NATL INST OF STAND & TECH PubHcations

AlllOl 7St3M0 ' AlllQb 176b11

NBSIR 77-1392

Interaction of Blood Proteins

With Solid Surfaces

L. E. Smith, W. H. Grant and R. E. Dehl

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D C. 20234

July 31, 1977

Annual Report for Period

August 16, 1976 to July 30, 1977

Issued december 1977

Prepared for Biomaterials Program

Division of Heart and Vascular Diseases

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement

QC

100

.U56

77-1392

1977

JAN 2 9 1980

NBSIR 77-1392

INTERACTION OF BLOOD PROTEINS <> V

WITH SOLID SURFACES

L. E. Smith, W. H. Grant and R. E. Dehl

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washmgton, D C. 20234

July 31. 1977

Annual Report for Period

August 16, 1976 to July 30, 1977

Issued December 1977

Prepared for Biomaterials Program

Division of Heart anc| Vascular Diseases

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement

U.S. DEPARTMENT OF COMMERCE, Juanita M. Kreps, S^crMry

Dr. Sidney Harman, Under Secntary

Jordan J. Baruch, Assistant Sacntary for Scianca and Taehnology

NATIONAL BUREAU OF STANDARDS, Ernest Ambler. Acting Diraetor

NOTICE

This is a draft of a report prepared as an account of progress of work

sponsored by the National Heart and Lung Institute. It is not to be

construed as a report, opinion, or recommendation of the National Heart

and Lung Institute. When produced in final form it will be available

from the National Technical Information Service, 5285 Port Royal Road,

Springfield, Virginia 22151.

Certain commercial equipment is identified in this report in order to

adequately specify the experimental procedure. In no case does such

identification imply recommendation or endorsement by the National

Bureau of Standards nor does it imply that the equipment identified

is the best available.
\
TABLE OF CONTENTS

Page

ABSTRACT i

SUMMARY li

INTRODUCTION v

EXPERIMENTAL

Protein Purity 1

Fibrinogen 1

Y-Globulin 1

Ellipsometry 2

Radiotracer Counting 3

Autoradiography ^ Secondary Ion Mass Spectrometry 5 NHLBI Contractors Biomaterials 6

RESULTS AND DISCUSSION 7

EXAMINATION OF NHLBI CONTRACTOR BIOMATERIALS 7

Ethylcellulose perfluorobutyrate 8

Polyalkylsulfone ^ Adsorption with Radiolabeled HSA(^23i) 10

Glow discharge coatings

Heparin- coated silastic '1

Polyetherure thanes

Carbon 16

CURRENT STATUS OF OTHER NHLBI CONTRACTOR BIOMATERIALS 19

CONFORMATION OF ADSORBED y-GLOBULIN ON SILICON OXIDE 23

Oxidized silicon substrate 23

Y- Globulin purity 23

Adsorption on hydrophilic silica 25

Adsorption on hydrophobic silica 27

Effect of pH on Y~globulin silica 30

RADIOTRACER ADSORPTION STUDIES 32

Separation of aggregates 32

Effect of labeling atom on protein adsorption 38

DISTRIBUTION OF ADSORBED PROTEINS ON SURFACES 40

REFERENCES 46
FIGURES

Page

Figure 1: Autoradiograph of polyetherure thane (A) with adsorbed

HSA(125i) 13

Figure 2: Autoradiograph of polyetherure thane (B) with adsorbed

HSA(125i) 14

125 Figure 3: Autoradiograph of carbon disc, with adsorbed HSA( I) . . 17

125

Figure 4: Autoradiograph of carbon disc, with adsorbed HSA( I) . . 18

Figure 5: SIMS spectrum of carbon- 10% Si disc surface 21

Figure 6: SIMS spectrum of a 200 nm carbon film ion-plated on stainless steel 22

Figure 7: Gel permeation chromatogram of bovine Y~globulin 24

Figure 8: Extension of bovine yglobulin on hydrophilic silicon

oxide, measured with the manual ellipsometer 26

Figure 9: Extension of bovine y-globulin on hydrophobic silicon

oxide, measured with the manual ellipsometer 28

Figure 10: Extension of bovine y-globulin on hydrophobic silicon

oxide, measured with the automatic ellipsometer 29

Figure 11: Gel permeation chromatogram of unlabeled HSA ....... 33

Figure 12: Gel permeation chromatogram of unlabeled HSA 34

125 Figure 13: Gel permeation chromatogram of I HSA 35

131 Figure 14: Gel permeation chromatogram of I HSA 36

3 Figure 15: Gel permeation chromatogram of H HSA 37

125

Figure 16: Autoradiograph of Ft slide with adsorbed HSA( I) 41

125 Figure 17: Autoradiograph of Pt slide with adsorbed HSA( I) .... 43

125 Figure 18: Autoradiograph of Pt slide with adsorbed HSA( I) .... 44

125 Figure 19: Autoradiograph of Pt slide with adsorbed HSA( I) .... 45
TABLES

Page

Table I: Effect of pH on Adsorbance and Molecular Extension of

Bovine Y~Globulin on Oxidized Silicon 31

125 131

Table II: Adsorption of HSA( I) and HSA( I) on Polyethylene ... 39

ABSTRACT

The purpose of this investigation is to help develop methods for

characterizing the surface properties of cardiovascular implant materials.

It is expected that these properties, which are related to the success or

failure of implants, will ultimately form the basis for in vitro testing

of prospective implant materials. The investigation is focused upon

three aspects of blood protein adsorption on surfaces, namely (1) the total

amount of protein adsorbed, (2) the spatial distribution of adsorbed protein

on the surface, and (3) the conformation of adsorbed protein molecules.

A number of materials produced by contractors to the NHLBI Biomaterials

Program have been examined by ellipsometry and by the adsorption of radiolabeled proteins. Autoradiography has been used to study the uniformity

of protein adsorption on several surfaces. Evidence for the migration of

proteins into biomaterials has been discovered. Adsorption studies of

y-globulin on a specially prepared oxidized silicon surface have contributed

to our basic understanding of the adsorption of blood proteins on surfaces.

i
SUMMARY

Work on this project is based on the premise that quantitative

measurements on adsorbed proteins can serve as methods of surface

characterization to aid in the selection and design of materials for

implant use and as the basis for the development of in vitro test methods.

Protein adsorption is the first in a complex series of events that

occurs when blood contacts the surface of an implanted device. The

detailed nature of this adsorption, i.e., the proteins involved, their

absolute and relative amounts, the conformational changes of the proteins,

the number and types of binding sites, and the spatial distribution of

the protein across the surface, potentially affect the later stages of

the interaction of the formed elements with the implant.

Accomplishments in this past year are described below.

