J. BIOMED. MATER. RES.
VOL. 9, PP. 4 8 7 4 9 9 (1975)
Platelet Adhesion and Contact Activation Time Tests on HEMA Coated Cellulose Acetate Membranes K. J. MUZYIIEWICZ,* Department of Chemical Engineering, E. B. CROWELL, JR., A. P. HART, and RI. SCHULTS, Department of Medicine, C . G. HILL, JR. and S. L. COOPER, Department of Chemical Engineering, University of Wisconsin, Madison, Wisconsin 53706
Summary Surface modification of cellulose acetate dialysis membranes was carried out by BOCo radiation induced graft copolymerization of the hydrogel, hydroxyethyl methacrylate (HEMA). The degree of grafting was controlled by varying the HEMA monomer concentration in the grafting solution and the radiation dose. A continuous flow platelet adhesion test was designed which allows testing under conditions more closely approximating hemodialysis than other small scale in vitro tests. Platelet adhesion on treated membranes fell substantially with increasing surface HEMA concentration. The presence of HEMA on the membrane surface did not affect the membrane activated clotting times significantly.
INTRODUCTION A major problem in the field of bioengineering involves devices which are thrombogenic when implanted within the human body or when used in extracorporeal situations. In order to characterize the interactions between constituents of the blood and the surfaces of these devices it is necessary to develop reproducible test methods which quantitatively describe the phenomena involved. This paper describes two test procedures used in evaluating the hemocompatibility of membrane surfaces created by irradiation grafting of HEMA (2-hydroxyethyl methacrylate) to a cellulose acetate substrate. * Present address: E. I. du Pont de Nemours & Co., Wilniington, Delaware. 487 @ 1975 by John Wiley & Sons, Inc.
488
MUZYKb:WICZ ET AL.
Platelet Adhesion Tests Ont. of the most important intcractions bdivwn blood and foreign materials involves thcb adhesion of platelets. Salzman’ has stated .‘It is now customary to view surface induced thrombosis as chiefly, if not exclusivcly, a platelet problem.” Many tests for platelet adhesiveness have been devised. These are based primarily on the rotating bulb method of Wright2 and the glass bead column of H e l l ~ m . These ~ methods make use of a standard surface such as glass beads and do not lend themselves to evaluation of the thrombogenic character of various foreign surfaccs. Lindsay ct al.4 have dcvised a system for the comparison of different hemodialysis membranes by the degree of platelet adhesion observed. Another test for platelet adhesion developed by Lyman et a1.j involves contacting a material with blood for a very short time and determining the density of platelets on the material surface. These tests, while valid for comparing two surfaces undcr the conditions of the test, do not compare membranes undcr conditions similar to those of hemodialysis where the blood is continuously flowing in responsc to a pulsatile pressure driving force. Thus a portion of this research was concerned with the design of a small scale in v i t ~ otest that could compare the adhesion of platelets to various surfaces under conditions of continuous blood flow for various time periods.
Contact Activation Time Tests A second major problem that arises from the interaction of blood with a foreign surface is activation of the intrinsic clotting niechanism. Most studics of the effects of a surface on the intrinsic clotting factors determine the partial thromboplastin times (PTT) or recalcification times for a control and for a membrane activated plasma6.’. In most cases to date, if a tube cannot be coated with the material in question, either whole blood or plasma is allowed to contact the material for a period of time. After this period of contact, the plasma or whole blood is separated from the material and the time for this contacted blood t o clot measured by determining a PTT in either a These methods involve extra hanglass or siliconizcd glass dling of thc blood and have inadvertent contact activation. I n this work, an in vitro test was developed to determine directly the role of
PLATELET ADHESION ON HEMA COATEI) MEMBRANES 489
the prepared membrantb surfaces in activating thc intrinsic clotting mechanism.
Hydrogel Materials Hydrogels are very lightly crosslinked networks of hydrophilic polymers. These hydrophilic networks arc capable of absorbing and retaining large quantities of water. Consequently, the surface that a hydrogel will present to blood proteins nil1 be a rather indefinite one which, like the exposed areas of the proteins, is hydrophilic. A hydrogel surface is easily deformable on the microscopic level and should provide little resistance to flowing blood rlcments. The interface which occurs between the blood and the gel would have minimal minimal interfacial free rnergy and should not greatly perturb the bioenvironment .g Therefore, such surfaces should preserve the existing conformation of blood proteins and minimizr surface platelet adhesion. Until recently, the major obstacle to the use of hydrogels as biomaterials has been their general lack of strength. This problem may be circumvented by the grafting of thin layers of the hydrogel polymrrs to polymer substrates having mechanical and transport properties more consistoent with biomedical applications. These thin layers (about 100 A thick) of hydrogel have bcen shown to yield surface properties identical to those of membranes composed entirely of the hydrogel material.1° Because cellulose acetate membranes have excellent permselectivity characteristics for water transport and are the material of choice for many ultrafiltration applications, this material was chosen as the substratr to which the HERIA was grafted.
