J. BIOMEL). MATER. RES. VOL. 3, PP.

191-206 (1969)

Initial Events in Interactions of Blood with a

Foreign Surface

ROBERT E. BAIER, Cornell Aeronautical Laboratory, Bu$alo,

New York l 4 S S l and ROBERT C. DUTTON,* Laboratory of Techni-
caE Development, National Heart Institute, Bethesda, Maryland ZOO14

Summary
The nature of the events occurring immediately after exposing foreign surfaces
t o fresh flowing blood was assessed using a combination of MAIR infrared
spectroscopy, ellipsometry, and contact angle measurements. Within the first 5
sec after contact with blood issuing from the jugular veins of lightly anesthesized
dogs, germanium prisms were uniformly coated with strongly adherent pro-
teinaceous films having an average optical thickness equivalent to 2 layers of
stearic acid (approx. 50 A) and having critical surface tensions of about 36
dyne/cm. A contact time of 60 sec led to a less uniform coatin5 of average
optical thickness equivalent to 5 layers of stearic acid (approx. 125 A), but with
similar MAIR spectrum and wettability. The internal reflection spectra were
consistent with the presence of either alpha-helical or random-chain configura-
tions, but not with the extended chain beta-structure for proteins. Films cast
from purified fibrinogen on platinum foils gave similar spectra and exhibited
similar wetting properties ; these observations are consistent with the hypothesis
that initial adsorptive events are dominated by fibrinogen deposition. The
involvement of small amounts of lipid or other protein remains a distinct
possibility, however.

INTRODUCTION
When blood first contacts a nonphysiologic, or foreign, interface,
a sequence of events is initiated which often ends in blood coagulation
or thrombus formation. These processes, representing steps-often
exaggerated-in the normal wound-healing mechanism, are generally
not desired with implanted prosthetic materials. In this study, we
* Present address: University of California Medical School, San Francisco,
California 94122.
191
192 R. E. BAIER AND R. C. DUTTON

attempt to define the earliest events occurring when selected ma-

terials are contacted by fresh, flowing blood. We have used, simul-
taneously, three powerful tools of surface chemistry t o investigate
the changes a t the surfaces of foreign materials within the first few
seconds of their exposure to blood. The methods are multiple
attenuated internal reflection (MAIR) infrared spectroscopy,
ellipsometry, and critical surface tension determination, applied in
this order to each of the samples investigated. MAIR infrared
spectroscopy is a relatively new optical technique’ which allows
micro-amounts of materials on foreign surfaces to be analyzed, result-
ing in the recording of a characteristic infrared “fingerprint” for the
material in question; ellipsometry is a related optical method2 which
allows an estimate of the thickness of adsorbed films on reflecting
surfaces; critical surface tensions, derived from a series of contact
angle m e a sur e m en t~ ,~ are indicative of the outermost chemical
constitution of organic surfaces and thus suggest the potential in-
fluence the modified surfaces will have on subsequent events such as
platelet a d h e ~ i o n . ~
Our application of these tools to the study of adsorption from
blood onto foreign surfaces has led to the conclusion that the earliest
common event is the formation of a predominantly proteinaceous film,
in which fibrinogen may be a major coniponent. A companion study
shows that such film formation precedes platelet adhesion and
microscopically identifiable thrombus formation.

MATERIALS AND METHODS

MAIR Spectroscopy
Internal-reflection prisms of optically polished germanium,
(50 mm x 20 mm x 1 mm) were obtained from Wilks Scientific
Corp., South Norwalk, Conn., as were Teflon holders and solid
sample clamping devices for these prisms. To record spectra, the
mounted prisms + samples were placed in a Wilks Model 9 internal
reflection mirror assembly situated in the sample beam of an infrared
spectrophotometer. Although Beckman spectrophotometers IR-5
and IR-12, and Perliin-Elmer spectrophotometers PE-21 and PE-457
were all used in various phases of the investigation, the spectra
reproduced here were traced from recordings produced on the last
named instrument.
 INTERACTIONS OF BLOOD 193

