Vous êtes sur la page 1sur 8

PERSPECTIVE

PUBLISHED ONLINE: 22 JULY 2011|DOI: 10.1038/NCHEM.1090

Exploring and exploiting chemistry at the


cell surface
Morgan D. Mager, Vanessa LaPointe and Molly M. Stevens*
Engineering the surface chemistry of a material so that it can interface with cells is an extraordinarily demanding task. The
surface of a cell is composed of thousands of different lipids, proteins and carbohydrates, all intricately (and dynamically)
arranged in three dimensions on multiple length scales. This complexity presents both a challenge and an opportunity to chemists working on bioactive interfaces. Here we discuss how some of these challenges can be met with interdisciplinary material
synthesis. We also review the most popular classes of functional molecules grafted on engineered surfaces and explore some
alternatives that may offer greater flexibility and specificity. Finally, we discuss the emerging field of dynamic surfaces capable
of stimulating and responding to cellular activity in real time.

n the aftermath of the Second World War, a physician named


Nicholas Ridley made a surprising observation. Many of the pilots
he treated had shards of poly(methyl methacrylate) (PMMA)
embedded in their eyes from shattered aircraft canopies, but, unlike
most materials, these fragments were not eliciting adverse biochemical responses1. This serendipitous finding led Ridley to conclude
that PMMA must be biocompatible, and today it is used in a variety of medical applications, from contact lenses to bone cements.
In the half-century since this discovery, we have made great progress in materials synthesis and, it is to be hoped, are approaching
a time when we can move past empirical engineering of surfaces
for biological applications. However, the formulation of overarching
design principles is an unmet challenge and many questions remain
unanswered. What does a cell perceive when it lands on a solid support? What cues are cells searching for and how do the components
of the cell membrane contribute to this search? These are not questions of purely academic interest. The formation and evolution of
the interface between cells and artificial materials is of profound
significance for a variety of applications, from routine cell culture
to tissue engineering.
From a systems biology perspective, there is nearly endless
complexity in the crosstalk between cells and dynamic functional
materials. Yet it may be possible to clarify the situation somewhat by
stepping back from biology and treating both the cell and the material surface as chemical systems. Extending this conceptual framework may allow the routine design of surfaces with specific cellular
activity. In the short term, a more modest goal that is already being
realized is the creation of chemically defined surfaces that eliminate
the need for animal-derived substrates and soluble factors. In addition to allowing simpler, better-defined experiments, this capability will significantly diminish the risks associated with the medical
application of engineered tissue and cell-based therapies2.

A physical model of the cell surface

As most cellmaterial interactions are mediated by the cell membrane, it is worth considering which molecules comprise the membrane and how they are arranged. There is enormous diversity among
cell types, especially when considering bacteria, which possess a cell
wall that is exterior to the cell membrane. In this Perspective, we will
focus only on cells from multicellular organisms, such as those that

may be found in human tissue. One of the most significant features


of the cell membrane is the incorporation of membrane proteins,
which can account for over a third of a cells surface area. Figure1a
illustrates the strikingly high protein density 3 (more than 30,000
molecules per square micrometre) of a typical cell membrane,
although this figure can vary significantly with cell type and disease state (Fig.1c). It is primarily through these proteins that cells
interact with surfaces, typically through the creation of attachment
points linking the cytoskeleton (the cells mechanical framework) to
extracellular binding sites.
When a cell begins to adhere to a surface, transient focal complexes are formed through the binding of a family of membrane
proteins called integrins. Given proper conditions, the cell will
flatten and spread on the surface. Eventually, if presented with
the proper ligands, focal complexes mature into focal adhesions,
the main attachment points between cells and two-dimensional
substrates4. This transition requires integrin clustering on tightly
regulated length scales, and stimulating this process in an artificial
system requires spatial as well as chemical specificity. Several studies
have demonstrated a critical spacing of 5070nm (ref. 5), beyond
which cells no longer recognize individual ligands as being clustered. However, it is not clear what happens mechanistically with
a ligand spacing of less than 1015 nm because individual integrins bind only one ligand and steric exclusion would prevent closer
approach of two integrins. These values impose tight limits on the
engineering of surfaces or molecules to encourage cell adhesion.
One simple method of studying ligand spacing is to use selfassembled monolayers (SAMs) with the ligand mixed at varying ratios with an inert filler molecule. Although this is a popular
method6, it can be difficult to control local ligand ordering and presentation precisely. Some of these limitations have been addressed
through the use of star polymer bundles that allow independent
control of bulk properties and ligand clustering. In one study, cells
recognized ligands within polyvalent bundles as being clustered
even though the bundles were spaced hundreds of nanometres
apart. This finding confirms that cells are capable of recognizing local order even on a sparsely functionalized surface. To study
this idea, researchers are using orthogonal synthetic strategies to
shape and functionalize surfaces. For instance, peptide SAMs have
been generated on gold nanoparticles that were then placed in a

Royal School of Mines, Prince Consort Road, Imperial College, London SW7 2AZ.
*e-mail: m.stevens@imperial.ac.uk
582

NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090

Glycocalyx
1 m

Cell membrane

Endothelial cell

HO

O
OH

AcHN

O
HO

COO
O

O3S

O
HO
O3SHN

RBC

Normal

Diseased

HO

COO
O

O
HO
AcHN

60 nm
Desulfated

OSO3

O
HO

20 nm

30 nm

10 nm

Normal

OH

Figure 1 | Representation of cell surface properties. a, Diagram of a 3535nm section of the red blood cell surface showing the lipids (pink), major
proteins (blue) and carbohydrates (orange). Different proteins and carbohydrates are indicated by different shapes and shading. Although there is also
considerable heterogeneity in the lipid component of the cell membrane, this is omitted both for simplicity and because the nanoscale organization is still
a matter of debate. The relative dimensions, concentrations and chemistries of all membrane components can vary drastically depending on cell type,
disease state and life cycle, as shown in bd. b, Relative sizes of the glycocalyx for a red blood cell (RBC; <10nm) and a cell lining the blood vessel wall
(>200nm). c, Relative concentration of HER2 protein (also known as ERBB2) at the surfaces of a normal cell and a cancerous cell. In certain tumour lines,
this protein alone can cover more than 15% of the cell surface area57. d, The carbohydrate coat plays a chemical as well as physical role in cell regulation.
Negatively charged domains resulting from sulfation of disaccharide chains are capable of specific protein interactions. Disrupting these domains with
strong oxidizing agents can drastically alter cell behaviour, even preventing embryonic stem cells from undergoing differentiation. Ac, acetyl. Insert
shows carbohydrate chain based on N-acetylglucosamine (brown) and glucaronic acid (tan) monomers. Green indicates N- (circle), 6-O- (square) or
2-O-sulfation (star).

precise gradient spacing using block copolymer nanolithography7.


In copolymer lithography, phase separation of amphiphilic block
copolymers drives ordering on the 110-nm scale, a range that is
difficult to access with conventional chemistry or top-down nanofabrication. One exciting possibility is that such techniques could
be integrated with recent advances in molecular-level templating in
which bioactive ligands are selectively grafted onto positions in a
preformed molecular backbone such as a cyclic peptide8. The resulting systems could provide a generic platform with defined chemical
features from the ngstrm level to the macroscopic level.
Ordering on the cell surface is also important in the vertical axis,
that is, away from the membrane plane. Many membrane lipids and
proteins are conjugated to polysaccharides, which comprise the glycocalyx, or cell coat, of all cells9. The glycome, or complement of
sugars in the cell, varies among cell types but also within cell types
owing to differences such as stage of differentiation and malignant
transformation10. This variation can in turn affect the dimensions of
the glycocalyx, which can be hundreds of nanometres thick in some
cells11 (Fig.1b). Even given the more typical cell dimension of a few

tens of nanometres, this layer can regulate the approach of the cell
to a solid support. Interference microscopy studies have shown that
for many cell types the membrane is initially held around 50 nm
away from a solid surface12,13. Over several minutes, this spacing
decreases12, allowing proteins in some regions of the cell membrane
to contact the support. Understanding how and why this process
occurs is of critical importance in determining which surfacegrafted ligands will be accessible to the cell, and when.
One way to study the effects of glycocalyx-mediated structuring
at the cell interface is by modifying ligand tethers. In one such investigation14, glycine spacers were systematically added to the end of
an adhesion-promoting peptide and it was found that, as expected,
adding a small spacer increased cell binding. Notably, spacers more
than nine amino acids in length actually decreased cell binding. It
was posited that this effect may be due to the peptide folding over,
underlining the need to consider the mechanical as well as dimensional properties of even a supposedly inert spacer. One study of this
effect looked at polymer brushes with a bimodal length distribution
(22- or 10-mer) where only one of the two chain populations bore

NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

583

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090

bioactive ligands15. Changing the length of the background chain


affected ligand presentation on the active chain (Fig.2b). Similar
studies using advances in grafting-from polymerization will be
an active research area in coming years. For instance, by applying recent techniques to generate gradient polymer brushes16,17,
researchers could create variable tether lengths across a substrate,
probing the vertical as well as planar effects of patterning.
It is tempting to view the glycocalyx in simple, structural terms
but, as usual in biology, it is now becoming clear that it is a multifunctional system. In addition to regulating bulk mechanical contact, the glycocalyx also has specific biochemical functions. The
extended conformation of the glycoproteins is enhanced by the
negative charge density borne by many chains owing to extensive
sulfation. Studies have shown that the distribution of sulfation on
chains is not random but is organized into domains that seem to
control specific binding with proteins18. The nature and degree of
this specificity are still a subject of investigation, but the maintenance of proper sulfation is clearly important for cell behaviour. In
one striking demonstration of this significance, a recent study 19 on
embryonic stem cells showed that chemically desulfated cells lost
the ability to differentiate (Fig.1d).
There is an important lesson for modern chemists in the multifunctionality of cellular structures: creating useful biologically
relevant systems will require continued crossing of disciplines.
Polymer chemistry alone may enable an elegantly structured tissue-engineering scaffold, but the complementary contribution of

peptide chemistry to functionalize that scaffold affords a much


more powerful material. Often the most efficient solutions will pack
multiple functions into a single system, as in the above example
of ligand tethers15. Through clever application of physical chemistry principles, simple tethers can become active components of
the cellmaterial interface. Figure 2 illustrates a few of the successful interdisciplinary investigations made so far; future work
will undoubtedly build on these concepts. Evolution has produced
remarkably elegant systems through the mixing, modification and
re-application of a comparatively limited biochemical toolbox. We
must now strive to do the same with the much greater array of
chemical methods at our disposal.

