Fisher John P. e.a. (ed.) Tissue Engineering
Подождите немного. Документ загружается.
mikos: “9026_c002” — 2007/4/16 — 21:43 — page4—#4
2-4 Tissue Engineering
2.6 Clinical Applications
Recombinant BMP 2 has been approved by the Food and Drug administration for spine fusion and open
fractures of tibia due to orthopaedic trauma. There have been several clinical applications of BMPs in
orthopaedic surgery [25–29]. The developing experience of BMPs will be of immense utility to the nascent
field of tissue engineering, the science of design-based manufacture of spare parts for human skeleton
based on signals, stem cells, and scaffolding [1,30] in medicine and dentistry. A prototype paradigm
has validated the proof of principle for tissue engineering based on tissue transformation by BMPs and
scaffolding [31].
2.7 Challenges and Opportunities
Despite the exciting advances in clinical applications of BMPs there remain many challenges. Foremost
among them is the need for developing synthetic scaffolds to deliver recombinant BMPs for skeletal tissue
engineering. The development of synthetic scaffolds with an ability to respond to biomechanical influences
that are known to be critical for musculoskeletal structures will lead to a quantum improvement of current
tissue engineering approaches to bone, cartilage, and meniscus. The remaining challenges make the field
of morphogen-based tissue engineering an exciting frontier with unlimited opportunities.
Acknowledgments
The research in the Center for Tissue Regeneration is supported by the Lawrence Ellision Chair in Mus-
culoskeletal Molecular Biology and grants from NIH and DOD. I thank Danielle Neff for the outstanding
help in completion of this article.
References
[1] Reddi, A.H., Role of morphogenetic proteins in skeletal tissue engineering and regeneration, Nat.
Biotechnol., 16, 247, 1998.
[2] Reddi, A.H. and Anderson, W.A., Collagenous bone matrix-induced endochondral ossification
hemopoiesis, J. Cell Biol., 69, 557, 1976.
[3] Weiss, R.E. and Reddi, A.H., Synthesis and localization of fibronectin during collagenous matrix-
mesenchymal cell interaction and differentiation of cartilage and bone in vivo, Proc. Natl Acad. Sci.
USA, 77, 2074, 1980.
[4] Reddi, A.H., Cell biology and biochemistry of endochondral bone development, Coll. Relat. Res.,1,
209, 1981.
[5] Sampath, T.K. and Reddi, A.H., Dissociative extraction and reconstitution of extracellular matrix
components involved in local bone differentiation, Proc. Natl Acad. Sci. USA, 78, 7599, 1981.
[6] Sampath, T.K. and Reddi, A.H., Homology of bone-inductive proteins from human, monkey,
bovine, and rat extracellular matrix, Proc. Natl Acad. Sci. USA, 80, 6591, 1983.
[7] Ma, S., Chen, G., and Reddi, A.H., Collaboration between collagenous matrix and osteogenin is
required for bone induction, Ann. NY Acad. Sci., 580, 524, 1990.
[8] Reddi, A.H., Extracellular matrix and development, in Extracellular Matrix Biochemistry, Piez, K.A.
and Reddi, A.H. Eds., Elsevier, New York, 1984, p. 247.
[9] Sampath, T.K., Wientroub, S., and Reddi, A.H., Extracellular matrix proteins involved in bone
induction are vitamin D dependent, Biochem. Biophys. Res. Commun., 124, 829, 1984.
[10] Wientroub, S. and Reddi, A.H., Influence of irradiation on the osteoinductive potential of
demineralized bone matrix, Calcif. Tissue Int., 42, 255, 1988.
[11] Wozney, J.M. et al., Novel regulators of bone formation: molecular clones and activities, Science,
242, 1528, 1988.
mikos: “9026_c002” — 2007/4/16 — 21:43 — page5—#5
Growth Factors and Morphogens 2-5
[12] Luyten, F.P. et al., Purification and partial amino acid sequence of osteogenin, a protein initiating
bone differentiation, J. Biol. Chem., 264, 13377, 1989.
