Available online at www.sciencedirect.

com

Advanced Drug Delivery Reviews 60 (2008) 215 – 228

www.elsevier.com/locate/addr

Biomaterials for stem cell differentiation☆

Eileen Dawson, Gazell Mapili, Kathryn Erickson, Sabia Taqvi, Krishnendu Roy ⁎
Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712, USA
Received 31 July 2007; accepted 11 August 2007
Available online 11 October 2007

Abstract

The promise of cellular therapy lies in the repair of damaged organs and tissues in vivo as well as generating tissue constructs in vitro for
subsequent transplantation. Unfortunately, the lack of available donor cell sources limits its ultimate clinical applicability. Stem cells are a natural
choice for cell therapy due to their pluripotent nature and self-renewal capacity. Creating reserves of undifferentiated stem cells and subsequently
driving their differentiation to a lineage of choice in an efficient and scalable manner is critical for the ultimate clinical success of cellular
therapeutics. In recent years, a variety of biomaterials have been incorporated in stem cell cultures, primarily to provide a conducive
microenvironment for their growth and differentiation and to ultimately mimic the stem cell niche. In this review, we examine applications of
natural and synthetic materials, their modifications as well as various culture conditions for maintenance and lineage-specific differentiation of
embryonic and adult stem cells.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Embryonic stem cells; Adult stem cells; Polymers; Metals; Ceramics; Bioreactors; Differentiation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
2. Biomaterials used for studying stem cells in 3-D culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.1. Scaffolds in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.2. Natural materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.3. Synthetic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2.3.1. Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2.3.2. Ceramics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2.3.3. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
3. Modifications to biomaterials for stem cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
3.1. Substrates with altered mechanical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
3.2. Modifications with chemical and biological stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
3.3. Patterned biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

☆
This review is part of the Advanced Drug Delivery Reviews theme issue on
“Emerging Trends in Cell-Based Therapeutics”. 1. Introduction
⁎ Corresponding author. Department of Biomedical Engineering, The
University Texas at Austin, ENS 610, C0800, 1 University Station, Austin,
TX 78712, USA. Tel.: +1 512 232 3477. Current therapies in modern medicine mostly involve
E-mail address: kroy@mail.utexas.edu (K. Roy). prevention, manipulation and control of diseases through
0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2007.08.037
216 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
chemical or biological molecules. More recently, the restoration
and direct replacement of diseased cells and tissues are
becoming a clinical possibility in large part due to parallel
advances in modifying biomaterials and understanding stem
cell (SC) behavior [1]. Complete tissue and organ replacement
using stem cells is still a distant milestone in which current
studies are laying the necessary groundwork. Recent review
articles have discussed how certain types of materials are used
as substrates to mimic the physico-chemical microenvironments
of cells and tissues [2,3]. Several papers have also carefully
examined materials that have been used to specifically study the
development of bone, cartilage, and skin [4–8]. Others have
reported studies on multiple pre-differentiated stem cell
populations and how to combine them together using
biomaterials to form hybrid constructs that closely mimic
native tissue [9,10]. In this review, we focus exclusively on
biomaterials and how they are revolutionizing studies in tissue
and organ replacements and providing a greater comprehension
of the underlying mechanism behind stem cell behavior and
differentiation within their physiological niche.
Stem cells are simply defined as a progeny of cells that have
the potential to differentiate into a variety of different lineages.
These cells can be isolated from a variety of sources including
embryos, umbilical cord blood, as well as from adult tissues, as
reviewed previously [11]. Since the isolation of mouse
embryonic stem cells (mESCs) in 1981 by David and Kaufman,
and the subsequent isolation of human embryonic stem cells
(hESCs) in 1998, their exploration in regenerative medicine
have received great interest. Embryonic stem cells (ESCs) are
isolated from the inner cell mass of the blastocyst during
embryological development [12,13]. Their pluripotent nature
gives them the ability to differentiate into any one of the three
germ layers: endoderm, ectoderm, and mesoderm. ESCs also
have the unique property of indefinite self-renewal i.e. they can
be cultured and maintained in an undifferentiated, pluripotent
state. Thus, ESCs have the potential of providing the biomedical
community with a continuous source of all cell types [13].
Although ESCs are attractive because of their pluripotency,
they are also difficult to work with because of this same
characteristic. Maintaining large number of ESCs in an undiffer-
entiated state and subsequently directing them to differentiate, in a
reliable and reproducible manner, into specific cell types are the
foremost complications in ESC-based cell therapy [14]. If ESCs
remain undifferentiated after implantation in the body, they will
spontaneously differentiate into multiple cell types and form a
type of tumor called teratoma [13,15]. To avoid teratoma
formation, ESCs must be guided to differentiate into particular
lineages prior to implantation [13]. On the other hand, ultimate
large scale therapeutic application of these cells necessitates their
maintenance in an undifferentiated state in vitro so that they can be
differentiated into specific lineages on-demand.
It is now well established that adult tissues carry a variety of
adult stem cells that are less pluripotent and are more committed
than ESCs. Adult stem cells are often referred to as progenitor
or multipotent cells since they have limited differentiation
potential. These cells have been found in bone marrow, cord
blood, adipose tissues, neural tissues etc. Bone-marrow derived
progenitor cells or mesenchymal stem cells (MSCs), hemato-
poietic stem cells (HSCs) from both bone marrow and cord
blood as well as adipose-derived stem cells have become
attractive cell populations for the field of tissue engineering
because of their ability to differentiate into variety of cell types
and relative ease in harvest, isolation and expansion in vitro.
In this review we have focused exclusively on the use of
biomaterials, both natural and synthetic, to maintain and
differentiate stem cells. Both adult and ESCs are discussed. A
major focus of this review is on the chemical and biological
modification of materials to better mimic the stem cell niche and
create microenvironments to control stem cell response.
2. Biomaterials used for studying stem cells in 3-D culture
2.1. Scaffolds in cell culture
Although classical 2-D cell cultures on flat surfaces have
provided us with majority of our knowledge in modern biology,
it is now well accepted that cells (including SCs) reside,
proliferate and differentiate inside the body within complex 3-D
microenvironments. Most of the current research in biomaterial-
directed SC manipulation is focused on such 3-D environments.
Hence this review will primarily focus on materials and
concepts that involve 3-D culture of SCs.
Biomaterial-based scaffolds have been the most important tool
in providing a 3-D environment to cells, both in culture or inside
the body. These 3-D structures provide an ideal platform for cell–
cell and cell–material communications and their properties can be
varied to promote differentiation of cells into specific lineages.
Scaffolds for tissue engineering serve numerous functions and
their role during tissue development is dependent upon specific
properties of the chosen biomaterial. 3-D systems have proven to
enhance osteogenic [16], hematopoietic [17], neural [18], and
chondrogenic [19,20] differentiation. They serve as biointeractive
stages promoting cell attachment, proliferation, and organization,
in addition to acting as delivery vehicles for bioactive molecules
during tissue formation.
Properties of biocompatible scaffolds, synthetic or natural, that
must be taken into careful consideration include optimal fluid
transport, delivery of bioactive molecules, material degradation,
cell-recognizable surface chemistries, mechanical integrity and
the ability to induce signal transduction. The overall success of
tissue organization and development is highly dependent upon
these properties, since they can ultimately dictate cell adherence,
nutrient/waste transport, matrix synthesis, matrix organization and
cell differentiation. Most scaffolding materials can be chemically
and physically modified to adjust all of these critical parameters,
and a variety of synthetic and natural materials have been used for
studying SC behavior through specifically manipulating these
properties. Several articles have reviewed the application of
scaffolds in tissue engineering in general [21,22].
2.2. Natural materials
Natural biomaterials used for developing scaffolds can
consist of components found in the extracellular matrix
 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
(ECM), such as collagen, fibrinogen, hyaluronic acid, glycosa-
minoglycans (GAGs), hydroxyapatite (HA) etc., and therefore
have the advantage of being bioactive, biocompatible, and of
similar mechanical properties as native tissue. Other natural
materials include those derived from plants, insects or animal
components (e.g. cellulose, chitosan, silk fibroin etc.) with
properties to provide favorable microenvironments for SC
culture. Disadvantages of using natural materials over synthetic
materials include limited control over physico-chemical prop-
erties, difficult to modify degradation rates, difficulty in
sterilization and purification as well as pathogen/viral issues
when isolating from different sources. However, in recent years,
several natural materials, e.g. chitosan, hyaluronic acid, silk etc.
have become commercially available, are well characterized,
and have reproducible, controlled properties.
Matrigel™, a product currently available commercially, is
comprised of a variety of ECM components including laminin,
collagen IV, and heparan sulfate proteoglycans [23,24] and has
been used extensively in cell culture [23,25]. In addition to
Matrigel's application in normal tissue culture, when injected
with cells isolated from the stromal vascular fraction (SVF) of
adipose tissue (i.e. adipose-derived SCs), improved neovascu-
lature formation was observed in an ischemic mouse model
[26]. Further studies indicated that cells pre-cultured under
proper conditions and then injected with Matrigel could produce
tubelike vascular structures [26].
Fibrinogen and fibrin are another class of tissue-derived natural
materials that can be utilized to create three-dimensional scaffold
materials [27]. Fibrin scaffolds have been optimized for neural
differentiation of mESCs [18]. These scaffolds, in conjunction
with various growth factors showed significant increase in neuron
and oligodendrocyte production as well as neuronal viability over
time [28]. Suggs and colleagues have also demonstrated the use of
fibrin as a material for mouse ESC culture [29]. Chondrogenesis
potential of MSCs derived from adipose tissue and bone marrow
has also been evaluated in fibrin gels, and while both cell types
demonstrated cellular functions indicative of chondrogenic
differentiation, bone marrow derived MSCs appeared to produce
differentiated cells that were more efficient in both collagen II
production as well as proteoglycan synthesis [27].
Hyaluronic acid is a highly attractive natural biomaterial due to
its participation in cell behavior and cell signaling. It is present in
tissue as a gel-like substance but can be chemically modified for
efficient processing into fibers, membranes, or microspheres. A
modified type of hyaluronic acid is commercially available as
Hyaff® [30]. Hyaff®-based scaffolds are biodegradable and
combine both the benefit of having a 3-D microenvironment
comprised of a natural material while allowing for cells to replace
the scaffold with their own ECM. Recently Gerecht et al. reported
the use of hyaluronic acid hydrogels for maintaining the
pluripotency and undifferentiated state of hESCs [31], and
showed that the addition of soluble growth factors to these
hydrogels successfully triggers lineage specific differentiation of
these hESCs. In addition, MSCs grown on this type of scaffolds,
modeled to resemble a tendon, have been shown in vitro to
express a variety of ligament proteins while suppressing
indicators of bone and cartilage differentiation [32].
