Introduction:
Cell adhesion to polymer surfaces has obvious implications in the field of tissue
engineering. Facilitating cellular adhesion, growth and differentiation onto a surface has
implications in wound healing and tissue growth. Polymers may be inserted into the
body and induce tissue repair or facilitate would healing. Cell adhesion has applications
in infection control of foreign bodies. If one could control the extent of bacterial cell
adhesion, one could prevent infection of prosthetics. One could also envision using
biodegradable polymers that would degrade in the body leaving behind the repaired
tissue. For these reasons and others, cellular adhesion to polymers has been an active
area of research among the scientific community. The purpose of this paper is to explore
the mechanism of cellular adhesion to polymers and how it can be controlled and
exploited for numerous applications.
1
Overview of a biosurface:
An experimental design of a possible biomaterial would have to view initial and
long term interactions from both a cellular (biological specificity) and a materials surface
perspective as mediated by the formation and chemistry of a conditioning film and the
extracellular matrix at the biomaterial/cell interface [1]. This interface is illustrated in
Fig. 1 below [1]:
The short and long term effects include cell health, competition with bacterial cells,
differentiation of cells and material degradation.
In order to study the effects of the interface in detail, one needs control over the
biomaterial in question, the medium and an understanding of cellular physiology is
necessary. There are three components in the understanding of a biomaterial/cellular
interaction [1]: (1) The role of the substrate and its surface chemistry, (2) the role of the
adsorbed macromolecule (i.e. polysaccharides, glycoproteins), (3) the role of the cellular
response.
The process of cellular adhesion, including protein adsorption, cell adhesion and
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spreading is illustrated in Fig. 2 below [9]:
Where % I is equal to the percent of inoculum attached.
Surface characterization is needed to develop correlation between the surface
energy, surface composition and the cellular response. Defining which functional groups
( i.e. carboxyl groups, hydroxyl groups, amines etc.) are present on the surface of the
biomaterial is crucial in understanding this correlation. Since functionality at the surface
of a polymer can be modified by various techniques, such as radio-frequency glow
discharge, an understanding of how the functionality effects adhesion can allow for better
engineered biomaterials. Surface energetics play a crucial role in the colloidal aspect of
protein adsorption which conditions the surface and makes it suitable for cellular
adhesion. Understanding qualitatively and quantitatively what is at the surface is
important in the experimental design of a biomaterial.
The macromolecules of the conditioning film play a crucial role in the adhesion of
cells to a biomaterial surface. Adhesion of animal cells to the extracellular matrix and
foreign surfaces and to other cells is mediated by the adhesion receptor proteins in the
cell’s membrane [2]. There are four such families of proteins, integrins, cadherins,
immunoglobulin superfamily and the selectins. The integrins are primarily responsible
for cell adhesion to extracellular proteins and foreign surfaces. The other three families
function primarily as cell/cell adhesion proteins. The ligands for the integrin adhesion
receptors are primarily extracellular and therefore mediate most substrate adhesion [2].
3
These include the matrix protein fibronectin, laminen, tenascin and thrombospondin.
There are also substrate absorbed forms of the normally fluid phase protein fibrinogen,
von Willebrand factor, vitronectin and plasma fibronectin that have adhesion properties.
Adsorbing these ligand proteins to a surface as a conditioning film can facilitate cell
adhesion and growth on the biofilm. Finally, the short and long term effects described
above will determine the viability of the biomaterial.
4
Surface characterization of substrate:
The surface energy of a substrate can be measured easily with tensiometric
measurements. The most common of which is contact angle measurements as illustrated
below in fig. 3 [3]:
By obtaining the angle of the curvature of the droplet, one can obtain the surface
energy of the solid substrate if one knows the properties of the droplet. This is done
utilizing the Young equation shown above. This understanding of the surface energy of
the polymer substrate is vital to determining protein adsorption to the substrate, as will be
described later.
The chemical composition is also important in determining the effect of the
substrate in the biomaterial/cellular interaction. Spectroscopy is especially useful in
determining the surface composition of a given polymer. The general scheme for surface
spectrometry is illustrated below in fig 4 [4]:
5
In general, a solid sample is irradiated with a primary beam of photons, electrons,
ions or neutral molecules. The impact of this beam results in the formation of a
secondary beam that consists of photons, electrons, ions or molecules. As one can see in
fig. 5 [4], the primary and secondary beams need not be the same type of particles:
There are three types of sampling methods. The first is to focus the primary beam
on a spot in the sample and then analyze the secondary beam. The second includes
scanning the surface with the primary beam in a raster pattern and then analyzing the
secondary beam for composition. The last method is called depth profiling. This is when
a beam of ions etch a hole, by sputtering, and then the primary beam is focused into the
center of the hole and the composition can be obtained as a function of depth.