Ellipsometry
Germanium prisms, with and without specimen films or calibration
films, were mounted-without handling them in any way or removing
them from their Teflon holders-with the aid of a small nylon
positioning jig in the sample space of a Rudolph Model 436-2003
photoelectric ellipsometer (0. C. Rudolph and Sons, Inc., Caldwell,
N. J.).
The positions of rotation of an analyzer and quarter-wave plate
necessary to produce minimum intensity of a polarized mercury-lamp
beam reflected from the prism faces were recorded as described by
Rothen.6 These values were plotted against known film thicknesses
of a series of long-chain carboxylic acids' in close-packed films
transferred to the prisms according to the method detailed by
Blodgett.* Contact angle measurements on each of the calibration
films verified their close packing, necessary to achieve reliable film
thicknesses, according to criteria outlined el~ewhere.~
Specimen films on germanium prisms were analyzed ellipso-
metrically by taking a series of eight readings on each of a t least three
1 mm2 areas in each region of the films. Diverse regions of each
sample were studied to determine the extremes of film thickness
formation. Film thicknesses were estimated in terms of the equiva-
lent thickness of close-packed stearic acid (an 18-carbon fatty acid)
film required to give the same optical parameters, using the calibra-
tion charts prepared with the model compounds. If one is willing
to accept that the refractive index of the specimen films was very
close to that of fatty acids, which is generally a good assumprion for
biological materials,'@ the equivalent thicknesses may be given a
geometric value by comparing them to a thickness of approximately
25 8 for stearic acid.

Critical Surface Tensions

Liquids used as sessile drops in the contact angle measurements
necessary to estimate critical surface tensions covered a wide range
of surface tensions and a variety of structural types. Sources,
methods of purification, and surface tensions of the pure compounds
may be found in the literature.11~12For convenience, the liquids used
and their liquid-vapor interfacial tensions are listed in the first two
columns of Table I. On the specimen surfaces, the slowly advancing
194 R. E. BAIER AND R. C. DUTTON

contact angles of each pure liquid were determined using the slow,
drop buildup methodI3 and a goniometer telescope (NRL Contact
Angle Goniometer, Rame-Hart, Inc., Mountain Lakes, N. J.). Each
contact angle recorded is that which occurred reproducibly within
the first 10-20 sec after slowly advancing the drop over a fresh
surface region. Contact angles exhibited by water, glycerol, forma-
mide, and thiodiglycol-all hydrogen-bonding and polar com-
pounds- changed after this time as the result of gradual penetration
of the liquids through the films. Contact angles exhibited by the
nonhydrogen-bonding organic liquids were generally constant for
many’minutes. All measurements were made with materials equili-
brated in a clean room maintained at 20°C and 50% relative humidity.

TABLE I
Contact Angles on Blood Films, Substrates, and Fibrinogen

Average contact angle, 0,. degrees

Water 72.8 66 78 62 11 72 80 92
Glycerol 63.4 67 63 76 20 64 69 64
Formamide 58.2 58 63 42 5 66 64 62
Thiodiglycol 54.0 47 51 47 8 54 47 48
Methylene iodide 50.8 46 45 31 25 46 41 43
Sym-Tetrabromoethane 47.5 41 39 27 21 39 - 36
1-Bromonaphthalene 44.6 32 32 10 16 32 16 26
0-Dibromobensene 42.0 28 28 0 8 35 - 24
1-Methylnaphthalene 38.7 24 31 0 4 25 0 19
Dicyclohexyl 33.0 0 13 0 0 19 0 0
n-Hexadecane 27.7 0 0 0 0 14 0 0
n-Tridecane 25.9 0 0 0 0 0 0 0

a Reported values are averages of at least 10 readings on at least two inde-

pendently prepared films of each type.
 INTERACTIONS OF BLOOD 195

The contact angle data were plotted in a Zisman format3 to obtain

approximate critical surface tensions.
Experimental Procedure
Germanium prisms were scrubbed thoroughly with an aqueous
solution of the detergent Tide, rinsed copiously with tap water, and
then with distilled water; this cleaning procedure resulted in
“cleaned” surfaces which produced blank MAIR spectral4 and con-
tact angles indicative of “cleaned” metal surfaces.16 The prisms
were mounted in Teflon holders throughout washing and rinsing, and
while still water-wet were exposed to fresh, flowing blood. The
jugular veins of 20-25 kg, lightly anesthetized dogs, were exposed,
transected, and the ends everted so that blood issuing from the veins
would not contact the transected ends. Blood flowed directly from
the veins and impinged against the prisms. Alternately, prisms or
polymethylmethacrylate windows were placed in a flow chamber
system that allowed only fresh blood t o reach the test surface.16
Considerable effort was made to prevent air-blood interfaces from
passing over the test surfaces. After a desired period of exposure,
the plates were removed and immediately flushed with a vigorous
flow of 500 cc of physiologic saline followed by extensive rinsing in
distilled water. By gross visual examination, no difference was
noted between the “exposed” and the initial “cleaned” appearance
of the test surfaces. The polymethylmethacrylate plates were
identical t o those upon which thrombus formation was observed.16
No difference in results was apparent in one experiment where the
blood was derived from the animal’s carotid artery.
Following air-drying in a large, covered, grease-free glass container,
the samples-still mounted-were analyzed sequentially by MAIR
IR spectrophotometry, ellipsometry, and contact angle techniques.
Formation of Fibrinogen Films
Thin films of commercially purified bovine fibrinogen” were formed
from water on polished platinum plates by a solvent-droplet spread-
ing technique developed earlier.14 The fibrinogen was not readily
solubilized in water but an air-water interfacial film was immediately
formed and spread to occupy the whole surface of the plate. Upon
drying, a clear, hard film of fibrinogen was obtained with residues of
the bulk material still present in the center of the plates.
196 R. E. BAIER AND R. C. DUTTON