217
Silk fibroin is another versatile natural material that is
isolated from silkworm cocoons. Silk has been developed into
porous scaffolds using gas foaming or salt leaching methods
[33–35] and is widely used as suture materials for surgical
applications. The use of silk as a biomaterial for SC culture has
been reviewed recently by Kaplan and colleagues [36].
Other natural materials used as scaffolds for studies in SC
differentiation include chitosan, HA, alginate and coralline.
Chitosan, isolated and processed from crustacean shells, is
hydrophilic in nature, biodegradable, biocompatible, and has
similar properties to GAGs. One particular study combined
chitosan with coralline, exoskeletons of marine species or coral,
as a composite scaffold to study MSC-osteogenesis since coral
is composed of calcium carbonate, a component found in bone
[37]. Another material that has been extensively used as a
composite for bone tissue engineering, both from osteoblasts as
well as MSCs, is HA, a natural, inorganic component of bone
mineral [38]. HA has the ability to encourage bone in-growth
and the inclusion of HA particles in a nanostructured self-
assembling peptide scaffold, encourages osteogenic differenti-
ation of mESCs [39]. Alginate (derived from algae cell walls) is
a natural polysaccharide that has been evaluated for the
encapsulation and differentiation of ESCs [40]. mESCs
encapsulated in alginate poly-L-lysine (PLL) was shown to
support cell proliferation [41]. Moreover, this microencapsu-
lated structure prevented embryoid body (EB) formation and
promoted differentiation towards a hepatic lineage without the
need for EB formation [41].
By crosslinking pullulan, dextran, and fucoidan at a ratio of
71:24:5 with a total concentration of 25% (w/v), homogenous
3-D hydrogels could be created and stored for up to 4 weeks
[42]. CD34+ human umbilical cord blood cells (hUCBCs)
previously differentiated towards the endothelial lineage,
CD133+ human bone marrow cells (hBMCs) also differenti-
ated into endothelial cells, and mature endothelial cells that had
been isolated from human saphenous veins, were all cultured
on this novel hydrogel [42]. While the scaffolds appear to
support cell adhesion, and lack cytotoxicity, further experi-
ments need to be performed before in vivo applications in
vascular tissue repair can be determined [42].
A classic natural material for tissue engineering is collagen
and its derivatives. Type II collagen, isolated from bovine
cartilage, has been used to culture human mesenchymal stem
cells (hMSCs) in pellet form when combined at the ratio of
1.25 mg type II collagen per 2.5 ⁎ 105 cells (altering the ratio of
collagen altered the structural integrity of the pellet) [43]. The
hMSCs cultured under these conditions not only assembled but
were able to reorganize the pellet structure as shown by the
degree of matrix contraction [43]. In vivo results indicate that
that this type of 3-D culture can provide a system capable of
maintaining chondrogenesis [43]. Collagen microbeads have
also been studied for the expansion of hUCBCs and shown to
not only improve expansion, as compared to a 2-D system, but
also improve overall cell viability [44]. Though these results
were not statistically significant, as compared to the 2-D system,
the cells isolated from these microbeads demonstrated more
favorable clonogenic ability, suggesting that collagen-beads
218 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
maintained cell hematopoietic functions for a longer duration in
culture [44]. Additional in vivo results suggested that cells
maintained within microbeads had the ability to engraft into the
bone marrow of NOD/SCID mice even after 12 days of culture,
whereas 2-D cultured hUCBCs failed to engraft in three out of
four mice [44].
2.3. Synthetic materials
2.3.1. Polymers
Polyglycolic acid (PGA), polylactic acid (PLA) and the
copolymer polylactide-co-glycolide (PLGA) have been exten-
sively used as synthetic 3-D scaffold materials for evaluating cell
behavior [45,46]. These materials are hydrolytically degradable
through bulk erosion due to the presence of ester bonds, and the
glycolic/lactic acid byproducts are physiologically removed via
metabolic pathways. Polymer molecular weight, copolymeriza-
tion ratio, and polydispersity can be easily adjusted to control the
degradation rate, making these attractive synthetic materials for
tissue engineering. Furthermore, standard methods (e.g., salt
leaching, sintering, porogen melting, and nanofiber electrospin-
ning) have been well established to prepare a wide variety of 3-D
scaffolds using these materials [45,47,48].
These scaffolds have been shown to support human ESC
growth as well as encourage 3-D tissue-like organization [49]. By
using this type of degradable scaffold, it may be possible to allow
for the structural support necessary to control growth of ESCs
while allowing the efficient transport of nutrients and other
growth factors. As ESCs form a 3-D tissue structure, the scaffolds
degrade, leaving only the differentiated cells. In such a system,
using appropriate growth cues, it may be possible to differentiate
ESCs into a number of different tissues without altering the initial
matrix material [49]. In our laboratory we have demonstrated that
the physical properties of the scaffold structure e.g. varying pore
sizes and polymer composition significantly influence mESC
differentiation into the hematopoietic lineage. Specifically,
scaffold porosity was found to be inversely related to ESC
hematopoiesis with an optimal pore size of less then 150 μm [50].
Hematopoietic progenitor cell (HPC) formation was also shown
to be directly proportional to polymer concentration in the
scaffold with optimal concentration being 20% w/v.
Non-woven fabrics developed from polyethylene tereptha-
late (PET), another widely used synthetic polymer, have been
studied with MSCs and human cord blood-derived HSCs to
evaluate seeding, proliferation, and aggregation for tissue
regeneration [51–53]. Nanofibrous scaffolds fabricated using
poly(ɛ-caprolactone) (PCL) have also been studied for
adipogenesis, chondrogenesis, and osteogenesis [54,55].
These electrospun polymers produce an interconnected porous
matrix capable of encouraging significant cell proliferation and
cell–cell interactions in both MSCs and mESCs [56]. The
introduction of adipogenic hormones into the system, resulted
in specific expression of a transcriptional factor, PPAR-γ,
associated with adipocyte differentiation [56].
Acrylated polymers that form hydrogels, such as poly
(ethylene glycol) diacrylates (PEGDA) and its derivatives, poly
(6-aminohexyl phosphate acryloyl) ((PPE-HA)-acryl) and
acrylated polyanhydrides or polyesters have been widely
studied for MSC and ESC cultures [57–59]. EBs from
mESCs encapsulated in a poly(ethylene glycol) (PEG) hydro-
gel, and exposed to transforming growth factor beta (TGF-β),
have been shown to up regulate the expression of chondrogenic
markers [19]. Furthermore, chondrogenic differentiation of
mESCs using PEG-diacrylate hydrogels can be augmented with
the addition of glucosamine, an amino monosacchaaride present
in GAGs [20]. Glucosoamine is known to increase the
production of chondrogenic proteoglycans; therefore its addi-
tion to the hydrogel structure may increase the mechanical
properties of the scaffold by synthesizing new ECM [20].
Recently Healy and colleagues demonstrated that photocros-
slinked hydrogels incorporating poly(N-isopropylacrylamide-
co-acrylic acid) [p(NIPAAm-co-AAc)] and an acrylated matrix
metalloproteinase sensitive peptide Gln-Pro-Gln-Gly-Leu-Ala-
Lys-NH2 (QPQGLAK-NH2) can support short term self
renewal and maintenance of hESCs [60].
Though synthetic materials provides the versatility of
creating 3-D microenvironments with tunable features (i.e.,
mechanical properties, degradation rates, porosities), disadvan-
tages for choosing such materials include poor inherent
bioactivity (e.g. PEG), acidic by-products (e.g. PLA or
PLGA), etc. It is thus critical to modify synthetic materials
with biological or chemical entities to achieve appropriate
cellular response (discussed in Section 3).
2.3.2. Ceramics
Calcium phosphates, bioactive glasses, and other biocera-
mics are desirable materials for studying SC differentiation,
especially osteogenesis since these materials have favorable
mechanical properties and can integrate with bone to a higher
degree than soft biomaterials, thereby enhancing mineralization
and matrix formation. Biphasic calcium phosphate ceramics can
be commercially purchased (Triosite™) and MSCs grown on
this material demonstrated continued osteoblastic phenotypic
properties even after an entire month of culture [61]. The use of
ceramics in tissue engineering has been extensively reviewed in
several articles [62,63].
2.3.3. Metals
Titanium, an attractive material due to its inertness and
biocompatibility, is used widely in orthopedic and dental
surgery [64]. The combination of titanium along with the
osteogenic differentiation potential of MSCs makes for an
attractive bone regeneration material. MSCs are able to attach
and proliferate on titanium dishes [64]. Furthermore, after
appropriate differentiation media is applied to the cultures,
titanium scaffolds are able to exhibit signs of bone matrix
formation [64]. Titanium fiber meshes seeded with rat MSCs
have been cultured in a perfusion bioreactor to study effects on
osteoblastic differentiation [65]. Levels of mineralization
formed by osteoblasts within the scaffolds were compared
between plain titanium meshes and pre-generated bone ECM-
deposited titanium meshes. Results indicated that synergistic
effects of both mechanical stimulation through shear stress and
the presence of ECM deposition onto the scaffold substrate
 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
profoundly enhanced osteoblastic differentiation. Layer by
layer (LbL) nano-assembly, a technique which allows for the
surface modification of materials can fabricate layered coatings
to modify any substrate material. Using nanoparticles of TiO2 it
is possible to produce a thin surface film which increases the
attachment of MSCs by creating a rougher surface [66].
Titanium nitride (TiN) can also be deposited on a surface in
the form of a thin film by using a DC magnetron sputtering
technique [67]. The TiN coating encouraged hMSC adherence
as compared with traditional implant material (TiAlV) [67].
Another metal based material suitable for SC differentiation is
Cytomatrix™, a tantalum based scaffold material. In studies
performed in our laboratory, Cytomatrix™ has been shown to be
able to effectively induce EB formation in mESCs in both static
conditions and dynamic culture conditions [17]. As compared to a
2-D culture, the EBs formed on Cytomatrix™ appeared to have
an ECM-like structure that covered not only the EB itself, but
extended onto the surface of the scaffold indicating both cell–cell
and cell–matrix interactions [17]. Additionally, EBs formed on
the Cytomatrix™ scaffolds appeared to be of a smaller size,
avoiding the formation of aggregates usually associated with 2-D
cultures [17]. HSC generation in 3-D cultures, especially those
cultured under dynamic conditions, was significantly higher as
compared to 2-D culture and revealed a higher potential for
further differentiation into the myeloid lineage [17]. Extensive
cDNA microarray analysis of mESCs differentiated in 2-D as
well as 3-D static and dynamic conditions demonstrated
significant differences in gene expression [68]. Cells differenti-
ated in the different culture conditions expressed genes unique to
their scaffold environment as well as to their culture conditions
[68].