[13] Celeste A.J. et al., Identification of transforming growth factor beta family members present in
bone-inductive protein purified from bovine bone, Proc. Natl Acad. Sci. USA, 87, 9843, 1990.
[14] Ozkaynak, E. et al., OP-1 cDNA encodes an osteogenic protein in the TGF-beta family, EMBO J., 9,
2085, 1990.
[15] Wang, E.A. et al., Recombinant human bone morphogenetic protein induces bone formation, Proc.
Natl Acad. Sci. USA, 87, 2220, 1990.
[16] Chen, P. et al., Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human
bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor beta 1
and beta 2, Exp. Cell Res., 195, 509, 1991.
[17] Yamaguchi, A. et al., Recombinant human bone morphogenetic protein-2 stimulates osteoblastic
maturation and inhibits myogenic differentiation in vitro, J. Cell Biol., 113, 681, 1991.
[18] Cunningham, N.S., Paralkar, V., and Reddi, A.H., Osteogenin and recombinant bone morphogen-
etic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor
beta 1 mRNA expression, Proc. Natl Acad. Sci. USA, 89, 11740, 1992.
[19] Luyten, F.P. et al., Natural bovine osteogenin and recombinant human bone morphogenetic
protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant
cultures, J. Biol. Chem., 267, 3691, 1992.
[20] Lietman, S.A. et al., Stimulation of proteoglycan synthesis in explants of porcine articular cartilage
by recombinant osteogenic protein-1 (bone morphogenetic protein-7), J. Bone Joint Surg. Am., 79,
1132, 1997.
[21] Khouri, R.K., Koudsi, B., and Reddi, A.H. M Tissue transformation into bone invivo a potential
practical application, JAMA, 266, 1953, 1991.
[22] ten Dijke, P. et al., Identification of type I receptors for osteogenic protein-1 and bone
morphogenetic protein-4, J. Biol. Chem., 269, 16985, 1994.
[23] Imamura T. et al., Smad6 inhibits signalling by the TGF-beta superfamily (see comments), Nature,
389, 622, 1997.
[24] Paralkar, V.M. et al., Interaction of osteogenin, a heparin binding bone morphogenetic protein,
with type IV collagen, J. Biol. Chem., 265, 17281, 1990.
[25] Einhorn, T.A., Clinical applications of recombinantBMPs: early experience and future development,
J. Bone Joint Surg., 85A, 82, 2003.
[26] Li, R.H. and Wozney, J.M. Delivering on the promise of bone morphogenetic proteins, Trends
Biotechnol., 19, 255, 2001.
[27] Ripamonti, U., Ma, S., and Reddi A.H. The critical role of geometry of porous hydroxyapatite
delivery system in induction of bone by osteogenin, a bone morphogenetic protein, Matrix, 12,
202, 1992.
[28] Geesink, R.G., Hoefnagels, N.H., and Bulstra, S.K. Osteogenic activity of OP1, bone morphogenetic
protein 7 (BMP 7) in a human fibular defect, J. Bone Joint Surg., 81, 710, 1999.
[29] Friedlaender, G.E. et al., Osteogenic protein 1 (bone morphogenic protein 7) in the treatment of
tibial non-unions, J. Bone Joint Surg. Am., 83A, S151, 2001.
[30] Govender et al., Recombinant human bone morphogenetic protein 2 for treatment of open tibiol
fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J. Bone
Joint Surg. Am., 84A, 2123, 2002.
[31] Nakashima, M. and Reddi, A.H. The application of bone morphogenetic protein to dental tissue
engineering, Nat. Biotechnol., 21, 1025, 2003.
