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Acta mater.

48 (2000) 263277


Massachusetts Institute of Technology, Department of Chemical Engineering and Division of
Bioengineering & Environmental Health, Room 66-466, 77 Massachusetts Ave., Cambridge,
MA 02139, USA
(Received 1 June 1999; accepted 15 July 1999)
AbstractThe revolution in the basic science of molecular cell biology, combined with advances in polymer science and engineering, is pushing the eld of biomaterials into new applications and an era of molecular design. New classes of degradable polymers and hydrogels are being developed and teamed with
molecular ligands to control specic cell behaviors. This paper reviews the basic science and technology for
applications in drug delivery and tissue engineering. # 2000 Published by Elsevier Science Ltd on behalf of
Acta Metallurgica Inc. All rights reserved.
Keywords: Polymers; Degradation; Tissue engineering; Drug delivery; Biomaterials


The term biomaterials has alternately been used to

describe materials derived from biological sources
or to describe materials used for therapies in the
human body. The focus of this paper is biomedical
applications of polymers, with an emphasis on
emerging new applications driving an expansion of
the polymeric biomaterials eld.
The use of polymers in medicine dates back
almost to the birth of the eld of polymer science;
virtually every early synthetic polymer found its
way into experimental surgical studies soon after its
invention and many endured to become staples of
clinical practice. Nylon sutures were reported in the
early 1940s and review articles on the use in surgery
of polymers such as nylon, poly(methylmethacrylate) (PMMA), Dacron polyester, and polyvinyl
chloride began appearing in prominent medical
journals in the mid-1940s [1, 2]. These original
materials have been joined by additional bulk commodity polymers (Teon, high-density polypropylene, polyurethanes) likewise adapted for use in
surgery after their initial uses for other purposes.
These polymers remain important in clinical
medicine as essential components of permanent
prosthetic devices including hip implants, articial
lenses, large diameter vascular grafts, catheters, etc.,

The Millennium Special Issue A Selection of Major

Topics in Materials Science and Engineering: Current
status and future directions, edited by S. Suresh.

and research continues to optimize the stability and

performance of these materials in vivo.
Whereas the original uses of polymers in surgery
centered primarily on replacements for connective
tissues, a host of new applications are emerging as
a result of major advances in the sciences of molecular cell biology and developmental biology. An
array of new protein and nucleic acid-based drugs,
which cannot be taken in classical pill form, are
providing impetus for new implantable polymers
for controlled drug delivery and gene therapy.
Applications in the relatively new eld of tissue engineering, where polymers are used to assist regeneration of three-dimensional tissue and organ
structures, are more nascent and are more integrated with biological demands. This paper begins
with background on fundamental aspects of biology
and polymer science relevant for these new areas of
polymeric materials. Specic approaches and challenges in drug delivery and tissue engineering are
then described.


2.1. Molecular cell biology

For roughly the past three or four decades, biology has been undergoing a revolution toward a
quantitative mechanistic understanding of cell and
organism behavior, driven by tremendous advances
in the tools available to identify and systematically
manipulate the molecular components within cells
and their environments. This revolution is leading

1359-6454/00/$20.00 # 2000 Published by Elsevier Science Ltd on behalf of Acta Metallurgica Inc. All rights reserved.
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Fig. 1. Schematic of receptor-mediated cell interactions with the external environment. Transmembrane
receptors bind ligands in the extracellular domain, and transmit signals to the interior of the cell via the
cytoplasmic domain inside the cell membrane. The receptor cytoplasmic domain may be an enzyme
itself which is activated on ligand binding, or it may increase its anity for molecules which become
active enzymes upon binding to the receptor. The boundaries between ``soluble ligands'' and ``physical
ligands'' are not strict, as some growth factors are found primarily in matrix-bound form.

to an industrialization of biological processes much

like a comparable mechanistic revolution in chemistry in the late 1800s led to broad industrialization
of chemical processes. With respect to biomaterials,
it is stimulating new ways to synthesize biomaterials, new ways to analyze the human body's response to materials, and rational design of
materials which interact with specic molecular
components in the body.
An overview of many of the basic molecular
players and interactions in regulation of mammalian cell functions relevant to biomaterials is shown
in Fig. 1. Cells interact with the external environment via transmembrane proteins, which transmit
both information and m. molecules from the outside of the cell to the inside. Many of these transmembrane proteins are receptors, characterized by
an extracellular ligand-binding domain and an intracellular signaling/regulatory domain. When a
receptor binds its cognate ligand (typically a protein
or peptide), a change in receptor conformation or
anity for other cellular molecules initiates an intracellular cascade of enzyme-mediated reactions
resulting in amplication of the signala process
termed ``signal transduction''. Signal transduction
ultimately aects gene regulation and may result in
translocation of the receptor from the cell surface

to intracellular compartments. Dozens of dierent

types of receptors are present on the average mammalian cell, each with the capacity to initiate unique
or overlapping signal transduction networks [3, 4].
Hence, cell functions including survival, proliferation, migration, and dierentiation are governed
by the integrated signals from many molecules, and
these processes are highly dynamic [4]. Many of the
specic molecular players are yet to be identied
denitively, but the wide array of molecules now in
hand provides the basis for manipulation of cell,
tissue, and organism behavior.
Two broad classes of extracellular ligands relevant for biomaterials are growth factors and extracellular matrix. Growth factors are typically
characterized as relatively soluble (diusible) peptide or proteins of molecular weight 500050,000
[5]. Growth factors acting on a given cell may be
produced by the same cell (autocrine stimulation),
by neighboring cells in contact (juxtacrine stimulation), by nearby but non-contacting cells (paracrine stimulation) or by cells in a distant tissue
(endocrine stimulation). Growth factor molecules
and their receptors are often internalized and
degraded by the cell following ligandreceptor binding, thus attenuating the response [3]. These molecules play a role in normal tissue homeostasis [e.g.


