimental groups were performed with a two-tailed Student's t test and Wilcoxon rank sums test. A p value of <0.05 was considered significant. Numeric values are reported as a mean value _+ the standard error (SEM). RESULTS Three animals in the experimental group died within 48 hours of stented graft placement. Two deaths were attributable to technical complications including an aortic dissection with perforation during delivery system
64
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Surgery July 1996
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Fig. 3. Mean length of endothelial cell ingrowth at proximal (A) a n d distal (B) anastomoses. Endothelial cell ingrowth is significantly greater in stent grafts, compared with controls, in each anastomotic region at both 4 a n d 8 weeks (p < 0.001). Error bars denote standard error of mean. passage a n d bilateral r e n a l artery occlusions f r o m inaccurate graft p l a c e m e n t . O n e a n i m a l was f o u n d d e a d o f a n u n d e t e r m i n e d cause in its cage 12 h o u r s after operation. All r e m a i n i n g grafts in the c o n t r o l a n d experim e n t a l g r o u p s were p a t e n t at the time o f d e a t h Perianastomotic endothelial cell ingrowth. Average l e n g t h of e n d o t h e l i a l cell ingrowth was calculated for the p r o x i m a l a n d distal anastomoses in each g r o u p (Fig. 3). I n 4-week c o n t r o l grafts (n = 6) e n d o t h e l i a l cell ingrowth was 0.42 +- 0.06 c m at the p r o x i m a l anastomosis a n d 0.47 +_ 0.08 c m at the distal anastomosis. I n 4-week i n t r a l u m i n a l grafts (n = 7) e n d o t h e l i a l cell ingrowth was 1.57 _+ 0.09 c m at the p r o x i m a l anastomosis a n d 1.54 -+ 0.12 c m at the distal anastomosis. At 8 weeks endothelial cell ingrowth i n c r e a s e d b o t h proximally a n d distally to 1.10 _+ 0.24 c m a n d 0.94 -+ 0.17 cm, respectively, in c o n t r o l grafts ( n = 6 ) ) . E n d o t h e l i a l cell in-
growth in 8-week i n t r a l u m i n a l grafts ( n = 8 ) were 1.88 + 0.06 c m for the p r o x i m a l a n d 2.11 + 0.25 c m for the distal a n a s t o m o t i c regions. At each o f 4 a n d 8 weeks a significant increase in e n d o t h e l i a l cell i n g r o w t h was e v i d e n t in s t e n t e d grafts at b o t h anastomotic sites ( p < 0 . 0 0 1 ) . W i t h i n each c o n t r o l a n d e x p e r i m e n t a l g r o u p n o difference in e n d o t h e l i a l cell ingrowth was identified b e t w e e n p r o x i m a l a n d distal anastamotic regions. I n all regions o f Evans b l u e dye exclusion, e n d o thelial cells were c o n f i r m e d with s c a n n i n g electron microscopy (Figs. 4 a n d 5). Characterization o f i n t i m a l thickness. I M H R was u s e d to quantify the i n t i m a l hyperplastic r e s p o n s e to each type o f graft (Fig. 6). I n 4-week c o n t r o l grafts the I M H R w a s 1 . 0 1 -+ 0.16 at the p r o x i m a l anastomoses a n d 0.90 - 0.12 at the distal anastomosis. I n 4-week intralum i n a l grafts the I M H R was 0.59 -+ 0.18 at the p r o x i m a l
Surgery Volume 120, Number 1
Ombrellaro et al.
65
Fig. 4. Scanning electron micrograph of 4-week control graft at proximal anastomosis. A, Endothelial cells along proximal graft surface; original magnification • B, Transition zone shows advancing edge of endothelial cells (arrows); original magnification • C, Unhealed central graft surface. Fibrin deposition and lack of endothelial cells are apparent along PTFE surface; original magnification x480.