[32] Khouri, R.K., Koudsi, B., and Reddi, A.H. Tissue transformation into bone in vivo: a potential
practical application. JAMA, 266, 1953, 1991.
mikos: “9026_c002” — 2007/4/16 — 21:43 — page6—#6
mikos: “9026_c003” — 2007/4/16 — 21:46 — page1—#1
3
Extracellular Matrix:
Structure, Function,
and Applications to
Tissue Engineering
Mary C. Farach-Carson
Roger C. Wagner
Kristi L. Kiick
University of Delaware
3.1 Introduction.............................................. 3-1
3.2 ECM and Functional Integration of Implanted
Materials .................................................. 3-2
3.3 Basement Membranes and Focal Adhesions ........... 3-2
3.4 Focal Adhesions as Signaling Complexes............... 3-4
3.5 ECMand Skeletal Tissues ............................... 3-5
3.6 Sources of ECM for Tissue Engineering Applications. 3-7
3.7 Properties of ECM ....................................... 3-8
3.8 Mining the ECM for Functional Motifs ................ 3-8
Collagen • Fibronectin • Laminin • Tenascin,
Thrombospondin, and Osteonectin/SPARC/BM-40 •
Proteoglycans and Glycosaminoglycans • Osteopontin
3.9 Summary of Functions of ECM Molecules ............ 3-10
3.10 Polymeric Materials and their Surface Modification .. 3-11
3.11 Formation of Gradient Structures ...................... 3-13
3.12 Delivery of Growth Factors ............................. 3-14
3.13 Summary and Conclusions ............................. 3-16
Acknowledgments............................................... 3-16
References ....................................................... 3-16
3.1 Introduction
A major goal of tissue engineering is to employ the principles of rational design to recreate appropriate
signals to cells that promote biological processes leading to production of new tissues or repair of damaged
ones. A key modulator of cell behavior is the ECM that provides individual cells with architectural
cues of time and space, modulates bioavailability of soluble growth and differentiation factors, and
organizes multicellular tissue development. This chapter will focus on key ECM components and their
functions, emphasizing relationships between natural matrices present in both hard and soft tissues and
cell function. The concept of “mining” the natural matrix for active motifs that are useful in tissue
3-1
mikos: “9026_c003” — 2007/4/16 — 21:46 — page2—#2
3-2 Tissue Engineering
engineering applications also is introduced. Finally, the utility of translating knowledge gained from
study of native ECM to controlled delivery of growth factors and deliberate modulation of cell and tissue
phenotype for engineering purposes is discussed.
3.2 ECM and Functional Integration of Implanted Materials
Cells sense and respond to a variety of signals that include those that are soluble such as growth factors,
differentiation factors, cytokines, and ion gradients. In addition, cell behavior and phenotype is governed
by responses to other types of signals that include mechanical forces, electrical stimuli, and various physical
cues. Immobilized protein matrices that generally are fixed in space also regulate cell function. The general
term that has come to denote the complex mixture of proteins on the outside of cells that governs their
behavior is ECM. Evolution has provided cells with surface receptors to ECM components that enable
them to recognize and decipher the signals that they encounter from the ECM and which influence cell
growth, division, and differentiation [1].
For descriptive purposes, cell adhesion is classified into categories of cell–substratum and cell–cell
attachment. Cell–cell interactions may occur between like cells (homotypic events) or between dissimilar
cells (heterotypic). Homotypic interactions stabilize epithelia, which typically lie upon a sheet of special-
ized ECM called the basement membrane, discussed in Section 3.2. Heterotypic interactions govern many
normal cellular phenomena including embryo–uterine implantation, immune surveillance, cell migration
during embryogenesis, and neurotransmission. They also characterize the pathological states of cancer
metastasis, rejection of transplanted tissues and organs, and inflammation [2,3]. In the context of tissue
engineering, both types of cellular interactions must be understood, and thus hopefully manipulated,
to ensure successful integration of transplanted materials.
There exists in tissues an exquisite balance between the anabolic process of ECM production and
the catabolic process of ECM turnover. Introduction of foreign materials inevitably disrupts this natural
homeostasis. A goal of tissue engineering is to successfully introduce replacement tissues that will, through
stimulation of anabolic processes, lead to ECM production and acceptance of engineered materials rather
than their immediate or eventual destruction via activation of catabolic pathways. To facilitate biointeg-
ration, both degradable and nondegradable foreign materials can be modified with native ECM or motifs
derived from proteins in the ECM. Achievement of this goal requires a thorough understanding of the
structural relationships among molecules in the ECM, their molecular interactions directing cell adhesion
events, their biosynthesis and turnover, and their natural functions.