Applying a range of biochemical interactions

Many cell types will not grow if floating freely, and must be attached
to a surface to function normally. However, some surfaces support cell growth much better than others. One important parameter is the materials wettability, which is determined mostly by the
charge, polarizability and polarity of the surface functional groups.
These factors do not, however, directly mediate cell response. The
effect of surface wettability is largely to alter the type (and state) of
proteins adsorbed from solution20, which in turn affects cell adhesion. Hydrophobic surfaces irreversibly adsorb large quantities of
albumin, an abundant serum protein that does not support cell
attachment. By contrast, moderately hydrophilic surfaces such as
glow-discharge-treated polystyrene (that is, tissue culture plastic)

a
f
b

Peptide chemistry

Polymer chemistry

Crystallography

Physical chemistry

c
Electrochemistry

Biochemistry

HO

O
HO

OH

Figure 2 | Interdisciplinary achievements and possibilities within chemistry for the development of bioactive surfaces. a, Grafting polymer scaffolds
(orange) with synthetic polypeptides (gray) allows for simultaneous control of structural and mechanical properties as well as incorporation of specific
cell-adhesion cues. b, Modifying the length and composition of a molecular tether can qualitatively alter functional presentation. A bimodal brush (shown
here) can increase ordering and promote ligand availability. c, With careful control of intermolecular spacing, it is possible to create a monolayer that
responds to electric fields with an altered conformation and consequently different surface properties. The example shown here undergoes a hydrophilic to
hydrophobic transition when the terminal carboxylic acid is electrostatically driven down onto the substrate. d, Through genetic engineering, it is possible
to introduce enzymes at the cell surface that will modify electroactive monolayers, enabling electronic transduction of biological activity, for instance
by conversion of 4-hydroxyphenyl valerate to quinones. e, The galactose-specific hepatic lectin has enabled carbohydrate-grafted tissue-engineering
scaffolds that promote liver cell growth. Many lectin-binding pockets are preformed, making them possible future targets for rational ligand design. f,
Cyclic RGD (shown here as part of a pentapeptide) is a more effective adhesion promoter than the linear sequence. Structural studies of native ligand
receptor pairs can reveal optimal conformations for synthetic peptides.
584

NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090


tend to adsorb adhesion-promoting proteins such as fibronectin.
It is unsurprising that fibronectin-rich surfaces would allow adhesion because fibronectin is a component of the extracellular matrix
(ECM), the fibrillar material that surrounds and connects cells in
most tissues. Surfaces coated with other ECM proteins, including
laminin and collagen, also promote adhesion and growth of many
cell types.
In light of the importance of ECM proteins, many studies have
focused on engineering protein adsorption at interfaces. Owing
to its simplicity and clinical applicability, this remains a popular
approach and there have been many studies using SAMs to generate
defined surfaces with known functional group identity and density.
Ideally, such surfaces will allow the development of quantitative
models to predict protein (and, thus, cell) response to a given material. At present, however, this challenge has not been met owing
to the complexity and dynamic nature of the processes involved.
Thus, there are still few general design principles21,22. An alternative approach is to synthesize and graft peptides and carbohydrates
onto surfaces to impart specific biofunctionality. Rational design of
surfaces using this concept may be a more immediately tractable
problem, as illustrated by some of successes so far (Fig.3).
The discovery that short peptide sequences can substitute for
whole ECM proteins to induce cell adhesion ushered in a new era
of biointerfaces. The use of short peptides is advantageous because
it provides a less variable system than do naturally derived proteins. Another advantage of synthetic peptides is that it is simple
to introduce a range of functional groups to aid in grafting. Adding
a cysteine, for instance, gives a thiol group that can be used to

create peptide SAMs on gold or, if added at both peptide termini,


to crosslink acrylates by means of a Michael addition. The most
widely used cell adhesion sequence is arginine/glycine/aspartic
acid6 (RGD). The RGD motif is found in many ECM proteins and is
recognized by around one-half of the known integrins. This binding
is extremely sensitive, being completely abolished by the substitution of glutamic acid for aspartic acid, a difference of only one
methylene23. Recognition seems to be conformationally dependent
as the flanking residues, which do not form direct bonds but can
alter the peptide conformation, affect binding affinity 24. Further evidence for conformation dependence is the fact that cyclic versions
are more effective than linear sequences25 (Fig. 2f). RGD-grafted
surfaces are now widely used for cell adhesion, but there remain
some limitations. First, precisely because RGD is so widely recognized, it offers little possibility for cell-specific adhesion. To address
this issue, researchers have created cell-specific surfaces by grafting
peptides from less generic adhesion-promoting proteins24. Another
limitation of RGD grafting is that it may be insufficient to promote
full spreading and focal adhesion formation for some cell types26.
Invivo, complementary receptors must be bound and activated in
addition to integrins27, implying that it may be necessary to consider some of these other binding motifs when designing growthpromoting surfaces.
The syndecans represent an interesting class of complementary
adhesion molecule because they act synergistically with integrins
as co-receptors for ECM proteins and because they demonstrate
the importance of membrane-bound carbohydrates. Each syndecan contains three to five glycosaminoglycan chains composed of
Peptidecarbohydrate

Peptidepeptide
Integrin

Immunoglobulin

Lectin

Syndecan

Cyclic RGD peptide for


generic cell adhesion
and spreading

10-mer NCAM-binding
peptide for neural
cell adhesion

Galactose-grafted
polymers for
hepatocyte culturing

HSBD-containing peptides
to simulate ECM protein
interactions better

Amino-acid sequence
Conformation (cyclization)
Modifications:
Methylation
Phosphorylation

Ligand
variables

Stereochemistry (epimers)
Chain length
Modifications:
Sulfation (domains)
Acetylation

Presentation variables
Material system
Polymer functionalization
Self-assembled monolayer

Tether properties

Co-functionalization

Density/clustering

Length
Flexibility

Synergystic binding
Protein-resistant background

Critical spacing and valence


Active reordering (tethers)