nerve growth factor (NGF) is required for proper

maintenance of nerve cell function]; in normal healing [e.g. bone morphogenetic protein (BMP) sends
signals to bone precursor cells to migrate to the
fracture site, proliferate, and form bone]; as well as
in pathological situations such as inammation and
cancer [e.g. overexpression of epidermal growth factor (EGF) is implicated in many cases of breast
cancer]. Although hundreds of such growth factors
have been identied, relatively few have been developed for clinical application, a situation likely arising from the combined eects of gaps in biological
understanding and lack of appropriate technologies
to deliver growth factors in physiologically relevant
The second broad class of extracellular molecules
which regulate cell behavior via receptor-mediated
pathways are the relatively insoluble ligands present
in extracellular matrix [3, 6]. Extracellular matrix
(ECM) is the gel of proteins, proteoglycans, and
charged polysaccharides which provides a physical
scaolding for cells and tissues, helps provide a permeability barrier between tissue compartments, and
enables polarization of tissue structures such as
skin. ECM molecules are typically quite large
Mw > 100,000 and contain several functional
domains or regions, including those involved in
receptor-mediated cell adhesion, and regions which
interact with other ECM molecules or growth factors. Of key interest to biomaterials scientists, molecular cell biology techniques have been used to
parse the structure/function properties of many
ECM molecules and map receptor-binding functions to small (320 amino acid) domains which are
conserved among many dierent proteins. The
prototypical adhesion domain is the tripeptide
arginineglycineaspartate (RGD, Mw 0400). The
RGD sequence was rst identied as a minimal
sequence required to mediate cell adhesion to the
ECM molecule bronectin Mw 1450,000 [7], and
has since been found to be present in and mediate
cell adhesion to a wide array of ECM molecules [8].
Following the discovery of RGD, many such small
adhesion-mediating peptide domains have been
identied and characterized within ECM molecules.
These peptides interact with a class of cell surface
adhesion receptors called integrins [810]. A functional integrin receptor comprises two subunit
chains, an alpha chain and a beta chain, drawn
from a family of 16 known alpha and eight beta
members [11], thus generating great diversity in the
specicity of receptorligand interactions. Like
growth factor receptors, integrins mediate many
aspects of cell behavior. Hence, manipulation of
integrin ligation by including peptide adhesion
domains in synthetic biomaterials is an area of
intense research and development in current biomaterials, both for improving function in existing applications such as vascular grafts as well as new
applications in tissue engineering.


2.2. Degradable materials

Permanent implants into tissue almost always elicit a chronic inammation called a foreign body response [1214]. This response is characterized by
formation of a poorly vascularized brous layer
analogous to a scar at the materialtissue interface.
The foreign body response is typically benign and
sometimes desirable to anchor devices into host tissue, but leads to enough clinical complications (e.g.
infection, tissue contraction) that it is considered a
risk to be avoided if possible in many applications.
For example, in controlled drug delivery, it is undesirable to leave an expired polymer device in the
patient permanently.
Emerging applications in tissue engineering and
drug delivery thus rely primarily on materials that
resorb or degrade in body uids so that the device
ultimately disappears with no ill eects. Degradable
polymers undergo extensive chain scission to form
small soluble oligomers or monomers. Degradation
may proceed by a biologically active process (e.g.
enzymes present in body uids participate) or by
passive hydrolytic cleavage. The term ``biodegradable'' typically refers to materials in which active
biological processes are involved. Resorbable polymers gradually dissolve and are eliminated through
the kidneys or other means. A wide variety of both
solid and hydrogel-type polymers have been developed and many serve dual applications in drug
delivery and tissue engineering.
2.2.1. Degradable polyesters. Synthetic degradable
polyesters were adopted in surgery 30 years ago as
materials for sutures and bone xation devices [15]
and remain among the most widely used synthetic
degradable polymers. Degradable polyesters derived
from three monomerslactide, glycolide, and
caprolactoneare in common clinical use and are
characterized by degradation times ranging from
days to years depending on formulation and initial
Mw. Lactic acid is a chiral molecule, existing in L
and D isomers (the L isomer is the biological metabolite), and thus ``polylactic acid'' actually refers to a
family of polymers: pure poly-L-lactic acid (L-PLA),
pure poly-D-lactic acid (D-PLA), and poly-D,L-lactic
acid (DL-PLA). Homopolymers of L-PLA and polycaprolactone (PCL) are used clinically. The range
of materials properties has been greatly extended,
however, by co-polymerization of the various
monomers. With the exception of polyglycolide
(PGA), the polymers in this family are soluble in
many common organic solvents and thus can be
processed by a variety of thermal and solvent-based
All polymers in this family are insoluble in water
but degrade by hydrolytic attack of the ester bond.
The mechanical and degradation properties are
aected by the combined eects of the crystallinity,
the molecular weight (Mw), the glass transition tem-



perature (Tg), and the monomer hydrophobicity

[1622]. With the exception of DL-PLA, all the
homopolymers can be crystallized; melting temperatures are > 1608C for PGA and L- or D-PLA and
about 608C for PCL [18, 21, 23, 24]. Co-polymerization reduces the crystallinity and most copolymers
are completely amorphous. Values of Tg for the
homo polymers range from well above body temperature (L-PLA0 558C) to just below body temperature
PCL0 608C); the most commonly used copolymers exhibit Tg > 378C: The dierences in degradation rates of dierent polymer compositions have
been attributed to dierences in the access of water
to the ester bond rather than to dierences in the
intrinsic rates of ester cleavage [20]. Water access to
the ester bonds is governed by the hydrophobicity
of the monomers [20], the crystallinity of the sample
[25], and the bulk sample dimensions [26]. Because
water rapidly plasticizes these polymers, degradation proceeds throughout the entire device simultaneously, often ultimately leading to mechanical
distortion, cracking, pitting, and ssure of the
device in uncontrolled ways [19].
Degradation is characterized in many waysloss
of Mw, loss of mass, or loss of mechanical
strengthand this can cause confusion when literature values of degradation for dierent polymer formulations are compared. The polymers degrade by
random chain scission. Since only the monomer
degradation products (lactic acid, glycolic acid, or
hexanoic acid) are soluble, a very large reduction in
both Mw and mechanical strength typically occurs
before a decrease in the weight of the sample is
observed. The crystallinity of L-PLA samples has
been observed to increase during degradation and
particles of highly crystalline material can be found
in vivo long after the bulk of the implant has
disintegrated [19].
The acidic degradation products of the classical
polyesters PLA, PGA, PCL, and their copolymers,
have been implicated in adverse tissue reactions,
particularly in bony sites [27, 28]. This, combined
with their somewhat limited range of physical properties, has led to synthesis of new block copolymers
with polyurethane linkages, allowing a far greater
range of mechanical properties to be achieved [29,
30]. These multiblock copolymers have crystallizable hard segments of PHB and non-crystallizing
oligoesters (adipic acid, ethylene glycol, 1,4-butanediol, and diol-terminated PCL) as soft segments.
Other degradable polyesters are also being developed for clinical applications, notably poly(hydroxybutyrate) (PHB) and copolymers of hydroxybutyrate with hydroxyvalerate [18]. These polymers
are produced by biological processes (typically in
micro-organisms) rather than classical synthesis
techniques and exhibit complex degradation patterns. Pure PHB is thermoplastic with properties
comparable to polypropylene [18], but its properties