Film Extraction
I n an attempt to remove soluble small molecules or lipids from the
films, a rigorous extraction procedure was followed :
A prolonged immersion of the samples in a rapidly flowing stream of
cold t a p water, followed by ( 1 ) a 2 hr immersion in acetone, ( 2 ) a 24
hr immersion in n-hexane, (3) a 2 hr immersion in benzene, and
ultimately, air-drying. All solvents were reagent grade.
An alternative procedure, less desirable because of the high proba-
bility of protein coagulation, included a hot water immersion step.
Film characteristics were monitored at each step.

RESULTS
Films on Germanium Prisms
Both the optical techniques were nondestructive, and were usually
performed within an hour of one another. During sample prepara-
tion, our most rapid manipulation resulted in a blood-prism contact
time of 5 sec, or slightly less. A MAIR infrared spectrum of such a
5 sec film is given as Figure 1. The diagnostic peaks for protein are
clearly seen, with the N-H absorption a t about 3300 cm-’ and the
amide I and amide I1 bands a t approximately 1650 cm-I and 1550
cm-l, respectively. A slight splitting of the amide I band suggests
the presence of differing protein conformations in the film; however,
the peak positions in the 1500-1700 wavenumber region, according to
diagnostic criteria outlined e l s e ~ h e r e , ~ are
~ J ~only
J ~ consistent with
the coiled protein configurations (e.g., alpha-helical, random tangle)
and not with the extended-chain beta-structure generally thought to
occur with interfacial protein films.20
The peaks in Figure 1 around 2800 cm-’ represent hydrocarbon
groups, probably substituents of the proteins; the absence of absorp-
tion in the region of 1750 cm-’ shows that free carbonyl groups, as
would be present with esters or carboxylic acids, are not present in
detectable amounts. The sharp “jags” in the spectral baseline,
notably near 2000 cm-’, are caused by grating changes in the record-
ing instrument, and appear to be quite exaggerated when germanium
prisms are used. The climbing baseline and sharp peaks beyond
1400 cm-I, or a t the higher wavelengths, are also typical cutoff effects
of the germanium prisms.
 INTERACTIONS OF BLOOD 197

These 5-sec adsorbed blood films had optical constants, determined

with the ellipsometer, equivalent to those which would be obtained
with 2 layers of stearic acid on the prisms. I n isolated spots, maxi-
mum film thickness was found to be equal to as much as 2% stearic
acid layers and minimum thickness obtained was indicative of 1%
stearic acid layers. Accepting the assumptions involved in trans-
lating these values to geometric thicknesses, the average film depth
was about 50 A, with extremes of about 35 and 65 8 in isolated spots.
The contact angles obtained for pure liquids placed on the surface
of 5 sec films, the same films whose spectra and optical constants had
been recorded, are given in the column labeled a in Table I. These
diita, when plotted as cos 8 vs ~ L inV a Zisman plot (see Fig. a), yield
an intercept with the cos 8 = 1 axis of about 36 dyne/cm; this is the
critical surface tension, y o for the new outermost surface created on
the prism face by the adsorbed blood components.
Exposure of the foreign surface to flowing blood for periods of about
1 min led t o the formation of slightly thicker, but less uniform, films,
perhaps incorporating some lipid components in addition to protein.
The MAIR spectra were qualitatively similar to that shown as
Figure 1, in the main. The protein peaks were all enhanced but not
shifted; the CH2 band around 2800 cm-’ was enhanced more than
proportionately, however, and a small absorption peak appeared a t
1750 cm-’ suggesting the inclusion of some fatty acid type material in
the films. Ellipsometry showed, with the qualifications mentioned
earlier, that the one minute film averaged 125 A inodepth, with ex-
tremes of 200 8 a t the maximum and as little as 25 A in one isolated
spot a t the minimum.
198 R. E. BAIER AND R. C. DUTTON