1. CHARACTERIZATION OF NHLBI CONTRACTOR'S SAMPLES

Adsorbed Protein Conformation Measurements

Samples from six NHLBI contractors have been examined. Fibrinogen

adsorption measurements have been made by ellipsometry on 3 of these samples

supplying data relating to the conformation of the adsorbed protein as well

as the amount adsorbed. Radiotracer measurements of labeled human serum

albumin have been used to clarify the ellipsometrlc data.

Measurement of the spatial distribution of adsorbed protein

Non-uniform protein adsorption can be diagnostic for sites of high

thrombogenetic potential that may be difficult to visualize in any other
manner. Certainly it can be used as a quality control method for evaluation

of surface properties. Inhomogeneous surfaces may be responsible for

failure in ±n vivo tests when the average properties of the surface seem

acceptable in in vitro criteria.

During the past year we have carried out a preliminary survey of the

adsorbed protein distribution on a number of NHLBI contractor's samples

using autoradiography. The method has been shown to be useful and is

capable of revealing subtle surface features.

Preliminaiy data on albumin adsorbance on polyurethanes has led us to

propose that such materials are significantly permeable to blood proteins.

Verification of this fact in regard to polyurethanes and other contractors

samples is currently in progress.

2. INVESTIGATION OF MODEL SURFACES

Investigation of radiotracer methods of protein adsorption measurements

The effects of protein purity have been investigated and suitable

separation and purification methods for labeled human serum albumin have

131 125 been developed. Different labeling isotopes, I or I have been used

131 and shown to yield the same results. The higher energy I decay has been

considered a likely cause of protein fragmentation by radiolysis. Such a

mechanism has been shown to be of no consequence for short term adsorption

measurements.

Investigation of the surface properties responsible for non-uniform adsorption

Strikingly non-uniform protein adsorption has been observed on a platinum

surface. Several potential causes for this distribution can be proposed.

iii
including microcrack formation and enhanced adsorption due to stress

history. These latter two mechanisms have implications for similar

effects in biomaterials . The technique of autoradiography is therefore

of significant potential value to the Biomaterials program and a major

part of next year's effort will be devoted to its application to contractors

samples and control studies of other reference surfaces.

Progress during the past year has been quite close to the milestones

outlined in the negotiated program plans. The only notable exception is

the secondary ion mass spectrometry (SIMS) investigation which is considerably

behind schedule due to severe equipment problems outside our control. These

problems have not been satisfactorily resolved and the SIMS work for next

year will be largely curtailed. Major emphasis will be placed on autoradiographic measurement of surface homogeneity and measurement of protein

penetration into the biomaterial. All other aspects of the negotiated

program plan will also be pursued on schedule.

iv
biomaterials with blood. A suitable set of experimental criteria

established by these studies may result in rapid in vitro screening tests

for candidate biomaterials.

vi
INTRODUCTION

The adsorption of plasma proteins is the first in a complex series of

events that occurs when a synthetic material is placed in the cardiovascular

system. It is kno\jn that this protein adsorption can alter the course of

subsequent reactions at the implant surface but the mechanism by which

this occurs is not well understood. If the detailed behavior of proteins

adsorbed at the solid surface were known and capable of control, the fate

of artificial materials placed in body could be predicted with greater

certainty and more successful materials perhaps could be designed. This

investigation is directed toward the identification of factors capable of

affecting protein adsorption and the development of a description of the

changes that occur in the protein upon interaction with a surface.

We have continued our examination of potentially useful synthetic

implant materials provided by contractors to the NHLBI Biomaterials Program.

In addition to the ellipsometric studies of fibrinogen adsorption on

materials whose optical properties were suitable for such investigation, we

have measured the adsorption of radiolabeled human serum albumin on several

of these materials. During the course of this work, it has become apparent

that some synthetic biomaterials may be permeable to proteins, which could

significantly affect our interpretation of the ultimate success or failure

of these materials in contact with the blood. Autoradiography studies have

also been initiated, both to serve as a qualitative confirmation of the

radiotracer counting method, and to test for non-uniform spatial adsorption

of proteins on surfaces. These studies are expected to lead to a better

understanding of surface properties governing the compatibility of synthetic
EXPERIMENTAL

Protein Purity

Fibrinogen

The human fibrinogen used in this work was purified by us according

to the Batt-Laki (1,2) method, as reported in our previous Annual Pjeports

(3-6), yielding a product which is greater than 96.7% clottable. Solutions

of the purified protein were quick-frozen in liquid nitrogen and stored

at low temperature until needed. Future experiments will utilize the

commercially purified fibrinogen supplied to all contractors to the NHLBI

Biomaterlals Program

y-Globulin

Bovine yglobulln (Cohn Fraction II) had an electrophoretic purity of

95% as stated by the supplier. The purity of this material was tested by

f ractionationation through a Sephadex G-200 gel permeation chroiratography

column, and the results are reported in the following section of this Report.

The radiotracer adsorption experiments utilized both tagged and untagged

125 131

HSA. The protein was tagged with a labeling atom of I or I by commercial

sources. Additional purification was conducted in our laboratory by passing

both labeled and un- labeled materials through a Sephadex G-200 gel permeation

chromatographic column which separated out the higher molecular weight

aggregates, dlmer, monomer and low molecular weight polypeptides. The

chromatographic separation of these materials is reported in a separate

A Certain commercial materials are identified in this report in order to

adequately specify the experimental procedures. In no case does such

identification imply recommendation or endorsement by the National Bureau

of Standards nor does it imply that the materials identified are the best

available.
section of this report.

Elllpsometry

Ellipsometric studies of fibrinogen adsorption on materials supplied

by contractors to the NHLBI Biomaterials Program was conducted in pH 7.4

buffer solution at 37''C. All y-globulin experiments, using both manual

and automatic ellipsometers , were carried out at ambient room temperature.

Both manual and automatic ellipsometers employ a He-Ne laser light

source (X = 632.8 nm) with an incident angle of 70°. For the manual

ellipsometer, determination of the polarizer (P) and analyzer (A) settings

necessary to produce extinction of the reflected light from the surface

was performed by the method of "swings", in which a photomultiplier with a voltmeter output is used to determine the null point by setting P or A

so as to produce equal light intensity on both sides of the null point. Since the

plot of reflected light intensity vs. P or A setting is parabolic about

the null point, a much more accurate null can be determined

by averaging the readings taken at the steep portion of the parabolic curve

on both sides of the null, than by simply measuring the null directly.