3.3 Basement Membranes and Focal Adhesions
Basement membranes, also called basal lamina, are sheets of highly organized ECM that are associated
with the basal membrane of epithelial cells, around muscle cells, below endothelial cells in blood vessels,
supporting fat cells, and associated with Schwann cells surrounding peripheral nerve axons [4,5]. In
addition to physically separating the cell layers comprising epithelia and stroma, they provide a diversity
of functions including structural support, selective filtration, and serve as barriers to invasion. They
also establish cell polarity, influence cell metabolism, induce cell differentiation, direct cell migration, and
organize membrane receptors. Figure 3.1 depicts scanning and transmission (SEM and TEM, respectively)
electron micrographs of natural basement membranes illustrating their typical functions and structural
roles in separating cells in tissues. Note that cells contact the basement membrane on a single face that
establishes cell polarity and creates the basal surface of attaching cell. The basement membrane exists as
a complex meshwork comprised of the proteins collagen IV, laminin, proteoglycans including perlecan,
agrin, and collagens XV and XVIII, fibulin, BM-40 (SPARC), and nidogen/entactin [4]. The spatial
relationships among these molecules were examined using atomic force microscopy where thin, threadlike
strands of heparan sulfate attached to certain proteoglycans were seen to protrude from the core meshwork
and proposed to function as an “entropic brush” that could filter proteins by their constant thermal
mikos: “9026_c003” — 2007/4/16 — 21:46 — page3—#3
Extracellular Matrix: Structure and Function 3-3
(a) (b)
(c)
FIGURE 3.1 (a) Transmission electron micrograph of a transverse section through a blood capillary consisting of a
single endothelial cell wrapped around to join upon itself to create a lumen. The endothelial cell is (as are all epithelia)
bordered by a basement membrane on its external aspect. The basement membrane is shared by pericapillary cells
called “pericytes” that are represented by the small bits of seemingly isolated cell material at the capillary periphery.
(b) High magnification TEM of the glomerular basement membrane separating the capillaries of the kidney glomerulus
and the “podocytes” on their surface. The capillary endothelium (on the right) contains apparent gaps which in three
dimensions represent “fenestrae” or “little windows” in the capillary wall. The podocyte “pedicles” on the left actually
are part of larger cell processes out of the plane of section. This panel highlights the function of the basement membrane
as part of a filtration barrier which sieves large molecules and those with cationic charges. (c) A SEM of the ciliated
respiratory epithelium of the trachea. Part of the epithelium has been denuded during preparation (bottom) to reveal
meshwork collagen IV fibers of the underlying basement membrane and connective tissue underneath it.
motion within the basement membrane [5]. Collagen IV and laminin within the basement membrane
form two overlapping polymeric networks [6,7] with which the other components interact in a highly
organized fashion. The basement membrane provides an anchor for cells, which adhere to it using specific
surface receptors called integrins [1]. Focal adhesions are the sites of cell–ECM attachment that were first
identified ultrastructurally as cell surface electron dense regions near sites of cell attachment to substrata
and then recognized as interfaces with the cytoskeleton [8]. Focal adhesions belong to the contractile
class of matrix contacts, distinct from protrusive contacts or those that provide mechanical support [9].
Figure 3.2 diagrams a cartoon of a focal adhesion consisting of both integrin and nonintegrin receptors
in the plasma membrane attached to ECM in the basement membrane. The processes of anchoring and
spreading of cells on substrata occur as multistep processes that involve, among other things, clustering
of surface receptors at sites of focal adhesions. Protein complexes link the cytoplasmic tails of surface
receptors to the cytoskeleton, facilitating cell adhesion and transmitting signals through the intracellular
network that ultimately signal to the nucleus to inform the cell that it has attached. This form of signaling
has come to be called “outside in” signaling and is a key component of engineering functional interfaces
between materials and living cells. It is important to note that cells seldom rely on one adhesion system
mikos: “9026_c003” — 2007/4/16 — 21:46 — page4—#4
3-4 Tissue Engineering
Integrin receptor
Nonintegrin receptor
Substratum/basement membrane
ECM
Plasma membrane
Linker proteins
Actin cytoskeleton
Surface
receptors
Continuum
FIGURE 3.2 Focal adhesion schematic. The diagram illustrates the components of the focal adhesion attaching
an adherent cell to an ECM substratum. Note the continuum of functional connections between the ECM and
the cytoskeleton, bridged by both integrin and nonintegrin transmembrane receptors.