can be modied by blending [31] or inclusion of

other monomers [32].
2.2.2. Other degradable polymers. A host of new
polymers have been developed specically for surgical applications, particularly drug delivery.
Polyanhydrides comprise monomers joined by hydrolytically labile anhydride bonds that yield two
carboxylic acids upon hydrolysis [33]. The prototype polyanhydride for medical applications is a
copolymer of 1,3-bis ( p-carboxyphenoxy) propane
and sebacic acid, a formulation which is used clinically for drug delivery [34, 35]. Polyanhydrides were
rst considered in the 1930s for commercial applications in textiles but were too unstable and were
not further developed until applications in medicine
for degradable polymers became compelling [33].
The anhydride bond is extremely hydrolytically unstable compared with esters, amides, and ethers.
Thus, degradation of polyanhydride devices is governed by access of water to the bonds and can be
controlled by the hydrophobicity of the monomers.
This property has been exploited to create polymers
that enable devices to degrade via surface erosion
like a bar of soaprather than by bulk erosion,
providing greater control over device properties.
Extensive engineering of copolymer anhydrides has
yielded formulations with a range of mechanical
properties, degradation times, and chemical reactivities [33, 36]. Polyanhydrides are currently under
clinical development for delivery of the anticancer
agent BCNU in treatment of brain tumors [35].
Poly(orthoesters), like poly(anhydrides), were
developed to address the issue of surface erosion to
improve the release of drugs from erodible matrices
[16, 37] and have been extensively developed for applications in drug delivery [3840]. One formulation
of polymers in this family produces acidic degradation products to yield an autocatalytic degradation of devices [41, 42] and an alternate
formulation is based on dialcohols which do not
exhibit autocatalysis [16]. These polymers have suitable mechanical and processing properties for drug
delivery devices but their role in tissue engineering
is less clear.
Pseudo-poly(amino acids) are a class of polymers
based on natural amino acids linked by nonamide
bonds including ester, iminocarbonate, urethane,
and carbonate [4347]. They oer improved mechanical properties, processing, stability, and ease of
synthesis over poly(amino acids) joined by traditional peptide bonds. The range of monomers and
bonds has enabled development of a combinatorial
approach to obtaining polymers with desired properties such as Tg, crystallinity, and hydrophobicity
[48, 49]. Among the many polymers in this family,
poly(tyrosine carbonates) show promise in orthopedic applications due to improved behavior of degradation products in bony sites [47].


2.2.3. Hydrogels. The move toward minimally

invasive procedures in all areas of surgery is driving
development of gels that can be formed in situ following injection of macromer components. Gelation
of precursors in the presence of cells or tissues may
be accomplished by a physicochemical reaction (addition of a divalent cation, change in temperature)
[5057], photochemical reaction (with addition of
an initiator) [5860], chemical reaction [61, 62], or
enzymatic reaction [63]. As with other areas of biomaterials, approaches range from adaption of existing materials to molecular design of new materials
[64]. For example, alginate, a seaweed-derived
charged polysaccharide which forms a gel in the
presence of supraphysiological concentrations of
calcium ions, is used widely in food products and
entered tissue engineering as an experimental material for encapsulating endocrine cells, such as
insulin-secreting pancreatic islets, for transplantation by injection [50, 51, 65]. Specic gel systems
are described in more detail in the following sections.

The eld of controlled drug delivery drives much

current innovation in biomaterials. Controlled drug
delivery applications include both sustained (over
days/weeks/months/years) delivery and targeted
(e.g. to a tumor, diseased blood vessel, etc.) delivery
on a one-time or sustained basis. Although the eld
of controlled drug delivery encompasses a wide
range of drugs, two key factors which stimulated
the emergence of the eld are the rise of proteins as
potential pharmaceuticals and the demonstration
that sustained release of active proteinsmacromolecules rather than small polymer-permeable
drugsfrom polymer matrices was possible.
Before the 1980s, protein-based drugs such as
insulin and growth hormone were produced by
extraction from tissues and few such drugs were in
wide clinical use. With the advent of molecular biology, once a protein was isolated and its sequence
determined, a gene encoding that protein could be
made synthetically and introduced into cells to
drive production of the desired protein in a relatively controlled fashion. In addition to advantages
in yield and safety, production of drugs by molecular biology-based techniques aords the possibility
of changing the molecular structure of the drug in a
controlled way via targeted changes in the amino
acid sequence encoded by the gene, to improve the
ecacy of the drug or provide enhanced patent protection. These advances have led to a great expansion in the identication and development of
protein and peptide therapeutics, ranging from antibodies for cancer treatment, to growth factors for
stimulation of blood cell proliferation, to enzymes
for control of cystic brosis symptoms [66].
Delivery issues still encumber clinical potential for