In most of these surface measurements, the sample is kept under ultra-high
vacuum conditions. Since air molecules can adsorb to the surface, ultra-high vacuum
(UHV) is used to prevent this contamination. The conditions are also necessary due to
interference on the part of air molecules in the secondary beam's travel to the detector.
Keeping the sample in pressures below 10-7 torr minimizes this problem. Some criticize
the use of UHV equipment in surface analysis of biomaterials since surfaces undergo
conformational changes at the water interface that are different than those at the vacuum
interface. Recent developments, though, have allowed for the hydration of a polymer for
a desired amount of time and subsequent analysis in a cryogenic state. This requires a
modification of the UHV equipment that can be adapted to almost any instrument as
stated by Gardella and Hawkridge [13].
XPS, x-ray photoelectron spectroscopy, is a technique used to elucidate the
surface composition of a polymer. In an XPS, a photon of a monochromatic x-ray beam
of a known energy, hv, displaces electrons form a K orbital with a binding energy of Eb.
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A + hv  A+* + e-
Eq. 1[4]
Where A is a molecule, atom or ion and A+* is an excited ion. The kinetic energy of the
electron EK is measured in the spectrometer and allows for the determination of Eb which
is its binding energy.
Eb = hv – EK – w
Eq. 2[4]
Where w is the work function and corrects for the electrostatic environment on the
surface.
The binding energy is characteristic of the atom and orbital of which it comes
from. A schematic of the XPS process is illustrated below in fig. 6 [4]:
Applications of XPS include qualitative analysis of the surface composition of the
polymer. A low resolution, wide scan XPS spectrum, or survey scan, is taken and
determines the elemental composition of the sample. The survey spectrum has a kinetic
energy range of 250 to 1500 eV, which corresponds to a binding energy of 0 to 1250eV.
Every element appears in this region. If one then uses a higher resolution, one can
elucidate chemical shifts that determine the surrounding environment of the atom,
oxidation states and physical structure [4].
Another useful tool is secondary ion mass spectrometry, SIMS. It is the most
highly developed of mass spectrometric surface measurements [4]. In this case, the
surface is bombarded with beams of 5 to 20 keV ions such as Ar+, Cs+, N2+, or O2+.
These are produced by an ion gun and are accelerated by a high dc potential. The surface
is then stripped or sputtered and the charged molecules that are sputtered off the surface
make it to the mass analyzer, such as a quadropole or flight tube.
7
The final technique to be discussed is scanning electron microscopy, SEM. In
this case, the surface is scanned in a raster pattern with a beam of energetic electrons.
Several types of signals are produced including: backscattered, secondary and auger
electrons, x-ray fluorescence photons and other photons with various energies. The two
most commonly used sources are backscattered and secondary electrons, which serve as
the basis of SEM, and X-ray emission which are used in EMS. SEM provides
morphologic and topographic information about the surface of solids and will give an
actual image as opposed to a spectra as the previous techniques described do.
These are only a few of surface techniques employed to fully understand the
substrate being used in the cell adhesion studies. Again, the energetic and chemical
properties of the surface are crucial for understanding the principles that effect
biomaterial/cellular interactions.
8
Conditioning film and protein adsorption:
The macromolecules involved in the conditioning film of the biomaterial have the
most impact on cellular adhesion. The adsorption of proteins to the polymer's surface is a
critical step in cell adhesion. One can take a colloidal treatment of this phenomenon into
account. The proteins are treated as particles suspended above the solid substrate and if
the energetics permit, will adsorb to the substrate. Adsorption of dissolved proteins to
lower-energy surfaces is common [3]. In the absence of significant electrostatic
interactions, adsorption is entirely due to hydrophobic interactions [3]. The protein acts
as a surfactant in which the hydrophobic domain exposes itself to the surface leaving the
hydrophilic domain pointing towards the liquid medium, water. The interfacial free
energy between the protein and the substrate can be described by the equation below:
GIF1w2 = GLW1w2 + GAB1w2 Eq 3. [3]
A favorable interaction between the substrate and protein corresponds to a negative value
for GIF1w2. These values can be obtained by tensiometric measurements as described
earlier. These measurements are extremely powerful in determining whether or not and
to what extent a protein will adsorb to a surface. Energy values for proteins with some
common polymers are given below in fig 7[3]:
When discussing protein adsorption to a surface, one must consider the Vroman
effect. The Vroman effect is a collection of observations that describes the behavior of
proteins during adsorption events. The Vroman effect consists of four principles. The
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first being that protein adsorption is essentially irreversible if no other proteins are
present in the solution. This was demonstrated by washing a surface coated with
adsorbed proteins and finding minimal desorption. The second observation is that
proteins can replace themselves at the surface. This behavior was shown by
radiolabelling a protein and allowing it to adsorb to a surface. An additional amount of
the same protein was added to the bulk and displacement of the labeled protein was
observed. Since different proteins have different affinities for a surface, proteins with a
stronger affinity can displace those with a weaker affinity. This is the third principle of
the Vroman effect. The final principle states that if proteins are allowed to adsorb to a
steady state an isotherm of either Langmuir or Freundlich type arises. All these
principles must be taken into account when trying to control the composition of a
conditioning layer. Exploiting the principles of the Vroman effect can allow one to
predict the type and amount of proteins that will adsorb to the surface.
