CHAPTER II.1.

Adsorbed Proteins on Biomaterials

PROBLEMS will not be present in high amounts on the surface

1. What are the three main driving forces that affect the if it is present in low bulk concentration compared
competitive adsorption of proteins to biomaterials to the other proteins, since the excess concentration
from complex mixtures such as blood plasma? of the competing proteins will overcome the affinity
2. Calculate the amount of adsorbed protein in a close differences.
packed monolayer for an average protein of molecu- 2. Close packing means that the molecules touch each
lar weight 100,000. Assume the protein is spherical other at their boundaries, so the problem can be
and has a partial specific volume of proteins being solved by calculating the cross-sectional area of one
0.73 ml/g. molecule of the protein and dividing that into square
3. When various biomaterials are implanted in the body, centimeter of area, which then gives the number of
the responses often depend on the chemistry of the molecules in that area. Using the molecular weight of
implanted material. Yet we know that the surface is the protein allows conversion of number of molecules
covered with adsorbed protein, and thus the biologi- into mass of molecule, and thus you end up getting
cal response is not due to direct interaction with the mass per unit area of a close packed monolayer.
surface. How then are the differences in responses
explained?
Protein Monolayer Calculation
4. What are three methods that show the specific effect
of adsorbed proteins on cellular interactions with The specific volume (volume per gram) of a protein
biomaterials? can be used to calculate the volume of an individual
5. As illustrated in Figure II.1.2.5, polystyrene and other protein molecule by using the molecular weight and
hydrophobic surfaces become more wettable after Avogadro’s number:
exposure to protein-containing media. However,
Vmolecule = Vspecific * M / NAvogadro
for certain materials, such as cleaned Germanium
= (0.73 ml / g) * (105 g / mole) / 6.02 × 1023
oxide prisms that initially have low contact angles = 1.22 × 10 − 19 ml / molecule
with water (11°) and are thus fairly wettable to begin
with, exposure to protein-containing solutions has The volume of an individual protein molecule
been reported to cause the contact angle to increase can be used to calculate the radius of the molecule r,
(to around 60°). How can this be explained? assuming it is spherical:
6. Proteins are tightly bound to many biomaterials, and ( )1 / 3
are considered irreversible in that the proteins are not r= 3 V
molecule
4Π
washed off in the buffer used for adsorption, even = 3.08 × 10 − 7 cm
after long soaking periods. Give a molecular explana-
tion of why proteins are so tightly bound. (This radius estimate can be compared to dimen-
sions of proteins measured by X-ray crystallogra-
phy. For proteins in the range of 100,000 molecular
SOLUTIONS TO PROBLEMS weight, average long dimensions are around 70 Å,
1. The three main factors that are believed to control giving a radius of around 35 Å, fairly close to what we
the outcome of competitive adsorption processes are: just calculated using the specific volume of proteins.)
(1) the intrinsic affinity of each protein for surfaces, The radius of the protein molecule can then be
meaning that some proteins are more surface active used to calculate the projected area of an individual
and more adsorptive than others; (2) the chemical protein molecule Amolecule, which is the “footprint” or
nature of the adsorbing surface, due to the fact that area occupied on the surface by the protein molecules:
the variations in surface affinity among proteins one
Amolecule = Πr2
sees for a given surface are only fixed for that sur- = 2.97 × 10 − 13 cm2
face, and will differ if another surface is used; (3) the
relative bulk concentration of each protein, meaning With the footprint area, you then can calculate
that even a protein with high affinity for a surface the number of protein molecules that can fit into a
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 square centimeter nprotein, assuming close packing of c. A
 ddition of antibodies specific to a given adhe-
circles representing the footprint area occupied by sion protein or to its receptor to a cell suspen-
each spherical protein molecule. (This is a small over- sion incubating with a biomaterial will result in
estimate of the occupyable area, because the tightest a decrease in adhesion that is proportional to the
packing of circles on a planar surface leaves about 9% concentration of added antibody. Because such
area unoccupied, according to a theory by Gauss.) antibody blocking studies can be done with sur-
faces adsorbed with mixtures of proteins such as
nprotein = 1 cm2 / Amolecule
= 3.36 × 1012 molecules
plasma, yet they selectively interfere in adhesion

mediated by only one adhesion protein, they pro-
Then convert the number of molecules per unit area vide information relevant to the functional role of
nprotein into mass of protein per unit area mprotein using the adhesion protein under conditions closer to
Avogadro’s number and the molecular weight of the the physiologic situation, where biomaterials are
protein: exposed to complex mixtures of proteins.
5. An increase in contact angle after exposure to pro-
mprotein = nprotein * M / NAvogadro
= 5.6 × 10 − 7 g
tein-containing media can be explained by adsorp-
= 0.56 microgram tion of protein if the adsorbed protein layer is less

wettable than the starting surface. For certain solid
Thus, according to this calculation, there should be materials, such as cleaned Germanium oxide prisms,
around 0.56 micrograms per square centimeter in the interaction is very strong with water, and so the
a close packed monolayer of a spherical protein of contact angle is low. After protein adsorption occurs,
molecular weight 100,000. the higher contact angle must mean that adsorbed
3. Due to differences in relative competitive affinity of proteins interact with water less strongly than the
proteins for various surfaces, the amount of adsorp- starting Germanium oxide surface. Thus, while pro-
tion of each protein varies with surface chemistry. For teins are much more wettable than polystyrene, they
example, some surfaces have more adsorbed fibrin- evidently are not as wettable as some solid surfaces.
ogen and others more adsorbed albumin, and since 6. The irreversible, tight binding of proteins to surfaces
platelets bind only to fibrinogen, surfaces enriched in is due to the large size of protein molecules, so that
fibrinogen are more platelet reactive. Thus, the effect many noncovalent bonds are formed between the
of surface chemistry is to vary the composition of the surface and each molecule, i.e., “multipoint” attach-
adsorbed protein, and this is why the reactivity of ment occurs. Although each bond is relatively weak
cells varies with surface chemistry. and reversible, the chance that all the bonds would be
4. Three methods to show the role of adsorbed proteins broken simultaneously is low, so multivalent bonding
are as follows. results in strong bonding.
a. If surfaces are preadsorbed with various purified Note: Latour (2008) adds another way to look at this
proteins, it is found that most inhibit cell adhe- issue. He notes that because of the large number of
sion to the surface, but a few such as fibronectin, contacts between functional groups of a protein and a
fibrinogen, and vitronectin cause the cell adhesion surface, even weak interactions will hold the protein.
to be much higher than for other proteins. Pro- For example, if the interactions with the surface are
teins that block adhesion are called passivating or equal in energy to their interactions with water in the
blocking proteins, while those that mediate it are bulk phase, and we assign the probability of a surface
called adhesion proteins. functional group being bonded to a protein versus a
b. Preadsorption with complex protein mixtures, water molecule to be 0.5, the probability for all of the
such as blood plasma selectively deficient in only functional groups of an adsorbed protein to dissociate
one protein, is a more physiologically relevant way from the protein at the same time would be P = (0.5)n,
to show if a protein is contributing an important with n being the number of functional group contacts
role to platelet adhesion in the presence of many between the protein and surface. So even if n is as small
other potential adhesion proteins in plasma. If cell as 20, the probability that they would all dissociate at
adhesion is greatly reduced when the surface is the same time is very small. Thus, even a hydrophilic
adsorbed with the protein mixture that is missing surface with OH groups (e.g., OH-SAM) will tend to
one protein such as fibronectin, it means this pro- irreversibly adsorb a large protein, while for hydro-
tein is playing an important role in that it is not phobic surfaces that have favorable thermodynamics
replaced by the action of other proteins still in the of adsorption for a nonpolar amino acid residue versus
mixture and on the surface. water, the likelihood of desorption is even lower.

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