Figure 3 | Molecular diversity of cell adhesion motifs available for surface engineering. The classic ligand-receptor pair of integrins and RGD has been
heavily optimized, for instance by cyclization (upper left) to increase affinity. More recently, alternative peptides have been explored that bind other
classes of molecules such as the immunoglobulin neural cell adhesion molecule (NCAM), potentially offering greater cell-type selectivity. In some cases
(such as syndecans, upper right), synthetic peptides (blue) can bind carbohydrates (orange) rather than the peptide portion of membrane components.
Conversely, some adhesion molecules such as lectins recognise carbohydrates, opening up a new class of surface functionalization possibilities. With this
potential comes a new set of molecular-level variables that can be tailored. However, many of the supramolecular considerations are the same. Regardless
of the type of molecule grafted on a surface, factors such as spacing and tether length will affect cellular response. Note that this is not a comprehensive
list of available classes of adhesion molecules; that subject has previously been covered in more detail58. Similarly, the applications given are intended only
as representative examples and the interested reader should consult more comprehensive reviews59. HSBD, heparan sulfate binding domain.
NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

585

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090

heparan sulfate, and it is through these carbohydrates that they


participate in adhesion. ECM proteins such as fibronectin contain peptide regions, known as heparan sulfate binding domains
(HSBDs), that interact with these chains. Initial studies indicated
a generic electrostatic interaction between the negatively charged
heparan sulfate and positively charged protein domains, but further work has shown that there is actually a more complicated
peptide binding sequence in HSBDs involving both basic and
hydrophobic amino acids28. By grafting HSBD peptide sequences
onto a surface together with integrin-binding domains such as
RGD, researchers have generated systems that bind cells through
multiple pathways, more fully replicating the natural interaction
between cells and the ECM29.
In addition to membrane-bound carbohydrates that bind specific peptide sequences, there are also membrane proteins known
as lectins that bind specific carbohydrates. This binding is sensitive
enough to distinguish between epimers such as glucose and galactose30 and may present an attractive target for rational design as the
recognition site seems to be preformed and not to undergo conformational changes during binding 31 (Fig.2e). Exploiting lectinsugar
interactions increases design flexibility by offering the possibility of
grafting active sugars as well as peptides. One successful exploitation of lectin binding is the creation of galactose-grafted scaffolds
to support hepatocyte (liver cell) growth32,33. It is notable that the
galactose receptor normally functions to bind soluble sugars, not to
mediate adhesion. Thus, it is clear that some molecules not naturally
involved with adhesion may be pressed into that service through
creative engineering. This is not a universal principle, however, and
care must be taken in interpreting results. Most molecules that bind
to a cell surface component (either specifically or nonspecifically)
will allow some degree of adhesion. However, many of these will
not induce subsequent spreading 32, and even some of those that do
result in spreading will not give rise to focal adhesions23,29. At present, there are few reliable rules predicting which ligandreceptor
pairs will yield which behaviour, and each new binding agent must
be experimentally validated for every cell type.
Given the dearth of guiding principles, it can seem a daunting
challenge to create novel cell-adhesive surfaces. However, methods have been developed that do not rely on a-priori functional
knowledge. One of the earliest examples of such a high-throughput
approach was the screening for cell growth capacity of more than
500 acrylate surfaces comprising 24 different monomers mixed in
varying ratios34. This idea was later extended to the screening of
peptide monolayer surfaces and, more recently, to the use of phage
display to screen millions of peptide sequences2. One exciting new
direction is the development of similar approaches using carbohydrate arrays. Printed glycan arrays have been created to identify lectin profiles on cell surfaces35, but this technology has mostly been
used for cell identification, not to screen for carbohydrates capable of supporting cell growth. Recent advances in the solid-phase
synthesis of carbohydrates36 should serve to facilitate this vein of
research by simplifying the creation of polysaccharide libraries analogous to those that exist for peptides.
Another significant development in this area has been the trend
towards adaptable systems for highly parallel quantitative immobilization, because molecular spacing and arrangement can drastically
affect the way cells interact with a surface. Recently, a novel system
giving greater precision than traditional self-assembly schemes was
demonstrated37. The method involves generating an alkanethiol
monolayer with hydroxyquinone termini that can subsequently react
with oxyamine-functionalized ligands to yield an oxime linkage. This
approach affords active control because the hydroxyquinone groups
must be electrochemically oxidized to quinones before they will
react. Furthermore, there is a built-in ability to quantify initial and
reacted groups because the quinone and oxime groups can be separately detected with cyclic voltammetry. Using such generic strategies
586

with an increasingly broad range of adhesion molecules will serve to


accelerate the discovery of underlying principles in cell adhesion.