EXPERIMENTAL METHODS Radiation Grafting Radiation grafting of the monomer 2-hydroxyethyl methacrylate, HEMA, (technical grade, Aldrich Chemical Co.) t o a solid membrane composed of cellulose acetate (Eastman Kodak IiP-98, 0.09 mm thick) was conducted using a 6oCoradiation source with a dose rate of 3500 rad/min. Typical doses used for the irradiations ranged from 0.05-0.25 Jlrad. Grafting was accomplished by mutual irradia-
MUZYKEWICZ ET AL.
490
tion of the cellulose acetate polymer and a solution of monomer in pure methanol (reagent grade). The grafting reaction was carried out in an annular reactor which could be placed in a large reaction vessel (Fig. 1). All components were made of aluminum. The reaction vessel was constructed so as to allow t h r removal of oxygen. A 20 min purge of the vessel with nitrogen gas provided good results. Prior to each grafting reaction, a portion of the crllulose acetate membrane (approximately 4 in. X 10 in.) was rinsed in distilled water and allowed to remain in a beaker of distilled water for several days. The membrane was then soaked in solvent for a t least 2 hr before irradiation. A small portion of membrane was measured while wet (after soaking in distilled water). It was superficially dried and weighed to determine the weight per unit area of untreated membrane. This small portion of dried membrane together with the larger solvent-soaked membrane was placed in the cylindrical container with a solution containing solvent and HERIA. The system was then purged of oxygen, sealed and irradiated. After the membrane sample was irradiated, it was removed from the reaction vessel and soaked in distilled water for a t least a day. The small section of predried membrane that was included in the reactor was soaked in methanol for a few hours and then for a t least a day in distilled water. This sample was then redried and weighed until a constant weight was achieved. The difference in pre- and postgrafting weight was recorded as the change in weight/arra and the yo change in weight relative to the initial value was then calculated.
n.
rig.
.
1.
. ..
irraaiation cell; A-cavity ror raaiation source, a-annular sample cell, C-vacuum connection.
Platelet Adhesion Test The devicc. shown in Figures 2 and 3 was usrd to pwform platclvt adhesion tests on thc mrmbranrs. Two shccts of mrmbranr rinsod in distilled water werr placed on thc Plexiglas blocks and air bubblcs removed. The gasket was placcd on one mcmbranr and thc halvrs then placcd together and clamprd firmly. Thc spaer bctwcen the mcmbranes was filled with distilled water and this was displaced with 0.9% salinc and thcn with hcparinizcd saline (HS)-sodium hrparin from intestinal mucosa, Minnesota hlining and Manufacturing Co.) containing 50 p heparin per ml. The tubing was flushcd with HS, connectcd t o the devicc., and HS was pumped through t h c systclm for 5 min. Blood was taken from a hralthy voluntrcr n h o had not had aspirin for a t lcast 2 w w k s and who had given his informrd consent. Twenty-five ml was drawn into a hcparinizcd plastic syringr (I'harmaseal Laboratories, Glendalc, California) to givc a final hcparin concentration of 50 p pcr ml. The system was flushrd sc.qucntially with two 10 ml aliquots of hcparinizcd blood and a timer bcgun with the entry of the second aliquot. Hcmatocrit determinations on thc blood before and after flushing showrd no apprcciable dilution effrct. The tubing was filled with blood and th(>blood pumprd through thc system a t a constant flow rate of 2 ml/min and an average lincar velocity across the membrane of 6 cm/min. Two-tcnth ml blood
\ PLEXIGLAS
BLOCK
GASKET
Fig. 2 . l)et,ail of platelet adhesion test device.
MUZYKISWICZ ET A L
492
d
SANDWICH
SlLlCONlZED
CIRCULATING
WOO0
Fig. 3. Platelet adhesion test device assembly.
samples for hematocrits and platelet counts were taken at 15, 30, and 60 min after the timer was begun. This blood was replaced by more heparinizcd blood to prcwxnt air bubblos in the system. All platelet counts n-(w done by phase microscopy by the samv technician.