This method allows us routine measurements of polarizer and analyzer

settings with an angular precision of + 0.01*. The automatic ellipsometer

employs a rotating analyzer to measure the ellipticity and azimuth of the

reflected light directly at a constant polarizer setting, rather than by

adjusting P and A for reflected null. Borh variables are presently being

recorded on a dual-channel strip chart recorder. All solution experiments

wrir- pipr fonmul ii\ n.t>c*i l ;j I I y d*-^H 1 jJ.iU'tl irlls, with wliulcwH f 1 1 I t'tl nl the 70°

incident nnglo in ordei to provide normal incidence of light upon the

windows and thus eliminate refraction of light by the cell. Further details

of the elllpsometry instrumentation and experimental proceedings may be

found in previous Annual Reports (3-6).
Analysis of the elllpsometry data to determine protein film thickness,

refractive index, and the amount adsorbed on the substrate was performed

with a computer program developed at NBS by Dr. F. L. McCrackin (9).

Radiotracer Counting

A highly efficient y-counting system was assembled with commercially

available components and used to count the human serum albumin labeled

125 131 with I and I. The detector was a high- resolution 76.2 x 76.2 mm NaT

crystal scintillation detector. Photomultiplier power was supplied by a

high voltage DC regulated power supply. Photocurrent from the photomultiplier

was amplified and fed to a single channel analyzer, with adjustable window

125 131

and baseline controls which were optimized for either I or I to yield

the maximum signal count and minimum background count.

With this simple and relatively inexpensive system, we have been able

to achieve a counting efficiency comparable to that of well known commercial

y-counters. In order to test the efficiency of our system, we counted a 125 calibrated sample of K I in carrier solution (SRM 4A07L-B) supplied by

the Office of Standard Reference Materials at NBS. In addition, through

the courtesy of Dr. Henriksen at the National Cancer Institute (NIH) , we

125 131

counted samples of human serum albumin labeled with I and with I, on

a commercial counting system. The efficiency of our system proved to be

somewhat greater than that of the commercial system.

The experimental techniques which we have developed for performing

quantitative adsorption studies of blood proteins, including the design of

the adsorption cell and the very important rinsing procedures required to

remove radioactive solution carry-out from adsorbates have been described

in the Annual Report for 1976 (6), and in previous Annual Reports (3-5).

3
Specific activity determinations were made as follows. Aliquots of the

radioactive solution were pipetted into small test tubes, weighed, and counted

with the Y~counter, to yield the sample activity in counts/min/wt . of

solution. The density of the solution was assumed to be 1. The solution

concentration was determined by measuring the optical density on a UV

spectrophotometer. In this way, the specific activity in counts/min/wt. of

protein was determined. In order to test for possible self- absorption of

radiation by the solution and its container, various experiments were

performed in which solutions were counted before and after drying, with and

without glass and plastic containers surrounding the sample. The count

for a given sample proved independent of any of these factors.

Most adsorption experiments were performed in duplicate, in order to

test the reproducibility of our results. We find that our current procedures

permit us to replicate a given adsorption experiment within + 2%. For many

purposes, therefore, it will not be necessary in the future to perform

duplicate experiments.

Autoradiography

A number of autoradiographic experiments have been performed during

the past year, after our valuable consultation with Dr. Curtis Harris at

the National Cancer Institute (NIH) . The experiments reported here were

performed by pressing x-ray film against both sides of a sample surface

which has been covered with radiolabeled protein by adsorption in the usual

manner. The surfaces were dried and clamped between glass plates to insure

good contact between film and specimen. The purpose of exposing film to

both sides, and to allow additional exposure time for one side, if the

developed film from the other side indicates insufficient exposure. All

autoradiographs described in this report were developed after a two-week

4
exposure at room temperature. In most cases, normal development times were

used, although for some samples, as noted in the fol] owing Discussion, the

film was deliberately over- or under-developed in order to bring out certain

features of the protein distribution on the surface. Optimum exposure times

depend upon the radioactivity of the sample, as well as the temperature

and humidity of the storage vessel.

Secondary Ion Mass Spectrometry (SIMS)

Secondary Ion Mass Spectrometry (SIMS) is a technique in vjhich a beam

of ionized gas as narrow as 1 ym is focussed upon a small surface area,

sputtering off a secondary beam of ions from the surface. The secondary

beam is collected and analyzed by a mass spectrometer, producing a spectrum

characteristic of the material on the surface. By scanning across the

surface, it is possible by this technique to determine the homogeneity

of the material on the surface, and by continuously sputtering one spot on

the surface or scanning a cross section of the material it is possible to

produce a depth profile of the atomic and/or molecular composition of the

substrate material. The intended uses of this technique in our work have

been to study the uniformity of distribution of adsorbed proteins on surfaces

and the depth of penetration of the protein into uncoated substrates,

hydrogel coatings, heparinized surfaces, etc.

The SIMS apparatus at NBS is located in the Analytical Chemistry

Division. Dr. K. F. J. Heinrlch, Chief of the Microanalysis Section, and

Dr. D. Newbury have agreed to examine polymer substrates with the SIMS

technique and have offered to perform preliminary experiments on biomaterial

substrates which are of interest to us and to the Project Officer.

Unfortunately, as noted in the follovdng discussion, instrumental breakdowns

have prevented us from exploiting this technique in a productive way thus

far.

5
NHLBI Contractor Bioniaterlals

The NHLBI Contractor Biomaterial samples which we have examined this

year by ellipsometry and radiotracer methods were all shipped to us in

sterile packages. All were shipped dry except for the polyalkylsulfone,

one sample of which was shipped under water in a plastic pouch. No surface

cleaning procedures were used other than a water rinse prior to ellipsometry

or radiotracer studies. The NHLBI contractors have been roost cooperative

in preparing samples which are suitably thick (1.5-2 mm) for ellipsometric

and radiotracer adsorption studies, even though such materials as the

membrane oxygenators would normally be fabricated in much thinner sheets

than is required for our measurements.

Characterization information and identification of actual samples

investigated has been transmitted to the Biomaterials Program, National

Heart, Lung and Blood Institute, NIH.

6
RESULTS AND DISCUSSION

EXAMINATION OF NHLBI CONTRACTOR MATERIALS

All of the candidate materials which we have received from contractors

to the NHLBI Biomaterials Program have been examined by ellipsometry , and

we have performed fibrinogen adsorption experiments upon all but one of

the materials whose optical properties allowed meaningful measurements to

be made. The only materials on which adsorption studies have not as yet

been performed are the carbon-coated samples from General Atomic Co, as

noted below. With this exception, each material listed in our 1976 Annual

Report under the heading "Current Status of Other NHLI Contractor Biomaterials"

has been studied by us during the past year, and the results are presented

below.

As a group, the dielectric substrates on which we have performed

ellipsometry studies during the past year present certain difficulties

in obtaining precise quantitative information about adsorbed protein films.