Adapting to interfacial dynamics

The surface of a living cell is not merely convoluted and hetero


geneous; it is also a site of constant, hectic motion and reordering.
Consider, for example, pinocytosis, or cell drinking, the process
through which cells sample their surroundings by pinching off small
folds in their membrane. This is not a slow and subtle process: some
cells internalize a membrane area equivalent to their entire surface
every thirty minutes38. Cell adhesion and growth can similarly be a
rapid, dynamic process, resulting in physical and chemical changes
to both the cell and the substrate. Here we discuss some examples
of how a cell actively modifies a surface and how materials can be
designed to accommodate these changes.
One of the first modifications that a cell performs after adhering is to pull on surface-bound biomolecules, often reorganizing
them. Such rearrangement is a critical part of natural growth but is
typically not possible with molecules covalently bound to a surface.
This immobility has limitations regarding the transition to active
clustering but can be overcome by incorporating flexible tethers
linking the ligand to the material surface. Even if RGD groups are
grafted farther apart than the critical cluster radius, when they are
attached to a long, flexible tether the cell can pull them together
into an active cluster 33. The critical parameter affecting cell adhesion
in such cases is not the absolute spacing of grafting points. Rather,
adhesion depends on the potential spacing accessible to the ligand
given a sampling volume determined by a random coil conformation of the tether (Fig. 4a). Thus, we again see the importance of
molecular-scale mechanical properties in addition to the known
dependence of cell behaviour on bulk material stiffness39. Future
studies will undoubtedly reveal further cooperative effects of ligand
presentation and substrate mechanics40.
Mechanical forces can sometimes change the chemical nature
of the molecules involved as well as their placement. In vivo, the
tension exerted by cells on the ECM can induce conformational
changes that expose cryptic binding sites41. Recently, scientists have
attempted to use the concept of mechanochemical transduction in
the design of actively switchable surfaces. One study 42 demonstrated
the use of electric fields to modify molecular conformation in a
monolayer. Applying a positive bias to the substrate attracted the
terminal carboxylate groups, burying them within the monolayer
and exposing the methylene backbone of the molecule (Fig.2c). The
resulting transition from a hydrophilic surface to a hydrophobic surface did not involve the participation of specific bioactive moieties,
but the techniques developed may be broadly applicable to a wide
range of chemistries. Specifically, a precise molecular packing was
achieved by incorporating a triphenyl spacer group with a cleavable
ester linkage, and the effects on spacing and conformation were verified throughout with sum frequency generation spectroscopy.
Apart from rearrangement and reordering, cells are also capable of covalently modifying their surroundings through enzymatic
cleavage. This is an important process invivo, as it allows the cell to
remodel the ECM to accommodate growth and migration (Fig.4b).
Originally, such remodelling was seen as an undesirable side effect
in artificial systems because carefully created protein coatings can
be degraded over a few days. This concern was in fact one of the
motivations for grafting peptides instead; short polypeptides are less
susceptible to proteolysis. However, it is also possible to design intentionally degradable materials that take advantage of cell-mediated
proteolysis. Incorporating oligopeptides that are recognized by cellsecreted proteases encourages migration and growth into artificial
substrates such as poly(ethylene glycol) hydrogels for tissue engineering applications43. Another advantage of using such gels is that
it is simple to incorporate integrin adhesion sites in the same material44, more closely mimicking the multifunctional natural ECM.
NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090


a

Natural role

Engineering challenge

Engineering opportunity and molecular realization

Physical
reordering

Proteolytic
cleavage

Protein
secretion
T

Figure 4 | Some of the pitfalls and possibilities associated with cellular microenvironment remodelling. a, Cells naturally pull on the ECM, maintaining a
state of tension (indicated by arrows). On adsorbed protein surfaces, this tension can result in rearrangement, leading to variable properties. But grafting
adhesion molecules (blue) to tethers (red) allows controlled reordering. The ligand initially samples a volume determined by the conformation of the
tether and, on binding, can move a distance determined by the ultimate tether length. b, Most cells secrete proteases that, in vivo, remodel the ECM (grey)
but, in vitro, can cleave grafted adhesion molecules (red/blue). However, this process can be exploited through the creation of specifically degradable
gels (orange) that encourage cell penetration. By crosslinking polymers with synthetic peptides (chains of spheres, arrows indicate point of cleavage),
it is possible to control cell-mediated scaffold degradation. c, Cells naturally secrete proteins to build the ECM, but in vitro the resulting layer (grey) can
cover an engineered surface (orange). With the right surface, however, this protein layer can also be used as a weak link to allow the gentle removal of
adherent cells. By switching the material surface properties, cells can be released (vertical arrows) without the use of harsh chemical agents. One class of
such smart materials is thermoresponsive polymers, that is, polymers which transition between a collapsed state (top) and a hydrated, extended state
(bottom) in a physiologically relevant range of temperatures (T).

Cells can also modify surfaces by synthesizing new proteins that


bind or cover ligands (Fig.4c). In one study of migrating fibroblasts,
the authors found that, rather than disassembling their anchor points,
cells simply broke off more than 80% of the integrins holding them
to the surface, essentially leaving a protein trail behind45. On a traditional material, this would permanently alter the surface, but recent
work on regenerable surfaces offers insight into a possible solution.
Using electrochemical grafting and release techniques, it is possible
to selectively remove a peptide group37 and any attached cells46. For
instance, an O-silyl hydroquinone linkage used to graft peptides can
be oxidized to a benzoquinone, releasing the peptide. A significant
benefit of this approach is that the benzoquinone is itself chemically
active and capable of immobilizing a diene-tagged second ligand
by means of a DielsAlder reaction. This capability opens the door
to reusable surfaces or even real-time reprogramming of biological
functionality. By incorporating photolabile protecting groups, it is
also possible to use lithographic processing to tune ligand density
and cell attachment. Such an approach can even achieve complex
patterns and ligand gradients within a single electrode47.
Direct chemical cleavage and regeneration can mediate shortterm, specific protein attachment but may not withstand sustained
cellular remodelling. In vivo, the ECM is created and renewed by
continual protein synthesis, export and assembly at the cell surface.
In long-term culturing experiments, cells perform the same process,
covering an engineered material and any attached bioactive ligands.
One common approach to minimizing this modification is to cograft the ligands within a background of protein-resistant molecules.
The protein-resistant molecule is typically an uncharged hydrophilic
polymer, most commonly poly(ethylene glycol), although some
work has been done using carbohydrate-based coatings to mimic
the natural protein resistance of the glycocalyx 48. These strategies

can help isolate specific effects temporarily, but evidence suggests


that even this approach may fail eventually, as large quantities of
cell-derived protein accumulate49. Ultimately, a dynamic material
solution may be required.
One class of dynamic material is that of smart surfaces whose
properties (such as wettability) change in response to external
stimuli. Much work in this area has focused on thermoresponsive polymers that transition from a collapsed state to a hydrated,
extended state that is protein resistant (Fig. 4c). When this
alteration occurs, the adsorbed protein layer acts as a weak link
between cells and the surface, gently releasing the cells without
aggressive chemical treatment or proteolytic cleavage50. Poly(Nisopropylacrylamide) is a popular thermoresponsive polymer
owing to its physiologically relevant transition temperature and
relative insensitivity to pH and salt content. However, recent studies have demonstrated some promising alternatives, in particular
based on co-polymers. For example, a co-polymer of di(ethylene
glycol) methacrylate and a 9-mer oligo(ethylene glycol) methacrylate prepared with atom transfer radical polymerization has
many of the same properties as poly(N-isopropylacrylamide)
but with additional resistance to nonspecific adsorption in the
extended state51. Furthermore, co-polymers offer the ability to
tune the transition temperature easily and, potentially, to incorporate a range of bioactive groups. Future studies of rationally
designed monomer blends will be critical to the development of
responsive surfaces. Temperature is just one possible environmental variable, and research is also ongoing into systems that respond
to changes in pH52, illumination53 or other triggers.
Most of the dynamic systems discussed thus far have focused
on controlling surface properties, but there is another aspect to
consider: measuring the effect of those changes. Ultimately, we

NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

587

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090

will need to create interfaces that read and react to cellular outputs
rapidly and specifically. One approach to this challenge is measurement using surface-sensitive optical techniques such as surface
enhanced Raman scattering or sum frequency generation spectroscopy. Alternatively, electrochemical measurements may offer
higher spatial resolution or greater possibility for rapid feedback.
One example54 of this approach was the generation of monolayers of 4-hydroxyphenyl valerate on an electrode and the genetic
engineering of a population of cells to express a membrane-bound
enzyme capable of oxidizing those groups to quinones (Fig. 2d).
By quantifying the quinone density with cyclic voltammetry, it
was thus possible to measure biochemical cellular activity. More
recently, this concept was extended55 by cloning an entire transmembrane electron transport chain into bacteria, creating cells
that were capable of direct electron transfer to inorganic electrodes.
Future advances in functional surfaces will probably increase the
range of transduction possible, perhaps even obviating the need for
genetically modified cells. Regardless, it will remain absolutely necessary to consider dynamic changes initiated by both the cell and
the artificial material.

Summary and outlook

Although we still lack a comprehensive set of rules for designing


biomaterial interfaces, it may be possible to define overarching concepts to be addressed. Research clearly supports the need to match
multiple spatial dimensions when engineering bioactive interfaces. In
addition to the established importance of lateral ligand clustering, it
is becoming increasingly clear that cells can sense signals in the third
dimension, out of the membrane plane. Continuing these investigations by systematically engineering tether length, ligand valency and
multiscale patterning will not only clarify design requirements, but
will also shed light on the spatial mechanisms of cell biology.
Another emerging theme is the need for a diverse and complementary set of binding schemes. Although the use of just a few
peptide sequences has yielded impressive results, there is clearly
room for innovation. Adhesion does not end with integrins, and
the incorporation of ligands for lectins, syndecans and other receptor classes offers possibilities for cell-specific surface design. Future
biomaterials will probably present a range of chemical motifs drawn
from different molecular classes including peptides, carbohydrates
and synthetic polymers. Conformational, mechanical and even
electrochemical properties must all be considered when designing
bioactive surfaces. In addressing this need, researchers will benefit
from crossing traditional boundaries between chemistry disciplines
and sharing knowledge bases from many fields.
Interdisciplinary approaches will be especially critical for designing dynamic systems because there is no such thing as a truly inert
(or even static) surface within a biological environment. Cells simply
have too many methods available to modify surfaces, from mechanical disruption to protein excretion and enzymatic degradation. The
next generation of cell-supporting materials will have to evolve with
or adapt to this rapidly changing microenvironment. An emerging
theme addressing this need is the use of hybrid materials that are
capable of multiple independent cell interactions. As with most of
the potential advances described in this Perspective, it remains to be
seen what balance can be struck between maximizing physiological
relevance and minimizing design complexity 56. We hope that defining both the cell and material systems in the common language of
surface chemistry will aid this optimization.

References

1. Apple, D.J. Nicholas Harold Lloyd Ridley. 10 July 1906 25 May 2001.
Biogr. Mem. Fellows R.Soc. 53, 285307 (2007).
2. Derda, R. et al. High-throughput discovery of synthetic surfaces that support
proliferation of pluripotent cells. J.Am. Chem. Soc. 132, 12891295 (2010).
3. Anstee, D.J. The nature and abundance of human red cell surface
glycoproteins. J.Immunogenet. 17, 219225 (1990).
588