to support cell attachment, a concept thought to confer some degree of protection to cells against single
mutations that would completely abolish adhesion-competence. Nonetheless, mutations in key adhesion
molecules found in focal adhesions frequently have extreme and adverse consequences on tissue and
organ development [10]. Guided tissue regeneration with artificial two-dimensional matrices such as
GORE-TEX® (expanded polytetrafluoroethylene or ePTFE) that resemble basement membranes has been
widely used for treatment of periodontal and bone defects and relies upon the membrane for physical
separation of cell layers during healing [11].
3.4 Focal Adhesions as Signaling Complexes
Attachment of cells to substrata provides signals for spreading, migration, survival, and proliferation.
As mentioned above, cell surface receptors recognize ECM components in a manner that is both specific
and reversible. The initial attraction and attachment often involves nonintegrin adhesion events that
may be related solely to charge or hydrophobic interactions between surfaces [12,13]. Examples include
recognition of polyanionic polymers such as those presented by the heparan sulfate or chondroitin sulfate
glycosaminoglycans (GAGS) attached to surface proteoglycans. Depending on the nature of the molecules
involved, these adhesive events may or may not be dependent on the presence of divalent cations such
as Ca
2+
[14–16]. In any case, these early events tend to rely on multiple, low affinity interactions similar
to the way that Velcro® functions as a two-sided hook and loop fastener. Once cells adhere, higher
affinity interactions such as those involving integrin receptors are stabilized during the spreading phase
of adhesion. A very interesting feature of integrin interactions with ECM proteins is that they display a
wide range of ligand affinities ranging from 10
−6
to 10
−9
l/M under different conditions [17]. It is this
quality that ensures reversibility of integrin-mediated cell adhesion and allows cells to both attach and
detach from biological substrata at sites of focal adhesions. In general, cells attach to differentiate and
carry out specialized cell functions such as secretion, absorption, or signal transmission. They detach
in order to migrate and proliferate. Interestingly, many epithelia undergo a form of programmed cell
death known as anoikis if forcibly detached from their substrata [18]. Elegant studies of migrating cells
have shown simultaneous formation of focal adhesions with substrata on the leading edge of the cell, and
dissolution of attachment sites on the trailing end [19].
Receptor clustering during adhesion triggers cascades of events involving protein phosphorylation and
dephosphorylation that carry intracellular signals through the cell from surface to nucleus, ultimately
leading to changes in gene transcription. Frequently, changes in cell shape mediated by the cytoskeleton
accompany and promote these signals. An emerging paradigm for signal transduction involves the forma-
tion of protein complexes or “signalosomes” held together by specific interactions of regulatory molecules
mikos: “9026_c003” — 2007/4/16 — 21:46 — page5—#5
Extracellular Matrix: Structure and Function 3-5
with scaffolding proteins that, like the electron transport chain of the mitochondrion, reduce diffusion
and speed up signal transmission. The interested reader is referred to one of several recent reviews on this
exciting subject [20–22].
Once activated, feedback loops within cells act quickly to attenuate signals, preventing desensitization
to extracellular signals and in some cases preserving cell viability. For example, prolonged Ca
2+
signals are
associated with activation of apoptotic pathways and cell death [23]. These loops also allow cells to return
to a responsive state in which they can respond to subsequent signals that they may encounter from the
ECM. Changes that occur within cells and that feed back to modulate the activity of surface receptors have
come to be called “inside out” signals and modulate activity of both integrin and nonintegrin receptors
in focal adhesions.