many such drugs because proteins must generally

be injected or infused and must often be delivered
to a specic site [66, 67].
Perhaps the strongest motivation for polymeric
delivery systems is that many of the more recently
discovered molecules require delivery at a very localized level due to their mechanism of action. For
example, the molecule vascular endothelial cell
growth factor (VEGF) is a cell-secreted protein that
induces the local formation of blood vessels (angiogenesis) [68]. Possible clinical uses of VEGF include
stimulation of blood vessel growth in patients with
such poor circulation in the legs they would ultimately require amputation, and stimulation of new
vessel growth in the heart where arteries are
clogged. A major challenge to clinical development
of VEGF and other growth factors is delivering
these fragile molecules directly to the sites they are
needed at the proper dose for the required amount
of time.
In the future, controlled protein delivery applications may yield in clinical importance to nucleic
acid delivery applications if gene therapy fullls its
promise. An alternative to delivering VEGF, for
example, is to deliver the VEGF gene to the site in
a manner it is transiently expressed. Trials using
this approach are underway [69]. The VEGF
example is somewhat unusual in that expression of
the gene is desired for a brief time, enough to
induce vessel formation. Most envisioned applications of gene therapy, such as curing cystic brosis or hemophilia, involve permanent modication
of gene expression. Clinical gene therapy trials,
which have been based primarily on using viral vectors to deliver the genes either in vitro or in vivo,
have been less successful than anticipated [70].
While many of the factors contributing to failure
are biological, the development of synthetic (nonviral) polymer conjugates is of emerging commercial
interest and is playing a role in illuminating the biological barriers to gene delivery [71].

3.1. Polymers and polymer systems for drug delivery

Controlled delivery devices are generally diusion-based release systems applicable to release of
drugs intended for the systemic circulation or for a
localized site. The classic polymeric drug delivery
system is the implantable contraceptive Norplant2,
which comprises a silicone rubber (polydimethylsiloxane) tube lled with a steroid dispersion. Drug
release is controlled by the permeability of the steroid in the tube wall and remains fairly constant
over several years after an initial transient. Few
drugs, though, are amenable to release by a
Norplant-like system and this drives development
of new polymers and release modes.
Langer demonstrated that even though proteins
are not permeable in solid polymers, implantable



devices could readily be used for controlled release

of macromolecules by incorporating small (0mm)
particles into a polymer matrix to form a network
of connected (touching) particles [72]. Drug in particles at the surface of the device becomes solubilized and diuses out of the matrix, leaving open
pores for drug deeper in the matrix to diuse out
when it is subsequently solubilized to enable sustained release over hours to days to weeks. The
prototype devices were fabricated from non-degradable materials including ethylene vinyl acetate
(EVA), a rubbery polymer at body temperatures, by
a solvent-based technique which allowed uniform
dispersion of the particles in the matrix.
The basic approach developed by Langer provided the foundation of a wide variety of implantable and injectible devices made primarily from
degradable materials [7375]. Key technical hurdles
in adapting this approach to clinical products
include controlling the release prole (i.e. achieving
``controlled'' as compared to just ``sustained''
release), stability of drugs within the device before
and following implantation, and producing delivery
congurations suitable for specic applications.
Control of the release prole is relatively straightforward in the case of Norplant-like systems as
solid drug is contained in a reservoir, maintaining a
constant equilibrium concentration of drug on one
side of a barrier. In diusion-based macromolecular
release systems, the diusion path length changes as
drug leaves the device, resulting in a diminishing
ux for slab or wafer-type devices. In theory and in
experimental devices, this problem can be addressed
by altering the device geometry. An alternative
approach is to employ relatively low drug loadings
and control release via degradation of the polymer
using surface eroding polymers, provided polymers
with appropriate processing properties are available
in the desired degradation range.
The choice of polymer is additionally constrained
by the potential for drugpolymer interactions, particularly for protein-based drugs. Water penetration
into devices following implantation hydrates drug
contained within the matrix, potentially enabling
adverse chemical reactions among the protein molecules or between protein and polymer and leading
to drug deactivation [76, 77]. Protein structure can
in some cases be altered to minimize occurrence of
amino acids susceptible to reaction without altering
the bioactivity of the protein, thus improving stability. Changes in the structure of a protein may lead
to subtle changes in other aspects of the protein
behavior in humans, and thus changes in the structure of a product already approved by the Food
and Drug Administration (FDA) generally require
a new clinical trial be conducted on the ``new'' molecule. The practical outcome of such competing
issues for development of protein-based pharmaceuticals is that the delivery mode must be identied
very early in the drug development process, so that

clinical trials are conducted with the appropriate

nal product.
Surgical implantation of devices is suitable for
applications such as implantation of BCNU-releasing wafers following removal of a brain tumor, but
surgery for the sake of device implantation alone is
not desirable compared to injection or other means
of minimally invasive delivery. Microspheres are a
clinically successful adaptation of the concept of
drug particles dispersed in a matrix to injectible format. Formation of microspherical drugpolymer
particles, with dimensions typically 00:1 mm, can
be accomplished by a variety of approaches [7883].
One of the simplest means is to dissolve ne
< 0:01 mm drug powder in a solution of polymer
in a volatile solvent such as methylene chloride,
form an emulsion of the drug/polymer solution in
an aqueous solution containing a stabilizer, and
maintain the emulsion until the solvent ultimately
evaporates, leaving solid microspheres. Microspheres may also be formed by spray-drying, freezing, and other techniques. A variation of this
approach is used to make the clinical product
Lupron Depot, a polylactide-co-glycolide microsphere formulation which releases gonadotropin
releasing hormone analog (GNRHa) for treatment
of prostate cancer and endometriosis [79], and
approaches for release of human growth hormone
are in development [82]. Although release from
these microspheres is sustained and reproducible,
the release prole is not well controlled, and injectible microspheres have found greatest application
for drugs with a large therapeutic window.
Microsphere delivery has recently been adapted for
delivery via the lung, using a combination of theoretical models of lung transport as a function of particle size and density and experimental verication
of these models to predict the microsphere properties required to reach the deep lungs via inhalation
The unique needs of protein and macromolecule
delivery have stimulated additional approaches to
controlled delivery. In applications such as delivery
of a protein growth factor or a gene to the wall of
a blood vessel, for example, it is desirable to have a
``device'' which conforms to the local anatomy and
is relatively inert [85]. Hydrogels formed in situ by
photopolymerization or other means provide an
approach which meets these constraints. Hydrogels
are also of growing interest as ``smart'' delivery systems which can respond to the local environment to
deliver drug [8689], for example by a change in
pH induced by an enzymatic reaction which occurs
only in the presence of a substrate, such as glucose,
which must be regulated by the release of the drug,
such as insulin. The ecacy of localized delivery of
protein-based drugs such as growth factors may
also be aected by the distance the drug can diuse
from the release site into tissue. Saltzman and coworkers have shown by a combination of math-


ematical modeling and experimentation that the

release of nerve growth factor in brain tissue results
in a steep concentration prole localized within a
few hundred micrometers of the device [90, 91], but
that penetration depths might be improved by changing the diusion coecient of the drug using polymer conjugation.
Many of the technical hurdles to protein delivery
are being overcome, particularly as new drugs coming down the pipeline are developed in concert with
delivery systems. The next frontier is delivery of
nucleic acidsgenes and antisense molecules. This
frontier presents an entirely new set of scientic and
technical issues, particularly when delivery is to be
targeted to a single tissue or cell type in the body
and long-term stable expression is desired. The
most ecient vectors for gene delivery are viruses,
which evolved over millions of years for this specic
purpose, and viral vectors have indeed been the
mainstay of genetic therapy research and development. Immunological reaction to and low specicity
of viral vectors continue to be issues in clinical realization of gene therapy approaches [70] and interest is growing in synthetic polymer conjugates for
gene delivery [71].
The essential components in a gene delivery vector for targeted administration are stable binding to
DNA, specic binding at the cell surface, internalization of the bound vector, tracking through cell
organelles to reach the nucleus, and nally, expression. For the DNA-binding component of the
vector, o-the-shelf positively charged polymers
such as polylysine and polyethyleneimine are currently most commonly used to complex negatively
charged DNA in a stable conguration. These polymers are potentially toxic and immunogenic, and
are not optimized to release the DNA at the most
appropriate site inside the cell; hence, new polymers
are now being developed to meet these specic criteria and include non-toxic proton sponges [92] and
cyclodextrins [93]. The molecular recognition component is generally a ligand for an internalizable
cell surface receptor such as the epidermal growth
factor receptor [94]. From the polymer science perspective, key issues in design include steric accessibility of the ligand for the receptor and valency,
which aects the binding [94].

The eld of tissue engineering has emerged over

the past decade, driven by a diverse range of clinical
needs for replacement of diseased or damaged tissue
and for delivering genetically engineered cells to
patients [95, 96]. Although many articial prosthetic
devices are available to replace connective tissues
such as joints, heart valves, blood vessels, and
breasts, few synthetic devices are able to perform
adequately over the lifetime of the patient and
devices vary greatly in their abilities to completely


replace all the functions of the native tissue. No

long-term replacements are available for some connective tissues, including heart, small diameter
blood vessels, and skin. The clinical needs are even
more dire in the case of organs such as liver, which
can now be replaced only by organ transplantation.
The goal of tissue engineers is to meet these clinical needs by creating living, three-dimensional tissues and organs using cells obtained from readily
available sources such as biopsy of the patient's
own (autologous) tissue or foreign tissue which
would be discarded after surgical procedures such
as circumcision. In many cases, the approach is to
break the donor material down to the level of individual cells, and then to coax the isolated cells into
forming a tissue structure of the appropriate size
and/or shape using a physical scaold to organize
cells on a macrosopic scale, and provide molecular
cues to stimulate appropriate cell growth, migration, and dierentiation. The process of tissue
growth may occur in vitro or in vivo. In some applications, such as bone and blood vessel engineering,
the ``donor material'' may be surrounding undiseased tissue containing progenitor cells which can
be stimulated to migrate into, multiply, and form
appropriate tissue structure within a scaold
implanted into the site [97101]. Finally, tissue engineering approaches are being used to make tissue
structures for non-clinical applications such as drug
development, where testing in animals is not always
predictive of outcomes in humans.
Tissue engineering presents enormous challenges
and opportunities for materials science from the
perspective of both materials design and materials
processing. Scaolds must direct the arrangement
of cells in an appropriate three-dimensional conguration and present molecular signals in appropriate spatial and temporal manner so that the
individual cells will form the desired tissue structuresand do so in a way that can be carried out
reproducibly, economically, and on a large scale.
At the molecular materials design level, there is a
substantial need for new materials that interact with
cells via highly specic receptor-mediated phenomena, controlling ligation of both adhesion and
growth factor receptors. Design of such materials is
proceeding along two parallel paths. The rst challenge is to understand quantitatively how cells
respond to molecular signals and integrate multiple
inputs to generate a given responsea signicant
challenge considering that the number of molecules
identied so far represents only a fraction of the
total which exists in the normal tissue environment.
Model polymeric and oligomeric systems, synthesized without constraints of in vivo biocompatibility or cost, and thus with potential for very
precise control of molecular and supramolecular
structure, are just emerging as tools to study these
issues. Model systems are needed to enable systematic investigation of the combined role of physical



and chemical aspects of signaling from the ECM

and growth factors by controlling the precise density and spatial organization of ligands in the cell
environment. For example, much evidence supports
the idea that integrin adhesion receptors require
aggregation for proper signal generation [102, 103].
Classical cell biology approaches are generally not
amenable to quantitative analysis of this phenomenone.g. determining how the size and number of
integrin receptor aggregates aects not only signaling but downstream responses such as cell growth
and migration. Model systems that allow these
quantitative, physical issues to be understood thus
provide the design basis for clinical implant materials, where design constraints include composition,
processibility, and cost.
At the meso- and macroscopic levels, new
approaches to polymer processing are required to
create scaolds with complex architectures and
macroscopic shapes, and which allow composition
variation to accommodate variations in evolving tissue structure. Ultimately, processing approaches
must be adaptable to manufacturing protocols that
are cost-eective and can meet FDA requirements
for Good Manufacturing Processes. These needs are
beginning to be met by approaches such as solid
free-form fabrication, in which complex threedimensional objects are formed as a series of thin
two-dimensional layers [104, 105].
A particular challenge in addressing materials
issues for tissue engineering is that the biological
processes are not yet understood well enough to
allow a clear set of design parameters to be specied a priori. Indeed, evolution of materials/devices
and knowledge of biological processes occur simultaneously. New materials/devices illuminate the
enormous complexity of biological responses
which then inform the improved design of materials
and scaolds.

4.1. Model materials for control of receptor-mediated

cell behavior
The rst key challenge in creating model systems
for control of receptor-mediated cell behavior is
controlling non-specic interactions between cells
and the material which presents the bioactive
ligand(s), so that cell responses can be attributed
solely to the specic receptorligand interactions
under study. Biological uids contain a wide variety
of proteins including many which mediate cell adhesion. The tendency of these proteins to adsorb
virtually irreversibly at interfaces, primarily due to
entropic and hydrophobic driving forces [106108],
has been exploited for years to enable cell adhesion
to in vitro cell culture substrates and many common
biomaterials. Indeed, adsorption of highly puried
proteins or protein fragments to cell culture sub-

strates is a classical, facile, model system in cell biology for studying the function of adhesion proteins
in receptor-mediated cell interactionsdespite the
obvious limitations in control over accessibility of
the active site(s) upon surface adsorption and subsequent protein denaturation, the relative instability
of small peptides toward adsorption, and the potential for cell-secreted proteins to adsorb to the substrate. Synthesis of new materials which allow
control over the amount and spatial organization of
ligand presentation and which are readily accessible
to the biology community should allow structure/
function studies to further evolve.
Highly hydrophilic, uncharged hydrogels such as
the polyacrylamide and agarose gels used commonly in electrophoresis and chromatography are
generally quite resistant to protein adsorption [55,
59, 109116] and are thus among the earliest model
systems used for presenting small bioactive ligands
against a (relatively) inert background [117120].
Hydrogels still play a prominent role in fundamental studies of cellsubstrate interactions [53, 121
123] as many gels allow the ligand concentration
(generally a volumetric concentration distributed
uniformly throughout a gel of macroscopic thickness) to be systematically varied over a wide range.
Polyacrylamide gels modied with small carbohydrate ligands were used, for example, to demonstrate cell adhesion by a non-classical adhesion
receptor, the asioaloglycoprotein receptor (ASGPR), a multimeric (i.e. containing several subunit
chainsfour to six in this case) receptor which
functions to internalize circulating damaged proteins from the blood and which has a low anity
for single (monovalent) sugar ligands and a very
high anity for branched ligands containing three
sugars (trivalent) [117]. The observation of cell
spreading occurring above a threshold concentration of ligand in the gel [124] suggested either
that a certain number of total receptors must be
occupied or that ligand spacing was important, perhaps to allow the simple sugars in the gel to simulate a high-anity branched ligand. Subsequent
studies using polyethylene oxide (PEO) gels in
which simple monovalent ligands were attached to
mobile tethers of systematically varied length (1
30 nm), allowing ligands to move freely to positions
01=3 tether length away from the statistically averaged spacing and thus allowing ligand spacing and
average concentration to be varied independently,
supported the idea that ligand spacing rather than
concentration is critical, as spreading occurred only
when it was physically possible for three adjacent
ligands to assume positions corresponding to spacings in a high-anity trivalent branched ligand
[125]. Despite the advantage gels have in terms of
inhibiting non-specic protein adsorption, the diffuse nature of the ligand-presenting interface makes
it dicult to discern the true amount of ligand
accessible to cellsurface receptors and thus impairs


the interpretation of results from the perspective of

quantitative molecular thermodynamics and kinetics
[126]. This may be addressed to some extent by
attaching macromolecular ligand conjugates to the
surface of a preformed gel, but alternative methods
based on polymer/oligomer surface modication of
rigid substrata oer more versatility in controlling
local ligand distribution over nmmm length scales
Among the wide variety of approaches used to
modify surfaces to accomplish ligand presentation
against an inert, protein-resistant background, most
involve creation of a hydrophilic polymer or oligomer brush at the uidsolid interface employing a
polymer formulation that will present a derivatizable end group at the surface of the brush.
Polyethylene oxide (PEO) is most commonly used
to create protein-resistant surfaces [128], but this
general problem remains an active area of research
and new polymerssuch as one which mimics the
hydrophilic, non-adhesive oligosaccharides protruding from the cell surface [129]are being developed
from basic design principles. The mechanism of
PEO brush inhibition of protein adsorption, which
can be achieved with a wide range of chain lengths,
grafting densities, and chain architectures, has been
studied extensively both theoretically and experimentally [128, 130138] and is attributed in part to
steric hindrance due to the self-repulsion of PEO in
water. No standard denition of ``protein adsorption resistance'' existsdierent labs use proteins
of dierent size and charge, dierent protein concentrations, and time spans ranging from minutes
to over a day, to assess resistance, which is typically
recorded as a percent reduction in adsorption compared to untreated control. Very few studies examine conditions at equilibrium, and it has been
predicted theoretically that one mechanism of PEO
inhibition is kinetic [138]. Thus, adopting a particular protocol for a new application generally requires
experimental verication that the surface remains
suciently inert for the specic conditions which
will be used.
In general, protein resistance increases with
longer chains and higher grafting densities. Endgrafting PEO from solution, though widely practiced and suitable for some applications, yields surfaces with moderate coverage [131, 136, 139, 140]
and thus several approaches have been developed to
increase the polymer segment density in the vicinity
of the surface. Alternative approaches include radiation grafting [141, 142], formation of interpenetrating networks [143, 144], and modication of one of
the polymer ends with an oligomeric or polymeric
segment which has a thermodynamic driving force
to assemble at the interface [129, 137, 145150].
Self-assembly approaches are becoming widely
used as they potentially oer control over ligand
spatial presentation over a continuum of length
scales ranging from a few nanometers (i.e. the scale


of a single receptor) to tens of micrometers (i.e. the

scale of a cell or group of cells) with dened spacer
lengths of the tether presenting the ligand.
Whitesides and co-workers developed a highly versatile approach based on monolayer self-assembly
of end-modied alkane thiols on gold-coated substrata [137, 145, 146, 151154]. Initial experiments
using monolayers containing systematically varied
ratios of unmodied (hydrophobic) alkanethiol
chains to alkanethiol chains modied with hydrophilic oligoethylene glycol (EG) segments (which
create a dense PEO brush) demonstrated protein
adsorptionand thus cell adhesioncould be
tuned by the degree of hydrophilicity (i.e. the fraction of hydrophilic-terminated alkanethiol chains).
Subsequent experiments illustrated that dierent
alkanethiols could be applied to selective regions of
a substrate via microcontact printing using a polydimethylsiloxane (PDMS) stamp cast from a photolithography-generated silicon mold [155, 156] to
create patterned protein/cell adhesive and non-adhesive domains of nely controlled geometries on
length scales down to the micrometer range; these
substrates have been used to control the degree of
cell spreading, which correlates in vitro with some
aspects of cell function. More recently, the technique has been further extended to presentation of
small ligands against the dense PEO brush [145,
146]. The potential to pattern more than one ligand
with these new approaches provides possibilities for
examining the role of spatial interactions between
dierent receptors by controlling placement of their
cognate ligands at subcellular length scales, or for
patterning larger regions with dierent ligands to
arrange two dierent cell types in precise patterns.
The favorable thermodynamics of the self-assembly
process are also being exploited to control ligand
spatial organization at the molecular level, for
example, inducing triple helix formation in collagen
domains and controlling the three-dimensional conguration of peptides [157159].
While length scales on the submicrometer level
are now accessible by microcontact printing, control
of individual ligand spacings over length scales of
10100 nm is an emerging area of need for model
studies of both homotypic and heterotypic interactions between receptors. Many biological
phenomena require aggregation of receptors [160],
and few methods are currently available to systematically and independently vary the number of receptors per aggregate and the total number of ligated
receptors per cell when very small aggregates < 10
receptors) are desired. So, for example, it is believed
that cells must rst form and then break integrin
aggregates in order to migrate [161]; an open question is whether the size of aggregates formed may
dictate the speed of the migration process. This
type of question might be addressed by the design
of block copolymers that can present a dened
number of ligands per molecule, with discrete spa-



cings between ligands. Such systems are now being

developed using both classic polymer synthesis
approaches [127, 147, 162] as well as molecular biology approaches [163].
Polymers also oer tools to study questions in
cell biology which are normally approached by molecular biology techniques, for example, the role of
growth factor/growth factor receptor internalization
on cell responses to dierent. Because dierent sets
of signaling molecules exist at the cell surface and
the cell interior, and many dierent growth factors
can stimulate the same signaling pathways, it has
been hypothesized that the location (surface/interior) of an active receptorligand complex dictates
the type of response. These ideas can be tested by
genetically engineering cells to have mutant growth
factor receptors or mutant molecules which control
internalization of the receptors, but molecular biology does not aord a way to test these hypotheses
in normal unperturbed cells. By tethering the molecule epidermal growth factor (EGF) to the substrate so that it is capable of binding and activating
the receptor, and yet cannot be internalized, it has
been demonstrated that in one cell type the soluble
and surface-restricted EGF elicit the same downstream cell responses in a primary cell type [164].
By appropriate polymer design, further questions
regarding interactions between growth factor receptors and adhesion receptors may be addressed.
New methods are also needed for characterization of these nanoscale soft materials, as the relatively low ligand density and low contrast with
surrounding material precludes analysis by classical
microscopy techniques.

4.2. Applied materials for tissue engineering

New implantable materials for tissue engineering
fall roughly into two categoriesthose used to surface-modify classical bulk materials such as the
degradable polyesters, and gels which are formed in
the presence of cells or tissues either in vitro or in
Most degradable polymers are at least moderately
cell adhesive [165170]. This intrinsic adhesiveness
is suitable for some applicationsfor example, a
skin product based on human broblasts cultured
on an unmodied degradable polyester matrix in
vitro is in clinical trials [166, 171, 172]but control
of adhesion is likely necessary for clinical development of most applications. Most approaches to tailored surface modication rely on addition of a
specic adhesion peptide to enhance the degree of
cell adhesion on poorly adhesive materials [173
178]. A simple approach to accomplishing this,
developed by Telios Pharmaceuticals, is attachment
of a long hydrophobic peptide tail and spacer to
the adhesion peptide to allow modication of generic hydrophobic substrates by adsorption of the