3. Modifications to biomaterials for stem cell culture
Chemical and biological modifications to biomaterials can
directly influence SC behavior by altering substrate properties,
surface interactions, scaffold degradation rate, microenviron-
ment architecture and ultimately manipulating the signal
transduction pathways in SCs. Biomaterials can be designed
to fine-tune their degradation kinetics, present specific ligand-
based signals and/or release of biological molecules in response
to their microenvironment. These influence cell–matrix inter-
actions and should lead to altered gene expression and lineage
specificity. Numerous studies have demonstrated how modified
biomaterials and scaffold surfaces introduce specific biological
responses in SCs. Ultimately, the goal of biomaterial-directed
SC culture is to mimic the properties, both physical and
biochemical, of the physiological SC niche. This section
provides a comprehensive review of the various concepts in
modifying biomaterials for maintenance and differentiation of
SCs.
3.1. Substrates with altered mechanical properties
The intrinsic mechanical properties of a biomaterial are of
significant interest since they influence the forces exerted by
cells on their substrates. For instance, chondrocytes exert
219
contractile forces when integrated onto substrates and can
change the overall microstructure of the scaffold during tissue
development depending on the mechanical properties of the
material [69,70]. Recently Discher and colleagues elegantly
demonstrated the importance of substrate mechanical properties
on SC differentiation using MSCs as a model [71]. Therefore
the overall mechanical integrity of scaffolding materials is a key
element that needs to be addressed when evaluating material
properties that effect differentiation pathways of SCs.
Bone marrow-derived MSCs are highly sensitive and
responsive to mechanical stimulation in vitro [71,72]. It is
speculated that mechanical stimuli activates cell surface
receptors and focal adhesion sites, which in turn triggers
intracellular signaling cascades. This leads to specific gene
activation that modulates ECM secretion. MSC differentiation
is influenced both by their physical microenvironment e.g. by
mechanical cyclic stresses applied directly to the cells [73] as
well as by the inherent biomaterial properties (i.e. material
integrity, crystallinity, crosslinking density, overall micro- and
macro-porosity etc.).
Substrates such as natural or synthetic hydrogels, closely
resemble the consistency of soft, native tissues, making them
attractive scaffold materials for soft tissue engineering. On the
other hand, MSC differentiation into connective tissue lineages
(i.e. bone, cartilage, ligaments, and tendons) require materials
with higher mechanical strength to closely mimic the tissue
mechanical properties. However, hydrogel-like materials, can
be modified to have increased modulus of elasticity, making
them more suitable for applications in connective tissue
engineering. For example, collagen gels can be adjusted to
have a higher modulus by adding HA, thereby mimicking the
composition of bone which is mostly composed of collagen
fibers and phosphate minerals. Adding HA to collagen at a 1:1
ratio increases the modulus from 0.392 MPa to 0.422 MPa,
which is comparable to trabecular bone (E = 0.443 MPa) [74].
Other studies have fabricated collagen composites to contain
PLA and chitin fibers to provide increased mechanical integrity
and have demonstrated higher human MSC attachment [75].
Silk-based materials have also been commonly used for
MSC culture [76–78]. Silk exhibits higher modulus of elasticity
over other natural materials, such as collagen. However de-
contamination and purification methods of silk, prior to their
use in vivo, are extremely critical in order to avoid inflammatory
and immunogenic reactions. Silk-based scaffolds seeded with
hMSCs were shown to induce bone formation in critical-sized,
cranial defects (larger than 4 mm) of nude mice, indicated by the
presence of bone sialoprotein, osteopontin, and osteocalcin
[76]. Furthermore, efficient cartilage formation was also seen
when differentiating MSCs into the chondrogenic pathway
within silk scaffolds [35].
In addition to natural materials, synthetic materials can also be
chemically modified to enhance mechanical properties. For
example, simply increasing the macromer concentration in photo-
crosslinked hydrogel scaffolds has been shown to increase the
modulus of elasticity, such as within (PPE-HA)-acryl hydrogels
[57]. A four times increase in the amount of acrylated-PEG
reacted with PPE-HA showed an almost 10-fold increase in shear
220 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
modulus (3 to 26 kPa) [57]. Additionally this biodegradable,
phosphate-based synthetic material is highly conducive to bone
tissue engineering due to the phosphate degradation product,
which could aid in overall scaffold mineralization during
osteogenesis [57].
Scaffolds have been increasingly applied to study the
differentiation of ESCs. While most studies focus on fluid
transport and other material properties of the scaffold, recent
studies suggest that the mechanical stiffness may also play a key
role in controlling differentiation [49,50,82]. Studies reported by
Battista et al. using scaffolds of collagen, fibronectin (FN) and
laminin, have indicated that alteration of mechanical properties
of scaffolds can drastically alter EB formation. When the elastic
modulus was increased from 16 to 34 Pa the formation of EBs
was severely inhibited suggesting that the increase in elastic
modulus resulted in an inhibition of apoptosis [82]. A second
possible explanation may be that the denser network of these
stiffer gels may have altered ESC growth. In our lab, however,
we were able to show similar trends with HSC differentiation
[50]. Our results indicated that scaffolds with higher modulus
were more conductive towards HSC generation.
Although not a focus of this review, MSC and ESC
differentiation induced by mechanical stimulation has also
been extensively investigated using bioreactors [65,79–81], or
in vitro systems that provide dynamic culturing conditions.
Cells in vivo consistently undergo fluid shear stress and
mechanical strain, thereby influencing cellular interactions and
responses. In our lab, the significance of mimicking this in vivo
environment was demonstrated with the differentiation of
mESCs into HSCs. Cytomatrix, a porous, tantalum-based
biocompatible scaffold was used to culture mESCs [68]. It was
shown that in comparison to 2-D culture, the 3-D tantalum
scaffold was more efficient at promoting HSC formation [68].
Furthermore, when combining 3-D environment with a dynamic
culture in a spinner flask, the efficiency of HSC formation was
even greater (1.4–2.2 times) than that of 3-D culture alone [68].
3.2. Modifications with chemical and biological stimuli
SC differentiation can be directly mediated by presenting
appropriate biological or chemical signals in their microenvi-
ronment. It is well established that specific growth factors,
hormones and cytokines can enhance proliferation and lineage-
specific differentiation of SCs. For example, fibroblast growth
factor-2 (FGF-2) has been shown to increase self-renewal of
MSCs and maintenance of their multi-lineage differentiation
potential [83–86]. Additionally, bone morphogenetic proteins
(BMPs), have shown to have significant role in the regeneration
of skeletal tissues, especially bone [87,88].
Propagation of ESCs without a feeder layer can be
maintained in the presence of a variety of cytokines including
leukemia inhibitory factor (LIF), stem cell factor (SCF), and
FMS-like tyrosine kinase 3 ligand (Flt3-ligand). Recently it was
also demonstrated that FGF-2 could allow long term self
renewal of hESCs and maintain their pluripotent status [89,90].
Addition of other growth factors can induce ES differentiation
and ultimately formation of a variety of different tissue types.
These growth factors include retinoic acid (RA), activin-A,
TGF-β, and insulin-like growth factor 1 (IGF-I). Activin-A has
been shown to promote the differentiation of mESC derived
EBs towards the endoderm germ layer [91]. Introducing activin-
A into EB cultures has been used in creating lung epithelial
progenitor cells [91]. Both RA and TGF-β are involved in
pancreatic formation [92]. The addition of RA and activin-A
have demonstrated success in promoting in vitro differentiation
of mESCs into α, β, γ, and δ cells, all of which are pancreatic
endocrine cells [92].
Growth factors, hormones, and chemicals have classically
been directly added into the culture medium. More recently
these bio-molecules have been directly incorporated within the
scaffold structure or into the scaffold biomaterial in a variety of
ways. Fig. 1 provides a schematic representation of some of the
methods used in incorporating these molecules into a 3-D
scaffold. Soluble growth factors can be directly encapsulated or
incorporated during the scaffold fabrication process [93,94] and
has been used widely. However, in order to mimic the patterned
distribution of growth factors and ECM molecules in the SC
niche, it is necessary to develop methods to sequester these bio-
factors in a localized microenvironment to prevent their
diffusion into other regions of the scaffolds. Heparan sulfates
have been known to bind and protect growth factors, especially
FGF-2, in the ECM. In our studies, we have successfully
demonstrated the effective binding of FGF-2 within photo-
polymerizable PEGDA scaffolds by covalently conjugating
acrylated-PEG moieties to heparan sulfate [95]. Using immuno-
histochemistry we effectively demonstrated that FGF-2 was
only localized or sequestered in a region of a multi-layered
scaffold that had heparin-PEG-acrylate.
A widely used method of growth factor delivery in tissue
engineering has been simple physical adsorption of biomole-
cules on the biomaterial or scaffold surface. Carstens et al.,
using absorbable collagen sponges as delivery vehicles for
recombinant human BMP-2 (rhBMP-2), showed in situ
osteogenesis in a craniofacial mandibular defect [96]. The
chemotactic effects of rhBMP-2, attracted MSCs to the vicinity
of the implants. These cells were characterized to be spindle-
shaped and having a pre-osteoblastic phenotype. In another
study titanium fiber mesh scaffolds were coated with arginine-
glycine-aspartic acid (RGD) [97], a cell adhesive, integrin-
binding peptide found in FN and laminin. MSCs were shown to
attach more strongly to these RGD-coated scaffolds, however
no change was observed in ECM secretion.
Although protein adsorption to scaffold surfaces can be an
effective route for presentation of bioactive molecules [98,99],
desorption of the protein during culture period and the inherent
poor reproducibility of adsorption processes limits its applica-
bility. Covalent conjugation of bioactive molecules to the
biomaterial surface should provide a more reproducible and
controlled method of presentation since both ligand amount and
ligand density can be controlled. By incorporating methacrylic
acid within a PEGDA macromer solution prior to photo-
polymerizing, we have shown that the surface of 3-D hydrogel
scaffolds can be functionalized with carboxylic acid groups.
These free carboxyl groups can be subsequently activated to
 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
Fig. 1. Schematic of biochemical modification and spatial patterning of scaffolds for stem cell culture.
attach FN or other ligands [100]. Murine MSCs seeded onto
such FN-functionalized scaffolds created by an LbL micro-
fabrication system, and cells effectively adhered and trans-
formed into osteoblasts.
RGD can be covalently conjugated to PEG-macromers using
the widely used N-hydroxysuccinimidyl-ester (NHS) chemistry
[101–104]. This has been reported for functionalization of
PEGDA scaffolds for hMSC differentiation into osteoblasts
[105]. Cell–matrix interactions were enhanced in RGD-
functionalized hydrogels leading to increased MSCs viability.
Nuttelman and colleagues showed that the viability of the
encapsulated hMSCs increases from 15% to 75% when RGD is
incorporated into PEGDA hydrogels [105]. It is also possible to
differentiate hESCs into chondrocytic like cells in an RGD
modified PEGDA hydrogel [106]. The hESC-derived cells not
only morphologically resembled chondrocytes but also RT-PCR
analysis indicated that the cells expressed a number of
chondrocytic markers and demonstrated the ability to produce
7% w/v of GAG after three weeks in culture [106]. MSCs
cultured under similar conditions without RGD produced 3.5%
w/v of GAG accumulation [59].
221
Semi-interpenetrating polymer networks (sIPN) which form a
hydrogel material can be chemically altered to mimic ECM in a
variety of ways, notably, the mechanical properties can be altered
as well as adding functional cell-adhesion ligands (at varied
densities). p(NIPAAm-co-AAc) was crosslinked with Gln-Pro-
Gln-GLY-Leu-Ala-Lys-NH2, which can be cleaved by matrix
metalloproteinase-13 [60]. The sIPN was further functionalized
with a polyacrylic acid-graft-Ac-CGGNGEPRGD-TYRAY-NH2
[p(AAc)-g-RGD] [60]. The p(AAc) chains were modified with
(Ac-CGGNGEPRGDTYRAY-NH2) which provides an active
RGD site to promote cellular adhesion [60]. hESCs cultured on
this fully synthetic ECM replacement, functionalized with 0 to
150 μM of RGD complexes, were shown to be morphologically
similar to hESCs cultured on an embryonic fibroblast feeder layer
and continued to produce markers characteristic of undifferenti-
ated hESCs [60].
In another study, incorporation of RGD in oligo(PEG-
fumarate) hydrogels enhanced alkaline phosphatase (ALP)
activity of osteoblasts when compared to non-functionalized
hydrogels [107]. Shin et al. compared RGD with an
osteopontin-derived peptide, and determined that hydrogels
222 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
modified with the osteopontin-derived peptide triggered
significantly higher osteoblast migration than RGD-modified
hydrogels. However, RGD remained the dominating peptide for
cell-attachment [108].
A study completed by Feng et al. compared the effect of
adsorbed, conjugated, and soluble FN on hHSC expansion in
PET non-woven scaffolds. Their study indicated that conjuga-
tion of FN directly to the scaffolds resulted in a higher
expansion percentage of CD34+ cells [51]. Because the FN was
directly conjugated to the scaffold material, detachment of FN
from the scaffold could be prevented, and perhaps aid in
stabilizing FN in the culture system [51].
In addition to presenting ligands to control differentiation via
a covalently linked hydrogel, it may be possible to utilize other
materials to mimic the cellular environment. HSCs located in
the thymus receive specific microenvironmental signals direct-
ing them toward the T lineage [111]. Traditionally, studies
focusing on T cell differentiation employ the usage of
retrovirally transfected stromal cells to present the necessary
cues to cause T lineage commitment. In our lab we have
employed a biomaterial based magnetic microbead system to
effectively present ligands necessary to promote T cell
formation (e.g. the notch ligand DLL4) [111]. Mouse
hematopoietic stem cells (mHSCs) isolated from the bone
marrow of mice and cultured with this microbead system
effectively produced Thy1.2+ early T cells [111]. This presents a
versatile biomaterial-based method to quantitatively present
ligands to SCs in order to direct lineage-specific differentiation
and study cellular processes during cell differentiation.
Scaffold mineralization during osteogenic differentiation of
MSCs can also be enhanced through chemical modification of
the biomaterial structure. For example, adding a phosphoester
group to photo-polymerizable PEG-based hydrogels not only
provides biodegradability but has also been shown to promote
mineralization of encapsulated MSCs. The use of such
phosphoester-containing hydrogels significantly increases
ALP and osteocalcin levels in differentiated cells [112,113]. It
was shown that cell–matrix interactions and the viability of
encapsulated MSCs increase in the presence of phosphate
molecules within these hydrogel scaffolds [105]. Phosphate
moieties contribute to the adsorption of osteopontin, a
sialoprotein that binds to bone mineralization and mediates
cell adhesion.
Natural materials can also be chemically altered to improve
specific properties for tissue engineering. For example, Zhang
and colleagues have created PEGylated fibrin patches to study
in vitro differentiation of MSCs into endothelial cell lineage
for potential use in myocardial repair [114]. Chitosan, a
polysaccharide, is another example of a natural biomaterial
that has been modified to be thermo-responsive [115,116] by
incorporating hydroxybutyl groups onto the polymer backbone.
The modified water soluble polymer undergoes sol–gel
transition when exposed to 37 °C [117]. This modified chitosan
has been used in encapsulation and in vitro culture of hMSCs
with the ultimate goal of developing an injectable cell-
biomaterial composite for degenerative disk diseases. MSCs
were effectively encapsulated within these chitosan gels, with
minimal cell toxicity, and showed gene expression of bone-
specific markers [117].
In addition to the supplementation of bio-chemical stimuli by
growth factors and other cytokines, it may be possible to
stimulate alterations in the micro-environment by modifying the
material surface chemistry. By altering hydrophobicity of
peptides used to create scaffolds it may be possible to encourage
a variety of cellular interactions [118]. Hydrophilicty of poly
(DL-lactide) (PDLLA), PLA, PGA and PLGA can be altered by
submerging the scaffolds in various concentrations of potassium
hydroxide [118]. As compared to non-surface treated scaffolds,
all poly(α-hydroxyl ester) scaffolds treated with potassium
hydroxide (0.1 M) were able to support a larger number of
mESCs [118] indicating that for each polymer used, there may
exist an optimized hydrophobicity that will best promote
cellular growth.
Konno et al. studied the effects of electrostatic charge on
mESCs by culturing mESCs on photoimmobilized polymers
with LIF [109]. It was found that only one polymer surface,
gelatin coupled with azidophenyl groups, improved the growth
and maintenance of mESCs [109]. The other polymers (coupled
with azidophenyl groups), poly(acrylic acid) and poly(2-
methacryloyl-oxyethyl phosphorylcholine-co-methacrylic
acid) (PMAc50) did not advance the growth of mESCs. In
fact, these polymers prevented cell adhesion resulting in EB
formation, and induced cell differentiation [109]. Carbonated
apatite surfaces may also have the potential to induce
differentiation as mESCs cultured on this material proliferated
in a differentiated state [110].
In addition to proteins, scaffolds can also operate as effective
delivery vehicles for small bioactive molecules that direct SC
growth and organization. Covalent conjugation and controlled-
release of dexamethasone, a synthetic corticosteroid crucial for
MSC osteogenesis in vitro, has been achieved through a
hydrolytically-labile lactide component incorporated within
PEG hydrogels [119]. Human MSCs were encapsulated within
these gels, which lead to their efficient osteogenic differenti-
ation. The gene expression levels for two common osteogenic
markers, ALP and core binding factor alpha 1 (Cbfa1),
significantly increased in MSCs cultured in dexamethasone-
functionalized scaffolds.
Degradable thin films may also act as an efficient delivery
vehicle of SCs [120]. Films coated with FN enabled adipose
derived SCs to adhere to poly(L-lactide-co-ɛ-caprolactone)
films as efficiently as cells adhering to tissue culture plastic
surfaces, with no significant difference in number of cells
attached even up to 24 hours after seeding [120]. The polymer
film itself degraded over a prolonged period of time, with a
sharp increase in degradation rate at week 6 [120].
Delivery of growth factors and chemicals can also be
mediated using degradable particles, entrapped within the
biomaterial scaffold, which could provide temporal release
kinetics for signaling biomolecules over a prolonged period of
time [121–124]. Osteogenic studies of rat MSCs using
recombinant human TGF-β1 encapsulated in polymer blends
of PEG-PLGA particles (sized at an average of 158 μm) has
been reported by Peter et al. [125]. Here they show that a
 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228 223

Table 1
Application of natural biomaterials in stem cell culture
Biomaterial Chemical modifications Application in stem cell culture Cell source References
Matrigel™ Cell culture applications [22,23,25]
Vascularization mADSC [26]
Fibrin Addition of growth factors Neural differentiation mESC [18,29]
Cell culture mESC [27]
Chondrogenesis hMSC
Hyaff® (hyaluron) Ligament formation Sheep MSC [32]
Hyaluron Maintenance of pluripotency hESC [31]
Addition of soluble growth factors Lineage specific differentiation
Modified with photoreactive groups Cell proliferation hESC [31]
Application of hyaluronidase Cell removal
Silk fibroin Medical suture material N/A [33–35]
Chitosan with coralline Osteogenesis Murine MSC [37]
Hydroxyapatite (HA) Osteogenesis mESC [39]
Alginate Cell encapsulation and differentiation ESC [40]
Pullulan, Dextran, and Addition of poly-L-lysine cross-linked with sodium Hepatic possible applications to vascular repair mESC [41]
Fucoidan trimetaphosphate hESC [42]
Collagen Type II Chondrogenesis hMSC [43]
Collagen microbeads Cell expansion and viability, hUCBC [44]
Hematopoiesis
Collagen Addition of HA MSC seeding and proliferation Rat MSC [74]
Addition of crosslinked chitin and PLA Cell attachment hMSC [75]
Addition of recombinant human BMP-2 (rhBMP-2) Osteogenesis Porcine MSC [96]
Silk Osteogenesis hMSC [76]
Chondrogenesis hMSC [35]
Chitosan Conjugation of hydroxybutyl groups Potential degenerative disk therapies hMSC [117]

loading of 6.0 ng TGF-β1/ mg of microparticle provided an
optimal dose for growth factor delivery for enhancing MSC
proliferation and transformation into osteoblasts [125]. Tables 1
and 2 summarizes the various natural and synthetic biomaterials
used in SC culture, their chemical modifications (as discussed in
Section 3) as well as their specific applications.
3.3. Patterned biomaterials
Recent developments in micro and nanofabrication techni-
ques have opened up a myriad of possibilities in studying and
controlling the differentiation of SCs. These techniques allow
for the controlled design of highly reproducible features on a
cellular level as well as the possibility of creating spatially and
temporally patterned scaffold structures that might ultimately
lead to generation of complex, hybrid tissue structures.
ESC lineage commitment of individual cells depends on a
number of variables. One important factor is the EB shape
and size [126]. The ability to effectively control the size of
EBs in a reproducible manner may have a large impact on the
ability to control and scale up ESC differentiation. One of the
approaches to culturing hESCs is to create a co-culture of
hESCs on top of inactivated murine embryonic fibroblasts
(MEFs). Using polydimethylsiloxane (PDMS) molds, Kha-
demhosseini et al. were able to create microwells on a glass
surface [127]. MEFs were then seeded onto the PDMS
surface and subsequently inactivated to allow for hESC seed-
ing. Results indicated that hESCs grew in highly homoge-
neous aggregates and demonstrated both high cell viability as
well as markers indicative of an undifferentiated cell state
[127].
By creating a microwell array system using photolithography
and plasma etching techniques, Mohr and colleagues were able
to create spatially uniform aggregates of undifferentiated hESCs
without the use of MEFs [128]. After culturing in microwells,
the hESCs could be removed from the microwells and further
cultured under standard conditions (plates coated with Matrigel)
showing little sign of prior differentiation [128]. EBs formed
from cells isolated from this microwell system ranged in size
from 200 to 359 μm, with a majority of EBs between 280 and
359 μm (78% of the total cell population) [128]. In comparison,
EBs that were derived on tissue culture polystyrene were more
variable in size, with only 31% of the total cell population
falling in a range of size between 280 and 359 μm in diameter
[128]. This system can be further altered by creating microwells
of different sizes and depths in order to form a variety of EB
structures allowing for the optimization of EB size [128].
Khademhosseini et al. reported micropatterned hydrogels of
hyaluronic acid, with photoreactive methacrylates [129]. A
composite of hyaluronic acid microwells was formed and
mESCs were either “docked” in these wells or encapsulated in
the hydrogel [129]. Both conditions promoted cell viability and
also illustrated how cells can be manipulated to colonize in
specific patterns and shapes based on the microarchitecture of
the wells [129].
The microenvironment comprising complex tissues includes
a variety of cellular signaling moieties presented in 3-D spatial
patterns. While many scaffolds incorporate various biomole-
cules into the scaffold construct, they do so in bulk, i.e. a
random distribution of factors throughout the scaffold. Using
LbL microfabrication and a digital micro-mirror (DMD)-based
system we have incorporated precise, pre-designed spatial
224 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228

Table 2
Application of synthetic biomaterials in stem cell culture
Biomaterial Chemical modifications Application in stem cell Cell References
culture source
poly(ɛ-caprolactone) (PCL) Cell propagation mESC [56]
Addition of adipogenic promoting factors Adipogenesis mESC [56]
Adipogenesis hMSC [54]
Chondrogenesis and Rat [55]
Osteogenesis MSC
Poly(L-lactic acid) (PLLA) Hematopoiesis mESC [50]
Polyglycolic acid (PGA), polylactic acid, Cell proliferation and 3-D hESC [49]
(PLA), polylactide-co-glycolide (PLGA) organization
Poly(ethylene glycol) diacrylates Cell and bio-chemical Goat [59]
(PEGDA) hydrogels MSC
molecule encapsulation hMSC [58]
Addition of glucosamine Chondrogenesis mESC [20]
Incorporation of RGD-PEG-acrylates Cell viability, Osteogenesis hMSC [105]
Modified with RGD Chondrogenesis hESC [106]
Release of dexamethasone Osteogenesis hMSC [119]
poly(N-isopropylacrylamide-co-acrylic acid) Incorporated into a photocrosslinked Cell self-renewal and maintenance hESC [60]
[p(NIPAAm-co-AAc)] hydrogel with a metalloproteinase sensitive peptide
PEGDA scaffolds Incorporation of methacrylic acid Osteogenesis Murine [100]
MSC
poly(6-aminohexyl phosphate acryloyl) Cell and bio-chemical Goat [57]
(PPE-HA-acryl) molecule encapsulation MSC
Polyethylene terephthalate (PET) Cell seeding, proliferation, and aggregation Rat [53]
MSC
hMSC [52]
hHSC [51]
Conjugated with FN CD34+ proliferation hHSC [51]
Poly(ethylene glycol) (PEG) hydrogel Exposed to TGF-β Chondrogenesis mESC [19]
Addition of a phosphoester Osteogenesis Goat [112]
MSC
Biphasic calcium phosphate(Triosite™) Osteogenesis hMSC [61]
Titanium Cell attachment and Rat [64]
proliferation MSC
Differentiation media Osteogenesis Rat [64,65]
MSC
Coated with RGD MSC attachment Rat [97]
MSC
Surface modification with TiO2 Cell adherence Murine [66]
MSC
Surface modification with TiO2 Cell adherence hMSC [64,67]
Tantalum (Cytomatrix) Hematopoiesis mESC [17,66,68]
PPE-HA-acrl Increase acrylated-PEG Osteogenesis Goat [57]
MSC
sIPN p(NIPAAm-coI-AAc) crosslinked with Cell propagation hESC [60]
Gln-Pro-Gln-GLY-Leu-Ala-Lys-NH2 and
functionalized with p(AAc) and RGD complexes
Oligo(PEG-fumarate) hydrogels Modified with osteopontin-derived peptide Osteoblast migration Rat [108]
MSC
Modified with RGD Cell attachment Rat [108]
MSC
Gelatin Coupled with azidophenyl groups Cell growth mESC [109]
Magnetic microbeads T cell formation mHSC [111]
Poly(α-hydroxyl ester) scaffolds Treated with potassium hydroxide Cell growth mESC [118]
Poly(L-lactide-co-ɛ-caprolactone) films Coated with FN ESC adherence hADSC [120]
PEG-PLGA polymer blends Encapsulation of recombinant human TGF-β1 MSC proliferation and Rat [125]
osteogenesis MSC
PDMS molds Seeded with MEFs Cell viability and hESC [127]
proliferation
Microwell array system Create spatially uniform hESC [128]
aggregates of undifferentiated cells
HA microwell system Cell viability, controlled cell patterning mESC [129]
and shaping
Biomaterial microarray Time efficient cell viability and hESC [130]
differentiation studies
 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
patterning of molecules into 3-D scaffolds to create complex
microenvironments more indicative of native tissues [95,100].
Using these techniques it may be possible to create a scaffold
containing regions conducive to directing a single stem
population to multiple, spatially organized tissues. By utilizing
ECM components capable of sequestering growth factors
(heparan), we demonstrated that the spatial pattern can be
maintained during the culture period. [95]. Fig. 1 provides a
schematic representation of such spatial patterning concept.
4. Concluding remarks
The use of SCs for cellular therapy offers enormous prospect
in replacing damaged or lost tissue function. By utilizing SCs,
the creation of an unlimited supply of fully differentiated cells
can be obtained, thus making cellular therapy a reality.
Ultimately there are a number of obstacles researchers must
overcome before this potential is reached. In addition to
overcoming the difficulty of obtaining a therapeutically
meaningful number of cells, a large variety of in vivo and in
vitro work is left to be done prior to the application of these cells
on a clinical level. Specifically, the safety of using SCs,
especially when considering the usage of allogenic or even
xenogenic donor cells must be carefully established before
using SC therapy in a clinical setting [11].
The cell microenvironment is known to play a significant
role in determining progenitor cell fate and function. The
precise coordination of interpreting spatial and temporal cues
from their microenvironment is highly essential for SCs to
create complex, functional tissues. Advanced and high-
throughput assays, such as extracellular microarrays and other
technologies extensively reviewed by Khademhosseini et al.,
[131–133], have uncovered specific cell interactions with ECM
components and polymers that can directly stimulate SC
signaling and response. As biomaterial research advances,
new materials as well as innovations in their manipulation and
usage continue. By utilizing high throughput arrays to
distinguish biomaterial functionality the assessment of these
materials for ultimate usage can occur in short periods of time,
wasting few materials and ultimately allowing for a more rapid
end product [130]. For SC based cellular therapy to be a viable
therapeutic option, major advances in both the understanding of
the local cues necessary for lineage commitment and the
biomaterials necessary to promote differentiation is necessary.
References
[1] J.P. Vacanti, R. Langer, Tissue engineering: the design and fabrication of
living replacement devices for surgical reconstruction and transplantation,
Lancet 354 (1) (1999) SI32–SI34.
[2] P.K. Yarlagadda, M. Chandrasekharan, J.Y. Shyan, Recent advances and
current developments in tissue scaffolding, Biomed. Mater. Eng. 15 (2005)
159–177.
[3] T. Ahsan, R.M. Nerem, Bioengineered tissues: the science, the
technology, and the industry, Orthod. Craniofac. Res. 8 (2005) 134–140.
[4] A. El-Ghannam, Bone reconstruction: from bioceramics to tissue
engineering, Expert. Rev. Med. Devices 2 (2005) 87–101.
[5] R.E. Horch, J. Kopp, U. Kneser, J. Beier, A.D. Bach, Tissue engineering
of cultured skin substitutes, J. Cell. Mol. Med. 9 (2005) 592–608.
225
[6] J.Y. Lim, H.J. Donahue, Biomaterial characteristics important to skeletal
tissue engineering, J. Musculoskelet. Neuronal. Interact. 4 (2004) 396–398.
[7] J. Raghunath, H.J. Salacinski, K.M. Sales, P.E. Butler, A.M. Seifalian,
Advancing cartilage tissue engineering: the application of stem cell
technology, Curr. Opin. Biotechnol. 16 (2005) 503–509.
[8] H. Yoshikawa, A. Myoui, Bone tissue engineering with porous
hydroxyapatite ceramics, J. Artif. Organs 8 (2005) 131–136.
[9] J. Elisseeff, C. Puleo, F. Yang, B. Sharma, Advances in skeletal tissue
engineering with hydrogels, Orthod. Craniofac. Res. 8 (2005) 150–161.
[10] M.N. Rahaman, J.J. Mao, Stem cell-based composite tissue constructs for
regenerative medicine, Biotechnol. Bioeng. 91 (2005) 261–284.
[11] M. Mimeault, S.K. Batra, Concise review: recent advances on the
significance of stem cells in tissue regeneration and cancer therapies,
Stem Cells 24 (2006) 2319–2345.
[12] N.D. Evans, E. Gentleman, J.M. Polak, Scaffolds for stem cells, Materials
Today 9 (2006) 26–33.
[13] M.T. Mitjavila-Garcia, C. Simonin, M. Peschanski, Embryonic stem
cells: meeting the needs for cell therapy, Adv. Drug. Deliv. Rev. 57 (2005)
1935–1943.
[14] Y. Li, D.A. Kniss, L.C. Lasky, S.T. Yang, Culturing and differentiation of
murine embryonic stem cells in a three-dimensional fibrous matrix,
Cytotechnology 41 (2003) 23–35.
[15] C. Chai, K.W. Leong, Biomaterials approach to expand and direct
differentiation of stem cells, Molec. Ther. 15 (2007) 467–480.
[16] E. Garreta, E. Genove, S. Borros, C.E. Semino, Osteogenic differentiation
of mouse embryonic stem cells and mouse embryonic fibroblasts in a
three-dimensional self-assembling peptide scaffold, Tissue Eng. 12
(2006) 2215–2227.
[17] H. Liu, K. Roy, Biomimetic three-dimensional cultures significantly
increase hematopoietic differentiation efficacy of embryonic stem cells,
Tissue Eng. 11 (2005) 319–330.
[18] S.M. Willerth, K.J. Arendas, D.I. Gottlieb, S.E. Sakiyama-Elbert,
Optimization of fibrin scaffolds for differentiation of murine embryonic
stem cells into neural lineage cells, Biomaterials 27 (2006) 5990–6003.
[19] N.S. Hwang, M.S. Kim, S. Sampattavanich, J.H. Baek, Z. Zhang, J.
Elisseeff, Effects of three-dimensional culture and growth factors on the
chondrogenic differentiation of murine embryonic stem cells, Stem Cells
24 (2006) 284–291.
[20] N.S. Hwang, S. Varghese, P. Theprungsirikul, A. Canver, J. Elisseeff,
Enhanced chondrogenic differentiation of murine embryonic stem cells in
hydrogels with glucosamine, Biomaterials 27 (2006) 6015–6023.
[21] H. Shin, S. Jo, A.G. Mikos, Biomimetic materials for tissue engineering,
Biomaterials 24 (2003) 4353–4364.
[22] J.L. Drury, D.J. Mooney, Hydrogels for tissue engineering: scaffold
design variables and applications, Biomaterials 24 (2003) 4337–4351.
[23] D.M. Bissell, D.M. Arenson, J.J. Maher, F.J. Roll, Support of cultured
hepatocytes by a laminin-rich gel. Evidence for a functionally significant
subendothelial matrix in normal rat liver, J. Clin. Invest. 79 (1987) 801–812.
[24] H.K. Kleinman, M.L. McGarvey, L.A. Liotta, P.G. Robey, K.
Tryggvason, G.R. Martin, Isolation and characterization of type IV
procollagen, laminin, and heparan sulfate proteoglycan from the EHS
sarcoma, Biochemistry 21 (1982) 6188–6193.
[25] C. Xu, M.S. Inokuma, J. Denham, K. Golds, P. Kundu, J.D. Gold, M.K.
Carpenter, Feeder-free growth of undifferentiated human embryonic stem
cells, Nat. Biotechnol. 19 (2001) 971–974.
[26] V. Planat-Benard, J.S. Silvestre, B. Cousin, M. Andre, M. Nibbelink, R.
Tamarat, M. Clergue, C. Manneville, C. Saillan-Barreau, M. Duriez, A.
Tedgui, B. Levy, L. Penicaud, L. Casteilla, Plasticity of human adipose
lineage cells toward endothelial cells: physiological and therapeutic
perspectives, Circulation 109 (2004) 656–663.
[27] G.I. Im, Chondrogenesis from mesenchymal stem cells derived from
adipose tissue on the fibrin scaffold, Curr. Appl. Phys. 5 (2005) 438–443.
[28] S.M. Willerth, T.E. Faxel, D.I. Gottlieb, S.E. Sakiyama-Elbert, The
effects of soluble growth factors on embryonic stem cell differentiation
inside of fibrin scaffolds, Stem Cells 25 (2007) 2235–2244.
[29] H. Liu, S.F. Collins, L.J. Suggs, Three-dimensional culture for expansion
and differentiation of mouse embryonic stem cells, Biomaterials 27 (2006)
6004–6014.
226 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
[30] P. Brun, G. Abatangelo, M. Radice, V. Zacchi, D. Guidolin, D. Daga
Gordini, R. Cortivo, Chondrocyte aggregation and reorganization into
three-dimensional scaffolds, J. Biomed. Mater. Res. 46 (1999) 337–346.
[31] S. Gerecht, J.A. Burdick, L.S. Ferreira, S.A. Townsend, R. Langer, G.
Vunjak-Novakovic, Hyaluronic acid hydrogel for controlled self-renewal
and differentiation of human embryonic stem cells, Proc. Natl. Acad. Sci.
U.S.A. 104 (2007) 11298–11303.
[32] S. Cristino, F. Grassi, S. Toneguzzi, A. Piacentini, B. Grigolo, S. Santi, M.
Riccio, E. Tognana, A. Facchini, G. Lisignoli, Analysis of mesenchymal
stem cells grown on a three-dimensional HYAFF 11-based prototype
ligament scaffold, J. Biomed. Mater. Res. A 73 (2005) 275–283.
[33] S. Hofmann, H. Hagenmuller, A.M. Koch, R. Muller, G. Vunjak-
Novakovic, D.L. Kaplan, H.P. Merkle, L. Meinel, Control of in vitro
tissue-engineered bone-like structures using human mesenchymal stem
cells and porous silk scaffolds, Biomaterials 28 (2007) 1152–1162.
[34] R. Nazarov, H.J. Jin, D.L. Kaplan, Porous 3-D scaffolds from regenerated
silk fibroin, Biomacromolecules 5 (2004) 718–726.
[35] Y. Wang, U.J. Kim, D.J. Blasioli, H.J. Kim, D.L. Kaplan, In vitro cartilage
tissue engineering with 3D porous aqueous-derived silk scaffolds and
mesenchymal stem cells, Biomaterials 26 (2005) 7082–7094.
[36] Y. Wang, H.J. Kim, G. Vunjak-Novakovic, D.L. Kaplan, Stem cell-based
tissue engineering with silk biomaterials, Biomaterials 27 (2006)
6064–6082.
[37] M. Gravel, T. Gross, R. Vago, M. Tabrizian, Responses of mesenchymal
stem cell to chitosan-coralline composites microstructured using coralline
as gas forming agent, Biomaterials 27 (2006) 1899–1906.
[38] F. Zhao, W.L. Grayson, T. Ma, B. Bunnell, W.W. Lu, Effects of hydroxyapatite
in 3-D chitosan-gelatin polymer network on human mesenchymal stem cell
construct development, Biomaterials 27 (2006) 1859–1867.
[39] E. Garreta, D. Gasset, C. Semino, S. Borros, Fabrication of a three-
dimensional nanostructured biomaterial for tissue engineering of bone,
Biomol. Eng. 24 (2007) 75–80.
[40] S.K. Dean, Y. Yulyana, G. Williams, K.S. Sidhu, B.E. Tuch,
Differentiation of encapsulated embryonic stem cells after transplanta-
tion, Transplantation 82 (2006) 1175–1184.
[41] T. Maguire, E. Novik, R. Schloss, M. Yarmush, Alginate-PLL
microencapsulation: effect on the differentiation of embryonic stem
cells into hepatocytes, Biotechnol. Bioeng. 93 (2006) 581–591.
[42] N.B. Thebaud, D. Pierron, R. Bareille, C. Le Visage, D. Letourneur, L.
Bordenave, Human endothelial progenitor cell attachment to polysac-
charide-based hydrogels: a pre-requisite for vascular tissue engineering,
J. Mater. Sci., Mater. Med. 18 (2007) 339–345.
[43] C.F. Chang, M.W. Lee, P.Y. Kuo, Y.J. Wang, Y.H. Tu, S.C. Hung, Three-
dimensional collagen fiber remodeling by mesenchymal stem cells requires
the integrin–matrix interaction, J. Biomed. Mater. Res. A. 80 (2007)
466–474.
[44] H.S. Kim, J.B. Lim, Y.H. Min, S.T. Lee, C.J. Lyu, E.S. Kim, H.O. Kim,
Ex vivo expansion of human umbilical cord blood CD34+ cells in a
collagen bead-containing 3-dimensional culture system, Int. J. Hematol.
78 (2003) 126–132.
[45] M.J. Mondrinos, S. Koutzaki, E. Jiwanmall, M. Li, J.P. Dechadarevian, P.I.
Lelkes, C.M. Finck, Engineering three-dimensional pulmonary tissue
constructs, Tissue Eng. 12 (2006) 717–728.
[46] C.S. Young, H. Abukawa, R. Asrican, M. Ravens, M.J. Troulis, L.B.
Kaban, J.P. Vacanti, P.C. Yelick, Tissue-engineered hybrid tooth and
bone, Tissue Eng. 11 (2005) 1599–1610.
[47] M. Borden, M. Attawia, C.T. Laurencin, The sintered microsphere matrix
for bone tissue engineering: in vitro osteoconductivity studies, J. Biomed.
Mater. Res. 61 (2002) 421–429.
[48] A.S. Lin, T.H. Barrows, S.H. Cartmell, R.E. Guldberg, Microarchitectural
and mechanical characterization of oriented porous polymer scaffolds,
Biomaterials 24 (2003) 481–489.
[49] S. Levenberg, N.F. Huang, E. Lavik, A.B. Rogers, J. Itskovitz-Eldor, R.
Langer, Differentiation of human embryonic stem cells on three-dimensional
polymer scaffolds, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 12741–12746.
[50] S. Taqvi, K. Roy, Influence of scaffold physical properties and stromal
cell coculture on hematopoietic differentiation of mouse embryonic stem
cells, Biomaterials 27 (2006) 6024–6031.
[51] Q. Feng, C. Chai, X.S. Jiang, K.W. Leong, H.Q. Mao, Expansion of
engrafting human hematopoietic stem/progenitor cells in three-dimen-
sional scaffolds with surface-immobilized fibronectin, J. Biomed. Mater.
Res. A 78 (2006) 781–791.
[52] W.L. Grayson, T. Ma, B. Bunnell, Human mesenchymal stem cells tissue
development in 3D PET matrices, Biotechnol. Prog. 20 (2004) 905–912.
[53] Y. Takahashi, Y. Tabata, Homogeneous seeding of mesenchymal stem cells
into nonwoven fabric for tissue engineering, Tissue Eng. 9 (2003) 931–938.
[54] W.J. Li, R. Tuli, X. Huang, P. Laquerriere, R.S. Tuan, Multilineage
differentiation of human mesenchymal stem cells in a three-dimensional
nanofibrous scaffold, Biomaterials 26 (2005) 5158–5166.
[55] M. Shin, H. Yoshimoto, J.P. Vacanti, In vivo bone tissue engineering
using mesenchymal stem cells on a novel electrospun nanofibrous
scaffold, Tissue Eng. 10 (2004) 33–41.
[56] X. Kang, Y. Xie, H.M. Powell, L. James Lee, M.A. Belury, J.J. Lannutti, D.A.
Kniss, Adipogenesis of murine embryonic stem cells in a three-dimensional
culture system using electrospun polymer scaffolds, Biomaterials 28 (2007)
450–458.
[57] Q. Li, J. Wang, S. Shahani, D.D. Sun, B. Sharma, J.H. Elisseeff, K.W.
Leong, Biodegradable and photocrosslinkable polyphosphoester hydro-
gel, Biomaterials 27 (2006) 1027–1034.
[58] C.R. Nuttelman, M.C. Tripodi, K.S. Anseth, In vitro osteogenic
differentiation of human mesenchymal stem cells photoencapsulated in
PEG hydrogels, J. Biomed. Mater. Res. A 68 (2004) 773–782.
[59] C.G. Williams, T.K. Kim, A. Taboas, A. Malik, P. Manson, J. Elisseeff,
In vitro chondrogenesis of bone marrow-derived mesenchymal stem
cells in a photopolymerizing hydrogel, Tissue Eng. 9 (2003) 679–688.
[60] Y.J. Li, E.H. Chung, R.T. Rodriguez, M.T. Firpo, K.E. Healy, Hydrogels
as artificial matrices for human embryonic stem cell self-renewal,
J. Biomed. Mater. Res. A 79 (2006) 1–5.
[61] J. Toquet, R. Rohanizadeh, J. Guicheux, S. Couillaud, N. Passuti, G.
Daculsi, D. Heymann, Osteogenic potential in vitro of human bone
marrow cells cultured on macroporous biphasic calcium phosphate
ceramic, J. Biomed. Mater. Res. 44 (1999) 98–108.
[62] H. Ohgushi, A.I. Caplan, Stem cell technology and bioceramics: from cell
to gene engineering, J. Biomed. Mater. Res. 48 (1999) 913–927.
[63] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biodegradable and
bioactive porous polymer/inorganic composite scaffolds for bone tissue
engineering, Biomaterials 27 (2006) 3413–3431.
[64] M. Maeda, M. Hirose, H. Ohgushi, T. Kirita, In vitro mineralization by
mesenchymal stem cells cultured on titanium scaffolds, J. Biochem.
(Tokyo) 141 (2007) 729–736.
[65] N. Datta, Q.P. Pham, U. Sharma, V.I. Sikavitsas, J.A. Jansen, A.G. Mikos,
In vitro generated extracellular matrix and fluid shear stress synergistically
enhance 3D osteoblastic differentiation, Proc. Natl. Acad. Sci. U. S. A.
103 (2006) 2488–2493.
[66] D.S. Kommireddy, S.M. Sriram, Y.M. Lvov, D.K. Mills, Stem cell
attachment to layer-by-layer assembled TiO2 nanoparticle thin films,
Biomaterials 27 (2006) 4296–4303.
[67] M. Manso-Silvan, J.M. Martinez-Duart, S. Ogueta, P. Garcia-Ruiz, J. Perez-
Rigueiro, Development of human mesenchymal stem cells on DC sputtered
titanium nitride thin films, J. Mater. Sci., Mater. Med. 13 (2002) 289–293.
[68] H. Liu, J. Lin, K. Roy, Effect of 3D scaffold and dynamic culture
condition on the global gene expression profile of mouse embryonic stem
cells, Biomaterials 27 (2006) 5978–5989.
[69] A. Subramanian, H.Y. Lin, Crosslinked chitosan: its physical properties
and the effects of matrix stiffness on chondrocyte cell morphology and
proliferation, J. Biomed. Mater. Res. A 75 (2005) 742–753.
[70] J.M. Zaleskas, B. Kinner, T.M. Freyman, I.V. Yannas, L.J. Gibson, M.
Spector, Contractile forces generated by articular chondrocytes in collagen-
glycosaminoglycan matrices, Biomaterials 25 (2004) 1299–1308.
[71] A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs
stem cell lineage specification, Cell 126 (2006) 677–689.
[72] G.H. Altman, R.L. Horan, I. Martin, J. Farhadi, P.R. Stark, V. Volloch, J.C.
Richmond, G. Vunjak-Novakovic, D.L. Kaplan, Cell differentiation by
mechanical stress, FASEB J. 16 (2002) 270–272.
[73] C.A. Simmons, S. Matlis, A.J. Thornton, S. Chen, C.Y. Wang, D.J.
Mooney, Cyclic strain enhances matrix mineralization by adult human
 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
mesenchymal stem cells via the extracellular signal-regulated kinase
(ERK1/2) signaling pathway, J. Biomech. 36 (2003) 1087–1096.
[74] L. Liu, L. Zhang, B. Ren, F. Wang, Q. Zhang, Preparation and charac-
terization of collagen-hydroxyapatite composite used for bone tissue
engineering scaffold, Artif. Cells Blood Substit. Immobil. Biotechnol. 31
(2003) 435–448.
[75] X. Li, Q. Feng, W. Wang, F. Cui, Chemical characteristics and
cytocompatibility of collagen-based scaffold reinforced by chitin fibers for
bone tissue engineering, J. Biomed. Mater. Res. B Appl. Biomater. 77 (2006)
219–226.
[76] L. Meinel, R. Fajardo, S. Hofmann, R. Langer, J. Chen, B. Snyder, G.
Vunjak-Novakovic, D. Kaplan, Silk implants for the healing of critical
size bone defects, Bone 37 (2005) 688–698.
[77] L. Meinel, S. Hofmann, V. Karageorgiou, L. Zichner, R. Langer, D.
Kaplan, G. Vunjak-Novakovic, Engineering cartilage-like tissue using
human mesenchymal stem cells and silk protein scaffolds, Biotechnol.
Bioeng. 88 (2004) 379–391.
[78] L. Meinel, V. Karageorgiou, S. Hofmann, R. Fajardo, B. Snyder, C. Li, L.
Zichner, R. Langer, G. Vunjak-Novakovic, D.L. Kaplan, Engineering
bone-like tissue in vitro using human bone marrow stem cells and silk
scaffolds, J. Biomed. Mater. Res. A 71 (2004) 25–34.
[79] M.E. Gomes, C.M. Bossano, C.M. Johnston, R.L. Reis, A.G. Mikos,
In vitro localization of bone growth factors in constructs of bio-
degradable scaffolds seeded with marrow stromal cells and cultured in
a flow perfusion bioreactor, Tissue Eng. 12 (2006) 177–188.
[80] H.L. Holtorf, N. Datta, J.A. Jansen, A.G. Mikos, Scaffold mesh size affects
the osteoblastic differentiation of seeded marrow stromal cells cultured in a
flow perfusion bioreactor, J. Biomed. Mater. Res. A 74 (2005) 171–180.
[81] H.L. Holtorf, J.A. Jansen, A.G. Mikos, Flow perfusion culture induces
the osteoblastic differentiation of marrow stroma cell-scaffold constructs
in the absence of dexamethasone, J. Biomed. Mater. Res. A 72 (2005)
326–334.
[82] S. Battista, D. Guarnieri, C. Borselli, S. Zeppetelli, A. Borzacchiello, L.
Mayol, D. Gerbasio, D.R. Keene, L. Ambrosio, P.A. Netti, The effect of
matrix composition of 3D constructs on embryonic stem cell differen-
tiation, Biomaterials 26 (2005) 6194–6207.
[83] G. Bianchi, A. Banfi, M. Mastrogiacomo, R. Notaro, L. Luzzatto, R.
Cancedda, R. Quarto, Ex vivo enrichment of mesenchymal cell progenitors
by fibroblast growth factor 2, Exp. Cell Res. 287 (2003) 98–105.
[84] I. Martin, A. Muraglia, G. Campanile, R. Cancedda, R. Quarto, Fibroblast
growth factor-2 supports ex vivo expansion and maintenance of osteogenic
precursors from human bone marrow, Endocrinology 138 (1997)
4456–4462.
[85] L.A. Solchaga, K. Penick, J.D. Porter, V.M. Goldberg, A.I. Caplan, J.F.
Welter, FGF-2 enhances the mitotic and chondrogenic potentials of
human adult bone marrow-derived mesenchymal stem cells, J. Cell.
Physiol. 203 (2005) 398–409.
[86] S. Tsutsumi, A. Shimazu, K. Miyazaki, H. Pan, C. Koike, E. Yoshida, K.
Takagishi, Y. Kato, Retention of multilineage differentiation potential of
mesenchymal cells during proliferation in response to FGF, Biochem.
Biophys. Res. Commun. 288 (2001) 413–419.
[87] A.H. Reddi, N.S. Cunningham, Initiation and promotion of bone
differentiation by bone morphogenetic proteins, J. Bone Miner. Res.
8 (2) (1993) S499–S502.
[88] J.M. Wozney, The bone morphogenetic protein family and osteogenesis,
Mol. Reprod. Dev. 32 (1992) 160–167.
[89] M.E. Levenstein, T.E. Ludwig, R.H. Xu, R.A. Llanas, K. VanDenHeuvel-
Kramer, D. Manning, J.A. Thomson, Basic fibroblast growth factor support
of human embryonic stem cell self-renewal, Stem Cells 24 (2006) 568–574.
[90] R.H. Xu, R.M. Peck, D.S. Li, X. Feng, T. Ludwig, J.A. Thomson, Basic
FGF and suppression of BMP signaling sustain undifferentiated
proliferation of human ES cells, Nat. Methods 2 (2005) 185–190.
[91] H.J. Rippon, J.M. Polak, M. Qin, A.E. Bishop, Derivation of distal lung
epithelial progenitors from murine embryonic stem cells using a novel
three-step differentiation protocol, Stem Cells 24 (2006) 1389–1398.
[92] M. Nakanishi, T.S. Hamazaki, S. Komazaki, H. Okochi, M. Asashima,
Pancreatic tissue formation from murine embryonic stem cells in vitro,
Differentiation 75 (2007) 1–11.
227
[93] J.A. Jansen, J.W. Vehof, P.Q. Ruhe, H. Kroeze-Deutman, Y. Kuboki, H.
Takita, E.L. Hedberg, A.G. Mikos, Growth factor-loaded scaffolds for
bone engineering, J. Control. Release 101 (2005) 127–136.
[94] T.P. Richardson, M.C. Peters, A.B. Ennett, D.J. Mooney, Polymeric system
for dual growth factor delivery, Nat. Biotechnol. 19 (2001) 1029–1034.
[95] G. Mapili, Y. Lu, S. Chen, K. Roy, Laser-layered microfabrication of
spatially patterned functionalized tissue-engineering scaffolds, J. Biomed.
Mater. Res. B Appl. Biomater. 75 (2005) 414–424.
[96] M.H. Carstens, M. Chin, X.J. Li, In situ osteogenesis: regeneration of 10-cm
mandibular defect in porcine model using recombinant human bone
morphogenetic protein-2 (rhBMP-2) and Helistat absorbable collagen
sponge, J. Craniofac. Surg. 16 (2005) 1033–1042.
[97] H.L. Holtorf, J.A. Jansen, A.G. Mikos, Ectopic bone formation in rat
marrow stromal cell/titanium fiber mesh scaffold constructs: effect of
initial cell phenotype, Biomaterials 26 (2005) 6208–6216.
[98] Y. Takahashi, M. Yamamoto, Y. Tabata, Enhanced osteoinduction by
controlled release of bone morphogenetic protein-2 from biodegradable
sponge composed of gelatin and beta-tricalcium phosphate, Biomaterials
26 (2005) 4856–4865.
[99] X.B. Yang, H.I. Roach, N.M. Clarke, S.M. Howdle, R. Quirk, K.M.
Shakesheff, R.O. Oreffo, Human osteoprogenitor growth and differen-
tiation on synthetic biodegradable structures after surface modification,
Bone 29 (2001) 523–531.
[100] Y. Lu, G. Mapili, G. Suhali, S. Chen, K. Roy, A digital micro-mirror device-
based system for the microfabrication of complex, spatially patterned tissue
engineering scaffolds, J. Biomed. Mater. Res. A 77 (2006) 396–405.
[101] B.K. Brandley, R.L. Schnaar, Covalent attachment of an Arg-Gly-Asp
sequence peptide to derivatizable polyacrylamide surfaces: support of fibro-
blast adhesion and long-term growth, Anal. Biochem. 172 (1988) 270–278.
[102] D.L. Hern, J.A. Hubbell, Incorporation of adhesion peptides into
nonadhesive hydrogels useful for tissue resurfacing, J. Biomed. Mater.
Res. 39 (1998) 266–276.
[103] U. Hersel, C. Dahmen, H. Kessler, RGD modified polymers: biomaterials for
stimulated cell adhesion and beyond, Biomaterials 24 (2003) 4385–4415.
[104] R.L. Schnaar, B.G. Langer, B.K. Brandley, Reversible covalent
immobilization of ligands and proteins on polyacrylamide gels, Anal.
Biochem. 151 (1985) 268–281.
[105] C.R. Nuttelman, M.C. Tripodi, K.S. Anseth, Synthetic hydrogel niches
that promote hMSC viability, Matrix Biol. 24 (2005) 208–218.
[106] N.S. Hwang, S. Varghese, Z. Zhang, J. Elisseeff, Chondrogenic
differentiation of human embryonic stem cell-derived cells in arginine-
glycine-aspartate-modified hydrogels, Tissue Eng. 12 (2006) 2695–2706.
[107] H. Shin, J.S. Temenoff, G.C. Bowden, K. Zygourakis, M.C. Farach-
Carson, M.J. Yaszemski, A.G. Mikos, Osteogenic differentiation of rat
bone marrow stromal cells cultured on Arg-Gly-Asp modified hy-
drogels without dexamethasone and beta-glycerol phosphate, Bioma-
terials 26 (2005) 3645–3654.
[108] H. Shin, K. Zygourakis, M.C. Farach-Carson, M.J. Yaszemski, A.G. Mikos,
Attachment, proliferation, and migration of marrow stromal osteoblasts
cultured on biomimetic hydrogels modified with an osteopontin-derived
peptide, Biomaterials 25 (2004) 895–906.
[109] T. Konno, N. Kawazoe, G. Chen, Y. Ito, Culture of mouse embryonic stem
cells on photoimmobilized polymers, J. Biosci. Bioeng. 102 (2006) 304–310.
[110] A.J. Melville, J. Harrison, K.A. Gross, J.S. Forsythe, A.O. Trounson, R.
Mollard, Mouse embryonic stem cell colonisation of carbonated apatite
surfaces, Biomaterials 27 (2006) 615–622.
[111] S. Taqvi, L. Dixit, K. Roy, Biomaterial-based notch signaling for the
differentiation of hematopoietic stem cells into T cells, J. Biomed. Mater.
Res. A 79 (2006) 689–697.
[112] D.A. Wang, C.G. Williams, F. Yang, N. Cher, H. Lee, J.H. Elisseeff,
Bioresponsive phosphoester hydrogels for bone tissue engineering,
Tissue Eng. 11 (2005) 201–213.
[113] J. Wang, H.Q. Mao, K.W. Leong, A novel biodegradable gene carrier
based on polyphosphoester, J. Am. Chem. Soc. 123 (2001) 9480–9481.
[114] G. Zhang, X. Wang, Z. Wang, J. Zhang, L. Suggs, A PEGylated fibrin
patch for mesenchymal stem cell delivery, Tissue Eng. 12 (2006) 9–19.
[115] J.H. Cho, S.H. Kim, K.D. Park, M.C. Jung, W.I. Yang, S.W. Han, J.Y.
Noh, J.W. Lee, Chondrogenic differentiation of human mesenchymal
228 E. Dawson et al. / Advanced Drug Delivery Reviews 60 (2008) 215–228
stem cells using a thermosensitive poly(N-isopropylacrylamide) and
water-soluble chitosan copolymer, Biomaterials 25 (2004) 5743–5751.
[116] E. Ruel-Gariepy, M. Shive, A. Bichara, M. Berrada, D. Le Garrec, A.
Chenite, J.C. Leroux, A thermosensitive chitosan-based hydrogel for the
local delivery of paclitaxel, Eur. J. Pharm. Biopharm. 57 (2004) 53–63.
[117] J.M. Dang, D.D. Sun, Y. Shin-Ya, A.N. Sieber, J.P. Kostuik, K.W. Leong,
Temperature-responsive hydroxybutyl chitosan for the culture of mesen-
chymal stem cells and intervertebral disk cells, Biomaterials 27 (2006)
406–418.
[118] J. Harrison, S. Pattanawong, J.S. Forsythe, K.A. Gross, D.R. Nisbet, H.
Beh, T.F. Scott, A.O. Trounson, R. Mollard, Colonization and
maintenance of murine embryonic stem cells on poly(alpha-hydroxy
esters), Biomaterials 25 (2004) 4963–4970.
[119] C.R. Nuttelman, M.C. Tripodi, K.S. Anseth, Dexamethasone-functiona-
lized gels induce osteogenic differentiation of encapsulated hMSCs, J.
Biomed. Mater. Res. A 76 (2006) 183–195.
[120] C.A. Burks, K. Bundy, P. Fotuhi, E. Alt, Characterization of 75:25 poly
(L-lactide-co-epsilon-caprolactone) thin films for the endoluminal
delivery of adipose-derived stem cells to abdominal aortic aneurysms,
Tissue Eng. 12 (2006) 2591–2600.
[121] T.A. Holland, Y. Tabata, A.G. Mikos, Dual growth factor delivery from
degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds for
cartilage tissue engineering, J. Control. Release 101 (2005) 111–125.
[122] Z. Lalani, M. Wong, E.M. Brey, A.G. Mikos, P.J. Duke, Spatial and
temporal localization of transforming growth factor-beta1, bone
morphogenetic protein-2, and platelet-derived growth factor-A in
healing tooth extraction sockets in a rabbit model, J. Oral Maxillofac.
Surg. 61 (2003) 1061–1072.
[123] H. Park, J.S. Temenoff, T.A. Holland, Y. Tabata, A.G. Mikos, Delivery of
TGF-beta1 and chondrocytes via injectable, biodegradable hydrogels for
cartilage tissue engineering applications, Biomaterials 26 (2005)
7095–7103.
[124] M.C. Wake, P.D. Gerecht, L. Lu, A.G. Mikos, Effects of biodegradable
polymer particles on rat marrow-derived stromal osteoblasts in vitro,
Biomaterials 19 (1998) 1255–1268.
[125] S.J. Peter, L. Lu, D.J. Kim, G.N. Stamatas, M.J. Miller, M.J. Yaszemski,
A.G. Mikos, Effects of transforming growth factor beta1 released from
biodegradable polymer microparticles on marrow stromal osteoblasts
cultured on poly(propylene fumarate) substrates, J. Biomed. Mater. Res.
50 (2000) 452–462.
[126] E.S. Ng, R.P. Davis, L. Azzola, E.G. Stanley, A.G. Elefanty, Forced
aggregation of defined numbers of human embryonic stem cells into
embryoid bodies fosters robust, reproducible hematopoietic differentia-
tion, Blood 106 (2005) 1601–1603.
[127] A. Khademhosseini, L. Ferreira, J. Blumling III, J. Yeh, J.M. Karp, J.
Fukuda, R. Langer, Co-culture of human embryonic stem cells with
murine embryonic fibroblasts on microwell-patterned substrates, Bioma-
terials 27 (2006) 5968–5977.
[128] J.C. Mohr, J.J. de Pablo, S.P. Palecek, 3-D microwell culture of human
embryonic stem cells, Biomaterials 27 (2006) 6032–6042.
[129] A. Khademhosseini, G. Eng, J. Yeh, J. Fukuda, J. Blumling III, R. Langer, J.
A. Burdick, Micromolding of photocrosslinkable hyaluronic acid for cell
encapsulation and entrapment, J. Biomed. Mater. Res. A 79 (2006) 522–532.
[130] D.G. Anderson, S. Levenberg, R. Langer, Nanoliter-scale synthesis of
arrayed biomaterials and application to human embryonic stem cells, Nat.
Biotechnol. 22 (2004) 863–866.
[131] D.G. Anderson, D. Putnam, E.B. Lavik, T.A. Mahmood, R. Langer,
Biomaterial microarrays: rapid, microscale screening of polymer–cell
interaction, Biomaterials 26 (2005) 4892–4897.
[132] C.J. Flaim, S. Chien, S.N. Bhatia, An extracellular matrix microarray for
probing cellular differentiation, Nat. Methods 2 (2005) 119–125.
[133] A. Khademhosseini, R. Langer, J. Borenstein, J.P. Vacanti, Microscale
technologies for tissue engineering and biology, Proc. Natl. Acad. Sci. U. S. A.
103 (2006) 2480–2487.