3.5 ECM and Skeletal Tissues
The ECM in cartilage is frequently described as part of a pericellular or “territorial” matrix that is both
avascular and noninnervated. Figure 3.3 depicts several views illustrating the architecture of cartilage at
the cellular level. Note that, unlike cells interacting with basement membranes, cells in mesenchyme are
surrounded on all sides by the territorial matrix. This is particularly evident in Figure 3.3c showing a single
chondrocyte in its lacunae. The chondrocyte has a very unique relationship with its microenvironment that
has been well described [24]. The cartilage matrix consists of type II collagen and various noncollagenous
(a)
(c)
(b)
FIGURE 3.3 (a) Section through hyaline cartilage of the trachea. Dark staining regions surrounding cells (chon-
drocytes) are pericellular or “territorial” matrix and represent most recently-deposited cartilage matrix. (b) SEM of
hyaline cartilage showing lacunae or “little lakes” in which chondrocytes reside. In this preparation some are occupied
by cells and others are empty because the cells have fallen out during cutting of the cartilage. The fibrous nature of the
matrix (due to the presence of type II collagen) is evident. (c) TEM of a chondrocyte within a lacuna. Collagen fibers
can be seen in the cartilage matrix surrounding the lacunae. These fibers are not seen in light microscopic preparations
such as those in panel a since they have the same refractive index as the collagen matrix material.
mikos: “9026_c003” — 2007/4/16 — 21:46 — page6—#6
3-6 Tissue Engineering
ECM molecules that differ depending on the type and differentiated state of cartilage. In articular cartilage,
type II, IX, and XI collagens form a meshwork that provides form and tensile stiffness and strength [25].
Type VI collagen is present in the matrix surrounding the chondrocyte; aggrecan provides resilience to
compressive forces, and small proteoglycans such as fibromodulin help to stabilize the matrix. Other
noncollagenous proteins that are present include cartilage oligomeric matrix protein (COMP), perlecan,
anchorin II, and tenascin. It has been proposed that the presence of tenascin-C in the articular cartilage
matrix, a protein which is absent from growth plate cartilage, may help articular chondrocytes avoid
endochondral ossification [26]. The role of the cartilage ECM in tissue engineering was reviewed recently
[27]. In the growth plate, a progressive series of events occur to produce a cartilaginous matrix that
gives way to bone matrix [28–30]. Early stages are marked by high rates of cell proliferation and ECM
production. The final stage of development of growth plate cartilage is hypertrophy, marked by turnover
of matrix, mineralization, invasion of marrow vasculature, and chondrocyte apoptosis. Type X collagen
is the classical marker for hypertrophic cartilage ECM, and is absent from articular cartilage [31].
The ECM of bone is distinct from that of cartilage. Bone tissue (Figure 3.4a) is greater than 95%
type I collagen based and includes noncollagenous proteins such as the acidic calcium binding proteins
osteocalcin, bone sialoprotein, osteopontin, and osteonectin/SPARC. Of these, osteocalcin and bone sialo-
protein can be considered specific to bone. It has been proposed that compact lamellar bone and woven
trabecular bone have distinct matrix protein compositions and ratios [32]. The marrow compartment
(Figure 3.4b) can be considered as structurally distinct from proper bone, although it houses cellular
precursors for bone cells. As shown in Figure 3.4c,d, osteocytes, like chondrocytes, are surrounded by
and embedded in matrix, but in this case the matrix normally remains fully mineralized. The processes
(a)
(c) (d)
(b)
FIGURE 3.4 (a) Transverse cut through the metaphysis of a calf femur. The bone shaft is compact bone and spongy
bone with bony trabeculae lining the interior. (b) Cross-section through a decalcified preparation of a fetal femur.
Compact bone encloses the marrow cavity. (c) Preparation of compact bone. Concentric lamellae of Haversian systems
surround blood vessels. Interstitial lamellae represent older Haversian systems replaced during bone remodeling.
(d) High magnification image of a compact bone preparation. India ink fills lacunae in which embedded bone cells
(osteocytes) are normally found. Channels connecting lacunae are called “canaliculi.” These are occupied by cell
processes and provide a means whereby osteocytes communicate with one another.
mikos: “9026_c003” — 2007/4/16 — 21:46 — page7—#7
Extracellular Matrix: Structure and Function 3-7
of bone remodeling that maintain structural integrity of bone and include a finely tuned balance between
osteoclastic bone resorption and osteoblastic bone formation have been reviewed recently [33]. ECM is
one of many influences on bone cell function that also include growth factors, physical stimuli, metabolic
demands, and structural responsibilities [34].
3.6 Sources of ECM for Tissue Engineering Applications
It is critical to choose an ECM that will support cell adhesion, and also ensure survival, growth,
and appropriate cell differentiation following adhesion. A number of alternatives exist for the tissue
engineer including commercial mixed matrices based upon the composition of basement membranes
(collagen IV, laminin, perlecan, nidogen/entactin). One example of such a commercial matrix is Mat-
rigel™ that is available in both growth factor replete and depleted forms. This matrix is extracted from
Engelbreth–Holm–Swam (EHS) mouse sarcoma and provides a material similar to the mammalian base-
ment membrane. Available from the global nonprofit ATCC (http://www.atcc.org/) is an ECM solution
also representing a solubilized basement membrane solution (30-2501) similar to Matrigel. This prepar-
ation contains tissue plasminogen activator as well as a number of growth factors including transforming
growth factor β, epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor 2, and
platelet derived growth factor. Both these products form a gel at room temperature that will support
growth and differentiation of cells grown on (or in) it as a two- or three-dimensional matrix.
Serum also provides a variety of adhesion factors including fibronectin and vitronectin, along with a
rich selection of soluble, circulating growth factors. The response of cells to serum components is highly
variable, as is the composition of serum itself. Because of the uncertainty in knowing the exact composition
of serum for use in tissue engineering applications, batch testing of serum for long-term applications is
encouraged when its use is necessitated. Purified ECM molecules provide a more reliable and predictable
source for engineering applications. Smaller molecules can be expressed as recombinant proteins in either
bacteria, insect, or mammalian cells in bioreactors [35–37]. Each of these expression systems provides a
set of advantages and disadvantages that have been reviewed [38]. For ECM molecules of large size or
complexity (collagens, many proteoglycans, noncollagenous glycoproteins), problems can occur related
to size, microheterogeneity, yield, and scalability. For most of the molecules in this class, function requires
posttranslational modifications that can only be acquired when expressed by mammalian cells. In this
case, some luck has been achieved by expressing recombinant constructs in cell lines such as the HEK-293
cell line or even in tissue systems [37,39]. For very large matrix molecules, such as perlecan, that are too
large to be expressed as full length recombinant proteins, it has been possible to create smaller expression
constructs that retain functions of individual protein domains [37]. Of interest, there appears to be
considerable cell type diversity in the manner and extent of posttranslational modification affecting the
fine structure of the carbohydrate chains. For example, domain I of perlecan that contains consensus
sites for both heparan and chondroitin sulfate chain addition can be produced in both active and inactive
forms depending on the cells producing it [40].
An alternative to the use of recombinant ECM proteins is the use of synthetic peptides or proteolytic
fragments of native molecules that retain selected functional properties of their parent molecules. Many
ECM molecules contain a consensus sequence recognized by integrin receptors that consists of the triplet
peptide, arginine–glycine–aspartate (RGD) sequence. The context in which this peptide is presented by
the ECM molecule containing it determines the strength and the specificity of the interaction. Examples
of common ECM molecules containing the RGD sequence include fibronectin, osteopontin, laminin,
vitronectin, some collagens, and tenascin. Current effort is focused on identifying other active motifs
present within ECM molecules that are small enough to be prepared as synthetic proteins. One promising
example of this is the laminin-derived cell binding YIGSR sequence that has been proposed for derivat-
ization of scaffolds for breast and soft tissue reconstruction [41]. Another is the tetrapeptide sequence
REDV which simulates an active motif in the CS5 domain of fibronectin [42]. This subject is discussed
further in Section 3.7 devoted to mining of the ECM.