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The Effect of Flow Shear Stress on Endothelialization of Impervious Dacron Grafts from Circulating Cells in the Arterial and Venous Systems of the Same Dog
The Effect of Flow Shear Stress on Endothelialization of Impervious Dacron Grafts from Circulating Cells in the Arterial and Venous Systems of the Same Dog Qun Shi, MD, Moses Hong-De Wu, MD, Yoko Onuki, MD, Yasuhiro Kouchi, MD, Rafi'k GhalL MD, Arlene R. Wechezak, PhD, and Lester R. Sauvage, MD, Seattle, Washington
The purpose of this report was to study effects of shear force and hemodynamic conditions that influence fallout healing in the arterial and venous systems of the same dog. Knitted Dacron grafts made impervious by a 1.5 mm thick coat of silicone rubber bonded to the external surface were implanted for 4 weeks during the same surgery in the descending thoracic aorta (DTA), abdominal aorta (AA) and inferior vena cava (IVC) of each of five dogs. Flow rates were measured during surgery and shear stresses calculated with the Hagen-Poiseuille formula. Full-wall thickness longitudinal tissue sections were embedded in resin and stained with hematoxylin and eosin for light microscopy, and in paraffin for immunocytochemistry studies with Factor VIII/von Willebrand factor, smooth muscle c~-actin, collagen IV, laminin, and proliferating cell nuclear antigen. Scanning electron microscopy and transmission electron microscopy studies were also performed. AgNO3 was used to determine percentage of endothelial-like cell coverage on the flow surface. All grafts were patent, without hematoma or seroma. Endotheliallike cell coverage was highest in the IVC grafts and lowest in the DTA. Shear stress and flow velocity were significantly lower in IVC grafts than DTA and AA. Proliferating cell nuclear antigen indicated extensive cellular proliferation in the intima and in the interstices of the inner portion of the graft wall. The degree of fallout healing in knitted Dacron grafts made impervious by an external coat of silicone rubber varies inversely with the sheer force of blood flow in these grafts. (Ann Vasc Surg 1998;12:341-348.)
INTRODUCTION In recent years we have reported the presence of endothelial cells on the flow surface of knitted Dacron grafts made impervious by an external coating of silicone rubber.1 We observed a higher percentage of endothelialization arising from ceils in the circulation when such impervious grafts were im-
From The Hope Heart Institute, the Providence Seattle Medical Center and the Department of Surgery, University of Washington School of Medicine, Seattle, WA. Correspondence to: L.R. Sauvage, MD, MedicalDirector, The Hope Heart Institute, 528 18th Avenue, Seattle, WA 98122, USA.
planted in the inferior vena cava (IVC) of dogs than in the abdominal aorta (AA) or the descending thoracic aorta (DTA), with the difference being greatest between the IVC and DTA grafts. We speculated that this difference was mainly attributable to the different shear stresses in the three locations, but this could not be confirmed, because a single graft had been implanted in a single location in each dog, and the fallout potential might differ between subjects. In order to eliminate confusing variables between individual dogs, we developed an experimental model in which knitted Dacron grafts made impervious with an external coating of silicone rubber were implanted in the DTA, AA, and IVC of the same animal during the same operative procedure. 341
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MATERIALS AND METHODS A tandem graft was created, consisting of a 3- or 4 cm-long knitted Dacron test segment, to which either 2.5- or 4 cm-long standard polytetrafluoroethylene (PTFE) segments were sutured to each end or the central Dacron graft to block pannus ingrowth. The entire composite graft was then made impervious to perigraft tissue ingrowth by an adherent 1.5 mm external coat of silicone rubber. This longer graft (12 cm) was implanted in the DTA (Fig. IA) and the shorter grafts (8 cm) were implanted in the AA and IVC of the same dog during the same surgery (Fig. IB). In order to match the calibers of the host vessels, diameters were 8 mm for the DTA and AA grafts and 10 mm for the IVC graft. Five adult mongrel dogs, weighing an average of 25.2 + 2.6 kg, were used. Care of the dogs throughout the study complied with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication #80-23, revised 1985).
Surgical Procedure The dogs received 0.25 mg/kg acepromazine and 0.01 mg/kg of atropine, after which general anesthesia was induced with 5-10 ml of 4% thiamylal intravenously and maintained with 0.5%-1.0% halothane and a 2:1 mixture of nitrous oxide and oxygen via a dosed-circuit respirator. A left thoracotomy was done through the seventh intercostal space. A 16 cm DTA segment was freed with ligation and division of the upper eight pairs of intercostal arteries. After 1 mg/kg of heparin was administered, a shunt from the left carotid to the left femoral artery was established, and the aorta was cross clamped just below the subclavian artery, and at about T9. A 10 cm aortic segment was then excised. The 12 cm-long composite graft was interpositioned into the DTA with running 6-0 Prolene. After completion of the anastomoses and removal of the cross clamp, i mg/kg of protamine was given. The chest wound was closed in a conventional manner.
After DTA implantation the dog was moved to the supine position, reprepped, and redraped. A midline abdominal incision was made. The infrarenal aorta was freed by dividing the lumbar branches and the inferior mesenteric artery; the IVC was mobilized by division of its tributaries. The aorta was then clamped proximally and distally. A 6 cm segment was resected, and the 8 cm-long tandem graft was implanted. The IVC graft was implanted in the
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same manner as the AA implantation. All dogs tolerated the lengthy operation and recovered well with no complications. Postoperatively, antibiotics were given for 10 days and antiplatelet therapy (162 mg aspirin/day) continued throughout the experiment until graft retrieval.
Flow Rate Measurement During surgery the flow rates in the DTA, AA, and IVC were measured with a Transonic flow meter (Model T-208, Transonic System Inc.; Ithaca, NY). Average shear stress on the graft wall, "r (dynes/ cm2), was calculated according to the HagenPoiseuille formula: T--
4~0 ~Tr3
where B is the viscosity of blood (assumed to be 0.035 poise), and Q is the mean flow rate. The velocity was also calculated according to the formula: V = Q / A , where A is the luminal area.
Specimen Harvest, Evaluation, and Data Collection The dogs were sacrificed at 4 weeks after induction of deep anesthesia. Ten thousand IU heparin were then administered, and the dog was exsanguinated. The three specimens were removed, gently flushed with Dulbecco's phosphate buffered saline solution, opened longitudinally, rinsed again, pinned flat, and photographed. The silicone-rubber coating was carefully removed with surgical instruments because it interfered with preparation of histology slides. Four full-wall thickness longitudinal tissue sections adjacent to each other, each approximately 10 x 3 mm, were taken from a white, thrombusfree area in the midportion of the impervious Dacron test graft, and from the PTFE and anastomotic areas. One block was embedded in resin, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopy study, and another block was embedded in paraffin and sectioned for immunocytochemistry study, for which the following antibodies were used: 1. Factor VIII/von Willebrand factor (FVIII/vWF) (DAKO, Code No. M-616; Carpinteria, CA) to identify endothelial cells. 2. R-actin (DAKO, Code No. M-85I , Carpinteria, CA) for smooth muscle cells.
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3. Monoclonal mouse antibody to h u m a n laminin to identify endothelial and smooth muscle cells (DAKO, Code M-638, Carpinteria, CA). 4. Collagen IV to identify endothelial and smooth muscle cells (DAKO, Code No. M85, Carpinteria, CA). 5. Monoclonal mouse antibody to h u m a n proliferating cell nuclear antigen (PCNA) (DAKO, Code No. M-879, Carpinteria, CA) to characterize the proliferating cell of the S-phase of the cell cycle. Concerning items 3, 4, and 5 above, three specim e n s w e r e studied for each graft location. The other two tissue blocks were used for scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The flow surface was evaluated by stereomicroscopy and m a p p e d on grid paper. A thrombus-free surface (TFS) score was obtained by calculating the total flow surface that was not covered with thrombus, and 0.3% silver nitrate staining was used to determine an endothelial-like cell coverage score for the remainder of the specimen. A paired t-test was used for comparing data from the three implant sites. Statistical significance was considered to be p < 0.05.
RESULTS Rheology Measurements The data on shear stress and velocity are given in Table I. There were statistically significant differences b e t w e e n the three implant sites for both factors.
Gross and General Observations There was no h e m a t o m a or seroma a r o u n d the graft, and no tissue adherence to the exterior surface of the silicone rubber coating. The adherence of the coating to the Dacron/PTFE t a n d e m graft was very tight, with no space b e t w e e n them. Representative gross specimens are s h o w n in Figure 1C. The IVC implants had the lowest TFS scores (Table II) and thicker fibrin-like deposition on the flow surface, whereas the AA and DTA were comparable to one another. Careful evaluation of samples from the PTFE segments of all specimens demonstrated that no endothelial cells were migrating across the PTFE from the host vessels onto the central impervious Dacron grafts.
Microscopy Studies
Stereomicroscopy after silver nitrate staining. Scattered islands of polygonal, endothelial-like cells
Table I. Average theologic m e a s u r e m e n t s (n = 5) Site
Calculated shear stress ( d y n e s / c i n 2)
Calculated velocity (cm/sec)
DTA AA IVC
38.7 _+ 13.7 6.4 + 2.2 1.5 + 0.3
55.2 + 18.6 9.1 _+3.2 4.2 + 0.8
DTA, descending thoracic aorta; AA, a b d o m i n a l aorta; IVC, inferior v e n a cava. Shear Stress: DTA versus AA, p < 0.003; DTA versus IVC, p < 0.003; AA versus IVC, p < 0.009. Velocity: DTA versus AA, p < 0.005; DTA versus IVC, p < 0.003; AA versus IVC, p < 0.0034.
delineated by silver nitrate staining were found on the flow surfaces of grafts from all three implant sites (Fig. 2A). In the IVC grafts, the endothelial-like cells were found on top of the red thrombus, as well as on the thrombus-free areas; however, they appeared only on the thrombus-free areas of the DTA and AA grafts. Remaining areas on the IVC grafts had a slightly thicker fibrin-like substance than was found on the DTA and AA graft flow surfaces. The endothelial-like cell coverage scores were highest in the IVC (35.8 + 8.9) and lowest in the DTA (5.18 + 6.67) (Table I).
Light microscopy after hematoxylin and eosin staining. In all three implant sites, there were scattered single layers of endothelial-like cells on the flow surfaces of these isolated, silicone-rubbercoated Dacron grafts (Fig. 2B). The IVC implants had the thickest intima, and the DTA the thinnest (Table II). In the IVC grafts, most of the substrate tissue consisted of m a n y red cells and a lesser degree of fibrinous coagulum matrix. The substrates of the AA and DTA grafts contained fewer red cells and more fibrinous coagulum matrix. SEM. Islands of cells with the characteristics of e n d o t h e l i u m , including surface microvilli, w e r e seen o n isolated s i l i c o n e - r u b b e r - c o a t e d Dacron grafts from all three implant sites (Fig. 2C). TEM. The ultrastructural characteristics of cells on the flow surface of isolated impervious Dacron grafts in the three implant sites were consistent with endothelial morphology, although the ultramicrocellular structure of the endothelial cell was not well demonstrated (Fig. 2D). Immunocytochemistry studies. The endotheliallike cells lining the flow surfaces were confirmed as e n d o t h e l i u m by FVIII/vWF staining in the three implant sites of each case (Fig. 2E). In one specimen each from the AA and IVC, H&E staining b e n e a t h these endothelial cells showed m a n y layers of fusiform cells that were positive to ~-actin s m o o t h
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Fig. 1. Experimental model showing the tandem impervious Dacron grafts implanted in the A descending thoracic aorta (DTA) and B abdominal aorta (AA) and inferior vena cava (IVC). {2 Gross DTA, AA, and IVC speci-
Annals of Vascular Surgery
mens; the IVC implants had the most, and the thickest, thrombus, and the AA and IVC were comparable to one another.
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T a b l e II. Thrombus-free surface scores, endothelial-like cell coverage, and average thickness of graft intima (n = 5) Endothelial-like
Thrombus-free
Average thickness
Site
cell coverage score (%)
surface score (%)
of graft intima (lam)
DTA AA IVC
5.18 _+6.67 i7.2 + 10.6 35.8 _+8.9
81.3 _+8 63.9 + 30 37.5 + 255
76.8 _+77.4 235.7 + 170.9 666.67 _+478.0
DTA, descending thoracic aorta; AA, abdominal aorta; IVC, inferior vena cava. Endothelial-like cell coverage: DTA versus IVC, p < 0.001; AA versus IVC, p < 0.028; DTA versus AA, p < 0.94. T h r o m b u s free surface: DTA versus IVC, p < 0.008; AA versus IVC, p < 0.113; DTA versus AA, p < 0.297. Average thickness of graft intima: DTA versus IVC, p < 0.0001; AA versus IVC, p < 0.001; DTA versus AA, p < 0.0001.
muscle cell staining. In three cases, collagen IV and laminin staining were used as markers to further confirm the presence of endothelium. On two DTA grafts and all AA and IVC grafts, cells were positive to collagen IV (Fig. 2F), two DTA grafts, one AA graft, and one IVC graft were positive to laminin staining, but results were not so conclusive as they were with collagen IV. M a n y PCNA-positive cells were concentrated in the intima and inner area of the interstices, but only a few positive cells in the outer interstices of these grafts (Fig. 3). Hematoxylin and eosin laminin, and ~-actin staining of the same IVC specimens demonstrated microvessels on the flow surface which continued into the intimal layer. DISCUSSION M a n y experimental and clinical efforts have been made to produce a better healed flow surface for decreased thrombogenicity and increased patency. Zilla et al. 2 reported significantly improved clinical femoropopliteal bypass patency at 32 m o n t h s for seeded grafts over controls. Another report 3 histologically confirmed endothelial cells on a seeded graft in the h u m a n 3 m o n t h s after implantation. We have reported endothelialization on h u m a n arterial explants. 4-6 In addition we h a v e d e m o n strated graft surface endothelialization in the dog, resulting from fallout healing. This appears to be a form of graft autoseeding, in that it is subject to m a n y theologic, biochemical, cellular, and i m m u nologic factors associated with the circulation. This study further confirms that fallout healing can take place in the DTA, AA, and IVC, and that rheologic
factors will affect the degree of healing in each of the three locations. In these impervious IVC implants, the flow surfaces w e r e c o v e r e d w i t h e n d o t h e l i u m and microvessels were identified in the intima, sometimes with connection to the flow surface, by c~-actin and l a m i n i n staining. It is possible t h a t the larger a m o u n t of thrombus which formed on the flow surface of the IVC grafts m a y elicit this angiogenic healing response. We also investigated proliferative activity in the graft healing process. Proliferating cell nuclear antigen clearly labelled nuclei of cells in the intima and inner part of interstices of these impervious grafts, indicating the most active cellular proliferation was a r o u n d the interface of the blood stream with the graft flow surface. In this study we have also focused on the possibility that increased flow rates might have an adverse effect on fallout healing. These effects would presumably be similar to those that influence the adhesion and proliferation of cells on grafts with preseeded endothelium. In vitro experiments immediately after seeding have indicated that blood flow rates had a significant influence on cell retention; more cells detached at higher flows. 7 Once a d h e r e n t to a surface, in vitro seeded endothelial ceils m u s t resist d e s q u a m a t i o n by shear stress. Rupnick et al. 8 subjected w o v e n Dacron grafts seeded at high density with microvascular endothelial cells to shear stress ranging from 10 to 20 dynes/ cm 2 and found a direct relationship b e t w e e n shear stress and cell desquamation. The endothelial cell (EC) densities decreased from the control values of 1.87 + 0.07 x 105 EC/cm 2 to 1.27 + 0.03 • 105 EC/cm x at I0 dynes/cm x and 1.13 _+ 0.04 x 105 EC/cm 2 at 20 dynes/cm 2 for 2 hours. 8 In vivo studies of endothelial regeneration evaluated within regions of high and low flow produced by coarctation of the abdominal aorta in rabbits have s h o w n that endothelial migration and repopulation were e n h a n c e d w h e n shear forces were estimated to be low and unidirectional. 9 In our laboratory, in vitro studies of mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress have demonstrated that d e t a c h m e n t of mitotic cells before completion of division was dependent on both the initial presence of intracellular connections and the magnitude of shear stress. The incidence of completed mitosis in nonisolated specimens increased w h e n shear stress decreased. 1~ This m a y indicate that u n d e r high shear stress there is less endothelial cell retention due to detachment of proliferating cells from graft flow surfaces. In porous grafts a high flow shear stress is associated with less thrombus on
346 Shi et al.
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Fig. 2. A Endothelial-like cells on the flow surfaces of grafts from all three implant sites. AgNO3; x70, x80, x80. B Scattered single layer of endothelial-like cells on the flow surfaces of grafts from all three implant sites. H&E; x570, x520, x540. C Islands of ceils with the typical morphology of endothelium on the flow surfaces of grafts from all three implant sites. SEM; x 1700, x 1400, x 1800. D Demonstration of the ultrastructural characteristics
of endothelial ceils on the flow surfaces of grafts from all three implant sites. TEM; x5100, x5500, x5800. E Factor VIII/von Willebrand factor-positive endothelial cells on the flow surfaces of grafts from all three implant sites; x520, x600, x510. F Positive collagen IV stained cells on the flow surfaces of grafts from all three implant sites; x380, x360, x380.
the surface a n d facilitation of transmural tissue ing r o w t h and surface endothelialization. However, in these experiments the fallout endothelial cells m a y be more d e p e n d e n t on a calmer rheologic climate in w h i c h to attach, than on a clean flow surface.
In this study, our results s h o w that shear stress was dramatically decreased from the DTA to the AA to the IVC, and the endothelial-like cell coverage scores were highest in the IVC and lowest in the DTA (Table 1). This higher percentage of cells on
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Endothelialization of impervious Dacrongrafts 347
Fig. 3. H&E, laminin and a-actin staining of an inferior vena cava (IVC) specimen. A Microvessels in the intima of the IVC implant site, x680. B Positive laminin stained microvessels in the intima of the IVC implant site, x420. C Positive c~-actin staining for smooth muscle cells and visualization of microvessels on the flow surface which continue into the intimal layer of the IVC implant site, x80.
t h e i m p e r v i o u s grafts in t h e IVC l o c a t i o n c o u l d p o s sibly b e e x p l a i n e d b y t h e l o w s h e a r stress, a n d l o w v e l o c i t y m a y p o s s i b l y facilitate a d h e r e n c e of cells to t h e f l o w surface, a l l o w i n g m i t o s i s , p r o l i f e r a t i o n , and spreading. The c h e m i c a l m a t e r i a l o n graft f l o w surfaces is u n d o u b t e d l y a n i m p o r t a n t f a c t o r in cell a t t a c h m e n t and adherence. Extracellular matrix molecules which promote the attachment, spreading, and mig r a t i o n of c u l t u r e d e n d o t h e l i a l cells g r o w n u n d e r n o - f l o w c o n d i t i o n s h a v e b e e n identified.~ 1,12 P o l y t e t r a f l u o r o e t h y l e n e grafts p r e c o a t e d w i t h f i b r o n e c t i n h a d s i g n i f i c a n t l y i m p r o v e d e n d o t h e l i a l cell a d herence,~3 a n d a sixfold i n c r e a s e in cell r e t e n t i o n . 1 4 Our study demonstrated that there was a thicker i n t i m a a n d m o r e t h r o m b u s in IVC grafts, c o m p a r e d to grafts i n t h e A A a n d DTA sites. P e r h a p s this t h r o m b u s , p l u s p r o t e i n s t h a t f o r m o n t h e f l o w surface, p r o v i d e t h e n e c e s s a r y g r o w t h factors a n d c h e m o a t t r a c t a n t s for cells to alight, a d h e r e , p r o l i f e r a t e , and spread. F u r t h e r s t u d i e s of t h e effects of s h e a r stress a n d f l o w surface c h e m i s t r y a r e n e e d e d , e s p e c i a l l y w i t h a p p l i c a t i o n of m o l e c u l a r b i o l o g y t e c h n i q u e s . This w o r k c o u l d l e a d to g r e a t l y i m p r o v e d p r o s t h e t i c vasc u l a r grafts. P a t e n c y rates a r e p o o r for s m a l l c a l i b e r a r t e r i a l grafts a n d v e n o u s grafts. ~5 This is l a r g e l y a t t r i b u t e d to t h e d i s a d v a n t a g e of l o w s h e a r stress in t h e a r e a s w h e r e t h e y a r e i m p l a n t e d . H o w e v e r , this s t u d y d e m o n s t r a t e d t h a t l o w s h e a r stress is b e n e f i cial to f a l l o u t h e a l i n g . If w e c o u l d c r e a t e s o m e w a y
to p r o m o t e f a l l o u t h e a l i n g , t h e p o o r p e r f o r m a n c e of grafts in u n f a v o r a b l e e n v i r o n m e n t s m i g h t b e i m proved.
We appreciate the assistance of Dorothy Mungin, HT (ASCP), David Criss, Medical Photographer, Mary Ann Sedgwick Harvey, Medical Editor, and Mary-Ann Nelson, Medical Hlustrator, for their contributions to this manuscript. REFERENCES 1. Shi Q, Wu H-D, 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;4:546-557. 2. Zilla P, Oppell UV, Deutsch M. The endothelium: A key to the future. J Cardiovasc Surg 1993;8:32-60 3. Herring MB, Baughman S, Glover J. Endothelium develops on seeded human arterial prostheses: A brief clinical note. J Vasc Surg 1985;2:727-730. 4. Sauvage LR, Berger K, Berlin LB, Smith JC, Wood SJ, Mansfield PB. Presence of endothelium in an axillary-femoral graft of knitted Dacron with an external velour surface. Ann Surg 1975;182:749-757. 5. Wu MH-D, Shi Q, Wechezak AR, Clowes AW, Gordon IL, Sauvage LR. Definitive proof of endothelialization of a Dacron arterial prosthesis in a human being. J Vasc Surg 1995; 21:863-867. 6. Shi Q, Wu MH-D, Onuki Y, Ghali R, Hunter GC, Johansen KH, Sauvage LR. Endothelium on the flow surface of human aortic Dacron vascular grafts. J Vasc Surg 1996;25:735-742. 7. Budd JS, Allen KE, Bell PRF. Effects of two methods of endothelial cell seeding on cell retention during blood flow. Br J Surg 1991;78:878-882. 8. Rupnik MH, Hubbard FA, Pratt K, Jarrell BE, Williams SK. Endothelialization of vascular prosthetic surface after seed-
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ing or sodding with h u m a n microvascular endothelial cells. J Vasc Surg 1989;6:788-795. Langille BL, Reidy MA, Kline RL. Injury and repair of endothelium at sites of flow disturbances near abdominal aortic coarctations in rabbits. Arteriosclerosis 1986;2:146-154. Wechezak AR, Viggers RF, Coan DE, Sauvage LR. Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress. J Cell Physiol 1994; 159:8391. Carey DJ. Control of growth and differentiation of vascular cells by extracellular matrix proteins. Ann Rev Physiol 1991; 53:161-177. Sage EH, Bornstein P. Extracellular proteins that modulate
Annals of Vascular Surgery
cell-matrix interactions. SPARC, tenascin, and thrombospondin. (Review) J Biol Chem 1991;266:14831-14834. 13. Seeger JM, Klingman N. Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts. J Surg Res 1985;38:641-647, 14. Ramalanjaona G, Kempczinski RF, Rosenman JE, Douville EC, Silberstein EB. The effect of fibronectin coating on endothelial cell kinetics in polytetrafluoroethylene grafts. J Vasc Surg 1986;2:264-272. 15. Dale WA. Synthetic grafts for venous reconstruction. In Bergan J J, ed. Surgery of the Veins. Orlando: Grune & Stratton, Inc., 1985, pp 233-239.
The purpose of this report was to study effects of shear force and hemodynamic conditions that influence fallout healing in the arterial and venous systems of the same dog. Knitted Dacron grafts made impervious by a 1.5 mm thick coat of silicone rubber bonded to the external surface were implanted for 4 weeks during the same surgery in the descending thoracic aorta (DTA), abdominal aorta (AA) and inferior vena cava (IVC) of each of five dogs. Flow rates were measured during surgery and shear stresses calculated with the Hagen-Poiseuille formula. Full-wall thickness longitudinal tissue sections were embedded in resin and stained with hematoxylin and eosin for light microscopy, and in paraffin for immunocytochemistry studies with Factor VIII/von Willebrand factor, smooth muscle c~-actin, collagen IV, laminin, and proliferating cell nuclear antigen. Scanning electron microscopy and transmission electron microscopy studies were also performed. AgNO3 was used to determine percentage of endothelial-like cell coverage on the flow surface. All grafts were patent, without hematoma or seroma. Endotheliallike cell coverage was highest in the IVC grafts and lowest in the DTA. Shear stress and flow velocity were significantly lower in IVC grafts than DTA and AA. Proliferating cell nuclear antigen indicated extensive cellular proliferation in the intima and in the interstices of the inner portion of the graft wall. The degree of fallout healing in knitted Dacron grafts made impervious by an external coat of silicone rubber varies inversely with the sheer force of blood flow in these grafts. (Ann Vasc Surg 1998;12:341-348.)
INTRODUCTION In recent years we have reported the presence of endothelial cells on the flow surface of knitted Dacron grafts made impervious by an external coating of silicone rubber.1 We observed a higher percentage of endothelialization arising from ceils in the circulation when such impervious grafts were im-
From The Hope Heart Institute, the Providence Seattle Medical Center and the Department of Surgery, University of Washington School of Medicine, Seattle, WA. Correspondence to: L.R. Sauvage, MD, MedicalDirector, The Hope Heart Institute, 528 18th Avenue, Seattle, WA 98122, USA.
planted in the inferior vena cava (IVC) of dogs than in the abdominal aorta (AA) or the descending thoracic aorta (DTA), with the difference being greatest between the IVC and DTA grafts. We speculated that this difference was mainly attributable to the different shear stresses in the three locations, but this could not be confirmed, because a single graft had been implanted in a single location in each dog, and the fallout potential might differ between subjects. In order to eliminate confusing variables between individual dogs, we developed an experimental model in which knitted Dacron grafts made impervious with an external coating of silicone rubber were implanted in the DTA, AA, and IVC of the same animal during the same operative procedure. 341
342
Shi et al.
MATERIALS AND METHODS A tandem graft was created, consisting of a 3- or 4 cm-long knitted Dacron test segment, to which either 2.5- or 4 cm-long standard polytetrafluoroethylene (PTFE) segments were sutured to each end or the central Dacron graft to block pannus ingrowth. The entire composite graft was then made impervious to perigraft tissue ingrowth by an adherent 1.5 mm external coat of silicone rubber. This longer graft (12 cm) was implanted in the DTA (Fig. IA) and the shorter grafts (8 cm) were implanted in the AA and IVC of the same dog during the same surgery (Fig. IB). In order to match the calibers of the host vessels, diameters were 8 mm for the DTA and AA grafts and 10 mm for the IVC graft. Five adult mongrel dogs, weighing an average of 25.2 + 2.6 kg, were used. Care of the dogs throughout the study complied with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication #80-23, revised 1985).
Surgical Procedure The dogs received 0.25 mg/kg acepromazine and 0.01 mg/kg of atropine, after which general anesthesia was induced with 5-10 ml of 4% thiamylal intravenously and maintained with 0.5%-1.0% halothane and a 2:1 mixture of nitrous oxide and oxygen via a dosed-circuit respirator. A left thoracotomy was done through the seventh intercostal space. A 16 cm DTA segment was freed with ligation and division of the upper eight pairs of intercostal arteries. After 1 mg/kg of heparin was administered, a shunt from the left carotid to the left femoral artery was established, and the aorta was cross clamped just below the subclavian artery, and at about T9. A 10 cm aortic segment was then excised. The 12 cm-long composite graft was interpositioned into the DTA with running 6-0 Prolene. After completion of the anastomoses and removal of the cross clamp, i mg/kg of protamine was given. The chest wound was closed in a conventional manner.
After DTA implantation the dog was moved to the supine position, reprepped, and redraped. A midline abdominal incision was made. The infrarenal aorta was freed by dividing the lumbar branches and the inferior mesenteric artery; the IVC was mobilized by division of its tributaries. The aorta was then clamped proximally and distally. A 6 cm segment was resected, and the 8 cm-long tandem graft was implanted. The IVC graft was implanted in the
A n n a l s of Vascular Surgery
same manner as the AA implantation. All dogs tolerated the lengthy operation and recovered well with no complications. Postoperatively, antibiotics were given for 10 days and antiplatelet therapy (162 mg aspirin/day) continued throughout the experiment until graft retrieval.
Flow Rate Measurement During surgery the flow rates in the DTA, AA, and IVC were measured with a Transonic flow meter (Model T-208, Transonic System Inc.; Ithaca, NY). Average shear stress on the graft wall, "r (dynes/ cm2), was calculated according to the HagenPoiseuille formula: T--
4~0 ~Tr3
where B is the viscosity of blood (assumed to be 0.035 poise), and Q is the mean flow rate. The velocity was also calculated according to the formula: V = Q / A , where A is the luminal area.
Specimen Harvest, Evaluation, and Data Collection The dogs were sacrificed at 4 weeks after induction of deep anesthesia. Ten thousand IU heparin were then administered, and the dog was exsanguinated. The three specimens were removed, gently flushed with Dulbecco's phosphate buffered saline solution, opened longitudinally, rinsed again, pinned flat, and photographed. The silicone-rubber coating was carefully removed with surgical instruments because it interfered with preparation of histology slides. Four full-wall thickness longitudinal tissue sections adjacent to each other, each approximately 10 x 3 mm, were taken from a white, thrombusfree area in the midportion of the impervious Dacron test graft, and from the PTFE and anastomotic areas. One block was embedded in resin, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopy study, and another block was embedded in paraffin and sectioned for immunocytochemistry study, for which the following antibodies were used: 1. Factor VIII/von Willebrand factor (FVIII/vWF) (DAKO, Code No. M-616; Carpinteria, CA) to identify endothelial cells. 2. R-actin (DAKO, Code No. M-85I , Carpinteria, CA) for smooth muscle cells.
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3. Monoclonal mouse antibody to h u m a n laminin to identify endothelial and smooth muscle cells (DAKO, Code M-638, Carpinteria, CA). 4. Collagen IV to identify endothelial and smooth muscle cells (DAKO, Code No. M85, Carpinteria, CA). 5. Monoclonal mouse antibody to h u m a n proliferating cell nuclear antigen (PCNA) (DAKO, Code No. M-879, Carpinteria, CA) to characterize the proliferating cell of the S-phase of the cell cycle. Concerning items 3, 4, and 5 above, three specim e n s w e r e studied for each graft location. The other two tissue blocks were used for scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The flow surface was evaluated by stereomicroscopy and m a p p e d on grid paper. A thrombus-free surface (TFS) score was obtained by calculating the total flow surface that was not covered with thrombus, and 0.3% silver nitrate staining was used to determine an endothelial-like cell coverage score for the remainder of the specimen. A paired t-test was used for comparing data from the three implant sites. Statistical significance was considered to be p < 0.05.
RESULTS Rheology Measurements The data on shear stress and velocity are given in Table I. There were statistically significant differences b e t w e e n the three implant sites for both factors.
Gross and General Observations There was no h e m a t o m a or seroma a r o u n d the graft, and no tissue adherence to the exterior surface of the silicone rubber coating. The adherence of the coating to the Dacron/PTFE t a n d e m graft was very tight, with no space b e t w e e n them. Representative gross specimens are s h o w n in Figure 1C. The IVC implants had the lowest TFS scores (Table II) and thicker fibrin-like deposition on the flow surface, whereas the AA and DTA were comparable to one another. Careful evaluation of samples from the PTFE segments of all specimens demonstrated that no endothelial cells were migrating across the PTFE from the host vessels onto the central impervious Dacron grafts.
Microscopy Studies
Stereomicroscopy after silver nitrate staining. Scattered islands of polygonal, endothelial-like cells
Table I. Average theologic m e a s u r e m e n t s (n = 5) Site
Calculated shear stress ( d y n e s / c i n 2)
Calculated velocity (cm/sec)
DTA AA IVC
38.7 _+ 13.7 6.4 + 2.2 1.5 + 0.3
55.2 + 18.6 9.1 _+3.2 4.2 + 0.8
DTA, descending thoracic aorta; AA, a b d o m i n a l aorta; IVC, inferior v e n a cava. Shear Stress: DTA versus AA, p < 0.003; DTA versus IVC, p < 0.003; AA versus IVC, p < 0.009. Velocity: DTA versus AA, p < 0.005; DTA versus IVC, p < 0.003; AA versus IVC, p < 0.0034.
delineated by silver nitrate staining were found on the flow surfaces of grafts from all three implant sites (Fig. 2A). In the IVC grafts, the endothelial-like cells were found on top of the red thrombus, as well as on the thrombus-free areas; however, they appeared only on the thrombus-free areas of the DTA and AA grafts. Remaining areas on the IVC grafts had a slightly thicker fibrin-like substance than was found on the DTA and AA graft flow surfaces. The endothelial-like cell coverage scores were highest in the IVC (35.8 + 8.9) and lowest in the DTA (5.18 + 6.67) (Table I).
Light microscopy after hematoxylin and eosin staining. In all three implant sites, there were scattered single layers of endothelial-like cells on the flow surfaces of these isolated, silicone-rubbercoated Dacron grafts (Fig. 2B). The IVC implants had the thickest intima, and the DTA the thinnest (Table II). In the IVC grafts, most of the substrate tissue consisted of m a n y red cells and a lesser degree of fibrinous coagulum matrix. The substrates of the AA and DTA grafts contained fewer red cells and more fibrinous coagulum matrix. SEM. Islands of cells with the characteristics of e n d o t h e l i u m , including surface microvilli, w e r e seen o n isolated s i l i c o n e - r u b b e r - c o a t e d Dacron grafts from all three implant sites (Fig. 2C). TEM. The ultrastructural characteristics of cells on the flow surface of isolated impervious Dacron grafts in the three implant sites were consistent with endothelial morphology, although the ultramicrocellular structure of the endothelial cell was not well demonstrated (Fig. 2D). Immunocytochemistry studies. The endotheliallike cells lining the flow surfaces were confirmed as e n d o t h e l i u m by FVIII/vWF staining in the three implant sites of each case (Fig. 2E). In one specimen each from the AA and IVC, H&E staining b e n e a t h these endothelial cells showed m a n y layers of fusiform cells that were positive to ~-actin s m o o t h
344 Shi et al.
Fig. 1. Experimental model showing the tandem impervious Dacron grafts implanted in the A descending thoracic aorta (DTA) and B abdominal aorta (AA) and inferior vena cava (IVC). {2 Gross DTA, AA, and IVC speci-
Annals of Vascular Surgery
mens; the IVC implants had the most, and the thickest, thrombus, and the AA and IVC were comparable to one another.
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T a b l e II. Thrombus-free surface scores, endothelial-like cell coverage, and average thickness of graft intima (n = 5) Endothelial-like
Thrombus-free
Average thickness
Site
cell coverage score (%)
surface score (%)
of graft intima (lam)
DTA AA IVC
5.18 _+6.67 i7.2 + 10.6 35.8 _+8.9
81.3 _+8 63.9 + 30 37.5 + 255
76.8 _+77.4 235.7 + 170.9 666.67 _+478.0
DTA, descending thoracic aorta; AA, abdominal aorta; IVC, inferior vena cava. Endothelial-like cell coverage: DTA versus IVC, p < 0.001; AA versus IVC, p < 0.028; DTA versus AA, p < 0.94. T h r o m b u s free surface: DTA versus IVC, p < 0.008; AA versus IVC, p < 0.113; DTA versus AA, p < 0.297. Average thickness of graft intima: DTA versus IVC, p < 0.0001; AA versus IVC, p < 0.001; DTA versus AA, p < 0.0001.
muscle cell staining. In three cases, collagen IV and laminin staining were used as markers to further confirm the presence of endothelium. On two DTA grafts and all AA and IVC grafts, cells were positive to collagen IV (Fig. 2F), two DTA grafts, one AA graft, and one IVC graft were positive to laminin staining, but results were not so conclusive as they were with collagen IV. M a n y PCNA-positive cells were concentrated in the intima and inner area of the interstices, but only a few positive cells in the outer interstices of these grafts (Fig. 3). Hematoxylin and eosin laminin, and ~-actin staining of the same IVC specimens demonstrated microvessels on the flow surface which continued into the intimal layer. DISCUSSION M a n y experimental and clinical efforts have been made to produce a better healed flow surface for decreased thrombogenicity and increased patency. Zilla et al. 2 reported significantly improved clinical femoropopliteal bypass patency at 32 m o n t h s for seeded grafts over controls. Another report 3 histologically confirmed endothelial cells on a seeded graft in the h u m a n 3 m o n t h s after implantation. We have reported endothelialization on h u m a n arterial explants. 4-6 In addition we h a v e d e m o n strated graft surface endothelialization in the dog, resulting from fallout healing. This appears to be a form of graft autoseeding, in that it is subject to m a n y theologic, biochemical, cellular, and i m m u nologic factors associated with the circulation. This study further confirms that fallout healing can take place in the DTA, AA, and IVC, and that rheologic
factors will affect the degree of healing in each of the three locations. In these impervious IVC implants, the flow surfaces w e r e c o v e r e d w i t h e n d o t h e l i u m and microvessels were identified in the intima, sometimes with connection to the flow surface, by c~-actin and l a m i n i n staining. It is possible t h a t the larger a m o u n t of thrombus which formed on the flow surface of the IVC grafts m a y elicit this angiogenic healing response. We also investigated proliferative activity in the graft healing process. Proliferating cell nuclear antigen clearly labelled nuclei of cells in the intima and inner part of interstices of these impervious grafts, indicating the most active cellular proliferation was a r o u n d the interface of the blood stream with the graft flow surface. In this study we have also focused on the possibility that increased flow rates might have an adverse effect on fallout healing. These effects would presumably be similar to those that influence the adhesion and proliferation of cells on grafts with preseeded endothelium. In vitro experiments immediately after seeding have indicated that blood flow rates had a significant influence on cell retention; more cells detached at higher flows. 7 Once a d h e r e n t to a surface, in vitro seeded endothelial ceils m u s t resist d e s q u a m a t i o n by shear stress. Rupnick et al. 8 subjected w o v e n Dacron grafts seeded at high density with microvascular endothelial cells to shear stress ranging from 10 to 20 dynes/ cm 2 and found a direct relationship b e t w e e n shear stress and cell desquamation. The endothelial cell (EC) densities decreased from the control values of 1.87 + 0.07 x 105 EC/cm 2 to 1.27 + 0.03 • 105 EC/cm x at I0 dynes/cm x and 1.13 _+ 0.04 x 105 EC/cm 2 at 20 dynes/cm 2 for 2 hours. 8 In vivo studies of endothelial regeneration evaluated within regions of high and low flow produced by coarctation of the abdominal aorta in rabbits have s h o w n that endothelial migration and repopulation were e n h a n c e d w h e n shear forces were estimated to be low and unidirectional. 9 In our laboratory, in vitro studies of mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress have demonstrated that d e t a c h m e n t of mitotic cells before completion of division was dependent on both the initial presence of intracellular connections and the magnitude of shear stress. The incidence of completed mitosis in nonisolated specimens increased w h e n shear stress decreased. 1~ This m a y indicate that u n d e r high shear stress there is less endothelial cell retention due to detachment of proliferating cells from graft flow surfaces. In porous grafts a high flow shear stress is associated with less thrombus on