The contact angles for the 60 sec films are recorded in the column
labeled b in Table I, and plotted (see the filled circles) in Figure 3.
Except for a higher water contact angle and the nonzero value ob-
tained with dicyclohexyl-which could be explained by the presence
of trace amounts of lipid-the wetting results are nearly identical to
those for the 5 sec film, as is the critical surface tension intercept
which quantitates the relative surface wettability. Water drops on
the 60 sec film occasionally showed a buildup effect-that is, as they
were incremented they increased in volume (and contact angle)
without advancing over a fresh region of the surface. This behavior
has been noticed previously with water drops on collagen films.21
Attempts to extract the 60 see films according to the regimen listed
in the methods section changed the films as follows: in the infrared
traces the CH, absorption band diminished, and the free carbonyl
band disappeared completely; the protein absorption bands were not
diminished, and in some cases appeared to sharpen slightly; no
significant change in average thickness occurred; the wettability
showed a net increase, as attested to by the generally lower contact
angles (column c in Table I) and the increased critical surface tension
(defined by the line through the filled squares on Fig. 3). An

SURFACE TENSION (yLv) IN DYNES/CM

Figure 2
 INTERACTIONS OF BLOOD 199

SURFACE TENSION (yLv) IN DYNES/CM

- I -
0
SURFACE TENSION CY,,) IN DYNES/CM

Figure 3

anomaly, yet to be explained, was the higher than anticipated con-

tact angle for the glycol compounds, glycerol and thiodiglycol.
When the extraction included a flowing hot water rinse, the
carbonyl absorption band at 1750 cm-’ showed temporary enhance-
ment-eliminated after the organic solvent immersions-probably
associated with protein denaturation due to heat coagulation.
The adsorbed films, of all exposures, were very tightly adherent to
the prism surfaces. A vigorous stream of water did not detach nor
significantly modify them, and solvent extraction did not markedly
deteriorate them. It was only after mechanical scrubbing, using a
soft brush and an aqueous detergent solution, that the MAIR spec-
trum once again became “blank” and the contact angles on the
prisms returned to about those listed in column d of Table I (which
are typical of “cleaned” metal surface^'^).
Films on Polymethylmethacrylate Plates
Windows of polymethylmethacrylate (PMMA) have shown a
typical interaction of blood with a foreign surface, culminating in the
buildup of thrombus, in companion e~ p erir n e n ts .~ ,’~
When these
windows were placed in a flow chamber and exposed to flowing blood
200 R. E. BAIER AND R. C. DUTTON

for 5 sec and for 60 sec, their surfaces were similarly modified by
adsorbed films.
With the films deposited on PXIRlA, R'IAIR infrared spectroscopy
required the use of auxiliary prisms. Operationally, the coated
PMRIA surfaces are pressed tightly against the optically polished
surface of a slightly deformable salt (KRS-5) prism which carries the
infrared signal; when films are on germanium prisms, the infrared
energy is channeled through the germanium itself and results in
greater sensitivity. The depth of penetration of the electromagnetic
evanescent wave into the bulk plastic, is also greater with KRS-5
prisms' and thus further limits sensitivity because of the larger con-
tribution of the plastic compared to the adsorbed film. With these
restrictions, the detection of protein bands in the 5 sec films on
PR4MA was only marginal, and only a little better with films ad-
sorbed after 60 sec contact with flowing blood. Protein peaks were
present, however, and were a t the same frequencies shown in Figure
1; this suggests that the same coiled conformations were present.
I n our experience, protein monolayers are only detectable when
coated directly on the internal reflection elements,l8and not detectable
when first coated on a plastic or metal substrate and then pressed
against KRS-5 prisms.22 Therefore, even the marginal detection of
protein absorption bands in films adsorbed on plastic suggests that
the film thicknesses were mof;ethan that common with protein mono-
layers (usually less than 50 A) and more likely of the order observed
with films on germanium and on Epon5 (about 50-200 A).
Ellipsometry was not attempted with the PMMA plates because
of their general unsuitability for these measurements.
Having only limited optical information, then, the contact angle
studies were of increased value. The column labeled e in Table I
records the wetting results of PMMA which had been exposed to
blood, and the column labeled f in that table records the data for an
unmodified PRIMA surface.23 Comparison of these values shows that
exposure to flowing blood led to a significant decrease in relative
surface energy of the plastic. Figure 4 is a Zisman plot of the data,
and the extrapolated line has been rather subjectively drawn to
intercept the cos 8 = 1 axis at a critical surface tension of about 36
dyne/cm; this intercept is the highest value compatible with the
results obtained, and may be compared with a value of 39 dyne/cm
characteristic of the unmodified p ~ l y m e r . ~A~ critical
,~~ surface
 INTERACTIONS OF BLOOD 201

