Quantification of gas denucleation and thrombogenicity of vascular grafts" Christopher V. Bemen>$ Richard D. Vann? Kim E. Koger,' and Bruce KlitzmantT5,' 'Plastic Surgery Research Laboratories, #E G. Hall Hyperbaric Laboratory, Department of Anesthesiology, and §Division of Physiology, Duke University Medical Center, Durham, North Carolina 27710
In vitro methods were developed to measure the air content of vascular graft walls and the thrombogenicity of this air. Gas content (volume %) of expanded polytetrafluoroethylene (ePTFE)grafts from different sources ranged from 75.5 a 0.4%to 61.8 k 0.3%.Exposure of Vitagraft ePTFE to a vacuum prior to saline immersion replaced 87.5% of the gas nuclei with saline (denucleation). Acetone and ethanol immersion produced 98.9% and 94.3% denucleation, respectively. Denucleation was essentially complete when vacuum exposure was followed by hydrostatic pressure
treatment at 500 psig or greater. The inf luence of gas content on thrombogenicity was determined by immersing graft samples in whole canine blood and weighing the adherent thrombus. Denucleation significantly reduced adherent thrombus weight compared with control grafts ( p < 0.001). Air in Vitagraft walls was responsible for 84% of the adherent thrombus weight at four minutes. The described methods could be employed to assess the hemocompatibility of various biomaterials.
INTRODUCTION
Biomaterials used in the production of vascular prostheses are inherently thrombogenic.' Heparin bonding, endothelial cell seeding, and the use of local and systemic antiplatelet agents are several methods that improve hem~compatibility.~~ One component of thrombogenicity that is of ten overlooked is the blood-gas interface. Gas was first found to cause blood damage in bubble oxygenators as the result of the interfacial denaturation of globular plasma protein^."^ This denaturation results in the activation of platelets, complement, and fibrinogen:-lo Madras et al. demonstrated that air trapped in surface imperfections will persist indefinitely on hydrophobic surfaces and may require prolonged times to dissolve on hydrophilic surfaces." Such air may be present at any material-blood interface. Expanded polytetraf luoroethylene (ePTFE)grafts were first used clinically by Soyer in 1972 and currently have widespread applicability in vascular re*Nobenefit of any kind will be received either directly or indirectly by the author(s), although this work was funded in part by Bard Cardiosurgery, Inc., Billerica, MA. 'To whom correspondence should be addressed at Plastic Surgery Research Laboratories, P. 0.Box 3906, Duke University Medical Center, Durham, North Carolina 27710. Journal of Biomedical Materials Research, Vol. 25, 373-386 (1991) CCC 0021-9304/91/030373-14$04.00 0 1991 John Wiley & Sons, Inc.
BENSEN ET AL.
374
construction. A limiting factor, however, is their high rate of thrombosis. Expanded PTFE grafts were found to have a gas content of 70% air by volume.’2 Thus, blood in contact with ePTFE is exposed to a composite surface of bubbles or ”gas nuclei” and ePTFE fibrils. The removal of these gas nuclei has been called denucleation. Several techniques, including immersion in ethanol, acetone, and vacuum, have been employed to denucleate biomaterials.8,11,12 Recent studies in rats have demonstrated a profound increase in patency of l-mm-I.D. ePTFE microvascular grafts denucleated by hydrostatic pres~ure.’”~ There is currently no method for systematically quantifying the efficacy of denucleation or assessing the consequent reduction of thrombogenicity. The purposes of these studies were to quantify the gas content of six types of vascular grafts, the efficiency of denucleation methods, and the effect of denucleation on the thrombogenicity of Vitagraf t. MATERIALS A N D METHODS