The surfaces which we receive are not always as flat or smooth as is

required to perform quantitative ellipsometry measurements. Further, the

refractive index of substrates such as these tends to differ less from that

of the overylying protein film than is necessary for precise quantitative

measurement of the film properties. Since some of these materials imbibe

aqueous solutions there is also the possibility that the refractive index

of the substrate may change during an ellipsometry run, which prohibits

quantitative interpretation of the observed data. For these reasons,

ellipsometry is, at best, a semiquantitative tool for measuring physical

properties of protein films on dielectric substrates. However, as we will

show, it can be very useful indicator of differences between the adsorptive

behavior of various substrates and, in conjunction with radiotracer

7
measurements can contribute significantly to our overall understanding

of the interaction of blood proteins with solid surfaces.

Ethylcellulose Perfluorobutyrate

In our Annual Report for 1976 (6) we reported that ethylcellulose

perfluorobutyrate was found to adsorb a much greater amount of fibrinogen

after 2-3 h than any other substrate thus far examined. However, the

2 adsorbances noted at 1/2 h and 1 h were about normal (3-5 mg/m ) as compared

with our measured fibrinogen adsorbance on other substrates. Another sample

2 of this material yielded an adsorbance of about 3 mg/m at 1/2 h and 1 h.

(For this sample, the experiment was terminated after 1 h) . The large

increase in adsorbance which we noted for the first 2 samples between 1 h and

2 h is very unusual, for a solution concentration as high as the 3 mg/rol

used in these experiments, and we suspect that the substrate refractive

index has changed significantly during this interval, leading to the very

high calculated adsorbance at 2-3 h.

Two circular samples, cut from the same sheet from which the ellipsometry

125

samples were taken, were equilibrated for 1 h in I-labeled human serum

albumin (HSA) solution, at a concentration of 7.02 mg/ml. The samples were

rinsed, dried and counted with the y-counter in the usual way. The average of

2 the two yielded an adsorbance of 9.99 mg/m . Another sample, equilibrated

131

tor 1 h In I-labeled HSA Holutlon, al « conci-nf r/iUon ol 7.9') myjml , yli-ldcd

2 an adsorbance of 3.99 mg/m . Since the adsorbance of HSA on polyethylene discs

8
from these 2 difference HSA solutions was found to be essentially the same

after 1 h equilibration, we do not feel that the differences noted here can

be due to specific isotope labeling effects, different for the 2 iodine isotopes.

12 S It is perhaps significant that the ratio of adsorbances for HSA( I)/

131

( I) is 2.50, while the corresponding ratio of solution concentrations used

in the adsorption experiments is about the same (2.38). These data suggest

to us either that the adsorbance on this substrate in the concentration range

used here is proportional to the solution concentration, or that there is

some permeation of the protein solutions into the substrate, entailing a migration of the protein molecules into the matrix. During the next year, we

expect to perform experiments which will test these hypotheses, and we

expect that the results will lead to a better understanding of the interaction

of protein solutions with biomaterial surfaces.

Polyalky Isulfone

Two samples of the polyalkylsulfone membrane oxygenator material have

been examined, and the results are quite different from any that we have

observed to date. The changes in reflection coefficients upon exchanging

the fibrinogen solution for buffer were so small as to indicate that virtually

no fibrinogen was deposited on the surface. While this would seem the most

likely explanation for the phenomenon, there is also a possibility that a

fibrinogen film was deposited, whose refractive index was essentially identical

to that of the underlying substrate. In this case, the incident light

9
would not "see" the film even though it is there. Since the index of refraction

of the substrate (1.47) is considerably higher than that of the protein

solution (1.33), the fibrinogen film would have to be quite dense, compared

with any other that we have observed thus far. In order to resolve this

question, an adsorption experiment was performed from a much more dilute

fibrinogen solution (0.12 mg/ml) . If a dense film was deposited from the

more concentrated solution, a less dense film should be deposited from

dilute solution, corresponding to a lower point on the adsorption isotherm,

which hopefully could be detected by the ellipsometer. This apparently has

proved to be the case. From the more dilute solution, the ellipsometer

measured an adsorbance of about 1.7 mg/ml, which increased only slightly

from 1/2 h to 2 h adsorption time. While this measurement tends to support

the "dense film" hypothesis, it must also be considered in the light of the

radiotracer experiments reported below.

125

Adsorption with Radiolabeled HSA( I)

IVo circular discs cut from the same sheet of PAS used in the ellipsometry

125

experiments were used to study the adsorption of HSA( I) . A solution

concentration of 7.02 mg/ml was used, and the adsorption experiments were

performed in the usual way, for 1.2 h. The average of 2 determinations of

2

the adsorbance was 19.7 mg/m . This result is in sharp constrast with the

ellipsometry results for fibrinogen adsorption, reported above, in which one

possible interpretation of the data was that no protein was adsorbed on the

material. The very high adsorbance of the HSA suggests the possibility that

the fibrinogen adsorbance from a solution concentration of 3 mg/ml may also

have been quite high.

10
Glov-Dlscharge Coatings

Unlike many other coated surfaces which we have examined by

ellipsometry , the tetramethyl disiloxane glow-discharge coated polyethylene sample from Research Triangle Institute gave a specular reflection,

and three fibrinogen adsorption experiments were performed on it. Two of

the three samples gave similar results, while the third gave

inconsistent results for the fibrinogen film thickness. The calculated

2

adsorbance for the first two samples were in the range 3.2-4.9 mg/m

during the first hour of adsorbance, with little change between 1/2 h and

1 h. One of the samples which was allowed to equilibrate for 2 h apparently

2 adsorbed another 9 mg/m in the interval between 1 h and 2 h. The same

phenomenon was noted above for the ethylcellulose perfluorobutyrate and,

as we have noted* the apparent adsorbance is probably due to changes in the

substrate which render the calculated adsorbance suspect.

Heparin-coated Silastic

Two samples of heparin, bonded to silastic-coated glass with APTES

,

were examined by ellipsometry. In both cases, light scattering from the

surface prohibited measurement of the reflection coefficients with sufficient

accuracy to warrant a fibrinogen adsorption measurement. This substrate is

a candidate for radiotracer studies, which will be performed in the near

future.

11
Polye the rure thanes

Our ellipsometric study of fibrinogen adsorption on two polyetherure thanes (A) and (B) was discussed in the Annual Report for 1976. We

reported that the average fibrinogen film thickness on (A) was 3C.7 + A. 6 nm,

and on (B) was 32.0 +5.7 nm. The average fibrinogen adsorbance on (A) and

2 2 (B) were found to be, respectively, 4.75 + 0.23 mg/m and 5.04 + 0.39 mg/m .

