Fisher John P. e.a. (ed.) Tissue Engineering

Подождите немного. Документ загружается.

mikos: “9026_c004” — 2007/4/9 — 15:50 — page 16 — #16

4-16 Tissue Engineering

[72] Gudi, S.R. et al., Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac

ﬁbroblasts, Am. J. Physiol., 274, C1424, 1998.

[73] Duncan, R.L. and Turner, C.H., Mechanotransduction and the functional response of bone to

mechanical strain, Calcif. Tissue Int., 57, 344, 1995.

[74] McIntire, L.V., 1992 ALZA Distinguished lecture: bioengineering and vascular biology, Ann.

Biomed. Eng., 22, 2, 1994.

[75] Papadaki, M. and Eskin, S.G., Effects of ﬂuid shear stress on gene regulation of vascular cells,

Biotechnol. Prog., 13, 209, 1997.

[76] Patrick, C.W., Jr. and McIntire, L.V., Shear stress and cyclic strain modulation of gene expression

in vascular endothelial cells, Blood Purif., 13, 112, 1995.

[77] Skalak, T.C. and Price, R.J., The role of mechanical stresses in microvascular remodeling,

Microcirculation, 3, 143, 1996.

[78] Liu, S.Q., Biomechanical basis of vascular tissue engineering, Crit. Rev. Biomed. Eng., 27, 75,

1999.

[79] Chien, S., Li, S., and Shyy, Y.J., Effects of mechanical forces on signal transduction and gene

expression in endothelial cells, Hypertension, 31, 162, 1998.

[80] Davies, P.F. and Tripathi, S.C., Mechanical stress mechanisms and the cell. An endothelial

paradigm, Circ. Res., 72, 239, 1993.

[81] Chen, B.P. et al., DNA microarray analysis of gene expression in endothelial cells in response to

24-h shear stress, Physiol. Genom., 7, 55, 2001.

[82] Davies, P.F. et al., The convergence of haemodynamics, genomics, and endothelial structure in

studies of the focal origin of atherosclerosis, Biorheology, 39, 299, 2002.

[83] Garcia-Cardena, G. et al., Biomechanical activation of vascular endothelium as a determinant of

its functional phenotype, Proc. Natl Acad. Sci. USA, 98, 4478, 2001.

[84] McCormick, S.M. et al., DNA microarray reveals changes in gene expression of shear stressed

human umbilical vein endothelial cells, Proc. Natl Acad. Sci. USA, 98, 8955, 2001.

[85] McCormick, S.M. et al., Microarray analysis of shear stressed endothelial cells, Biorheology, 40, 5,

2003.

[86] Segev, O. et al., CMF608-a novel mechanical strain-induced bone-speciﬁc protein expressed in

early osteochondroprogenitor cells, Bone, 34, 246, 2004.

[87] Lee, R.T. et al., Mechanical strain induces speciﬁc changes in the synthesis and organization of

proteoglycans by vascular smooth muscle cells, J. Biol. Chem., 276, 13847, 2001.

[88] Karjalainen, H.M. et al., Gene expression proﬁles in chondrosarcoma cells subjected to cyclic

stretching and hydrostatic pressure. A cDNA array study, Biorheology, 40, 93, 2003.

[89] Carinci, F. et al., Titanium–cell interaction: analysis of gene expression proﬁling, J. Biomed. Mater.

Res., 66B, 341, 2003.

[90] Schwarz, G. et al., Shear stress-induced calcium transients in endothelial cells from human

umbilical cord veins, J. Physiol., 458, 527, 1992.

[91] Segurola, R.J., Jr. et al., Cyclic strain is a weak inducer of prostacyclin synthase expression in bovine

aortic endothelial cells, J. Surg. Res., 69, 135, 1997.

[92] Rosales, O.R. et al., Exposure of endothelial cells to cyclic strain induces elevations of cytosolic

+ concentration through mobilization of intracellular and extracellular pools, Biochem. J.,

326, 385, 1997.

[93] Reich, K.M. and Frangos, J.A., Effect of ﬂow on prostaglandin E2 and inositol trisphosphate levels

in osteoblasts, Am. J. Physiol., 261, C428, 1991.

[94] Geiger, R.V. et al., Flow-induced calcium transients in single endothelial cells: spatial and temporal

analysis, Am. J. Physiol., 262, C1411, 1992.

[95] Shen, J. et al., Regulation of adenine nucleotide concentration at endothelium–ﬂuid interface by

viscous shear ﬂow, Biophys. J., 64, 1323, 1993.

[96] Kuchan, M.J. and Frangos, J.A., Role of calcium and calmodulin in ﬂow-induced nitric oxide

production in endothelial cells, Am. J. Physiol., 266, C628, 1994.

mikos: “9026_c004” — 2007/4/9 — 15:50 — page 17 — #17

Mechanical Forces on Cells 4-17

[97] Frangos, J.A. et al., Flow effects on prostacyclin production by cultured human endothelial cells,

Science, 227, 1477, 1985.

[98] Donahue, S.W., Donahue, H.J., and Jacobs, C.R., Osteoblastic cells have refractory periods for

ﬂuid-ﬂow-induced intracellular calcium oscillations for short bouts of ﬂow and display multiple

low-magnitude oscillations during long-term ﬂow, J. Biomech., 36, 35, 2003.

[99] Hung, C.T. et al., Real-time calcium response of cultured bone cells to ﬂuid ﬂow, Clin. Orthop.,

256, 1995.

[100] Lee, M.S. et al., Effects of shear stress on nitric oxide and matrix protein gene expression in human

osteoarthritic chondrocytes in vitro, J. Orthop. Res., 20, 556, 2002.

[101] Osanai, T. et al., Cross talk between prostacyclin and nitric oxide under shear in smooth muscle

cell: role in monocyte adhesion, Am. J. Physiol. Heart Circ. Physiol., 281, H177, 2001.

[102] Sharma, R. et al., Intracellular calcium changes in rat aortic smooth muscle cells in response to

ﬂuid ﬂow, Ann. Biomed. Eng., 30, 371, 2002.

[103] Yellowley, C.E. et al., Effects of ﬂuid ﬂow on intracellular calcium in bovine articular chondrocytes,

Am. J. Physiol., 273, C30, 1997.

[104] Smalt, R. et al., Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not

mechanical strain, Am. J. Physiol., 273, E751, 1997.

[105] Smalt, R. et al., Mechanotransduction in bone cells: induction of nitric oxide and prostaglandin

synthesis by ﬂuid shear stress, but not by mechanical strain, Adv. Exp. Med. Biol., 433, 311, 1997.

[106] McAllister, T.N. and Frangos, J.A., Steady and transient ﬂuid shear stress stimulate NO release in

osteoblasts through distinct biochemical pathways, J. Bone Miner. Res., 14, 930, 1999.

[107] Johnson, D.L., McAllister, T.N., and Frangos, J.A., Fluid ﬂow stimulates rapid and continuous

release of nitric oxide in osteoblasts, Am. J. Physiol., 271, E205, 1996.

[108] Abulencia, J.P. et al., Shear-induced cyclooxygenase-2 via a JNK2/c-Jun-dependent pathway

regulates prostaglandin receptor expression in chondrocytic cells, J. Biol. Chem., 278, 28388,

2003.

[109] Alshihabi, S.N. et al., Shear stress-induced release of PGE2 and PGI2 by vascular smooth muscle

cells, Biochem. Biophys. Res. Commun., 224, 808, 1996.

[110] Williams, B., Mechanical inﬂuences on vascular smooth muscle cell function, J. Hypertens., 16,

1921, 1998.

[111] Lee, T. and Sumpio, B.E., Cell signalling in vascular cells exposed to cyclic strain: the emerging

role of protein phosphatases, Biotechnol. Appl. Biochem., 39, 129, 2004.

[112] Shyy, J.Y. and Chien, S., Role of integrins in cellular responses to mechanical stress and adhesion,

Curr. Opin. Cell Biol., 9, 707, 1997.

[113] Shyy, J.Y. and Chien, S., Role of integrins in endothelial mechanosensing of shear stress, Circ. Res.,

91, 769, 2002.

[114] Davidson, R.M., Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-

like cells, J. Membr. Biol., 131, 81, 1993.

[115] Berk, B.C. et al., Protein kinases as mediators of ﬂuid shear stress stimulated signal transduction in

endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated

by ﬂow, J. Biomech., 28, 1439, 1995.

[116] Berk, B.C. et al., Atheroprotective mechanisms activated by ﬂuid shear stress in endothelial cells,

Drug News Perspect., 15, 133, 2002.

[117] Helmke, B.P. et al., Spatiotemporal analysis of ﬂow-induced intermediate ﬁlament displacement

in living endothelial cells, Biophys. J., 80, 184, 2001.

[118] Jalali, S. et al., Integrin-mediated mechanotransduction requires its dynamic interaction with

speciﬁc extracellular matrix (ECM) ligands, Proc. Natl Acad. Sci. USA, 98, 1042, 2001.

[119] Li, S. et al., The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial

cells, Proc. Natl Acad. Sci. USA, 99, 3546, 2002.

[120] Shiu, Y.T. et al., Rho mediates the shear-enhancement of endothelial cell migration and traction

force generation, Biophys. J., 86, 2558, 2004.

mikos: “9026_c004” — 2007/4/9 — 15:50 — page 18 — #18

4-18 Tissue Engineering

[121] Stamatas, G.N. and McIntire, L.V., Rapid ﬂow-induced responses in endothelial cells, Biotechnol.

Prog., 17, 383, 2001.

[122] Davies, P.F., Zilberberg, J., and Helmke, B.P., Spatial microstimuli in endothelial mechanosignal-

ing, Circ. Res., 92, 359, 2003.

[123] Helmke, B.P. and Davies, P.F., The cytoskeleton under external ﬂuid mechanical forces:

hemodynamic forces acting on the endothelium, Ann. Biomed. Eng., 30, 284, 2002.

[124] Davies, P.F. et al., Spatial relationships in early signaling events of ﬂow-mediated endothelial

mechanotransduction, Annu. Rev. Physiol., 59, 527, 1997.

[125] Ando, J. et al., Wall shear stress rather than shear rate regulates cytoplasmic Ca

+ responses to

ﬂow in vascular endothelial cells, Biochem. Biophys. Res. Commun., 190, 716, 1993.

[126] Ando, J. et al., Flow-induced calcium transients and release of endothelium-derived relaxing factor

in cultured vascular endothelial cells, Front. Med. Biol. Eng., 5, 17, 1993.

[127] Koller, A., Sun, D., and Kaley, G., Role of shear stress and endothelial prostaglandins in ﬂow- and

viscosity-induced dilation of arterioles in vitro, Circ. Res., 72, 1276, 1993.

[128] Ando, J., Komatsuda, T., and Kamiya, A., Cytoplasmic calcium response to ﬂuid shear stress in

cultured vascular endothelial cells, InVitroCellDev.Biol., 24, 871, 1988.

[129] Dull, R.O. and Davies, P.F., Flow modulation of agonist (ATP)-response (Ca

+) coupling in

vascular endothelial cells, Am. J. Physiol., 261, H149, 1991.

[130] Dull, R.O., Tarbell, J.M., and Davies, P.F., Mechanisms of ﬂow-mediated signal transduction in

endothelial cells: kinetics of ATP surface concentrations, J. Vasc. Res., 29, 410, 1992.

[131] Mo, M., Eskin, S.G., and Schilling, W.P., Flow-induced changes in Ca

+ signaling of vascular

endothelial cells: effect of shear stress and ATP, Am. J. Physiol., 260, H1698, 1991.

[132] Nollert, M.U. and McIntire, L.V., Convective mass transfer effects on the intracellular calcium

response of endothelial cells, J. Biomech. Eng., 114, 321, 1992.

[133] Bancroft, G.N. et al., Fluid ﬂow increases mineralized matrix deposition in 3D perfusion culture

of marrow stromal osteoblasts in a dose-dependent manner, Proc. Natl Acad. Sci. USA, 99, 12600,

2002.

[134] Sikavitsas, V.I. et al., Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion

culture increases with increasing ﬂuid shear forces, Proc. Natl Acad. Sci. USA, 100, 14683, 2003.

[135] Nerem, R.M., Role of mechanics in vascular tissue engineering, Biorheology, 40, 281, 2003.

[136] Kanda, K. and Matsuda, T., Mechanical stress-induced orientation and ultrastructural change of

smooth muscle cells cultured in three-dimensional collagen lattices, Cell Transplant., 3, 481, 1994.

[137] Seliktar, D., Nerem, R.M., and Galis, Z.S., Mechanical strain-stimulated remodeling of tissue-

engineered blood vessel constructs, Tissue Eng., 9, 657, 2003.

[138] Davisson, T. et al., Static and dynamic compression modulate matrix metabolism in tissue

engineered cartilage, J. Orthop. Res., 20, 842, 2002.

[139] Park, S., Hung, C.T., and Ateshian, G.A., Mechanical response of bovine articular cartilage under

dynamic unconﬁned compression loading at physiological stress levels, Osteoarthr. Cartil., 12, 65,

2004.

[140] Hung, C.T. et al., A paradigm for functional tissue engineering of articular cartilage via applied

physiologic deformational loading, Ann. Biomed. Eng., 32, 35, 2004.

[141] Huang, C.Y. et al., Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow

derived mesenchymal stem cells, Stem Cells, 22, 313, 2004.

[142] Tran-Son-Tay, R. Techniques for studying the effects of physical forces on mammalian cells and

measuring cell mechanical properties, in Physical Forces and the Mammalian Cell, Frangos, J.A.

(Ed.), Academic Press, San Diego, 1993.

[143] Konstantopoulos, K., Kukreti, S., and McIntire, L.V., Biomechanics of cell interactions in shear

ﬁelds, Adv. Drug Deliv. Rev., 33, 141, 1998.

mikos: “9026_c005” — 2007/4/16 — 21:47 — page1—#1

Cell Adhesion

Aaron S. Goldstein

Virginia Polytechnic Institute and

State University

5.1 Introduction.............................................. 5-1

5.2 Adhesion Receptors in Tissue Structures............... 5-2

Integrins • Cadherins • