Kenneth E. Gonsalves, Craig R. Halberstadt, Cato T. Laurencin, Lakshmi S. Nair. Biomedical Nanostructures

Подождите немного. Документ загружается.

modulate signaling pathways. This group of complex carbohydrates is

classiﬁed based on the nature of their repeating disaccharide units and

includes chondroitin sulfate, dermatan sulfate, heparan sulfate, hyaluronan,

and keratan sulfate [57]. Due to their high sulfate content and/or the presence

of uronic acid, GAGs bare many negative charges that enable them to

associate with numerous ligands by electrostatic interactions. All GAGs are

found in tissues as covalently attached components of proteoglycans, with the

exception of hyaluronan. Hyaluronan is unique since it is secreted directly

into the extracellular space by the cell membrane bound enzyme hyaluronan

synthase and is able to function as a f ree carbohydrate [58, 59]. It is ﬂexible,

bending and twisting into many conformations forming random coils, and

can self-associate to form networks. It has a strong afﬁnity to water due to the

large number of anionic residues on its surface and can bind cations by COO



groups on its surface. Additionally, it can form superaggregates with a

number of proteoglycans including aggrecan.

9.2.2.3 Proteoglycans Proteoglycans are a diverse group of molecules

that are abundant in the ECM and expressed on cell surfaces. They represent

a glycoprotein subset in which structure and function are mediated by protein

cores and covalently linked GAG side chains. Numerous peptide motifs exist

within these molecules and many of these are often found in the same protein

core including epidermal growth factor (EGF) repeats, hyaluronan-binding,

immunoglobulin-like, leucine-rich repeats, and sugar-binding lectin domains

[60]. Several classes are present in the ECM and these may be divided into

two subfamilies based on the typical features of the protein core arrangement

(Table 9.2) [61].

The ﬁrst family, the small leucine-rich proteoglycans, includes proteoglycans

like biglycan, decorin, ﬁbromodulin, and lumican. While the second family, the

modular proteoglycans, can be further divided into the nonhyaluronate-

binding proteoglycans like perlecan and agrin and the hyaluronan/lectin-

binding proteoglycans like aggrecan, versican, and CD44 [2]. This diversity in

protein core domains and GAG side chains enables proteogl ycans to have a

high afﬁnity to binding of various ligands and accounts for many speciﬁc

interactions in the matrix. Perlecan, for instance, is embedded in basement

membranes and gives them a ﬁxed negative electrostatic charge, which is

responsible in part for their charge-selective ﬁltration properties [63]. Whereas

the small leucine-rich proteoglycan, decorin, can bind and regulate the

ﬁbrillogenesis of collagen [64, 65], other proteoglycans, like heparan sulfate

proteoglycans, participate in ﬁbroblast growth factor (FGF) signaling by

directly interacting with FGFs and its receptors on the cell surface,

subsequently increasing FGF’s afﬁnity for its associated receptors [57, 66].

9.2.2.4 Glycoproteins With the ability to organize collagens, elastins,

proteoglycans, and cells into ordered structures, glycoproteins are also essential

building blocks of the ECM. These macromolecules vary in size, structure, and

236 BIOMEDICAL NANOSTRUCTURES

TABLE 9.2 General Properties of Selected Proteoglycans

Proteoglycan

Protein core

(kDa)

GAGs

(number)

Representative

tissue Function

Small leucine-rich proteoglycans

Biglycan 3840 Chondroitin

sulfate/

dermatan

sulfate (2)

Muscle, bone,

cartilage,

epithelia

Associated

with cell

surfaces

Decorin 3640 Chondroitin

sulfate/

dermatan

sulfate (1)

Collagenous

matrices, bone,

teeth

Binds collagen

ﬁbrils and

modiﬁes

their assembly;

binds to

TGF-beta

Fibromodulin 4243 Keratan

sulfate (24)

Cartilage,

skin, tendon

Binds collagen

I and II;

reduces size

of collagen

ﬁbrils

Modular proteoglycans

Nonhyaluron

an binding

Agrin 200250 Heparan

sulfate (3)

Synaptic

sites of

neuromuscular

junctions,

renal

basement

membranes

Involved in

the aggregation

of acetylcholine

receptors

Perlecan 400467 Chondroitin

sulfate/

heparan (3)

Basement

membranes,

cell surfaces,

cartilage

Self-associates;

binds laminin

in basement

membranes;

binds basic

ﬁbroblast

growth

factor

Hyaluronan

and

lectin

binding

Aggrecan 220250 Chondroitin

sulfate (>150)

and Keratin

sulfate

(100

150)

Cartilage,

brain,

blood

Binds

hyaluronan,

hydrates and

ﬁlls

ECM

Versican 260370 Chondroitin

sulfate/

dermatan

sulfate

(1030)

Blood

vessels,

brain,

cartilage,

skin

May bind

hyaluronan

Data complied from multiple references [2, 40, 61, 62].

ECM INTERACTIONS WITH CELLS FROM THE MACRO- TO NANOSCALE 237

distribution and are composed of a protein and covalently linked oligosac-

charide(s). Glycoproteins are made up of multiple subunits colored with an

assortment of distinct peptide motifs that interact with not only themselves, but

also cells and other matrix proteins [6]. They may exist as large molecules with

extended conformations that span distances of several hundred nanometers. A

variety of glycoproteins are found in the ECM including ﬁbronectin and

laminin.

Fibronectin is one of the best studied glycoproteins, has multiple

isoforms, and is composed of three modules, repeating motifs of type I, II,

and III, separated by connecting sequences [67]. It is a large glycoprotein

with numerous binding sites for collagens, proteoglycans, and cell surface

receptors like integrins (Fig. 9.1). The type III module is the most abundant

module in its structure and includes many aspartate-containing sequences

such as RGD, which mediate cellular adhesion through integrins [68, 69].

Fibronectin is critical for numerous cellular functions and is one of the

earliest macromolecules to be laid down in the ECM. It is required for the

assembly of several ECM proteins like collagen I [70] and plays a role in

regulating the incorporation of morphogens and growth factors into the

ECM [71]. Its distribution in areas of skeletogenesis suggests that it may be

involved in early stages of bone development [72] as well as continued

mature bone homeostasis by regulating the differentiation and survival of

osteoblasts [73].

Laminins are the primary adhesive matrix proteins in the basement

membrane, exist in several isoforms, and are large heterotrimeric glycoproteins

constituted by three genetically distinct polypeptide alpha, beta, and gamma

FIGURE 9.1 Schematic representation of ﬁbronectin dimer. Depicts individual modules

as well as a variety of binding domains specialized for particular macromolecules.

Alternate splicing accounts for the differences in the two similar chains, which are joined

by two disulﬁde bonds near the C-termini.

238

BIOMEDICAL NANOSTRUCTURES

chains that assemble to form a cruciform structure with globular domains at

each arm end (Fig. 9.1) [74, 75]. They contain multiple binding domains that

participate in numerous biological activities (network and ﬁlament formation,

cell binding through cell surface receptors, and binding to other matrix

proteins in supramolecular assemblies), with the ECM structure-forming

domains generally located on the short arms of all three subunits and cell-

interacting domains mapped onto the N- and C-terminal domains of the alpha

subunits [74]. For example, within the ba sement membrane, all three short

arms (alpha1, beta1, and gamma1) of laminin-1 are involved in laminin self-

assembly [76], while binding with entactin/nidogen is restricted to the short arm

region of the gamma1 chain [77], and integrin cell surface receptors to the

laminin G-domain-like (LG) modules of the alpha subunits [78, 79]. Such

binding interactions are essential for proper assembly and function of the

basement membrane, where type IV collagen, laminins, and entactins are

thought to be the primary basement membrane scaffold, ﬁbrous two-

dimensional supramolecular networks where entactin binds the laminin and

collagen networks with stable noncovalent bridges [78, 80].

9.2.3 Putting It All Together—Hierarchical Assembly

Hierarchy of scales is apparent in the biological structures formed during tissue

development and homeostasis. Within the ECM, the multifunctional nature of

matrix components is a direct reﬂection of their modular and hierarchical

architecture. The mult iple binding domains, chemistry, and topology of the

various ECM constituents help regulate matrix assembly, stabilize ECM

architecture, and modulate cellmatrix interactions. Some examples include

ﬁbrous structures that provide strength, elasticity, or cell permissivity,

proteoglycans that act as reservoirs for morphogens or regulate osmotic

pressure, and glycoproteins that stabilize ﬁbrous structures or act as binding

sites for cells. All of these structures combine together to give tissues their

specialized properties. The signiﬁcance of this relationship between ECM

hierarchy and function is apparent in tissues like articular cartilage.

Articular cartilage is an avascular tissue containing a relatively sparse number

of chondrocyte cells that covers the subchondral bone in diarthrodial joints [22].

In association with synovial ﬂuid, it forms a wear-resistant, friction-reduced,

load-bearing surface only a few millimeters thick [81, 82]. Its matrix and cells are

organized into distinct zones: superﬁcial, middle, deep, and calciﬁed zones

(Fig. 9.2) and these zones exhibit variations in biochemical composition, cell

morphology, and cellmatrix structural organization [81, 83].

The superﬁcial zone consists of a densely packed collagen ﬁbrillar network

with ﬂattened chondrocytes that are alig ned tangentially with the articulat-

ing surface [84]. Collagen ﬁbril alignment within this zone enables the

accommodation of high shear and tensile forces and type II collagen form s the

bulk of the ﬁbrils. Microﬁbril assembly is mediated in part by type XI collagen

and decorin with the former regulating lateral growth and the latter regulating

ECM INTERACTIONS WITH CELLS FROM THE MACRO- TO NANOSCALE 239

ﬁbril diameter [41]. Fibrils are thinnest in this zone and measure approximately

20 nm in diameter [83].

The middle zone represents a transitional region from the tangential ﬁber

orientation of the superﬁcial zone to the radial ﬁber orientation of the deep

FIGURE 9.2 Hierarchical organization of articular cartilage over different length

scales. Articular cartilage forms a wear-resistant, load-bearing surface that covers bone

in diarthrodial joints (a). It is organized into distinct zones (b), where the organization

of the collagen structures varies between zones (c). Resident chondrocytes are encased in

pericellular regions (d), which are surrounded by a well-deﬁned matrix nanoarchitecture

of aggrecan/hyaluronic acid superaggregates and macroﬁbrillar collagen networks (e).

240

BIOMEDICAL NANOSTRUCTURES

zone. Cells exhibit a round morphology and their density is lower than that in

the superﬁcial zone [85, 86]. The ECM within this region is aggrecan rich and

the collagen ﬁbrils are larger in diameter, randomly arranged, and loosely

packed [84, 87].

Within the deep zone, the collagen ﬁbers are perpendicularly oriented

with the subchondral bone and attain a maximum diameter size that ranges

from 70 to 12 0 nm [83]. Cell density is lowest in this region [86], whereas

aggrecan content is at a maximum. Cartilage compressive stiffness is provided

by the swelling pressure of hydrated aggregates of aggrecan and hyaluronan

bound to the porous collagen II structure within this zone, where swelling is

restricted by the collagen network. This hydrated ﬁbrous proteoglycan

structure, in conjunction with synovial ﬂuid and articulating joint movements,

allows nutrients to circulate and nourish cells [24].

The calciﬁed zone provides a buffer region between the uncalciﬁed

cartilage and subchondral bone. The chondrocytes that reside in this region

are in a hypertrophic phenotypic state. These cells secrete the hexagonal sheet

forming collagen type X and participate in calcifying the ECM within this

region [83].

Additionally, within the different zones a pericellular matrix region,

approximately 2 mm wide, surrounds the chondrocytes [83]. This region varies

in its structure compared to that of the surrounding matrix. Type VI collagen

and decorin are concentrated in this region and assemble to form a

microﬁlamentous network. The cell surface proteoglycan perlecan is also

present within this region. It cross-links with ECM components such as type VI

collagen, thereby linking cell surfaces with matrix organization.

Therefore, the highly specialized composi tion and structure of articular

cartilage has evolved to enable joint mobility and gives an excellent example of

the relationship between the ECM hierarchy and tissue function.

9.3 CELLECM INTERACTIONSTHE MULTIDIMENSIONAL MAP

Beyond its role in providing structural support and organization for resident

cells and imparting tissues with speciﬁc biofunctional properties, the ECM also

serves in an instructive capacity. A vast amount of information is encoded in

the matrix architecture and this forms a multidimensional map (Fig. 9.3). Cells

use this map to guide their behavior. The information within it is expressed in

the form of signaling gradients, which may exist as chemical distributions or

differential expressions of ECM components. Cell surface receptors interpret

these gradie nts and respond accordingly with directionality being dictated by

signaling gradient changes. A bidirectional ﬂow of information between the

cells and the ECM ensues where changes in the local microenvironment result

in dynamic releases of information. This map may be perceived in a variety of

different ways and its context is dependent on external events as well as speciﬁc

cell types.

ECM INTERACTIONS WITH CELLS FROM THE MACRO- TO NANOSCALE 241

9.3.1 Signaling Gradients

In order to direct cells into the desired mode of behavior, signaling cues need to

be display ed within an appropriate context. This requires both spatial and

temporal positioning of signals from different sources. Consequently, each cell

type has evolved to produce their own network of precisely encoded ECM

proteins. This enables the conﬁguration of the structural properties and

information content of distinct ECM microenvironments. The three-dimen-

sional matrix architecture assembled from ECM constituents regulates the

spatial and temporal signals that are derived from localized biochemical

distributions and mechanical forces. Biochemically, the ECM interacts with

cells to provide information on the microenvironment through soluble and

insoluble effectors, while physically it achieves this action through structural

and mechanical restraints. The mechanisms to establish gradients of signaling

molecules within the matrix architecture coincide with the deposition of ECM

constituents. This may involve sequestering effector molecules from cells,

secreting them in activated or deactivated states, or varying concentrations

within the local microenvironment. Autoregulation of these mechanisms

occurs through incremental changes in which different levels of regulation help

maintain a homeostatic balance between ECM remodeling and signaling

molecule release during processes like morphogenesis and tissue remodeling.

A variety of extrinsic factors control cellular behavior. The major sources of

these external signals are soluble factors, ECM components, environmental

stresses, and cellcell interactions (Fig. 9.4) [88]. The modulation and

FIGURE 9.3 Multidimensional tissue map. By providing signaling gradients to cells,

natural tissues effectively provide multiple behavioral maps, whose context is dependent

on external events. These gradients present themselves in the form of different signaling

molecules or binding opportunities that result from changes in the ECM through

processes such as stretching and proteolytic cleavage. (Modiﬁed from Reference 7 with

permission from AAAS).

242

BIOMEDICAL NANOSTRUCTURES

presentation of these external factors within the local ECM microenvironments

is of equal importance to the presence of the external signals themselves. This

chapter, however, will primarily focus on those sources associated with ECM

structures.

9.3.2 Soluble Factors

Secreted by cells, soluble factors (Fig. 9.4) feed into co mplex and overlapping

signaling pathways that often share common receptors. The modulation of

cellular activity by soluble factors is complex. Not only is the presence of the

factors required, but they must be present at the appropriate times,

concentrations, and ratios. These secreted molec ules have a tendency to

diffuse great distances, which potentially makes them unavailable or available

in ineffective concentrations [89]. Consequently, a number of mechanisms have

evolved to modulate their spatial and temporal distributions. These mechan-

isms include the production of molecules in an inactive form such as

Transforming growth factor TGF-beta [90], which activates only at the

appropriate site of action; the requirement of cofactors such as heparan sulfate

for recept or activation [57], and the localization of effector molecules like Ca

by bind ing to speciﬁc matrix constituents [91]. A direct or indirect relationship

FIGURE 9.4 Extrinsic factors inﬂuencing cellular behavior. Four major sources of

external signals regulate cell behavior: soluble factors, ECM components, environ-

mental stresses, and cellcell interactions. Soluble factors include growth factors,

cytokines, morphogenetic proteins, peptides, hormones, proteases, and ions. ECM

components include proteins, GAGs, proteoglycans, and glycoproteins. Environmental

stresses include mechanical forces (dynamic and static), oxygen tension, and pH effects.

Cellcell interactions can be either homophilic or heterophilic and are regulated by cell

adhesion molecules. These extrinsic factors in turn extend profound inﬂuences on gene

expression and cell behavior, forming a dynamic cell microenvironment essential for

tissue development and function.

ECM INTERACTIONS WITH CELLS FROM THE MACRO- TO NANOSCALE 243

can exist between ECM components and many of these soluble factors. For

instance, interactions with GAGs and proteoglycans can lead to dynamic

concentrations of free signaling molecules [88]. Additionally, signaling

synergies often exist with ECMsoluble factor interactions. For example,

cell adhesion has been shown to enhance autophosphorylation of the EGF

receptor in response to its associated ligand [92]. Of the numerous soluble

factors, growth factors are indicative of the necessity for spatial and temporal

distribution in the modulation of cellular activities.

9.3.3 Growth Factors

Growth factors are specialized polypeptide molecules that bind to cell surface

receptors and trigger intracellular signaling pathways [12]. The binding of growth

factors to ECM structures is a major mechanism for regulating growth factor

activity. Growth factors are essential for establishing signaling gradients during

matrix synthesis. ECMgrowth factor association enables the storage of large

quantities of signals in readily available form. This association with matrix

structures means the signals can prevent or slow down diffusion, thereby

decreasing the loss of information [93]. Additionally, ECMgrowth factor

binding enables the transmission of signals to cells in contact with the same matrix

region later, which is essential for the development of complex cellular structures.

Not all growth factors found wi thin the matrix architecture are in an active

state. This inertness may be attributed to a variety of factors. The growth

factors may be concealed within the matrix only to be revealed during matrix

degradation or cell movement. For instance, proteolysis of ECM-bound

vascular endothelial growth factor (VEGF) by matrix metalloproteinase

MMP-9 releases and increases VEGF bioavailability [94]. Similarly, an

epidermal growth factor-like domain DIII, released throu gh cleavage of

laminin-5 by MMP-2, stimulates cell migration and changes in gene expression

only upon its degradation [95]. A group effect may also occur where multiple

signals from the matrix counteract each other and signaling activity would

depend on the ratios of various growth factors (i.e. biological activity woul d

require perturbations). For example, during angiogenesis, antiangiogenic

factors dominate until the release of angiogenic growth factors by ECM

proteinases causes a proangiogenic environment to ensue [96–100]. Finally, the

growth factors can be latent with limited biological activity. For instance,

TGF-beta is secreted by the majority of cells as latent complexes that are

unable to interact with their corresponding signaling receptors until activated

by processes like proteolysis [101].

Growth factor activity is prim arily controlled by two biochemical

mechanisms, release from the matrix architecture and activation. Release

involves the dissociation of growth factors from matrix binding sites, while

activation involves the dissociation of mature growth factors from dormant

proteins or the induction of conformal changes that allow its binding to

244 BIOMEDICAL NANOSTRUCTURES

receptors [93]. The release of growth factors from the matrix into biologically

active form may occur via proteolytic enzymes. For example, cell surfa ce

localized matrix metalloproteinases such as MMP-9 and MMP-2 can release

and activate TGF-beta by proteolytically cleaving its latent form [102]. This

proteolytic release and activation of stored growth factors generates rapid

localized signals. Proteolytic modulation is faster than sim ple control of gene

expression, which is of particular importance in wound healing and immune

system control [93].

9.3.4 ECM Components

ECM components can serve as landmarks for cells by leaving spatial and

temporal cues. As discussed earlier, as cells migrate through the microenviron-

ments within developing tissue, each with different ECM landmarks, they

modulate their behavior accordingly, adjusting gene expression in response to

the environmental cues. This creates a feedback loop as the responding cells

also inﬂuence the surrounding ECM by differentially expressing particular

ECM components and/or proteases. The stage- and tissue-speciﬁc assembly of

matrix components therefore requires an intricate interplay between the

residing cells and the signaling cues. With respect to the ECM components,

these cues may emanate from the molecular composition, that is, multiple

binding domains that may be apparent on the protein surface or concealed as

cryptic sites or from the surface topography.

9.3.4.1 Binding Domains Numerous proteins are expressed in the ECM

and each contains multiple motifs. These motifs are made up of speciﬁc amino

acid sequences that present multiple binding sites for cell interactions with many

distinct cell surface receptors. These interactions may directly or indirectly

inﬂuence cellular activities such as adhesion, migration, proliferation, differ-

entiation, and apoptosis. For instance, alpha5-beta1 integrin cell surface

receptors bind directly to RGD sites on ﬁbronectin molecules (10th type III

repeating unit) [103], whereas tenascin indirectly interferes with cellular adhesion

by binding to ﬁbronectin [104], thus altering its ability to promote cell adhesion.

The spatial proximity and regularity of binding domains has also been found to

elicit different cellular responses. The migration of ﬁbroblasts has been found to

be a function of the average surface binding site density where cell speed varies

with the spatial presentation of RGD binding sites and affected cell adhesion

[105]. Colocalization of synergistic binding sites can also modulate cellular

responses. For example, the synergistic Pro-His-Ser-Arg-Asn (PHSRN) site in

the central cell-adhesive domain of ﬁbronectin (9th type III repeating unit),

although itself not biologically active, has been found to substantially enhance

the cell-adhesive activity mediated by the alpha5-beta1 integrin cell surface

receptor to its associated RGD binding site (10th type III repeating unit) [106].

The spacing between the PHSRN and RGD sites in the native conformation of

ﬁbronectin is approximately 4 nm [107].

ECM INTERACTIONS WITH CELLS FROM THE MACRO- TO NANOSCALE 245