Methods of gas denucleation Graft samples measuring 4 mm I.D. X 1.0 cm long were weighed to the nearest 0.0001 g and placed in 10-mL Vacutainer (Becton Dickinson, Rutherford, NJ) tubes. For acetone or ethanol treatment, tubes were filled with 5 mL of liquid for 15 min and vigorously rinsed with distilled water. For vacuum treatment, a small vacuum pump reduced the absolute pressure in the Vacutainer to 4 mm Hg as a “pretreatment” to eliminate most of the gas. The pump was connected to a 21-gauge needle inserted in the rubber stopper. After vacuum exposure for 1 min, the tube was filled with physiologic saline degassed by boiling through a water seal as described by Do~g1as.l~ For pressure denucleation, tubes were exposed to vacuum and then placed in a hydrostatic pressure chamber and subjected to 100, 200, 300, 400, 500, 1000,2000,4000,6000, or 20,000 psig for a period of 1min. Determination of degree of denucleation 1. Qualitative analysis
Graft samples in tubes filled with degassed saline were subjected to a
4 mm Hg vacuum and observed for surface air bubbles. Bubbling was interpreted as an indication that grafts were not totally denucleated. If no bubbles appeared on the graft surfaces, denucleation was considered complete. 2. Quantitative analysis
(a) Buoyancy method: Graft samples were suspended from a support tower (Fig. 1)by a single strand of 5-0 nylon suture and submerged in a beaker of
QUANTIFICATION OF DEN UCLEATION A N D T HROMBOGENICITY
375
Support Tower lcm Graft
,
Analytical Balance
- w
_(Turntable
Laboratory Jack
Figure 1. Schematic diagram of the apparatus for gravimetric assessment of thrombogenicity.
physiologic saline. Grafts were anchored by a small washer. The apparatus was weighed on an analytical balance accurate to 0.0001 g. The washer weight was subtracted to obtain the underwater weight of the graft. The graft was weighed before and after denucleation and the volumes of the graft material and air were determined by Archimedes’ principle (see Appendix). Air volume could be resolved to 100 nL, which corresponds to 0.15% denucleation in a 1-cm length of graft. (b) Photometry: The transmittance of white light was measured through 10 control grafts and 10 grafts denucleated at 20,000 psig. Graft samples were placed on microscope slides on the stage of a Zeiss ACM microscope and illuminated from below hith a 100-W halogen lamp. Light transmittance was measured through a UD20 objective at a magnification of approximately 300X using a Hamamatsu model 1P21 photomultiplier tube.
Gas content of vascular prostheses For comparison with Vitagraft ePTFE, the gas content was determined for naked and albumin-coated Dacron (Bard Cardiosurgery, Inc., Billerica, MA), naked polyurethane (Corvita, Inc., Miami, FL), and other ePTFE grafts made by Impra, Inc. (Tempe, AZ) and W. L. Gore, Inc. (Flagstaff, AZ).
Renucleation Renucleation is the regeneration of gas nuclei on the surface or in the matrix of a denucleated graft. Renucleation occurs if the graft is exposed to air or immersed in a gas-saturated solution. Quantitatively, renucleation is the percentage of original air regained following complete denucleation. Renucleation was measured in the conditions described below.
376
BENSEN ET AL.
1. Shelf life
Five 2-cm lengths of denucleated graft were stored at atmospheric pressure in 10-mL Vacutainer tubes filled with distilled water. The volume of air in each sample was measured after eight months of storage using Archimedes’ principle. 2. Intraoperafive renucleation
To simulate various conditions that might be encountered during surgical implantation, three groups of 10 I-cm denucleated grafts were removed from their saline-filled tubes and placed on a lab counter in room air. The temperature was 22°C and the relative humidity was approximately 70%. The percentage renucleation was measured after 2 h. The three conditions under which renucleation was assessed included intermittently immersed, nonimmersed, and gauze wrapped. The intermittently immersed grafts were submerged in normal saline for 30 s every 15 min, nonimmersed grafts were allowed to dry in air with no saline immersion, and gauze-wrapped grafts were covered with wet 2 x 2 gauze pads and irrigated with normal saline every 15 min.