From these data we concluded that there is no significant difference in the

adsorbed amount or moleculai- extension of fibrinogen on the 2 substrates,

as measured by el lipsometry . These findings were not unexpected, given the

chemical similarity of the two materials, which would be expected to lead

to essentially the same protein adsorption affinity. Certain physical

differences were noted between the two materials, however. (B) is mechanically

less flexible than (A) , and, unlike the latter, does not appear to imbibe

water after immersion for several hours. These differences may be responsible

for the considerable differences in affinity for radiolabeled albumin

solution, as reported below.

Two circular discs of (A) and (B) were cut from the same sheets as

those from which the ellipsometry samples were taken. One sample of each

material was flexed by bending sharply across the disc several times. This

flexure prior to protein adsorption was done to test the possibility that the

adsorption might be affected by partial orientation of the polymer molecules,

resulting from stress orientation. The four samples were equilibrated for

125

1/2 h in 1-3 rivaled human serum albumin, at a concentration of 7.02 mg/ml,

in our usual way. They were then rinsed, dried overnight in air, and pressed

between x-ray film for two weeks, as we have discussed above. Enlarged

photographs of the 2-week autoradiographic films of (A) and (B) are shown in

Figs. 1 and 2 . The light areas correspond to the radiotracer-activated areas

of the film, and both prints were made at the same exposure time. Clearly,

12
125 Figure 1. Auto radio graph of polyetherurethane (A) with adsorbed HSA( I)

13
Autoradiograph of polyetherurethane (B) with adsorbed HSAC"'"^^

lA
the (A) substrate has a greater amount of adsorbed HSA than the (B) substrate.

There was found to be no discernible difference between the autoradlographs

of flexed and unflexed samples of each material, nor was any significant

difference noted between the autoradlographs of the 2 sides of a given sample.

These two substrates clearly have considerably more adsorbed radiolabeled

protein than any other material that we have studied by autoradiography thus

far, a result which is confirmed by radiotracer counting as reported below.

125 131 Adsorption of both HSA( I) and HSA( I) was carried out on circular

disc samples of (A) and (B). Adsorption was performed for about 1 h on each

sample, followed by our usual rinsing, drying, and counting procedures. The

125 concentration of the HSA( I) solution was 7.02 mg/ml, while that of the

131

HSA( I) was 2.95 mg/ml. Average results for two samples of each substrate

125 2 are as follows. For substrate (A), adsorbance of HSA( I) was A2.01 mg/m , 131 2 and adsorbance of HSA( I) was 10.98 mg/ra . For substrate (B) , adsorbance

3 2'i 9 1 "^1 2

of HSA( I) was 24.19 mg/m , while HSA( I) adsorbed 5.54 mg/m . For both

isotopes, therefore, it is clear that adsorbance on the (A) substrate is about

twice that of the (B) , confirming the autoradiographic results above, which

indicated considerably higher adsorbance on (A) than on (B)

It is useful to compare these findings with those reported above for the

ethylcellulose perfluorobutyrate. Although the latter substrate adsorbed

125 131 less HSA( I) and HSA( I) than the polyetherurethanes , the adsorbance ratio

of the two HSA's was roughly the same for all three materials. We believe that

125 the possibility of a specific isotope labeling effect, different for I and

131

I, has been eliminated by the adsorption experiments on polyethylene, as

reported below. The only significant difference between the adsorption

experiments involving the two isotopes, as noted above, was the concentration

of the solutions. Since the higher apparent adsorbance in each case

corresponded to the higher HSA concentration, and since the equilibrium

15
adsorbance from both solutions is expected to be about the same, we are led

to the conclxision that the "adsorbances" measured here represent, in fact, an

entrapment of some protein solution by the materials during the 1/2 h immersion

in HSA solution. Solution is trapped by the substrate in a way which prevented

its being rinsed off during the brief (2-3s) rinsing time, which has been

shown to be adequate for removal of protein solution from nonporous surfaces

such as polyethylene or glass.

Carbon

One sample of a graphitized carbon heart-valve disc alloyed with 10%

125 Si was studied by a 2-week autoradiographic exposure, after HSA ( I)

adsorption following our usual procedure. Figs. 3 and k are the autoradiographs of both sides of the disc. One side (Fig, 3), which was developed

for the normal 4.5 minutes, shows an intense line due to a scaratch which

was placed on the surface as an aid in orienting the sample prior to HSA

adsorption. Enhanced protein adsorption, and possibly incomplete removal

of protein solution during the normal rinse time, are responsible for the

high radiolabeled protein concentration in the scratch. Except for a small

ring of adsorbed protein near the outside rim of the disc, there is very little

evidence of adsorbed protein on this side of the disc. The film on the other

side (Fig. 4 ) was developed for only 2-1/2 minutes. Although the light

intensity is low, there is clear evidence of protein adsorption over the

entire surface, and a number of enhanced adsorption regions. The word

"Reject" which was etched into the surface has clearly enhanced protein

adsorption, and there is a relatively bright ring near the edge on which

adsorption is enhanced.

16

i 9 Figure ^>

. Autoradiograph of carbon disc, with adsrrbec' H'-W'^'IK Film

Linder developed.

18
CURRENT STATUS OF OTHER NHLBI CONTRACTOR BIOMATERIALS

All of the NHLBI Contractor Biomaterials which we have received and

agreed with the Project Officer to examine have been studied by ellipsoraetry

,

except for certain carbon-coated substrates. We are presently awaiting

the receipt of fresh samples of these materials.

As noted in the above Discussion, radiotracer and autoradiography are

providing valuable information about amounts and distribution of radiolabeled

proteins on surfaces. We expect to continue to use both of these techniques

to study biomaterial surfaces. Each of the Contractor Biomaterials listed

in the report will be further examined, using both of these techniques,

with special emphasis on samples such as the heparinized Silastic, which

cannot be examined by ellipsometry

In our last work statement we proposed to use secondary ion mass

spectrometry (SIMS) to study protein distribution on surfaces of interest

to the Biomaterials Program. This work was to be performed on a spectrometer

in the Analytical Chemistry Division at NBS. We have been able to obtain

preliminary spectra from a pyrolytic carbon disc alloyed with 10% Si, and

of a stainless steel planchette "ion-plated" with carbon. The spectra

obtained are shown in Figs. 5 and 6. Clearly, numerous surface impurities

are present on these materials, although some additional work would be

required to obtain a quantitative analysis of surface impurities by this

method. Unfortunately, numerous instrumental breakdowns have prevented further

use of the SIMS equipment, and we have not been able to obtain any information

as yet on the protein-coated substrates which are of interest to this

program.

19
While the SIMS technique appears less than promising at this time, we believe as a result of recent findings that most of the information that

would have been obtained by SIMS about protein distribution on surfaces and

depth of penetration into substrates will be furnished by radiotracer

techniques, including autoradiography and radiotracer studies of protein

migration into biomaterials . As noted below, our preliminary results

obtained by these techniques are most encouraging.