Fig. 5. Scanning electron micrographs of 4-week intraluminal stented graft at proximal anastomosis. Images are from same relative position along stent arm with increasing magnification. Reendothelialized surface is shown over both the stent and PTFE graft materials, a, Original magnification x45; b, original magnification x90; C, original magnification •
and 0.47 _+ 0.13 at the distal anastomosis. At 8 weeks control IMHRs were 1.42 + 0.16 for the proximal and 0.84 + 0.12 for the distal anastomoses, respectively. IMHRs in 8-week intraluminal grafts were 0.50 _+ 0.14 for proximal and 0.42 -+ 0.1 for distal anastomotic regions. The intimal hyperplastic response at the proximal anastomosis was lower in stented grafts when compared with controls. At 8 weeks the difference achieved statistical significance (p < 0.001). At the distal anastomosis intireal thickness was significantly less in intraluminal stented grafts at both 4 and 8 weeks (p < 0.05). Lesion composition. Immunohistochemical stains for specific cell surface and intracellular antigens were used to characterize intimal lesion evolution and composition and to quantitate the various cellular constituents along control and stented grafts. Factor VIII and the more specific CD31 antigens were used to identify
endothelial cells (Fig. 7). Smooth muscle cells and macrophages were identified by using antibodies against ~l-actin and CD44, respectively (Fig. 8). Proliferating cells were quantified by using antibodies against PCNA. Mean counts for each cell type are reported for proximal and distal anastomotic sites (Table). The quantity of endothelial cells as identified by CD31 staining was greater in stented grafts, when cornpared with controls, at the 4-week distal and both 8-week anastomotic sites (p< 0.001). Factor VIII positive ce.ll content was significantly higher in control grafts at all sites and time intervals except at the 8-week distal anastomosis (p < 0.02). At the proximal anastomosis no difference in smooth muscle cell content was noted between control and experimental grafts at any time. At the distal anastomosis smooth muscle cell content was greater in stented grafts at both time intervals (p < 0.01).
66
Ombrellaro et al,
Surgery July 1996
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Fig. 6. Mean IMHRs at proximal (A) and distal (B) anastomoses. At 8 weeks intimal thickness was significantly lower in stented grafts at proximal anastomosis (p < 0.001). At 4 weeks this difference was not statistically significant, lnfimal thickness was significantly lower in stented graft group, when compared with controls, at both 4 and 8 weeks at distal anastomosis (p < 0.05). Error bars denote standard error of mean.
At 4weeks the macrophage content in each anastomotic region was lower in intravascular graft specimens when compared with controls (p < 0,0005). By 8 weeks macrophage content remained stable in stented grafts and a significant macrophage efflux occurred in control s. As a result of the decline in control macrophage content, macrophages were more prevalent in stented graft specimens at 8 weeks. At the distal anastomosis this difference in 8-week macrophage content achieved statistical significance (p < 0.003). The n u m b e r of actively proliferating cells (PCNA positive) were significantly less in stented grafts at each anastomosis at up to 8 weeks (p < 0.05).
DISCUSSION The inability of h u m a n beings to repave a prosthetic graft luminal surface with functional endothelial cells remains an unexplained p h e n o m e n o n . After implancation a perianastomotic intimal accumulation of smooth muscle cells, extracellular matrix, and endothelial cells occurs that typically extends I to 2 cm beyond the suture line onto the graft surface. 2 Beyond this region the graft is laminated with a pseudointima of platelets, red blood cells, and compacted fibrin and is unhealed. Furthermore, a variety of animal species exhibit a great propensity to heal prosthetic grafts, but the mechanisms responsible for these observed differ-
Surgery Volume 120, Number 1
Ombrellaro et al.
67
Fig. 7. Composite p h o t o m i c r o g r a p h shows reendothelialization of proximal, mid-graft, and distal anastomotic sites along control and stented grafts at 8 weeks. Arrows denote cells along luminal surface staining positive for endothelial cell CD31 antigens. Mid-graft region is layered with endothelial cells in intraluminal grafts and fibrin and debris in controls. CD 31 antibodies with alkaline phosphatase chromagen; original magnification x400
T a b l e . P e r i a n a s t o m o t i c cell counts (cells p e r h i g h - p o w e r field; x l 0 0 0 m a g n i f i c a t i o n )
Antigen Endothelial cells Group
CD 31
A (4-wk control) (n = 15) PA 10 • 0.6 DA 8 • 0.5 B (4-wk stented graft) (n = 12) PA 10 • 0.6 DA 11 • 0.6* C (8-wk control) (n = 18) PA 8 + 0.6 DA 7 + 0.5 D (8-wk stented graft) (n : 24 PA 11 • 0.5]DA 10 • OAt
Macrophage
Factor VIII
SMC actin
11 _+ 0.5 11 _+ 0.6 7 _+ 0,7* 7 • 0.8* I2 +- 0.5
10• 7 -+ 0.5t 9-+0.6
CD44
PCNA
108 • 8 88 + 6
23 • 2 32 • 4
30 -+ 4.5 21 +- 2.5
129 • 9 108 • 7*
10 • 2* 9 • 2*
126 • 7 108•
12 -+ 1.5 7-+ 1
121 + 6 146•
9 • 1.5 10-+ 1t
8 • 5* 9 • 2.5* 20 -+ 4 11 • 7 _+ 3.5t 7_+2t
Data shown as mean -+ standard error.
PA, Proximal anastomosis; DA, distal anastomosis; SMC, smooth muscle cells. *p < 0.05 compared with group A. tP < 0.05 compared with group C.
e n c e s are u n k n o w n , z' 18 It is h y p o t h e s i z e d that an intact, i n t r a l u m i n a l e n d o t h e l i a l cell layer is i m p o r t a n t in p r e v e n t i n g graft failure. E n d o t h e l i a l cell d a m a g e o r dysfunction f r o m the bypass p r o c e d u r e itself o r indirectly t h r o u g h the effects o f graft material, shear stress, a n d c o m p l i a n c e m i s m a t c h o n h e m o d y n a m i c s can
c o n t r i b u t e to a m i c r o e n v i r o n m e n t o f c h r o n i c arterial injury a n d i n d u c e p e r i a n a s t o m o t i c intimal hyper-plasia. 1'1926 T h e m e t a b o l i c r e s p o n s e o f m o n o c y t e s , m a c r o p h a g e s , T lymphocytes, a n d s m o o t h m u s c l e cells to a c h r o n i c injury stimulus f u r t h e r p e r p e t u a t e s this process.20, 27
68
Ombrellaro et al.
Surgery July 1996
Fig. 8. Composite photomicrograph shows smooth muscle cell (SMC) content of proximal, mid-graft, and distal anastomotic sites along control and stented grafts at 8 weeks. Mid-graft region appears covered with thin SMC layer in intraluminal grafts, whereas fibrin and debris are present in controls. SMC ix-1 actin antibodies with alkaline phosphatase chromagen; original magnification x400.
Whereas the internal graft environment may affect patency, the importance of the external graft environment and its potential role in graft healing are now being appreciated. Recent animal investigations have shown enhanced endothelial cell deposition and capillary ingrowth within PTFE arterial grafts wrapped with omentum. 2s Because endothelial cell ingrowth is characteristically limited to within 1 to 2 cm of the anastomotic regions of grafts placed in a nonendothelialized environment, placing these conduits in a milieu with a high concentration of endothelial cells may improve graft healing. Sources of endothelial cells for in situ graft resurfacing include the native vessel in contact with the ends of the graft, capillary ingrowth through the graft interstices, and circulating endothelial microemboli.1, 2, 29, a0 The relative contribution to graft resurfacing and clinical significance of each mechanism to graft reendothelialization remains undefined. The concept of intraluminal placement of an arterial bypass conduit is a recent development made possible by the introduction of stented grafts. Early animal investigations have described the technique, as applied to aortic aneurysm repair, and the presence of endothelial-like cells along the intraluminal surface, alluding to a healed graft. 8-n These devices make it possible to study the relationship between external graft environm e n t and prosthetic graft healing with regard to the specific effects of a completely intraarterial location.
In our study intraluminal prosthetic (stented) grafts were associated with a significant increase in graft surface reendothelialization and decrease in perianastc~ motic intimal hyperplastic response when compared with interposition control grafts. The presence of endothelial cells along most of the stented graft surface, including the central graft region, was shown by use of scanning electron microscopy and, unequivocally, by using antibodies against endothelial cell specific CD31 surface antigens. Although yon Willibrand factor VIII surface antigen is often used to identify endothelial cells, these epitopes are also present on fibroblasts and other cell types. For this reason we also tested for the presence of endothelial cells by using the more specific CD31 cell marker. Factor VIII data were included in this report because of its acceptance in the literature. The significant increase in factor VIII positive cell content (in relation to CD31 cell staining) in control grafts may be a manifestation of its lower specificity and reflect cross-reaction with other cell types. In stented grafts a significant increase in smooth muscle cell content was observed at the distal anastomosis, whereas IMHR remained unchanged. Differences in intimal thickness and smooth muscle cell content may be attributable to cell migration, as well as changes in extracellular matrix volume. An increase in cellular concentration in the presence of diminished or unchanged lesion thickness suggests a reduction of ex-
Surgery Volume 120, Number 1
tracellular m a t r i x v o l u m e has o c c u r r e d . R e d u c e d peria n a s t o m o t i c synthetic a n d m e t a b o l i c activity m a y be s u p p o r t e d by t h e persistently low m a c r o p h a g e c o n t e n t , in a d d i t i o n to the o b s e r v e d d e c r e a s e in actively proliferating cells, as d e t e r m i n e d by P C N A staining. A l t h o u g h a low, stable m a c r o p h a g e c o n t e n t is o b s e r v e d in stented grafts, a significant effiux o f m a c r o p h a g e s is i d e n t i f i e d in c o n t r o l grafts by 8 weeks. T h e cause a n d significance o f t h e shift in c o n t r o l m a c r o p h a g e c o n t e n t are u n c e r tain. O n the basis o f the i m m u n o h i s t o c h e m i c a l c h a n g e s in s m o o t h muscle, m a c r o p h a g e , a n d p r o l i f e r a t i n g cell c o n t e n t in this m o d e l , i n t r a l u m i n a l grafts a p p e a r to be associated with a q u i e s c e n t m e t a b o l i c state, suggestive o f c o m p l e t e healing. O n e c o n c e r n with any a n i m a l study is the applicability o f e x p e r i m e n t a l l y o b s e r v e d p h e n o m e n a to the clinical arena. A n i m a l s d o n o t typically d e v e l o p atherosclerosis, n o r are grafts p l a c e d in n o r m a l vessels. N o r m a l vessels w e r e p u r p o s e l y c h o s e n to assess the effect o f the e x t e r n a l graft e n v i r o n m e n t o n prosthesis healing. Alt h o u g h o p i n i o n s vary r e g a r d i n g the m o s t a p p r o p r i a t e m o d e l for studying p r o s t h e t i c graft healing, we conc l u d e d that a m o d e r a t e - s i z e d PTFE graft p l a c e d in a d o g for 4 to 8 weeks simulates the characteristic, n o n h e a l e d state f o u n d in h u m a n beings, z' 9-11,18 A s t a n d a r d porosity P T F E graft was u s e d because it is the m o s t clinically relevant. In this study i n t r a l u m i n a l location e n h a n c e s graft h e a l i n g a n d suggests that t h e role o f the e x t e r n a l graft e n v i r o n m e n t m a y be i m p o r t a n t . T h e m e c h a n i s m s r e s p o n s i b l e for e n h a n c e d e n d o t h e l i a l cell ingrowth, r e d u c e d intimal thickness, a n d t h e b e h a v i o r o f intral u m i n a l grafts in diseased vessels r e m a i n to b e determ i n e d . A l t h o u g h e n d o t h e l i a l d a m a g e occurs in r e g i o n s o f c o m p l e x a t h e r o s c l e r o t i c plaques, r e c e n t investigations suggest that m a n y vascular lesions o c c u r in the p r e s e n c e o f an intact e n d o t h e l i a l cell layer. E n d o t h e l i a l cell c o n t a c t with the i n t r a l u m i n a l graft m a y be a significant factor affecting graft healing. A l t h o u g h the longt e r m results o f i n t r a l u m i n a l s t e n t e d grafts in h u m a n beings is u n d e f i n e d , in a d o g m o d e l significant graft heali n g o c c u r s as the result o f an intravascular location. We thank W. L. Gore & Associates, Inc, for the PTFE graft material, Johnson & Johnson Interventional Systems for the Palmaz stents, and Boston Scientific Corp. for the balloon catheters. We also thank Dorcas Schaeffer, DVM, MS, and Sonia Doss for technical assistance with the animals,Jeni Scirrotta for specimen processing, Dr. Esteban Walker for statistical analysis, and Paride Ombrellaro for the illustrations.
REFERENCES
1. Clowes AW, Gown AM, Hanson SR, Reidy MA. Mechanisms of arterial g'raft failure: 1. Role of cellular proliferation in early healing of PTFE prostheses. Am J Pathol 1985;118:43-54. 2. Berger K, Sauvage LR, Rao AM, Wood SJ. Healing of arterial
OmbreUaro et al.
69
prostheses in man: its incompleteness. Ann Surg 1972;175:11827. 3. Schmidt SP, Hunter TJ, Sharp WV, Malindzak GS, Evancho MM. Endothelial cell-seeded four-millimeter Dacron vascular grafts: effects of blood flow manipulation through the grafts.J Vasc Surg 1984;1:434-41. 4. Douville EC, Kempczinski RF, Bitinyi LK, Ramalanjaona GR. hnpact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft. J Vasc Surg 1987;5:544-50. 5. Herring M, Smith J, Dalsing M, et al. Endothelial seeding of polytetrafluoroethylene femoral popliteal bypasses: the failure of low-density seeding to improve patency. J Vasc Surg 1994;20: 650-5. 6. PalmazJC. Balloon-expandable intravascular stent. AJR 1988;150: 1263-9. 7. Katzen BT, Becker GJ. Intravascular stents. Surg Clin North Am 1992;72:941-57. 8. Balko A, Piasecki GJ, Shah DM, et al. Transfemoral placement of intraluminal polyurethane prosthesis for abdominal aortic aneurysm. J Surg Res 1986;40:305-9. 9. Lawrence DD Jr, Charnsangavej C, Wright KC, Gianturco C, Wallace S. Percutaneous endovascular graft: experimental evaluation. Radiology 1987;163:357-60. 10. Mirich D, Wright KC, Wallace S, et al. Percutaneously placed endovascular grafts for aortic aneurysms: feasibilitystudy. Radiolo~,y 1989;170:1033-7. 11. Laborde JC, Parodi JC, Clem MF, et al. Intraluminal bypass of abdominal aortic aneurysm: feasibility study. Radiology 1992; 184:185-90. 12. parodi JC, Palmaz JC, Barone HD. Transfemoral intraluminal graft implantation for abdominal aortic aneurysms. Ann Vasc Surg 1991;5:491-9. 13. Lazarus HM. Endovascular grafting for the treatment of abdominal aortic aneurysms. Surg Clin North Am 1992;72:959-68. 14. Chuter TAM, Green RM, Ouriel K, Fiore WM, DeWeese JA. Transfemoral endovascular aortic graft placement. J Vasc Surg 1993;18:185-97. 15. May J, White G, Waugh R, Yu W, Harris J. Transluminal placement of a prosthetic graft-stent device for treatment of subclavian artery aneurysm. J Vasc Surg 1993;18:1056-9. 16. Matin ML, Veith FJ, Panetta TF, et al. Transfemoral endoluminal stented graft repair of a popliteal aneurysm. J Vasc Surg 1994;19:754-7. l 7. Moore WS, Vescera CL. Repair of abdominal aortic aneurysm by transfemoral endovascular graft placement. Ann Surg 1994;220: 33141. 18. Sauvage LR, Berger KE, Wood SJ, et al. Interspecies healing of porous arterial prostheses: observations, 1960-1974. Arch Surg 1974;109:698-705. 19. Ojha M, Cobbold RSC,Johnston KW. Hemodynamics of a sideto-end proximal arterial anastomosis model. J Vasc Surg 1993;17: 646-55. 20. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med 1994;330:1431-8. 21. Galt SW, Zwolak RM, Wagner RJ, Gilbertson .[J. Differentia][ response of arteries and vein grafts to blood flow reduction.J Vasc Surg 1993;17:563-70. 22. Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and ath-. erosclerosis in the human carotid bifurcation: positive correlation between plaque location and low and oscillating shear stress. Arteriosclerosis 1985;5:293-302. 23. Zarins CK, Giddens DP, Bharadvaj BK, Sottiural VS, Mabron RF, Glagov S. Carotid bifurcation atherosclerosis: quantitative correlation of plaque localization with flow velocity profiles and wall shear stress. Circ Res 1983;53:502-14.
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O
28. Sterpetti AV, Hunter WJ, Schultz RD, Farina C. Healing of high-porosity polytetrafluoroethylene arterial grafts is influenced by the nature of the surrounding tissue. Surgery 1992;111: 677-82. 29. Clowes AW, Kirkman TR, Reidy MA. Mechanisms of arterial graft healing: rapid transmural capillary ingrowth provides a source of endothelium and smooth muscle in porous PTFE prostheses. Am J Pathol 1986;123:220-30. 30. Shi Q, Wu MH, Hayashida N, Wechezak AR, Clowes AW, Sauvage LR. Proof of fallout endothelialization of impervious Dacron grafts in the aorta and inferior vena cava of the dog. J Vasc Surg 1994;20:546-57.
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