Scanning electron microscopy Structural changes in ePTFE that occurred as a result of denucleation were examined with scanning electron microscopy. Grafts denucleated by either acetone immersion or vacuum followed by 20,000 psig were immersed in ethanol, and allowed to dry. Control and dried denucleated grafts were sputter coated with gold. Micrographs of the graft sample luminal surface were taken at magnifications of 1 0 0 0 ~and 2 0 0 0 ~ .
Gravimetric assessment of thrombogenicity The thrombogenicity of vascular graft materials was assessed using a method in which the weight of an adherent thrombus was measured as a function of time. A wooden support tower (Fig. 1) was constructed and covered with several coats of polyurethane sealant to minimize changes in hydration and weight. A small plexiglass rod hung from a cantilevered wooden arm connected to the top of the tower. A segment of graft material 4 mm I.D. x 1.0 cm long was placed over the rod leaving only the exterior graft surface exposed. The support structure rested on an analytical balance accurate to 0.0001 g. Directly under the graft on a platform was a 1-mL polycarbonate test tube that rotated at 6 rpm to prevent thrombus from simultaneously adhering to both the graft and the sides of the tube. The platform could be raised or lowered to completely immerse the graft in the tube.
QUANTIFICATION OF DENUCLEATION AND THROMBOGENICITY
377
A thrombogenicity test began by weighing the graft and support structure in air. The graft was then immersed in saline for 15 s, removed, and touched to the side of the test tube to remove loose fluid adhering to the graft. The weight was again recorded. The saline immersion was performed to ensure that all easily wetted surfaces were exposed to water. A similar submersion in canine whole blood immediately followed the saline immersion. The graft was immersed for 15 s, removed, and the scale was tared to establish an initial weight. The graft was again immersed in blood and subsequently removed for 10 s and weighed at 4,5,6, 7, and 8 min. Preliminary experiments indicated that measurable thrombosis occurred after 4 min. Blood was drawn from 20-kg mongrel dogs into standard blue-top Vacutainer tubes containing buffered sodium citrate. Experiments were performed within 24 h of collections. The citrate was reversed just prior to the assay by adding 0.07 mL of 0.025M calcium chloride to 0.7 mL of blood for a 1:10 ratio. The thrombogenicity of Vitagraft ePTFE was determined at four degrees of denucleation: undenucleated (CONTROL, n = lo), denucleated by 4 mm Hg vacuum only (VACUUM, n = 10) partially denucleated at 100 psig (PARTIAL, n = lo), and completely denucleated at 20,000 psig (TOTAL, n = 10).
Statistics Differences between groups were evaluated using a Wilcoxon Rank Sum test with a p value of 0.05 or less required for statistical significance. RESULTS
Gas denucleation All denucleation methods caused grafts to change from opaque white to translucent gray as previously rep~rted.”””’~ Acetone or ethanol treatments produced a more uniform change to a translucent appearance than pressure treatments. However, small bubbles formed on these grafts when placed in water. Preliminary studies indicated that vacuum and vacuum plus pressure denucleation caused a 10-20% decrease in graft length due to a reduction of the internodal distance. No measurable shortening occurred in grafts denucleated in acetone or ethanol. Grafts used in this study had various pretreatment lengths in order to assure uniform 1-cm lengths following treatment.
Determination of degree of denucleation 1. Qualitative analysis
When exposed to vacuum (4 mm Hg), untreated grafts bubbled violently. Partially denucleated grafts (100 psig-500 psig) produced only small bubbles.
BENSEN ET AL.
378
No bubbles were observed on the completely denucleated grafts (500 psig or greater). 2. Quantitative analysis (a) Buoyancy method (Archimedes’ Principle): Graft buoyancy decreased as the denucleation pressure increased (Fig. 2). Denucleation was 97.4% complete with a pressure treatment of 100 psig and indistinguishable from 100% with pressure treatments of 500 psig or greater. Grafts denucleated by ethanol immersion had a mean denucleation of 94.3%. Grafts denucleated by acetone immersion had a significantly higher denucleation of 98.9% ( p < 0.05). (b) Photometry: The light transmittance of control grafts was 8.88 f 0.70%. After denucleation at 20,000 psig, transmittance significantly increased to 41.70 t 0.81% ( p < 0.001).
Gas content of vascular prostheses These results indicate that there is a wide range in gas content of commonly used vascular prostheses on submersion in water. Vitagraft ePTFE was 75.5 f 0.4% air (n = 20), Gore-tex ePTFE 69.2 f 0.3% (n = 5), Impra ePTFE 61.8 t 0.3% (n = lo), Corvita naked polyurethane 30.2 f 0.3% ( n = 5), naked Dacron 7.6 4 0.7%(n = lo), and albumin-coated Dacron 2.4 -+ 0.3% (n = 10). Renucleation 1. Shelflife
The data indicate that an average of 0.01 f 0.01% renucleation occurred over the 8-month test period. Since the resolution of the method was 0.15%,no detectable renucleation occurred when grafts were immersed during storage. C
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QUANTIFICATION OF DEN UCL EATION A N D THROMBOGENICITY
2. In t raoperat ive renucleation
The results of the simulated surgical setting are summarized in Figure 3. These data demonstrate that intermittent immersion dramatically reduced graft renucleation from 89% to 3.4%.Wrapping the grafts in wet gauze further reduced renucleation significantly to only 1.4%( p < 0.001). Scanning electron microscopy Figure 4 shows that denucleation caused a reduction in internodal distance. This is consistent with the observed overall shortening of the grafts. The closer nodes appear to allow the fibrils to clump together into bundles, resulting in large, irregularly spaced pores between the nodes. The large pores appear to be more numerous in the pressure denucleated grafts. Gravimetric assessment of thrombogenicity Figure 5a indicates that both partially (100 psig) and totally (20,000 psig) denucleated grafts had significantly less adherent thrombus than the control and vacuum denucleated grafts. Differences between partially and totally denucleated grafts were not significant. At 4 min, vacuum denucleated grafts had significantly less thrombus than control grafts. In Figure 5b, the thrombus weights of Figure 5a are plotted against the percentage denucleation from Figure 2. Figure 5b shows that the removal of air from the graft results in progressive and significant reduction in adherent thrombus weight. DISCUSSION
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In vitro methods were developed to measure the air content of vascular graft walls and the thrombogenicity of this air. Gas content (volume %) of expanded polytetrafluoroethylene (ePTFE)grafts from different sources ranged from 75.5 a 0.4%to 61.8 k 0.3%.Exposure of Vitagraft ePTFE to a vacuum prior to saline immersion replaced 87.5% of the gas nuclei with saline (denucleation). Acetone and ethanol immersion produced 98.9% and 94.3% denucleation, respectively. Denucleation was essentially complete when vacuum exposure was followed by hydrostatic pressure
treatment at 500 psig or greater. The inf luence of gas content on thrombogenicity was determined by immersing graft samples in whole canine blood and weighing the adherent thrombus. Denucleation significantly reduced adherent thrombus weight compared with control grafts ( p < 0.001). Air in Vitagraft walls was responsible for 84% of the adherent thrombus weight at four minutes. The described methods could be employed to assess the hemocompatibility of various biomaterials.
INTRODUCTION
Biomaterials used in the production of vascular prostheses are inherently thrombogenic.' Heparin bonding, endothelial cell seeding, and the use of local and systemic antiplatelet agents are several methods that improve hem~compatibility.~~ One component of thrombogenicity that is of ten overlooked is the blood-gas interface. Gas was first found to cause blood damage in bubble oxygenators as the result of the interfacial denaturation of globular plasma protein^."^ This denaturation results in the activation of platelets, complement, and fibrinogen:-lo Madras et al. demonstrated that air trapped in surface imperfections will persist indefinitely on hydrophobic surfaces and may require prolonged times to dissolve on hydrophilic surfaces." Such air may be present at any material-blood interface. Expanded polytetraf luoroethylene (ePTFE)grafts were first used clinically by Soyer in 1972 and currently have widespread applicability in vascular re*Nobenefit of any kind will be received either directly or indirectly by the author(s), although this work was funded in part by Bard Cardiosurgery, Inc., Billerica, MA. 'To whom correspondence should be addressed at Plastic Surgery Research Laboratories, P. 0.Box 3906, Duke University Medical Center, Durham, North Carolina 27710. Journal of Biomedical Materials Research, Vol. 25, 373-386 (1991) CCC 0021-9304/91/030373-14$04.00 0 1991 John Wiley & Sons, Inc.
BENSEN ET AL.
374
construction. A limiting factor, however, is their high rate of thrombosis. Expanded PTFE grafts were found to have a gas content of 70% air by volume.’2 Thus, blood in contact with ePTFE is exposed to a composite surface of bubbles or ”gas nuclei” and ePTFE fibrils. The removal of these gas nuclei has been called denucleation. Several techniques, including immersion in ethanol, acetone, and vacuum, have been employed to denucleate biomaterials.8,11,12 Recent studies in rats have demonstrated a profound increase in patency of l-mm-I.D. ePTFE microvascular grafts denucleated by hydrostatic pres~ure.’”~ There is currently no method for systematically quantifying the efficacy of denucleation or assessing the consequent reduction of thrombogenicity. The purposes of these studies were to quantify the gas content of six types of vascular grafts, the efficiency of denucleation methods, and the effect of denucleation on the thrombogenicity of Vitagraf t. MATERIALS A N D METHODS
Methods of gas denucleation Graft samples measuring 4 mm I.D. X 1.0 cm long were weighed to the nearest 0.0001 g and placed in 10-mL Vacutainer (Becton Dickinson, Rutherford, NJ) tubes. For acetone or ethanol treatment, tubes were filled with 5 mL of liquid for 15 min and vigorously rinsed with distilled water. For vacuum treatment, a small vacuum pump reduced the absolute pressure in the Vacutainer to 4 mm Hg as a “pretreatment” to eliminate most of the gas. The pump was connected to a 21-gauge needle inserted in the rubber stopper. After vacuum exposure for 1 min, the tube was filled with physiologic saline degassed by boiling through a water seal as described by Do~g1as.l~ For pressure denucleation, tubes were exposed to vacuum and then placed in a hydrostatic pressure chamber and subjected to 100, 200, 300, 400, 500, 1000,2000,4000,6000, or 20,000 psig for a period of 1min. Determination of degree of denucleation 1. Qualitative analysis
Graft samples in tubes filled with degassed saline were subjected to a
4 mm Hg vacuum and observed for surface air bubbles. Bubbling was interpreted as an indication that grafts were not totally denucleated. If no bubbles appeared on the graft surfaces, denucleation was considered complete. 2. Quantitative analysis
(a) Buoyancy method: Graft samples were suspended from a support tower (Fig. 1)by a single strand of 5-0 nylon suture and submerged in a beaker of
QUANTIFICATION OF DEN UCLEATION A N D T HROMBOGENICITY
375
Support Tower lcm Graft
,
Analytical Balance
- w
_(Turntable
Laboratory Jack
Figure 1. Schematic diagram of the apparatus for gravimetric assessment of thrombogenicity.
physiologic saline. Grafts were anchored by a small washer. The apparatus was weighed on an analytical balance accurate to 0.0001 g. The washer weight was subtracted to obtain the underwater weight of the graft. The graft was weighed before and after denucleation and the volumes of the graft material and air were determined by Archimedes’ principle (see Appendix). Air volume could be resolved to 100 nL, which corresponds to 0.15% denucleation in a 1-cm length of graft. (b) Photometry: The transmittance of white light was measured through 10 control grafts and 10 grafts denucleated at 20,000 psig. Graft samples were placed on microscope slides on the stage of a Zeiss ACM microscope and illuminated from below hith a 100-W halogen lamp. Light transmittance was measured through a UD20 objective at a magnification of approximately 300X using a Hamamatsu model 1P21 photomultiplier tube.
Gas content of vascular prostheses For comparison with Vitagraft ePTFE, the gas content was determined for naked and albumin-coated Dacron (Bard Cardiosurgery, Inc., Billerica, MA), naked polyurethane (Corvita, Inc., Miami, FL), and other ePTFE grafts made by Impra, Inc. (Tempe, AZ) and W. L. Gore, Inc. (Flagstaff, AZ).
Renucleation Renucleation is the regeneration of gas nuclei on the surface or in the matrix of a denucleated graft. Renucleation occurs if the graft is exposed to air or immersed in a gas-saturated solution. Quantitatively, renucleation is the percentage of original air regained following complete denucleation. Renucleation was measured in the conditions described below.
376
BENSEN ET AL.
1. Shelf life
Five 2-cm lengths of denucleated graft were stored at atmospheric pressure in 10-mL Vacutainer tubes filled with distilled water. The volume of air in each sample was measured after eight months of storage using Archimedes’ principle. 2. Intraoperafive renucleation
To simulate various conditions that might be encountered during surgical implantation, three groups of 10 I-cm denucleated grafts were removed from their saline-filled tubes and placed on a lab counter in room air. The temperature was 22°C and the relative humidity was approximately 70%. The percentage renucleation was measured after 2 h. The three conditions under which renucleation was assessed included intermittently immersed, nonimmersed, and gauze wrapped. The intermittently immersed grafts were submerged in normal saline for 30 s every 15 min, nonimmersed grafts were allowed to dry in air with no saline immersion, and gauze-wrapped grafts were covered with wet 2 x 2 gauze pads and irrigated with normal saline every 15 min.
Scanning electron microscopy Structural changes in ePTFE that occurred as a result of denucleation were examined with scanning electron microscopy. Grafts denucleated by either acetone immersion or vacuum followed by 20,000 psig were immersed in ethanol, and allowed to dry. Control and dried denucleated grafts were sputter coated with gold. Micrographs of the graft sample luminal surface were taken at magnifications of 1 0 0 0 ~and 2 0 0 0 ~ .
Gravimetric assessment of thrombogenicity The thrombogenicity of vascular graft materials was assessed using a method in which the weight of an adherent thrombus was measured as a function of time. A wooden support tower (Fig. 1) was constructed and covered with several coats of polyurethane sealant to minimize changes in hydration and weight. A small plexiglass rod hung from a cantilevered wooden arm connected to the top of the tower. A segment of graft material 4 mm I.D. x 1.0 cm long was placed over the rod leaving only the exterior graft surface exposed. The support structure rested on an analytical balance accurate to 0.0001 g. Directly under the graft on a platform was a 1-mL polycarbonate test tube that rotated at 6 rpm to prevent thrombus from simultaneously adhering to both the graft and the sides of the tube. The platform could be raised or lowered to completely immerse the graft in the tube.
QUANTIFICATION OF DENUCLEATION AND THROMBOGENICITY
377
A thrombogenicity test began by weighing the graft and support structure in air. The graft was then immersed in saline for 15 s, removed, and touched to the side of the test tube to remove loose fluid adhering to the graft. The weight was again recorded. The saline immersion was performed to ensure that all easily wetted surfaces were exposed to water. A similar submersion in canine whole blood immediately followed the saline immersion. The graft was immersed for 15 s, removed, and the scale was tared to establish an initial weight. The graft was again immersed in blood and subsequently removed for 10 s and weighed at 4,5,6, 7, and 8 min. Preliminary experiments indicated that measurable thrombosis occurred after 4 min. Blood was drawn from 20-kg mongrel dogs into standard blue-top Vacutainer tubes containing buffered sodium citrate. Experiments were performed within 24 h of collections. The citrate was reversed just prior to the assay by adding 0.07 mL of 0.025M calcium chloride to 0.7 mL of blood for a 1:10 ratio. The thrombogenicity of Vitagraft ePTFE was determined at four degrees of denucleation: undenucleated (CONTROL, n = lo), denucleated by 4 mm Hg vacuum only (VACUUM, n = 10) partially denucleated at 100 psig (PARTIAL, n = lo), and completely denucleated at 20,000 psig (TOTAL, n = 10).
Statistics Differences between groups were evaluated using a Wilcoxon Rank Sum test with a p value of 0.05 or less required for statistical significance. RESULTS
Gas denucleation All denucleation methods caused grafts to change from opaque white to translucent gray as previously rep~rted.”””’~ Acetone or ethanol treatments produced a more uniform change to a translucent appearance than pressure treatments. However, small bubbles formed on these grafts when placed in water. Preliminary studies indicated that vacuum and vacuum plus pressure denucleation caused a 10-20% decrease in graft length due to a reduction of the internodal distance. No measurable shortening occurred in grafts denucleated in acetone or ethanol. Grafts used in this study had various pretreatment lengths in order to assure uniform 1-cm lengths following treatment.
Determination of degree of denucleation 1. Qualitative analysis
When exposed to vacuum (4 mm Hg), untreated grafts bubbled violently. Partially denucleated grafts (100 psig-500 psig) produced only small bubbles.
BENSEN ET AL.
378
No bubbles were observed on the completely denucleated grafts (500 psig or greater). 2. Quantitative analysis (a) Buoyancy method (Archimedes’ Principle): Graft buoyancy decreased as the denucleation pressure increased (Fig. 2). Denucleation was 97.4% complete with a pressure treatment of 100 psig and indistinguishable from 100% with pressure treatments of 500 psig or greater. Grafts denucleated by ethanol immersion had a mean denucleation of 94.3%. Grafts denucleated by acetone immersion had a significantly higher denucleation of 98.9% ( p < 0.05). (b) Photometry: The light transmittance of control grafts was 8.88 f 0.70%. After denucleation at 20,000 psig, transmittance significantly increased to 41.70 t 0.81% ( p < 0.001).
Gas content of vascular prostheses These results indicate that there is a wide range in gas content of commonly used vascular prostheses on submersion in water. Vitagraft ePTFE was 75.5 f 0.4% air (n = 20), Gore-tex ePTFE 69.2 f 0.3% (n = 5), Impra ePTFE 61.8 t 0.3% (n = lo), Corvita naked polyurethane 30.2 f 0.3% ( n = 5), naked Dacron 7.6 4 0.7%(n = lo), and albumin-coated Dacron 2.4 -+ 0.3% (n = 10). Renucleation 1. Shelflife
The data indicate that an average of 0.01 f 0.01% renucleation occurred over the 8-month test period. Since the resolution of the method was 0.15%,no detectable renucleation occurred when grafts were immersed during storage. C
-n=5
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n=5
n.5
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379
QUANTIFICATION OF DEN UCL EATION A N D THROMBOGENICITY
2. In t raoperat ive renucleation
The results of the simulated surgical setting are summarized in Figure 3. These data demonstrate that intermittent immersion dramatically reduced graft renucleation from 89% to 3.4%.Wrapping the grafts in wet gauze further reduced renucleation significantly to only 1.4%( p < 0.001). Scanning electron microscopy Figure 4 shows that denucleation caused a reduction in internodal distance. This is consistent with the observed overall shortening of the grafts. The closer nodes appear to allow the fibrils to clump together into bundles, resulting in large, irregularly spaced pores between the nodes. The large pores appear to be more numerous in the pressure denucleated grafts. Gravimetric assessment of thrombogenicity Figure 5a indicates that both partially (100 psig) and totally (20,000 psig) denucleated grafts had significantly less adherent thrombus than the control and vacuum denucleated grafts. Differences between partially and totally denucleated grafts were not significant. At 4 min, vacuum denucleated grafts had significantly less thrombus than control grafts. In Figure 5b, the thrombus weights of Figure 5a are plotted against the percentage denucleation from Figure 2. Figure 5b shows that the removal of air from the graft results in progressive and significant reduction in adherent thrombus weight. DISCUSSION
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