peptide from solution [176, 179]. This approach, as

well as some involving covalent linkage of peptides
to the polymer backbone [173, 175], introduce additional sites for adhesion without necessarily
blocking non-specic protein adsorption and adhesion or allowing precise control over the surface
density of ligand. Because some cell behaviors, such
as migration and dierentiation, can be inhibited by
strong cellsubstrate adhesion induced by high
ligand surface densities [161, 180], interest is
growing in implantable polymers which can
surface-modify bulk materials to present ligand at
controlled densities against a relatively inert
background. Established PEOpolypropylene oxide
(PPO)PEO triblock copolymers are one approach
to this [181, 182]. Enhanced stability and ability to
control ligand density and spacing may come from
use of branched or comb PEO-containing polymers
which can be blended with the bulk phase and
induced to surface segregate [147, 162].
Interest in natural and synthetic hydrogel systems
is growing rapidly. Natural polymers such as alginate and agarose are some of the rst materials
employed in tissue engineering. Alginate is relatively
non-interactive with cells and serves primarily as a
mechanical support to hold cells or tissue fragments
in place and as a partial barrier to the surrounding
environment. In endocrine applications, injectible
microcapsules are typically formed by dispersing
cells in an alginate solution, which is then dropped
into a calcium solution for gelation and subsequently coated with an immuno-isolating layer of
one or more additional polymers (e.g. polylysine)
via complexation. While biological issues with endocrine cells have largely precluded success of this
approach in the clinic, alginate has been fairly successfully adapted to culture of cartilage cells (chondrocytes) to form cartilage tissue, both for in vitro
study of cartilage behavior [183, 184] as well as for
in vivo implantation by injection [185]. In the latter
case, the objective is not to form cartilage at its
original site in vivo, but to use cartilage as a de
facto biomaterial to ll space (here, pushing a urinary tube back into an anatomically correct place),
replacing synthetic polymers in applications where
injectible synthetic polymers have adverse long-term
side eects. Cartilage has an unusual ability to form
cartilage-like tissue from aggregates of dispersed
cells; for more general tissue engineering applications, approaches are being developed to modify
alginate and the comparable thermally induced gelforming polysaccharide agarose with adhesion peptides [186, 187].
Natural polymer gels oer little control over gelation kinetics, gel degradation/resorption, or gel
structural properties such as molecular permeability
and thus several approaches are being developed to
tailor such properties, in addition to cell adhesive
properties, in new synthetic polymers. Hubbell and
co-workers developed a highly versatile gel family


based on photopolymerization of end-modied

PEO macromers [58, 59, 85, 115, 188191]. In this
family of gels, the basic macromer unit is a linear
or branched PEO end-capped with acrylate groups,
which are hydrophobic and tend to associate in
aqueous solution. Free radical polymerization of
the monomers to form a hydrogel is induced by
non-toxic visible light in the presence of an initiator; by coating the initiator on a tissue surface,
such as the lining of a blood vessel, and controlling
the irradiation time, the location and thickness of
the gel can be precisely controlled [192]. This system
is particularly exible for tissue engineering as it is
intrinsically non-adhesive for cells and the gel properties can be tailored: the permeability of the gel
can be controlled by the size of the monomers and
the gel thickness [192]; controlled degradation has
been demonstrated by including hydrolyzable polyester segments or enzyme-cleavable peptides at the
chain ends [188, 191, 193, 194]; and adhesion peptides can be included in the gel at a range of concentrations to control cell interactions [115, 195].
This exibility in both biological and physical properties is being used to address at least one complex
and long-standing problem in tissue engineering
blood vessel healing following angioplasty, which
denudes the vessel wall of the endothelial lining.
Healing is often accompanied by growth factormediated hyperproliferation of the smooth muscle
underlying the endothelium, leading to ultimate reocclusion of the vessel, a process partly mediated
by proteins in the blood diusing into the damaged
vessel. Photopolymerizable gels oer the means to
address several facets of this problem: formation of
a gel on the luminal surface of the vessel can provide a non-clotting barrier to blood-borne stimulators of hyperproliferation [196198]; proteins can
be delivered from the gel to further control the
behavior of the underlying tissue [198]; and endothelial cell-specic adhesion peptides can be incorporated into the gel to induce a normal
repopulation of the vessel surface with endothelium
[115, 199].
Other desirable features are being incorporated
into new gel systems to enhance the applicability
in minimally invasive applications. Photopolymerization generally requires introduction of a light
source and is thus perceived as more invasive than
a simple injection, but highly suitable for applications where other ``invasion''such as angioplastyis employed; gelation upon simple injection
is highly desirable in applications such as the repositioning of the ureter described earlier. Although
ionically induced gelation has been used for injection, the kinetics are rapid (0seconds) and dicult
to control; further, inhomogeneous gelation can
result if the ion is not uniformly distributed
throughout the initial macromer solution.
Thermally induced gelation is one approach provided a physiologically relevant temperature range


for the solgel transition can be identied [56, 200].

A potentially versatile approach to controlling gelation kinetics over a wide range while allowing incorporation of biologically active peptides is use of
enzymatic gelation [63], where gelation kinetics can
be controlled by the choice of substrate, the concentration of enzyme, and the concentration of the
relative substrates. This approach was rst demonstrated using PEO macromers modied with small
peptide substrates for transglutaminase, an enzyme
chosen because it is naturally present in extracellular matrix in humans and is available in recombinant form [63]. Gelation times from minutes to
hours can be achieved with this approach, allowing
adequate time for mixing and injection of cellpolymer solutions. Barriers to injectible photopolymerizable polymers are also being overcome using
monomers sensitive to light that penetrates tissue
Finally, the cells themselves may be viewed as a
``macro materials'' component of forming a complete tissue structure via self-assembly [201].
Pioneering work by Steinberg suggests that the sorting out of intermixed embryonic cells, the spreading
of one tissue over another, and the specic inside/
outside tissue stratication that arises by any of
these processes are guided in large part by the
dierential intercellular adhesivities of the cell types
involved [202, 203]. Thus, barring kinetic barriers,
the number and strength of cellcell and cell
substrate bonds formedparameters which can be
systematically modulated by solid or soluble biomaterialswill dictate the nal arrangement of a
cell mixture into a tissue-like structure [201]. This
perspective is being used to overcome kinetic barriers to forming neural cell aggregates, for example,
by the design of bi-functional PEO macromers
bearing cell adhesion ligands [204, 205] and to control formation of liver cell structures via modulation
of cellsubstrate adhesion strength [201, 206].


Traditional applications in polymeric biomaterials

continue to be robust and derive new directions
from advances in materials science. A growing number of applications require intimate integration of
polymer science with molecular cell biology, both
for developing new approaches to human therapies
and as basic tools in biological research. The age of
using polymeric materials to controllably manipulate cell function is still in its infancy, and expansion of this eld will likely enable development of
new therapies.
AcknowledgementsThis work was partially supported by
NSF BES96-32714 and by Therics, Inc. The assistance of
Gargi Maheshwari with the literature search is gratefully




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