4. Chen, W.T. & Singer, S.J. Immunoelectron microscopic studies of the sites
of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol.
95, 205222 (1982).
5. Arnold, M. et al. Activation of integrin function by nanopatterned adhesive
interfaces. Chemphyschem 5, 383 (2004).
6. Hersel, U., Dahmen, C. & Kessler, H. RGD modified polymers: biomaterials for
stimulated cell adhesion and beyond. Biomaterials 24, 43854415 (2003).
7. Hirschfeld-Warneken, V.C. et al. Cell adhesion and polarisation on
molecularly defined spacing gradient surfaces of cyclic RGDfk peptide patches.
Eur. J.Cell Biol. 87, 743750 (2008).
8. Boturyn, D., Coll, J.L., Garanger, E., Favrot, M.C. & Dumy, P. Template
assembled cyclopeptides as multimeric system for integrin targeting and
endocytosis. J.Am. Chem. Soc. 126, 57305739 (2004).
9. Martins, M.D. F. & Bairos, V.A. Glycocalyx of lung epithelial cells.
Int. Rev. Cytol. 216, 131173 (2002).
10. Tateno, H. et al. A novel strategy for mammalian cell surface glycome profiling
using lectin microarray. Glycobiology 17, 11381146 (2007).
11. Nieuwdorp, M. et al. Measuring endothelial glycocalyx dimensions in humans:
a potential novel tool to monitor vascular vulnerability. J.Appl. Physiol.
104, 845852 (2008).
12. Pierres, A., Benoliel, A.M., Touchard, D. & Bongrand, P. How cells tiptoe on
adhesive surfaces before sticking. Biophys. J. 94, 41144122 (2008).
13. Zeck, G. & Fromherz, P. Repulsion and attraction by extracellular matrix
protein in cell adhesion studied with nerve cells and lipid vesicles on silicon
chips. Langmuir 19, 15801585 (2003).
14. Beer, J.H., Springer, K.T. & Coller, B.S. Immobilized Arg-Gly-Asp
(RGD) peptides of varying lengths as structural probes of the platelet
glycoprotein-IIb/IIIa receptor. Blood 79, 117128 (1992).
15. Kuhlman, W., Taniguchi, I., Griffith, L.G. & Mayes, A.M. Interplay between
PEO tether length and ligand spacing governs cell spreading on RGD-modified
PMMA-g-PEO comb copolymers. Biomacromolecules 8, 32063213 (2007).
16. Harris, B.P. & Metters, A.T. Generation and characterization
of photopolymerized polymer brush gradients. Macromolecules
39, 27642772 (2006).
17. Tomlinson, M.R. & Genzer, J. Formation of grafted macromolecular
assemblies with a gradual variation of molecular weight on solid substrates.
Macromolecules 36, 34493451 (2003).
18. Lamanna, W.C. et al. The heparanomethe enigma of encoding and decoding
heparan sulfate sulfation. J.Biotechnol. 129, 290307 (2007).
19. Lanner, F. et al. Heparan sulfation-dependent fibroblast growth factor signaling
maintains embryonic stem cells primed for differentiation in a heterogeneous
state. Stem Cells 28, 191200 (2009).
20. Curtis, A.S.G. The competitive effects of serum proteins on cell adhesion.
J.Cell. Sci. 71, 1735 (1984).
21. Gray, J.J. The interaction of proteins with solid surfaces.
Curr. Opin. Struct. Biol. 14, 110115 (2004).
22. Stevens, M.M. & George, J.H. Exploring and engineering the cell surface
interface. Science 310, 11351138 (2005).
23. Massia, S.P. & Hubbell, J.A. Covalent surface immobilization of Arg-Gly-Aspcontaining and Tyr-Ile-Gly-Ser-Arg-containing peptides to obtain well-defined
cell-adhesive substrates. Anal. Biochem. 187, 292301 (1990).
24. Pierschbacher, M.D. & Ruoslahti, E. Influence of stereochemistry of
the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion.
J.Biol. Chem. 262, 1729417298 (1987).
25. Kolhar, P., Kotamraju, V.R., Hikita, S.T., Clegg, D.O. & Ruoslahti, E.
Synthetic surfaces for human embryonic stem cell culture. J.Biotechnol.
146, 143146 (2010).
26. Kam, L., Shain, W., Turner, J.N. & Bizios, R. Selective adhesion of astrocytes to
surfaces modified with immobilized peptides. Biomaterials 23, 511515 (2002).
27. Massia, S.P. & Hubbell, J.A. Immobilized amines and basic amino acids as
mimetic heparin-binding domains for cell-surface proteoglycan-mediated
adhesion. J.Biol. Chem. 267, 1013310141 (1992).
28. Cardin, A.D. & Weintraub, H.J.R. Molecular modeling of proteinglycosaminoglycan interactions. Arterioscler. Thromb. Vasc. Biol. 9, 2132 (1989).
29. Rezania, A. & Healy, K.E. Biomimetic peptide surfaces that regulate adhesion,
spreading, cytoskeletal organization, and mineralization of the matrix
deposited by osteoblast-like cells. Biotechnol. Prog. 15, 1932 (1999).
30. Weis, W.I. & Drickamer, K. Structural basis of lectin-carbohydrate recognition.
Annu. Rev. Biochem. 65, 441473 (1996).
31. Sharon, N. & Lis, H. in Molecular Immunology of Complex Carbohydrates 2
(ed. Wu, A. M.) 116 (Springer, 2001).
32. Oka, J.A. & Weigel, P.H. Binding and spreading of hepatocytes on synthetic
galactose culture surfaces occur as distinct and separable threshold responses.
J.Cell. Biol. 103, 10551060 (1986).
33. Griffith, L.G. & Lopina, S. Microdistribution of substratum-bound ligands
affects cell function: hepatocyte spreading on PEO-tethered galactose.
Biomaterials 19, 979986 (1998).
NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