20

22
CONFORMATION OF ADSORBED y-GLOBULIN ON SILICON OXIDE

Oxidized Silicon Substrate

In previous Annual Reports to NHLI (197A, 1975), it has been suggested

that confoirmational changes may occur with increasing surface concentration,

for bovine yglobulin and B-lactoglobulin adsorbed on a fused silica surface.

Because the results were not entirely conclusive, we have pursued the ellipsometry

work in more detail, using as the substrate a specially prepared polished

silicon surface covered with a 200 nm film of silicon oxide. This surface was

chosen because (1) it is chemically the same as the fused silica previously

used in this work, (2) it has superior ellipsometric sensitivity as compared

with ordinary fused silica, and (3) it offers the possibility of studying the

effects of surface energy on the conformation of adsorbed protein. This last

possibility arises because the oxide film, which is hydrophobic, is easily hydro lyzed

in hot water, yielding enough SiOH groups to reduce the contact angle of water

to zero.

y-Globulin Purity

Proteins received from commercial sources have sometimes been found to be

quite contaminated with Impurities, as reported in our Annual Report for 1976.

Thus, even though the electrophoretic purity of our commercially prepared yglobulin

was stated by the manufacturer to be 95%, a sample of this protein was fractionated

by gel permeation chromatography using Sephadex G-200. The elution chromatogram for tliis

material is shown in Figure 7. There appears to be a small fraction of higher

molecular weight (probably dimer) present as a shoulder on the main peak, which

was assumed to be monomeric y-globulin. Fractions 31-AO (mostly dimer) were

collected for adsorption experiments in comparison with fractions 50-57 (monomer).

Both the adsorbance and molecular extension of the adsorbed "dimer" on hydrophobic

21
o

o

24
silicon oxide were found by ellipsometry to be significantly higher than the

values obtained for the monomer. However, the monomer values were the same,

within experimental error, as the values obtained for the unf ractionated protein.

We therefore concluded that it was not necessary to fractionate our yglobulin

in order to obtain meaningful adsorption measurements.

Adsorption on Hydrophllic Silica

Hydrophilic silica was prepared by heating the oxidized silicon wafers in

hot water for 20 hours. Thirteen separate yglobulin adsorption experiments

were performed on this substrate with the manual ellipsometer , using a wide

range of protein concentrations, from 0.016 to 10.1 mg/ml. The data obtained

from these experiments is shown in Figure 8 where the calculated film thickness

(molecular extension) is plotted against the calculated adsorbance. Each

adsorption experiment yielded 3-5 data points at 1/2 h and 1 h, and at 1 h intervals

until the experiment was concluded. All data points are included in the figure.

Adsorption from very dilute solution produced low adsorbances and greatly

scattered extension values, as seen in the left portion of the plot. The scatter

is due to the small differences in refractive index between these very dilute

films and the supernatant solution. Clearly, little can be said about the

2 molecular extension in this region. At higher adsorbances, between 2-3 mg/m ,

the data points for individual runs are connected by lines. With only one

exception, there is an overall increase in extension with

adsorbance within each run, although the magnitufi' of the increase is variable.

2 The plateau adsorbance, about 3 mg/m , is in goo igreement with the value 3.A ±

0.6 previously reported for Y~8lot)ulin on fused silica. However, the extension

reported on fused silica at the plateau adsorbance was 32 ± 10 nm, which is

considerably higher than the average value obtained in this work. Despite the

25
*

0) T3

•H

»<oGOO

•H

iH

•H

0]# 1 \j •HfH

•H43

O.OM •

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26
apparent increase in extension with adsorbance within individual runs, the

average extension does not appear to increase significantly with adsorbance.

It is likely that these data are more directly comparable with the fused silica

results previously reported than with the following results for the hydrophobic

silica, since the fused silica was also hydrophilic.

Adsorption on Hydrophobic Silica

Thirteen separate adsorption experiments were performed on hydrophobic

silicon oxide with the manual ellipsometer, and the results are summarized in

Figure 9. As compared with the hydrophilic substrate, the plateau adsorbance

2 on the hydrophobic surface is higher by about 1 mg/m , and the adsorption isotherm

is much steeper. For example, the adsorbance attained on the hydrophobic

2 substrate after 3 h from a concentration of 0.015 mg/ml was 2.8 mg/m , whereas

a comparable experiment on the hydrophilic substrate produced an adsorbance of

2 only 0.16 mg/m . As with the hydrophilic substrate, there was an overall

increase in extension with adsorbance within each individual run, although

again the magnitude of the increase was not constant.

The results of two adsorption experiments on the hydrophobic surface

using the aucomatic ellipsometer are shown in Figure 10. Very dilute solutions

(0.10 and 0.018 mg/ml) were used in these experiments, in order to observe the

initial stages of the adsorption process. Maximum probable error bars on three

of the data points indicate that .he experimental uncertainty in the calculated

extension decreases with increasing adsorbance, but is so great at low adsorbances

that care must be used in extracting quantitative information. Over the adsorbance

2 range of about 1.2-2.5 mg/m , there is no clear trend in the extension with adsorbance.

27
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29

derived from these data, however. It is apparent that little, If any,

significant difference exists between the hydrophobic and hydrophilic surface

with respect to their overall net interaction with the protein. Neither the

extension nor the adsorbance seems much affected by the surface free energy.

However, both extension and adsorbance appear to be significantly lower at th

lowest pH, which corresponds to the highest net (positive) molecular charge.

It remains to be seen whether a corresponding negative charge produces the

same effect. Further work is in progress to expand and replicate these resul

TABLE I

EFFECT OF pH ON ADSORBANCE AND MOLECULAR EXTENSION

OF BOVINE Y-GLOBULIN ON OXIDIZED SILICON

Molecular

Extension (nm)

Adsorbance

(mg/m )

(b)

pH

(a)

3.9 (1) 4.8

(2) 5.4

2.49

2.18

4.9 (1) 12.6

(2) ]4.0

4.57

3.68

6.4 (1) 13.7

(2) 16.0

4.17

3.98

(a) (1) is for hydrophobic and (2) is for hydrophilic

silicon oxide. Results are average of 1/2 h and

1 h values.

(b) Results are for 1 h adsorption time.

31
RADIOTRACER ADSORPTION STUDIES

Separation of Aggregates

Before a definitive study of the adsorption of blood proteins on substrates

can be undertaken, the degree of purity of these adsorbates must be ascertained.

Both labeled and unlabeled proteins have been found by us (6 ) to contain

varying amounts of dimers and higher aggregates as well as low molecular weight

polypeptide fragments. Competitive adsorption from such mixtures can lead

to apparent protein adsorbances which are quite different from the desired

value of the monomer alone.

Commercially available human serum albumin (HSA) has been previously

analyzed and separated into fractions by us (6). We have continued to use a

gel permeation chromatographic column packed with Sephadex G-200 to separate

the various molecular weight components. Figure 11 shows the results for

unlabeled HSA ( 6 ) fractionation. Portions of the monomer fraction were used

125

to increase the protein concentration of the labeled I HSA used for

adsorption studies thereby allowing us to conserve valuable labeled protein.

The unlabeled sample has less than 0.5% low molecular weight polypeptide,

approximately 72% monomer and 28% dimer and higher aggregates. In Figure 12

a similar separation is shown for unlabeled HSA supplied by another commercial

source. This material is clearly contaminated with more dimer and higher

aggregates. This material was not used in any of our work presented here.

125

In Figure 13 the same, separation into fractions Is shown for I labeled

HSA. As shown, a relatively small amount of contaminants are found in this

material from supplier (s). The central peak is for the monomer fraction,

131

The other labeled material, HSA( I), from supplier (n) in Fig. 14 also

3 showed essentially no contamination. However, the H HSA, shown in Fig. 15,

also from supplier (N) has had little or no contamination by dimer or higher

aggregates. There is a small amount of low molecular weight polypeptides

present, however.

32
33
ELUTION VOLUME

Figure 12. Gel permeation chromatogram of unlabeled HSA.

34
UJO

2

<

CDo

<

oc

UJO

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•

• •

•

•

•

• •

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9

•

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ELUTION VOLUME

ch

35

125 Figure 13. Gel periDeation chromatogran. of I KSA.

33NVflU0SaV

37
Effect of Labeling Atom on Protein Adsorption

Some of the possible errors associated with the use of radiotracers to

study protein adsorption have been discussed by us In a recent paper ( 8 )

One must consider the possibility that the labeling atom may be specifically

adsorbed to a surface, thus enhancing the adsorption of the protein.

125 131

Isotopes of the same atom, such as I and I have very different decay

schemes, and the energy spectra associated with the decays are quite different.

There is a possibility that autoradiolysls resulting from the emitted

radiation may lead to an undesirable degradation of the protein, thereby

producing molecular fragments which compete for surface adsorption sites

with the undegraded protein. The end products of radioactive decay are also

different for different isotopes, which Introduces further possible

conq) 11 cations in the interpretation of the adsorption data. For these reasons,

it is necessary to test the effects of various labeling atoms on the adsorptive

behavior of labeled compounds. In the present work, we report the effect of

125 131

two labeling iodine isotopes, I and I, on the adsorption of human

serum albumin on polyethylene.

Linear polyet liy lonti , a well characterized polymer, Standard Reference

form of solid pellets and molded into flat sheets from which small discs with

a surface area of 5.81 cm were cut. The adsorption procedure for this

study has been described previously in the Annual Report for 1976. Adsorption

38
of HSAC"*"^^!) (7.66 mg/ml) was carried out for 30 minutes, 1 h, 2h and 3h and

131

the adsorption of HSA( I) for a time period of only 1 h followed by our

standard quick rinse, drying and counting procedure. The adsorption results

are given in Table II.

TABLE II

125 131

ADSORPTION OF HSA( I) AND HSA( I) ON POLYETHYLENE

2 Adsorbance (mg/mg ) Adsorption

Time HSAC^^^I) HSAC'''-'l)

30 min 3.58

1 hour 4.68 3.71

2 hour 5.00

3 hour 5.04

Initially, for the adsorption of HSA( I), as observed in Table II

there is an increase in the adsorbance between 30 min and 1 h, with little

change between Ih and 3h. The small difference in the 1 h results for the

two Isotopes is probably due to the difference in concentration of the

solutions. The rate of adsorption, and perhaps the equilibrium adsorbance

131

as well, would likely be somewhat less for the less concentrated HSA( I)

solution, which is in qualitative accord with the observed results. From

these data, we conclude that there is apparently no significant difference

in the adsorbance of HSA tagged with the different iodine isotopes.

3

We are now about to find out how the iodine label compares with a H

3

label, in its effect on HSA adsorbance. The H labeled HSA has been fractionated

by chromatography, according to our usual procedure, and the elution chromatogram

is shown in Fig. 15. The fractionated protein will be used to study

adsorbance on polyethylene and other surfaces.

39
DISTRIBUTION OF ADSORBED PROTEINS ON SURFACES

125 Autoradiographic measurements of HSA( I) adsorption have been performed

on a variety of surfaces, some of which have been noted in earlier sections

of this report. It is clear that this technique can yield much valuable

information about the distribution of adsorbed proteins, as well as semiquantitative

information about the total amount adsorbed. The other surfaces which we have

thus far examined by this technique are polyethylene, glass, oxidized silicon,

and platinum. Two week autoradiographic exposures were clearly not sufficient

for the first 3 of these substrates. Additional exposure time and/or the

use of higher-activity labels will be necessary in order to derive useable

information about these surfaces. The platinum surface, however, has shown

some interesting and unexpected patterns of protein adsorption, as we will

show.

Figs. 16 through 19 are the autoradiographs of Pt slides, which were

subjected to different surface treatments. Fig. 16 shows a very intricate

125 four- fold symmetry pattern in its adsorption of HSA( I). The surface was

treated only by acid cleaning and rinsing. While the exact cause of this

adsorption pattern is not understood as yet, it could result from stressinduced crystallization or microcrack. formation propagated by the shearing

of the metal plate, producing high energy sites on the surface and leading to

enhanced protein adsorption. Since the presence of high energy sites on

biomaterials is often postulated as a cause of enhanced adsorption of blood

proteins, the opportunity to test this idea, using a material on which the

surface energy may be altered by the introduction or removal of high energy

sites is intriguing and potentially of considerable use in understanding the

mechanism of protein adsorption.

40
Figure 16. Autoradiograph of Pt slide with adsorbed HSA( I). Acid

cleaned only.

41
Fig. 17 is the autoradiograph of another Pt slide, which was acid-cleaned

and heated in a muffle furnace at 500**C for 10 min. Enhanced adsorption is

apparent in the mosaic light pattern, which suggests the presence of highenergy sites at grain boundaries between metal crystals. We will pursue this

line of investigation, to determine by surface etching and other techniques

whether the enhanced protein adsorption can be correlated with known physical

properties of the metal surface.

Figs. 18 and 19 are the autoradiographs of the two sides of the same

Pt slide, which was acid-cleaned, heated at 500*0 for 10 min, and flamed to

red heat for 20-30 s, prior to protein adsorption. As in Fig. 16, mosaic

patterns are seen, but the areas of enhanced adsorption are much larger,

suggesting the possibility of metal crystal growth during the intense heat

treatment. These ideas will require further testing and corroboration by

other physical means.

42


Figure 19. Autoradiograph of Pt slide with adsorbed KSA( I). Surface

acid cleaned and flamed to red heat.

45
REFERENCES

1. Batt, C. W., Mikulka, T. W. , Mann, K. G. , Gaurraclno, C. L. Altiere,

R. J., Graham, R. G. , Qulgley, J. P., Wolf, J. W. , and Zafonte,

C. W. , J. Biol. Chem. , 245, 4857 (1970).

2. Laki, K. , Arch. Biochem. Biophys., 32^ 317 (1951).

3. Fenstermaker , C. A., Grant, W. H. , Morrissey, B. W. , Smith, L. E. , and

Stromberg, R. R. , "Interaction of Plasma Proteins v/lth Surfaces," PB

232 629/6 (Available from the National Technical Information Service),

Annual Report prepared for the Biomaterials Program, National Heart

and Lung Institute, NIH, Bethesda, Md., March 1974.

4. Stromberg, R. R. , Morrissey, B. W. , Smith, L. E., Grant, W. H. , and

Fenstermaker, C. A., "Interaction of Blood Proteins with Solid Surfaces,"

PB 241 267/4SL (Available from the National Technical Information

Service) , Annual Report prepared for the Biomaterials Program, National

Heart and Lung Institute, NIH, Bethesda, Md., January 1975.

5. Stromberg, R. R. , Morrissey, B. W. , Smith, L. E. , Grant, W. H. , and

Dehl, R. E., "Interaction of Blood Proteins with Solid Surfaces,"

NBSIR 76-1017, Annual Report prepared for the Biomaterials Program,

National Heart and Lung Institute, NIH, Bethesda, Md. , March 1976.

6. Smith, L. E. , Dehl, R. E. , Grant, W. H. , Stromberg, R. R. , and Morrissey,

B. W. , "Interaction of Blood Proteins with Solid Surfaces," NBSIR 76-1128,

Annual Report prepared for the Biomaterials Program, National Heart, Lung

and Blood Institute, NIH, Bethesda, Md., August 1976.

7. See, for example. West, E. S., Todd, W. R. , Mason, H. S., and van Bruggen,

J. T. , "Textbook of Biochemistry," p. 326, Macmillan Co., New York (1966).

8. Grant, W. H. , Smith, L. E. , and Stromberg, R. R. , J. Biomed. Mater.

Res. Symposium 8^, 33 (1977).

9. McCrackin, F. L. , "A Fortran Program for Analysis of Ellipsometric

Measurements," NBS Technical Note 479, Washington, D. C. 20234 (1969).

46
BIBLIOGRAPHIC DATA

SHEET

1 . Report No. 3. Recipient's Accession No.

4. Title and Subtitle

INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

5. Report Date July 31, 1977

6.

7. Author(s) L. E. Smith, W. H. Grant, R. E. Dehl

8. Performing Organization Rept.

NBSIR 77-1392

9. Performing Organization Name and Address

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington. D. C. 20234

10. Project/Task/Work Unit No.

11. Contract/Grant No.

2 YD 1-HV- 30014

12. Sponsoring Organization Name and Address

Devices & Technology Branch, Biomaterials Program

Division of Heart and Vascular Diseases

National Heart, Lung, and Blood Institute

Bethesda, Maryland 20014

13. Type of Report & Period

Covered

Annual

14. August 16, 1976 - July 30, 1977

15. Supplementary Notes

16. Abstracts The purpose of this investigation is to help develop methods for characterizing the

surface properties of cardiovascular implant materials. It is expected that these

properties, which are related to the success or failure of implants, will ultimately

form the basis for in vitro testing of prospective implant materials. The investigatior

is focused upon three aspects of blood protein adsorption on surfaces, namely (1) the

total amount of protein adsorbed, (2) the spatial distribution of adsorbed protein

on the surface, and (3) the conformation of adsorbed protein molecules. A number of

materials produced by contractors to the NHLBI Biomaterials Program have been examined

by ellipsometry and by the adsorption of radiolabeled proteins. Autoradiography has

been used to study the uniformity of protein adsorption on several surfaces. Evidence

for the migration of proteins into biomaterials has been discovered. Adsorption

studies of yglobulin on a specially prepared oxidized silicon surface have contributed

to our basis understanding of the adsorption of blood proteins on surfaces.

17. Key Words and Document Analysis. 17a. Descriptors

Adsorption

Autoradiography

Biomaterials

Blood Proteins

Ellipsometry

Polymer Adsorption

Protein Adsorption

17b. Identif lers/Opcn-Ended Terms

17c. COSATI Field Group

18. Availability Statement 19. Security Class (This

Report

)

UNCLASSIFIED

20. Security Class (This

Page

UNCLASSIFIED

21. No. of Pages

22. Price

FORM NTis-39 iREv. 10-73) ENDORSED BY ANSI AND UNESCO. THIS FORM MAY BE REPRODUCED USCOMM-DC 8285-^74


II

II
ORGANIZATION

Polymers Division

Institute for Materials Research

ADDRESS

National Bureau of Standards

REPORT TITLE

INTERACTION OF BLOOD PROTEINS WITH

SOLID SURFACES

Washington, D. C. 20234

CONTRACT NO. 2 Y01-HV-3001A

Reimbursable

Agreement

AUTHORS

L. E. Smith, W. H. Grant, R. E. Dehl

REPORT NO. 5 TYPE OF REPORT

Annual

ORGANIZATION

DATE OF REPORT

PERIOD COVERED

August 16, 1976 - July 30, 1977

REPORT SUBJECT INDEX

J BLOOD PUMPS

MATERIALS

ENERGY

CONTROLS C3

INSTRUMENTATION

OXYGENATORS

EMKRGENCY SYSTEM

TEMPORARY SYSTEM

PERMANENT SYSTEM

TOTAL REPLACEMENT

PHYSIOLOGY

BIOLOCjY

8

CHEMISTRY

PHYSICAL SCIENCE

ENGINEERING

•

I ' Willi

i WBtiiw II iiTiiiiiiiii»riiiii irii'ii'«iaiiiifririiiriiifi!'f«iinnffriTiir[(if'Ti---i TEST AND EVALUATION

SYSTEMS

RESPIRATORY DEVICES

Polymers Division

Institute for Materials Research

National Bureau of Standards

PREPARED FOR biomaterials program

DEVICES AND TECHNOLOGY DRANCH

DIVISION OF HEART Mi VASCULAR DISEASES

NATIONAL HEART AND LUNG INSTITUTE

AETHESDA. MARYLAND 20014

U. S. DEPARTMENT OF

HEALTH, EDUCATION, AND WELFARE

PUBLIC HEALTH SERVICE

NATIONAL INSTITUTES OF HEALTH

mH-l556 (Rev. 11-72)