Membrane Activated Partial Thromboplastin Time The dcvice depicted in Figurc 4 was devcloped to allow clotting time trsts t o be performed on blood t h a t is in direct contact with the membrane material. It consists of 3 sections of I’lexiglas constructed such that they may be fastcned together to form 2 vials, 1.4 em X 0.5 cm X 7.5 cm. When separated, thc areas of thc Plexiglas sections that form the vials may be coated with the mc.mbrane to be tested. This was done by laying sections of membrane flat on the 2 outer Plexiglas parts and folding 3 other portions of membrane to coat the sides and bottom of the vial as illustrated in Figure 4(b). This procedure results in 2 narrow containers whose surfaces are composed solely of test material. The ratio of the surface area of membrane to tht. liquid volumc in the vials is 0.7 cm2/cm3. The test is conducted by first rinsing test mcmbrancs in distilled watvr, cutting thcm to size and coating the. vials with them. In each case, one vial was coated with untrc.ated membrane while the other was coatcd with the trst membrane. The vials were filled with distilled water and then empticd by shaking until no water droplets were visible on the mcmbrancs. Partial thromboplastin clotting times, I’TT’s, were then pcrformrd on uncontacted plasma (9 parts blood to 1 part 3.87, sodium citratv centrifuged a t 3000 X y for 15
CLEAR PLEXIGLAS
CLOTTING VIALS
Y Fig. 4.
Membrane activated clotting device.
min) in the membrane coated vials. 0.5 ml of cephalin in 0.025 M CaClz was added to the vials in a water bath a t 37°C. An rqual volume of plasma was added to each vial and a timer startrd for each vial. The vials w.v(vtiltcd a t 15-30 scc intervals until the mixturr no longer flowd. This point was taken as the end of the PTT. The plasma used had been previously storod in a plastic tube (Falcon Plastics, Oxnard, California) and transferred with a siliconized glass pipet. Thc diffrrcncc in the clotting timrs for the test and untreated
494
*MUZYKEWICZ ET AL.
mclmbrancs \\-as dividcd by the clotting timcb for t hc untrcL:ttc.d sample and reported as perccnt of control. The us(’ of a devicci containing 2 vials permitted the amount of agitation for control and test samples to be identical.
RESULTS AND DISCUSSION Radiation Grafting The results for the radiation induced graft copolymerization of 2-hydroxyethyl methacrylatc (HERIA) on cellulose acetate are shown in Table I. I n most cases, the w i g h t increases for HERIA grafts correlate with the HEAIA concrntration in the methanol grafting solution and with the radiation dose. I n cases in which the radiation dose was constant, increased HENIA concentration in the grafting solution generally resulted in increased grafted weight gains. In the few cases in which the HEMA concentration was held constant and the dose varied, a gcnrral increase in yield was seen for incrrased radiation dose. There is a fair amount of scatter, however. It could be attributed to the fact that the time for swelling of the membrane in the grafting solution prior to irradiation varied from sample to sample. This time was not measured for most samples but was generally 1 to 2 hr. Some samples were swollen for longer times, however. One sample that was swollen in solution for slightly more than 7 hr (sample no. .i in Table I) showed a t least a 25% higher yield than other samples of equal HERIA solution concentration a t the same radiation dosr.
Blood Compatibility Results The main thrust of this aspect of the research program involved the dcvelopment of improved zn vztl-o testing proccdures for determining the thrombogenic character of the materials synthesized. Two tests were performed as described in Methods. One measured the specific interaction of platelets with the membrane surface and the other measured the intcraction of blood contact factors with membranes. Each will be discussed in turn. Platelet Adhesion Test and Results.-The platelet adhesion test designed in this work is advantageous in that it is simple, can simulate the actual flow conditions of hemodialysis and can provide a good means of comparing different membranes. Care must be taken when
TABLL I Experimental Data for (;rafting of HEJIA on Celliilo\e Acetate -
7;HEMA in Run
Grafting Solution
It adiat ion I h e (nlrad)
Wr/Area mg/un*
0.0 1.0 2 .5 -5.0 . .i 0 .5 .0 10.0 10.0 10.0 10.0 12.6 15.0 22.0 28.0 32.0
0.21 0.21 0.11 0.21 0.21 0.21 0.11 0.11 0.21 0.2;i 0.11 0.11 0.11 0.11 0.07
0.0 0.20 0.31 0.45 0.91 0.77 0 . .58 0.76 1 .2.5 1.01 1.07 0..% 1.91 4.32 3.41
Invreahe in Wt ~~
1 2 3 4 3
6 7 8 9 10 11 12 13 14 15
0.0 7.4 11.1 13..5 32.0 2.5.6 18..i 25.3 3.5. 1 s1.9 83.8 19.2 56.0 140.0 112.0
conducting this test howw'r, as the blood flow rat", the degree of blood-air contact, surface wrinkling leading to blood channeling, and the degree of hemolysis should all affect the interaction of platelets with a surface. In order to minimize these c.ffccts the following procedures were employed : The blood flow ratc was kept constant for each run by regulation of the roller pump. Blood-air contact was virtually eliminated in that the only air contact with the blood occurred a t the tiny areas a t the tip of the syringc. prior to entry into the device and a t the exit port during sampling. Air bubbles were removed from the device itself prior to introduction of the blood. In one case, the membrane surface becamcb ribbcd (giving a washboard appearance) during a long irradiation a t high HERtA concentration. When tested for platelet adhesion, visible channeling of the blood occurred with thc resultant very high hemolysis and high platelet adhesion. The surface of all samples described below appeared equally smooth and displayed even blood distribution. The introduction of the ellipse-shaped gasket maintained constant blood height during flow and reduced hemolysis. Hematocrits determined for the blood a t each stage of sampling, showed blood dilution and hemolysis to be minimal in most cases.