PERSPECTIVE

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1090


34. Anderson, D.G., Levenberg, S. & Langer, R. Nanoliter-scale synthesis
of arrayed biomaterials and application to human embryonic stem cells.
Nature Biotechnol. 22, 863866 (2004).
35. Blixt, O. et al. Printed covalent glycan array for ligand profiling of diverse
glycan binding proteins. Proc. Natl Acad. Sci. USA 101, 1703317038 (2004).
36. Plante, O.J., Palmacci, E.R. & Seeberger, P.H. Automated solid-phase synthesis
of oligosaccharides. Science 23, 15231527 (2001).
37. Luo, W., Chan, E.W.L. & Yousaf, M.N. Tailored electroactive and
quantitative ligand density microarrays applied to stem cell differentiation.
J.Am. Chem. Soc. 132, 26142621 (2010).
38. Steinman, R.M., Brodie, S.E. & Cohn, A.Z. Membrane flow during
pinocytosis: a stereologic analysis. J.Cell. Biol. 68, 665687 (1976).
39. Discher, D.E., Janmey, P. & Wang, Y-l. Tissue cells feel and respond to the
stiffness of their substrate. Science 310, 11391143 (2005).
40. Deeg, J. et al. Impact of local versus global ligand density on cellular adhesion.
Nano Lett. 11, 14691476 (2011).
41. Zhong, C. et al. Rho-mediated contractility exposes a cryptic site
in fibronectin and induces fibronectin matrix assembly. J.Cell. Biol.
141, 539551 (1998).
42. Lahann, J. et al. A reversibly switching surface. Science 299, 371374 (2003).
43. Raeber, G.P., Lutolf, M.P. & Hubbell, J.A. Molecularly engineered PEG
hydrogels: a novel model system for proteolytically mediated cell migration.
Biophys. J. 89, 13741388 (2005).
44. Lutolf, M.P. et al. Synthetic matrix metalloproteinase-sensitive hydrogels for
the conduction of tissue regeneration: engineering cell-invasion characteristics.
Proc. Natl Acad. Sci. USA 100, 54135418 (2003).
45. Palecek, S.P., Schmidt, C.E., Lauffenburger, D.A. & Horwitz, A.F.
Integrin dynamics on the tail region of migrating fibroblasts. J.Cell. Sci.
109, 941952 (1996).
46. Yeo, W.S., Yousaf, M.N. & Mrksich, M. Dynamic interfaces between cells
and surfaces: electroactive substrates that sequentially release and attach cells.
J.Am. Chem. Soc. 125, 1499414995 (2003).
47. Lee, E-J., Chan, E.W.L. & Yousaf, M.N. Spatio-temporal control of cell
coculture interactions on surfaces. ChemBioChem 10, 16481653 (2009).
48. Holland, N.B., Qiu, Y., Ruegsegger, M. & Marchant, R.E. Biomimetic
engineering of non-adhesive glycocalyx-like surfaces using oligosaccharide
surfactant polymers. Nature 392, 799801 (1998).

49. Koo, L.Y., Irvine, D.J., Mayes, A.M., Lauffenburger, D.A. & Griffith, L.G.
Co-regulation of cell adhesion by nanoscale RGD organization and mechanical
stimulus. J.Cell. Sci. 115, 14231433 (2002).
50. Wischerhoff, E. et al. Controlled cell adhesion on PEG-based switchable
surfaces. Angew. Chem. Int. Ed. 47, 56665668 (2008).
51. Lutz, J.F., Akdemir, O. & Hoth, A. Point by point comparison of two
thermosensitive polymers exhibiting a similar LCST: is the age of
poly(NIPAm) over? J.Am. Chem. Soc. 128, 1304613047 (2006).
52. Yu, X., Wang, Z.Q., Jiang, Y.G., Shi, F. & Zhang, X. Reversible pH-responsive
surface: from superhydrophobicity to superhydrophilicity. Adv. Mater.
17, 12891293 (2005).
53. Renner, C. & Moroder, L. Azobenzene as conformational switch in model
peptides. ChemBioChem 7, 868878 (2006).
54. Collier, J.H. & Mrksich, M. Engineering a biospecific communication
pathway between cells and electrodes. Proc. Natl Acad. Sci. USA
103, 20212025 (2006).
55. Jensen, H.M. et al. Engineering of a synthetic electron conduit in living cells.
Proc. Natl Acad. Sci. USA 107, 1921319218 (2010).
56. Place, E.S., Evans, N.D. & Stevens, M.M. Complexity in biomaterials for tissue
engineering. Nature Mater. 8, 457470 (2009).
57. Aguilar, Z. et al. Biologic effects of heregulin/neu differentiation factor on
normal and malignant human breast and ovarian epithelial cells. Oncogene
18, 60506062 (1999).
58. Juliano, R.L. Signal transduction by cell adhesion receptors and the
cytoskeleton: functions of integrins, cadherins, selectins and immunoglobulinsuperfamily members. Annu. Rev. Pharmacol. Toxicol. 42, 283323 (2002).
59. Shin, H., Jo, S. & Mikos, A.G. Biomimetic materials for tissue engineering.
Biomaterials 24, 43534364 (2003).

Acknowledgements

We thank J. Weaver for constructive reading of this manuscript. M.M.S. thanks ERC
starting investigator grant Naturale for funding.

Additional information

The authors declare no competing financial interests. Reprints and permissions


information is available online at http://www.nature.com/reprints. Correspondence
should be addressed to M.M.S.

NATURE CHEMISTRY | VOL 3 | AUGUST 2011 | www.nature.com/naturechemistry

2011 Macmillan Publishers Limited. All rights reserved

589

Vous aimerez peut-être aussi