SURFACE TENSION (yLv) IN DYNESICM

+I 0 +I0

09 09

08 08

07 07
Q)
,
al 0.6 06%
-
z- t05:
g+o 5
0
04 0.4

03 03

02.
!FILM ADSORBED ON PMMA FROM FLOWING 8LOODlI
, 1 1 ,
I \ -0 2
- I 1111 i 1 1 I I) I i I I 1 ’ -
+01 ’
,.I111 I I ‘ l l , l , I I
I +01
I -
0
SURFACE TENSION (yLv) IN DYNESICM

Figure 4

tension intercept substantially lower than 36 dyne/cm might actually

be more appropriate for the “filmed” PRIMA, as indicated by the
nonzero contact angles obtained with liquids of surface tension as low
as 27 dyne/cm.
This lower y c intercept, if correct, would favor the possibility that
lipids were also present in the films on PMRIIA.
Films of Fibrinogen
Because the spectra obtained with the adsorbed blood films are
consistent with the presence of highly alpha-helical proteins, and
because one such protein, fibrinogen, has been implicated in the
initial adsorptive events at interfaces of blood and foreign ma-
teria1,5~25we studied thin films of fibrinogen to determine whether
or not their properties were similar to those films adsorbed from blood.
Figure 5 is the MAIR infrared spectrum typical of these fibrinogen
films; its resemblance to Figure 1, obtained with a 50 A thick film
adsorbed from blood, is quite good. Qualitative differences between
Figure 5 and Figure 1 are explained as follows: the lesser amount of
“noise” in the fibrinogen trace results from using the recording
instrument at lower gain, which was possible because of the greater
202 R. E. BAIER AND R. C. DUTTON

MICRONS
25 30 40 50 60 70 80 90 10 12 14 1 6 1 8 2 0 2 5 3 0 4 0

40----L- - - - -

20 -- --

0 __ 1
4& 3500 3000 25W 2000 18W 16W 1400 1200 1000 800 6W 4W 250
WAVENUMBER ICM ’I

Figure 5

sample thickness; the flatter baseline and resolved bands beyond

1400 cm-l, and the minimization of baseline “jags” around 4 and 5 p
are the result of the use of KRS-5 prisms rather than germanium
internal reflection elements. The fibrinogen spectrum, in addition
to strong diagnostic protein bands, shows a slight enhancement of the
CH, absorption and the suggestion of some absorption in the free
carbonyl region; some lipid moieties may have been present in the
commercially purified material used. Nonetheless, by spectral
criteria, adsorbed films from blood are closely matched by at least one
protein, fibrinogen, known t o be present in circulating blood.
Figure 6 is the Zisman plot for the wetting data (recorded in the
last column of Table I) for fibrinogen films. With the exception of
the water contact angle-not plotted-the data are remarkably
similar to those for the adsorbed films from blood. Indeed, the plots
in Figure 2 and Figure 6 are nearly superposable. The anomalously
high water contact angle on fibrinogen resulted from the drop buildup
effect previously m e n t i o ~ i e d and
, ~ ~ since the effect was also present
with 60-sec adsorbed films from blood, it is a further point in favor of
their similarity to one another.