346 Shi et al.
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Fig. 2. A Endothelial-like cells on the flow surfaces of grafts from all three implant sites. AgNO3; x70, x80, x80. B Scattered single layer of endothelial-like cells on the flow surfaces of grafts from all three implant sites. H&E; x570, x520, x540. C Islands of ceils with the typical morphology of endothelium on the flow surfaces of grafts from all three implant sites. SEM; x 1700, x 1400, x 1800. D Demonstration of the ultrastructural characteristics
of endothelial ceils on the flow surfaces of grafts from all three implant sites. TEM; x5100, x5500, x5800. E Factor VIII/von Willebrand factor-positive endothelial cells on the flow surfaces of grafts from all three implant sites; x520, x600, x510. F Positive collagen IV stained cells on the flow surfaces of grafts from all three implant sites; x380, x360, x380.
the surface a n d facilitation of transmural tissue ing r o w t h and surface endothelialization. However, in these experiments the fallout endothelial cells m a y be more d e p e n d e n t on a calmer rheologic climate in w h i c h to attach, than on a clean flow surface.
In this study, our results s h o w that shear stress was dramatically decreased from the DTA to the AA to the IVC, and the endothelial-like cell coverage scores were highest in the IVC and lowest in the DTA (Table 1). This higher percentage of cells on
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Endothelialization of impervious Dacrongrafts 347
Fig. 3. H&E, laminin and a-actin staining of an inferior vena cava (IVC) specimen. A Microvessels in the intima of the IVC implant site, x680. B Positive laminin stained microvessels in the intima of the IVC implant site, x420. C Positive c~-actin staining for smooth muscle cells and visualization of microvessels on the flow surface which continue into the intimal layer of the IVC implant site, x80.
t h e i m p e r v i o u s grafts in t h e IVC l o c a t i o n c o u l d p o s sibly b e e x p l a i n e d b y t h e l o w s h e a r stress, a n d l o w v e l o c i t y m a y p o s s i b l y facilitate a d h e r e n c e of cells to t h e f l o w surface, a l l o w i n g m i t o s i s , p r o l i f e r a t i o n , and spreading. The c h e m i c a l m a t e r i a l o n graft f l o w surfaces is u n d o u b t e d l y a n i m p o r t a n t f a c t o r in cell a t t a c h m e n t and adherence. Extracellular matrix molecules which promote the attachment, spreading, and mig r a t i o n of c u l t u r e d e n d o t h e l i a l cells g r o w n u n d e r n o - f l o w c o n d i t i o n s h a v e b e e n identified.~ 1,12 P o l y t e t r a f l u o r o e t h y l e n e grafts p r e c o a t e d w i t h f i b r o n e c t i n h a d s i g n i f i c a n t l y i m p r o v e d e n d o t h e l i a l cell a d herence,~3 a n d a sixfold i n c r e a s e in cell r e t e n t i o n . 1 4 Our study demonstrated that there was a thicker i n t i m a a n d m o r e t h r o m b u s in IVC grafts, c o m p a r e d to grafts i n t h e A A a n d DTA sites. P e r h a p s this t h r o m b u s , p l u s p r o t e i n s t h a t f o r m o n t h e f l o w surface, p r o v i d e t h e n e c e s s a r y g r o w t h factors a n d c h e m o a t t r a c t a n t s for cells to alight, a d h e r e , p r o l i f e r a t e , and spread. F u r t h e r s t u d i e s of t h e effects of s h e a r stress a n d f l o w surface c h e m i s t r y a r e n e e d e d , e s p e c i a l l y w i t h a p p l i c a t i o n of m o l e c u l a r b i o l o g y t e c h n i q u e s . This w o r k c o u l d l e a d to g r e a t l y i m p r o v e d p r o s t h e t i c vasc u l a r grafts. P a t e n c y rates a r e p o o r for s m a l l c a l i b e r a r t e r i a l grafts a n d v e n o u s grafts. ~5 This is l a r g e l y a t t r i b u t e d to t h e d i s a d v a n t a g e of l o w s h e a r stress in t h e a r e a s w h e r e t h e y a r e i m p l a n t e d . H o w e v e r , this s t u d y d e m o n s t r a t e d t h a t l o w s h e a r stress is b e n e f i cial to f a l l o u t h e a l i n g . If w e c o u l d c r e a t e s o m e w a y
to p r o m o t e f a l l o u t h e a l i n g , t h e p o o r p e r f o r m a n c e of grafts in u n f a v o r a b l e e n v i r o n m e n t s m i g h t b e i m proved.
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