Figure 7 illustrates that with relatively low-energy polymers, strong adhesion
occurs because of the large negative values of G. Polymers of medium surface energy,
s = 30 to 33 mJ/m2, generally have maximum adsorption at the surface. Adsorption of
proteins are generally illustrated in the form of adsorption isotherms where the log
adsorption is plotted versus the log of bulk concentration.
Despite these conclusions, in vivo cells adhere least to these medium surface
energy polymers as shown by Baier [10]. This is explained by taking into account that
extracellular proteins, polysaccharides and other components adhere to the polymer
surface quickly and strongly. The hydrated surface of the adsorbed protein is hydrophilic
in nature and thus results in minimal cell adhesion. Polymers with such high adsorption
properties are not selective to adhesive proteins and can be coated with non-adhesive
proteins, which will inhibit cell adhesion. Other approaches must be taken to avoid this
problem.
One such approach was employed by Busscher [6] in using colloidal science to
predict cell adhesion. His work concluded that the higher the surface energy, the higher
the cell growth and spreading. The author used a variety of polymer surfaces with
different values for s.
10
The cell growth and spreading went from poor to good at around s = 55 mJ/m2 as
shown in Fig 8. [6] Below:
Although one cannot generalize this to all cell types, it seems to work well with
fibroblasts, smooth muscle cells and epithelial cells in vitro and in vivo [6]. This shows a
powerful use of colloidal phenomena to explain and predict cellular adhesion to
substrates.
Vogler [8] also stresses the need for colloidal science in the field of biomaterials.
He states that the equilibrium cell adhesion is controlled by arithmetic combination of
substrate and cell wetting tension t. Wetting tension, t, is derived from contact angle
measurements and can directly predict whether adsorption will occur.
Although Vogler’s [8] assertion that colloidal principles need to be employed
more often in the field of biomaterials is valid, it still does not give a complete picture of
the interactions at the biomaterial surface. The colloidal approach treats the proteins as
particles in solution and although it is extremely powerful in predicting degree of
adsorption in does not explain the chemical interactions between the cell adhesion
receptor proteins and the adhesion proteins at the conditioning film's surface.
The ability of adsorbed proteins to influence cell adhesion depends on the
substrate to which it is adsorbed to [2]. Upon adsorption, proteins undergo
conformational and orientational changes that make the bioactive regions of the protein
be expressed differently. It is shown that a protein can adsorb to two different substrates
at virtually the same rate and amount, yet have different activity depending on which
substrate it is adsorbed to [2]. Therefore, not only a colloidal understanding of the
protein adsorption, but a also a chemical understanding of how the substrate effects the
11
conformation and activity of the protein must be understood as well. The community
needs to understand how the functionality at the surface effects the conformation of the
proteins and then develop guidelines on how to control the conformation by controlling
the functionality. The analytical methods describe above can help correlate the surface
chemistry with the activity of the biofilm macromolecules, since hydrophobicity cannot
explain it alone.
The work of van Oss [14] is an example of relating microscopic properties to the
adhesion of protein to a surface. In the case of H.S.A. (human serum albumin) and glass,
surface tension measurements reveal that a net repulsion exists between the protein and
the glass. This would lead one to believe that the protein would not adsorb to the surface,
but in fact the protein does indeed adsorb to the surface. It was determined that a portion
of the protein, “elbow”, was able to overcome the net repulsion to form a local point of
contact between itself and the glass surface. This point of attachment is via a divalent
metal cation, Ca2+, electron acceptor interacting with an electron donor in the protein.
This was shown by observing desorption with the addition of EDTA, which will complex
with the cation and release the protein. It is believed that very little conformational
changes exist because of the small point of contact. Although glass is not a conventional
polymer surface, this work is a good example of relating microscope interactions with
adsorption events. Below, in fig. 9 [14], is an illustration of the interaction between the
protein and the surface:
One way investigators have tried to avoid the problems of conformational
changes is to use minimal peptide sequences to induce cell adhesion. It has been
determined that the most influential portion of many adhesion proteins is the RGDS
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segment corresponding to arginine-glycine-aspartic acid-serine [2]. This minimal peptide
sequence is thought to be the major contributor in cell adhesion. Modifying a polymer
with this peptide sequence can induce cell adhesion and spreading and has been used
successfully.
One such case is the work of Brandley and Schnaar [7]. This group used the
sequence (tyr-ala-val-gly-arg-gly-asp-ser) which contains the crucial RGD sequence.
They covalently immobilized this polypeptide onto a chemically well-defined
polyacrylamide gel surface. The gel was coated with the peptide at a density of 2 nmol
peptide/cm2 and supported long-term fibroblast growth at a rate and an extent
comparable to that of tissue culture plastic. Although this example displays much
promise, the secondary structure of the entire protein probably has some role in mediating
cell adhesion. The absence of this secondary structure has most likely been a source of
problems for other studies.
13
Biomaterial #1:
Busscher [11, 12] studied the effect of modified flouro-polymers in a cell
adhesion study. The investigator studied the adhesion and spreading on hydrophobized,
hydrophilized FEP-Teflon, untreated FEP-Teflon and tissue grade polystyrene. Using
profilometry, SEM and contact angle measurements, the surface was characterized upon
cell adhesion. Cell spreading increased on hydrophilic FEP, decreased on
superhydrophobic FEP and increased on tissue culture PS relative to the untreated FEP.
Considering this data, an in vivo test was done.
Superhydrophobic ePTFE, (-CF2-CF2-), similar to FEP, was used for vascular
grafts implanted in rabbit carotid arteries. SEM and TEM were used to analyze the
explanted material. The lumen of the modified polymer had a mixed population of
platelets and most importantly the platelets did not cause a regular thrombic cascade
which would result in clotting [1]. Below, in Fig. 10 [12], are SEM images of the lumen
of the modified and unmodified e-PTFE graft:
Image 3 is that of the modified surface and image 4 is that of the unmodified
surface. A large clot can be seen on the unmodified surface and is labeled C. Platelet
adhesion was much more pronounced on the unmodified surface.
14
The SEM of the anastomic side is pictured below in Fig. 11 [12]:
The anastomic side, which was unmodified, had some endothelial overgrowth as
desired. The image also illustrates a smooth transition from the original blood vessel,
labeled BV, and the graft, labeled G. This material was successful on both fronts and is
an illustration of the final step in evaluating a biomaterial with a specific goal [1].
15
Biomaterial #2:
The work of Sipehia [15] illustrates the use of biodegradable polymers in cell
adhesion of human and rabbit endothelial cells. Biodegradable polymers, such as PLLA,
poly(l-lactic acid), PGA, poly(glycolic acid) and PLGA, poly(dl-lactic-co-glycolic acid),
degrade by a hydrolysis mechanism when exposed to water. This author cites the use of
these polymers in vascular grafts. The polymer would be shaped into a vascular graft,
serving as a scaffold for tissue ingrowth, the polymer would then degrade in the presence
of water leaving behind the newly formed tissue. The author studied human endothelial
cell, HUVEC, and rabbit endothelial cell growth, RbMVEC on unmodified and modified
PLLA surfaces.
Four surfaces were used to seed the endothelial cells, unmodified control-PLLA,
ammonia plasma modified PLLA, Fn (fibrinogen)-coated PLLA and ammonia modified
Fn-coated PLLA. The ammonia plasma modifies the surfaces by introducing amino and
amide functionality. Fig. 12 [15] is a chart illustrating the cell growth on these various
surfaces15:
The control PLLA did not support cell spreading, yet when it was coated with fibrinogen
a slight increase in spreading was observed. The most successful surface was that which
was modified with ammonia plasma treatment and then pre-coated with fibrinogen.
16
Similar results were seen with the rabbit cells as shown below in fig. 13 [15]:
This study demonstrates the use of a biodegradable polymer in cell adhesion studies. The
modified surface proved successful in with both types of cells.
17
Conclusion:
There are four aspects that need to be addressed to adequately understand a
biomaterial/cellular interaction[1]:
1. Biomaterial substrate must be well characterized including its chemical
composition, morphology and structure.
2. A firm understanding of macromolecular film conditioning structure,
conformation and interaction with the substrate.
3. Cell processes such as retention, adhesion, differentiation and growth
should be quantitatively measured.
4. Short and long term effects should be evaluated.
The above criteria will pose a large challenge to those in the field. The subject matter is
truly interdisciplinary drawing from the fields of colloidal science, chemistry and cell
biology. It will continue to be, as it has been, an active and challenging area of research.
18
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