DISCUSSION
These results confirm and generalize the supposition that the initial
common event in the interaction of blood with foreign surfaces is the
rapid deposition of strongly adherent proteinaceous films. Similarity
of the spectral and wetting properties of the adsorbed films to those of
purified fibrinogen suggests that fibrinogen might be a major com-
 INTERACTIONS OF BLOOD 203

Figure 6

ponent of the naturally deposited layers, but these results do not by

themselves confirm the presence of fibrinogen nor give cause for ex-
clusion of other components. The antibody-antigen study of
Vroman shows that fibrinogen does deposit rapidlyz5and the electron
microscopic demonstration of fibrin strands emerging from the
“conditioning” adsorbed surface layers5 also suggests the involvement
of fibrinogen in these layers. The observation of a wettability in-
crease (coupled with disappearance of carbonyl peaks and decrease in
CH2 absorption) after extracting films with organic solvents argues
for the inclusion of some lipid components in the thicker films.
Changes in protein conformation during extraction cannot be com-
pletely excluded as an alternative explanation.
A critical, or perhaps limiting, thickness of the adsorbed films
appears t o be in the neighborhood of 200 A, sincc platelets have been
seen deposited on such films during initial thrombus generation5 and
must change the nature of the adsorption process in their vicinity by
their presence. The idea that the initially adsorbed layer is required
for, or greatly influences, platelet adhesion and thrombus formation is
supported by the “threshold” time noted before platelets first deposit
on foreign surfaces from fresh, circulating blood16,26-which is of the
204 R. E. BAIER AND R. C. DUTTON

order of a minute or two. But platelets do not a t any time up to 15

min (enough time for the surface to be coated with thrombus)
entirely cover the surface. Therefore, the fact that platelets might
prevent further protein deposition in some areas does not eliminate the
possibility of additional protein deposition in others.
The thicknesses obtained here, by ellipsometry, agreed well with
those determined by electron microscopy; although encouraging, such
good agreement may be in large measure fortuitous. The layers
measured by electron microsocpy were chemically fixed, stained, and
embedded in resin;5 the layers measured by ellipsometry were air-
dried and affixed to germanium prisms. In both instances, many
assumptions are involved in accepting the measured film depths as
representative of the actual films deposited in vivo.
It is also difficult to assume that the film surfaces investigated here
are adequate models for the films deposited in vivo. A major dif-
ference, for instance, is th at all films included in the present study
were allowed t o dry a t the air-water interface; during this process
interfacial forces might have modified the film structure or favored
the exposure of certain atomic groupings and the burying of others
within the film matrix. Nevertheless, since all films prepared here
were treated identically, relative comparisons of their surf ace chemical
constitutions in the dry state are legitimately made.
The critical surface tensions obtained, in the high 30’s dyne/cm,
are representative of organic surfaces of relatively high en erg^;^,^ for
comparison, the critical surface tensions of waxes, fatty acids, and
silicone rubbers usually fall in the low 20’s dyne/~rn.~r*-~4 Thus
relatively high energy groups are available in the adsorbed protein
films; supposing that such groups are not masked in the films de-
posited on foreign surfaces in vivo, they should contribute to better
adhesion, and perhaps surface modification, of platelets which con-
tact them.* If, as other workers have and as our experi-
ments suggest,29the intimal surface of blood vessels is of intrinsically
low surface energy, the higher surface energy protein films may “con-
dition” implanted surfaces to favor platelet adhesion and subsequent
thrombogenesis. Following this reasoning, damage to the blood
vessel itself-exposing a collagenous matrix of higher surface energy
than the vessel wall-would induce the same sequence of events
observed with nonphysiologic implants. On the other hand, it was
shown here that adsorbed films from blood lowered the critical surface
 INTERACTIONS OF BLOOD 205

tension of polymethylmethacrylate ; if surface energy arguments are

relevant, this means that cell adhesion to “filmed” PR/1R!IA--although
still of relatively high surface energy and still thrombogenic-should
be less than that on the clean polymer surface. Experiments of
A. C . T a y l ~ r ~and
~ v ~of~ Weiss and Blumenson3* on cell adhesion
in tissue culture appear to support this contention, since in the
absence of protein supplements cell adhesion and spreading could be
correlated with the relative surface energies of the solid substrates
while in the presence of protein supplements (horse serum) cell
behavior a t most interfaces became similar.
Needless to say, the surface chemical arguments just offered are
highly speculative without additional experimental support.

This collaborative study was initiated while R. E. B. was NRC-NAS Post-

doctoral Research Associate in Surface Chemistry a t the Naval Research Labo-
ratory, Washington, D. C., continued while he was a guest worker in the
Laboratory of Technical Development, National Heart Institute, Bethesda, Md.,
and continues under Cornell Aeronautical Laboratory’s Internal Research Pro-
gram. Support and encouragement from colleagues at these laboratories are
greatly appreciated.

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206 R. E. BAIER AND R. C. DUTTON

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Bovine Fibrinogen Powder.
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Received October 14, 1968