Thcl facat that hlood also rontacts othvr s u r f a w ~(i.v., thcb tubing, the gask(1t and the syringv) should not affvrt thc rc~lativc~ rcdts significantly. The area of thr tubing and thv gaskvt comt)inc)d is Icss t h a n 3% of the total surfarcs area nhich t h c blood rontacts. JIorrovcir, th(b slight c+frct of thcw other surfaccis 11as constant from run to run. The sampling method, n hich rcyuirvd substqucmt introduction of fresh blood into the systcm to displace. air from the tubing, also Yhould have vrry little effwt on thc rcsults. Thr total blood added ovcr thc coursr of the test was only about 5% of the total volume of the device. Oncr again, any effect of this procedure was constant from run to run. The degrec of platc4et adhcsion was determined by the drop in platelrt count obscrvcd for each blood samplc. The yc adhwion a t time t was determined as follows:
% Adhesion
(Initial Platelets - I’latelets at time t) Initial Platelets
= --~---_____
The rcsults of the platelvt adhcsion tests are shown in Figures 5 and 6. Figure 5 shows the percent platelet adhesion versus time of blood-membrane contact for various surface conccntrations of HEMA. This data indicates that as the surface concentration of HEMA increases, the degree of platclet adhesion t o the surface decreases. This result is shown more clearly in Figure 6 for a bloodI
TIME OF BLOOD -MEMBRANE
Fig. 5.
I
CONTACT ( min
I
Platelet adhesion vs. time of blood-membrane contact for various surface concentrations of HEMA.
SURFACE HEMA
Fig. 6.
Platelet adhesion
VS.
CONCENTRATION (mg/cr$)
burface concentration of HEMA after 60 min of blood contact.
membrane contact time of 60 min. This trend can probably be attributed to a grrater uniformity and incrrmed thickness of the poly(HE1IA) layer resulting in a greater degree of hydrogel character. For untreated mcmbranrs the rrsults represent an average of 2 runs. All other results represent single runs. Although identical replications were not performed, the trend in the data supports its validity . Membrane Activated Partial Thromboplastin Time.-The other in vitl-o test developed in this work involved a measurement of the membrane surface activation of the intrinsic clotting system. The test utilizes the partial thromboplastin timc (PTT) to eliminate thc effect of red blood cells and platelets on the clotting time. This procedure has the advantage that it allows testing of thin, flexible transparent films of materials without the necessity for such films to be cast on the inside of glass tubes. Furthermore, the presence of the 2 test vials allows testing of the treated samples and controls under identical conditions. The results of the membrane activated PTT tests arc shown in Table 11. It lists the effects of HEMA concentration on the clotting time. While there appears to be a slight increase in percent of control clotting time (that is, an improvement in blood compati-
MUZYKISWICZ E T AI,.
498
bility) I\ ith HISJIA conccwtration, thct diffwcwcm I\ mi slight. Thcsch timcs are rc3lativdy long and arc’ comparablr to t h o w for a silironc1 rubber surface. TABLE I1 Effect of HEMA Surface Concentration o n Clotting Tirneh
Run no. 1
3 12 6 5 9 13
Wt/Area mg/cm2
0 0.31 0.59 0.77 0.91 1.2,; 1.91
,Membrane activated partial thromboplastin clotting y6 of contiol time side rlotting time 8 min 8 min 8 min 8 min 8 min 10 min 9 min
50 sec 5 1 sec. 20 her 40 sec 25 sec 2.5 sec 8 sec
-
I 00(/, 97% 107% 120yo I2.50/, 104
cx
CONCLUSIONS Surface modification of cellulose acetate dialysis membranes was carried out by radiation induced graft copolymerization of hydroxyethyl mrthacrylate, HERIA. The degree of grafting was found to depend on the HERIA concentration in thc grafting solution, the radiation dose, the swelling time and the solvent used. Two in vitro tests were developed as a means of comparing thc thrombogenicity of thc membranes prepared. The tests are a continuous flow platelet adhesion test and a membrane activated partial thromboplastin clotting time test. The former is advantageous in that it allows testing under conditions more closely approximating hemodialysis than other small scale in vztro tests. The results from thew tests indicate that a surface coating of HEMA may decrease thrombogenicity. Platelet adhesion on treated membranes fell substantially with increasing surface HEMA concentration. However, t h r presence of HEMA on the membrane surface did not affect thc membrane activated clotting timcs significantly. The authors wish to acknowledge support of this research from National Institutes of Health Grants HL-GM 15668-02 and HL14786-02.
References I . I
VOL. 9, PP. 4 8 7 4 9 9 (1975)
Platelet Adhesion and Contact Activation Time Tests on HEMA Coated Cellulose Acetate Membranes K. J. MUZYIIEWICZ,* Department of Chemical Engineering, E. B. CROWELL, JR., A. P. HART, and RI. SCHULTS, Department of Medicine, C . G. HILL, JR. and S. L. COOPER, Department of Chemical Engineering, University of Wisconsin, Madison, Wisconsin 53706
Summary Surface modification of cellulose acetate dialysis membranes was carried out by BOCo radiation induced graft copolymerization of the hydrogel, hydroxyethyl methacrylate (HEMA). The degree of grafting was controlled by varying the HEMA monomer concentration in the grafting solution and the radiation dose. A continuous flow platelet adhesion test was designed which allows testing under conditions more closely approximating hemodialysis than other small scale in vitro tests. Platelet adhesion on treated membranes fell substantially with increasing surface HEMA concentration. The presence of HEMA on the membrane surface did not affect the membrane activated clotting times significantly.
INTRODUCTION A major problem in the field of bioengineering involves devices which are thrombogenic when implanted within the human body or when used in extracorporeal situations. In order to characterize the interactions between constituents of the blood and the surfaces of these devices it is necessary to develop reproducible test methods which quantitatively describe the phenomena involved. This paper describes two test procedures used in evaluating the hemocompatibility of membrane surfaces created by irradiation grafting of HEMA (2-hydroxyethyl methacrylate) to a cellulose acetate substrate. * Present address: E. I. du Pont de Nemours & Co., Wilniington, Delaware. 487 @ 1975 by John Wiley & Sons, Inc.
488
MUZYKb:WICZ ET AL.
Platelet Adhesion Tests Ont. of the most important intcractions bdivwn blood and foreign materials involves thcb adhesion of platelets. Salzman’ has stated .‘It is now customary to view surface induced thrombosis as chiefly, if not exclusivcly, a platelet problem.” Many tests for platelet adhesiveness have been devised. These are based primarily on the rotating bulb method of Wright2 and the glass bead column of H e l l ~ m . These ~ methods make use of a standard surface such as glass beads and do not lend themselves to evaluation of the thrombogenic character of various foreign surfaccs. Lindsay ct al.4 have dcvised a system for the comparison of different hemodialysis membranes by the degree of platelet adhesion observed. Another test for platelet adhesion developed by Lyman et a1.j involves contacting a material with blood for a very short time and determining the density of platelets on the material surface. These tests, while valid for comparing two surfaces undcr the conditions of the test, do not compare membranes undcr conditions similar to those of hemodialysis where the blood is continuously flowing in responsc to a pulsatile pressure driving force. Thus a portion of this research was concerned with the design of a small scale in v i t ~ otest that could compare the adhesion of platelets to various surfaces under conditions of continuous blood flow for various time periods.
Contact Activation Time Tests A second major problem that arises from the interaction of blood with a foreign surface is activation of the intrinsic clotting niechanism. Most studics of the effects of a surface on the intrinsic clotting factors determine the partial thromboplastin times (PTT) or recalcification times for a control and for a membrane activated plasma6.’. In most cases to date, if a tube cannot be coated with the material in question, either whole blood or plasma is allowed to contact the material for a period of time. After this period of contact, the plasma or whole blood is separated from the material and the time for this contacted blood t o clot measured by determining a PTT in either a These methods involve extra hanglass or siliconizcd glass dling of thc blood and have inadvertent contact activation. I n this work, an in vitro test was developed to determine directly the role of
PLATELET ADHESION ON HEMA COATEI) MEMBRANES 489
the prepared membrantb surfaces in activating thc intrinsic clotting mechanism.
Hydrogel Materials Hydrogels are very lightly crosslinked networks of hydrophilic polymers. These hydrophilic networks arc capable of absorbing and retaining large quantities of water. Consequently, the surface that a hydrogel will present to blood proteins nil1 be a rather indefinite one which, like the exposed areas of the proteins, is hydrophilic. A hydrogel surface is easily deformable on the microscopic level and should provide little resistance to flowing blood rlcments. The interface which occurs between the blood and the gel would have minimal minimal interfacial free rnergy and should not greatly perturb the bioenvironment .g Therefore, such surfaces should preserve the existing conformation of blood proteins and minimizr surface platelet adhesion. Until recently, the major obstacle to the use of hydrogels as biomaterials has been their general lack of strength. This problem may be circumvented by the grafting of thin layers of the hydrogel polymrrs to polymer substrates having mechanical and transport properties more consistoent with biomedical applications. These thin layers (about 100 A thick) of hydrogel have bcen shown to yield surface properties identical to those of membranes composed entirely of the hydrogel material.1° Because cellulose acetate membranes have excellent permselectivity characteristics for water transport and are the material of choice for many ultrafiltration applications, this material was chosen as the substratr to which the HERIA was grafted.
EXPERIMENTAL METHODS Radiation Grafting Radiation grafting of the monomer 2-hydroxyethyl methacrylate, HEMA, (technical grade, Aldrich Chemical Co.) t o a solid membrane composed of cellulose acetate (Eastman Kodak IiP-98, 0.09 mm thick) was conducted using a 6oCoradiation source with a dose rate of 3500 rad/min. Typical doses used for the irradiations ranged from 0.05-0.25 Jlrad. Grafting was accomplished by mutual irradia-
MUZYKEWICZ ET AL.
490
tion of the cellulose acetate polymer and a solution of monomer in pure methanol (reagent grade). The grafting reaction was carried out in an annular reactor which could be placed in a large reaction vessel (Fig. 1). All components were made of aluminum. The reaction vessel was constructed so as to allow t h r removal of oxygen. A 20 min purge of the vessel with nitrogen gas provided good results. Prior to each grafting reaction, a portion of the crllulose acetate membrane (approximately 4 in. X 10 in.) was rinsed in distilled water and allowed to remain in a beaker of distilled water for several days. The membrane was then soaked in solvent for a t least 2 hr before irradiation. A small portion of membrane was measured while wet (after soaking in distilled water). It was superficially dried and weighed to determine the weight per unit area of untreated membrane. This small portion of dried membrane together with the larger solvent-soaked membrane was placed in the cylindrical container with a solution containing solvent and HERIA. The system was then purged of oxygen, sealed and irradiated. After the membrane sample was irradiated, it was removed from the reaction vessel and soaked in distilled water for a t least a day. The small section of predried membrane that was included in the reactor was soaked in methanol for a few hours and then for a t least a day in distilled water. This sample was then redried and weighed until a constant weight was achieved. The difference in pre- and postgrafting weight was recorded as the change in weight/arra and the yo change in weight relative to the initial value was then calculated.
n.
rig.
.
1.
. ..
irraaiation cell; A-cavity ror raaiation source, a-annular sample cell, C-vacuum connection.
Platelet Adhesion Test The devicc. shown in Figures 2 and 3 was usrd to pwform platclvt adhesion tests on thc mrmbranrs. Two shccts of mrmbranr rinsod in distilled water werr placed on thc Plexiglas blocks and air bubblcs removed. The gasket was placcd on one mcmbranr and thc halvrs then placcd together and clamprd firmly. Thc spaer bctwcen the mcmbranes was filled with distilled water and this was displaced with 0.9% salinc and thcn with hcparinizcd saline (HS)-sodium hrparin from intestinal mucosa, Minnesota hlining and Manufacturing Co.) containing 50 p heparin per ml. The tubing was flushcd with HS, connectcd t o the devicc., and HS was pumped through t h c systclm for 5 min. Blood was taken from a hralthy voluntrcr n h o had not had aspirin for a t lcast 2 w w k s and who had given his informrd consent. Twenty-five ml was drawn into a hcparinizcd plastic syringr (I'harmaseal Laboratories, Glendalc, California) to givc a final hcparin concentration of 50 p pcr ml. The system was flushrd sc.qucntially with two 10 ml aliquots of hcparinizcd blood and a timer bcgun with the entry of the second aliquot. Hcmatocrit determinations on thc blood before and after flushing showrd no apprcciable dilution effrct. The tubing was filled with blood and th(>blood pumprd through thc system a t a constant flow rate of 2 ml/min and an average lincar velocity across the membrane of 6 cm/min. Two-tcnth ml blood
\ PLEXIGLAS
BLOCK
GASKET
Fig. 2 . l)et,ail of platelet adhesion test device.
MUZYKISWICZ ET A L
492
d
SANDWICH
SlLlCONlZED
CIRCULATING
WOO0
Fig. 3. Platelet adhesion test device assembly.
samples for hematocrits and platelet counts were taken at 15, 30, and 60 min after the timer was begun. This blood was replaced by more heparinizcd blood to prcwxnt air bubblos in the system. All platelet counts n-(w done by phase microscopy by the samv technician.
Membrane Activated Partial Thromboplastin Time The dcvice depicted in Figurc 4 was devcloped to allow clotting time trsts t o be performed on blood t h a t is in direct contact with the membrane material. It consists of 3 sections of I’lexiglas constructed such that they may be fastcned together to form 2 vials, 1.4 em X 0.5 cm X 7.5 cm. When separated, thc areas of thc Plexiglas sections that form the vials may be coated with the mc.mbrane to be tested. This was done by laying sections of membrane flat on the 2 outer Plexiglas parts and folding 3 other portions of membrane to coat the sides and bottom of the vial as illustrated in Figure 4(b). This procedure results in 2 narrow containers whose surfaces are composed solely of test material. The ratio of the surface area of membrane to tht. liquid volumc in the vials is 0.7 cm2/cm3. The test is conducted by first rinsing test mcmbrancs in distilled watvr, cutting thcm to size and coating the. vials with them. In each case, one vial was coated with untrc.ated membrane while the other was coatcd with the trst membrane. The vials were filled with distilled water and then empticd by shaking until no water droplets were visible on the mcmbrancs. Partial thromboplastin clotting times, I’TT’s, were then pcrformrd on uncontacted plasma (9 parts blood to 1 part 3.87, sodium citratv centrifuged a t 3000 X y for 15
CLEAR PLEXIGLAS
CLOTTING VIALS
Y Fig. 4.
Membrane activated clotting device.
min) in the membrane coated vials. 0.5 ml of cephalin in 0.025 M CaClz was added to the vials in a water bath a t 37°C. An rqual volume of plasma was added to each vial and a timer startrd for each vial. The vials w.v(vtiltcd a t 15-30 scc intervals until the mixturr no longer flowd. This point was taken as the end of the PTT. The plasma used had been previously storod in a plastic tube (Falcon Plastics, Oxnard, California) and transferred with a siliconized glass pipet. Thc diffrrcncc in the clotting timrs for the test and untreated
494
*MUZYKEWICZ ET AL.
mclmbrancs \\-as dividcd by the clotting timcb for t hc untrcL:ttc.d sample and reported as perccnt of control. The us(’ of a devicci containing 2 vials permitted the amount of agitation for control and test samples to be identical.
RESULTS AND DISCUSSION Radiation Grafting The results for the radiation induced graft copolymerization of 2-hydroxyethyl methacrylatc (HERIA) on cellulose acetate are shown in Table I. I n most cases, the w i g h t increases for HERIA grafts correlate with the HEAIA concrntration in the methanol grafting solution and with the radiation dose. I n cases in which the radiation dose was constant, increased HENIA concentration in the grafting solution generally resulted in increased grafted weight gains. In the few cases in which the HEMA concentration was held constant and the dose varied, a gcnrral increase in yield was seen for incrrased radiation dose. There is a fair amount of scatter, however. It could be attributed to the fact that the time for swelling of the membrane in the grafting solution prior to irradiation varied from sample to sample. This time was not measured for most samples but was generally 1 to 2 hr. Some samples were swollen for longer times, however. One sample that was swollen in solution for slightly more than 7 hr (sample no. .i in Table I) showed a t least a 25% higher yield than other samples of equal HERIA solution concentration a t the same radiation dosr.
Blood Compatibility Results The main thrust of this aspect of the research program involved the dcvelopment of improved zn vztl-o testing proccdures for determining the thrombogenic character of the materials synthesized. Two tests were performed as described in Methods. One measured the specific interaction of platelets with the membrane surface and the other measured the intcraction of blood contact factors with membranes. Each will be discussed in turn. Platelet Adhesion Test and Results.-The platelet adhesion test designed in this work is advantageous in that it is simple, can simulate the actual flow conditions of hemodialysis and can provide a good means of comparing different membranes. Care must be taken when
TABLL I Experimental Data for (;rafting of HEJIA on Celliilo\e Acetate -
7;HEMA in Run
Grafting Solution
It adiat ion I h e (nlrad)
Wr/Area mg/un*
0.0 1.0 2 .5 -5.0 . .i 0 .5 .0 10.0 10.0 10.0 10.0 12.6 15.0 22.0 28.0 32.0
0.21 0.21 0.11 0.21 0.21 0.21 0.11 0.11 0.21 0.2;i 0.11 0.11 0.11 0.11 0.07
0.0 0.20 0.31 0.45 0.91 0.77 0 . .58 0.76 1 .2.5 1.01 1.07 0..% 1.91 4.32 3.41
Invreahe in Wt ~~
1 2 3 4 3
6 7 8 9 10 11 12 13 14 15
0.0 7.4 11.1 13..5 32.0 2.5.6 18..i 25.3 3.5. 1 s1.9 83.8 19.2 56.0 140.0 112.0
conducting this test howw'r, as the blood flow rat", the degree of blood-air contact, surface wrinkling leading to blood channeling, and the degree of hemolysis should all affect the interaction of platelets with a surface. In order to minimize these c.ffccts the following procedures were employed : The blood flow ratc was kept constant for each run by regulation of the roller pump. Blood-air contact was virtually eliminated in that the only air contact with the blood occurred a t the tiny areas a t the tip of the syringc. prior to entry into the device and a t the exit port during sampling. Air bubbles were removed from the device itself prior to introduction of the blood. In one case, the membrane surface becamcb ribbcd (giving a washboard appearance) during a long irradiation a t high HERtA concentration. When tested for platelet adhesion, visible channeling of the blood occurred with thc resultant very high hemolysis and high platelet adhesion. The surface of all samples described below appeared equally smooth and displayed even blood distribution. The introduction of the ellipse-shaped gasket maintained constant blood height during flow and reduced hemolysis. Hematocrits determined for the blood a t each stage of sampling, showed blood dilution and hemolysis to be minimal in most cases.
Thcl facat that hlood also rontacts othvr s u r f a w ~(i.v., thcb tubing, the gask(1t and the syringv) should not affvrt thc rc~lativc~ rcdts significantly. The area of thr tubing and thv gaskvt comt)inc)d is Icss t h a n 3% of the total surfarcs area nhich t h c blood rontacts. JIorrovcir, th(b slight c+frct of thcw other surfaccis 11as constant from run to run. The sampling method, n hich rcyuirvd substqucmt introduction of fresh blood into the systcm to displace. air from the tubing, also Yhould have vrry little effwt on thc rcsults. Thr total blood added ovcr thc coursr of the test was only about 5% of the total volume of the device. Oncr again, any effect of this procedure was constant from run to run. The degrec of platc4et adhcsion was determined by the drop in platelrt count obscrvcd for each blood samplc. The yc adhwion a t time t was determined as follows:
% Adhesion
(Initial Platelets - I’latelets at time t) Initial Platelets
= --~---_____
The rcsults of the platelvt adhcsion tests are shown in Figures 5 and 6. Figure 5 shows the percent platelet adhesion versus time of blood-membrane contact for various surface conccntrations of HEMA. This data indicates that as the surface concentration of HEMA increases, the degree of platclet adhesion t o the surface decreases. This result is shown more clearly in Figure 6 for a bloodI
TIME OF BLOOD -MEMBRANE
Fig. 5.
I
CONTACT ( min
I
Platelet adhesion vs. time of blood-membrane contact for various surface concentrations of HEMA.
SURFACE HEMA
Fig. 6.
Platelet adhesion
VS.
CONCENTRATION (mg/cr$)
burface concentration of HEMA after 60 min of blood contact.
membrane contact time of 60 min. This trend can probably be attributed to a grrater uniformity and incrrmed thickness of the poly(HE1IA) layer resulting in a greater degree of hydrogel character. For untreated mcmbranrs the rrsults represent an average of 2 runs. All other results represent single runs. Although identical replications were not performed, the trend in the data supports its validity . Membrane Activated Partial Thromboplastin Time.-The other in vitl-o test developed in this work involved a measurement of the membrane surface activation of the intrinsic clotting system. The test utilizes the partial thromboplastin timc (PTT) to eliminate thc effect of red blood cells and platelets on the clotting time. This procedure has the advantage that it allows testing of thin, flexible transparent films of materials without the necessity for such films to be cast on the inside of glass tubes. Furthermore, the presence of the 2 test vials allows testing of the treated samples and controls under identical conditions. The results of the membrane activated PTT tests arc shown in Table 11. It lists the effects of HEMA concentration on the clotting time. While there appears to be a slight increase in percent of control clotting time (that is, an improvement in blood compati-
MUZYKISWICZ E T AI,.
498
bility) I\ ith HISJIA conccwtration, thct diffwcwcm I\ mi slight. Thcsch timcs are rc3lativdy long and arc’ comparablr to t h o w for a silironc1 rubber surface. TABLE I1 Effect of HEMA Surface Concentration o n Clotting Tirneh
Run no. 1
3 12 6 5 9 13
Wt/Area mg/cm2
0 0.31 0.59 0.77 0.91 1.2,; 1.91
,Membrane activated partial thromboplastin clotting y6 of contiol time side rlotting time 8 min 8 min 8 min 8 min 8 min 10 min 9 min
50 sec 5 1 sec. 20 her 40 sec 25 sec 2.5 sec 8 sec
-
I 00(/, 97% 107% 120yo I2.50/, 104
cx
CONCLUSIONS Surface modification of cellulose acetate dialysis membranes was carried out by radiation induced graft copolymerization of hydroxyethyl mrthacrylate, HERIA. The degree of grafting was found to depend on the HERIA concentration in thc grafting solution, the radiation dose, the swelling time and the solvent used. Two in vitro tests were developed as a means of comparing thc thrombogenicity of thc membranes prepared. The tests are a continuous flow platelet adhesion test and a membrane activated partial thromboplastin clotting time test. The former is advantageous in that it allows testing under conditions more closely approximating hemodialysis than other small scale in vztro tests. The results from thew tests indicate that a surface coating of HEMA may decrease thrombogenicity. Platelet adhesion on treated membranes fell substantially with increasing surface HEMA concentration. However, t h r presence of HEMA on the membrane surface did not affect thc membrane activated clotting timcs significantly. The authors wish to acknowledge support of this research from National Institutes of Health Grants HL-GM 15668-02 and HL14786-02.
References I . I
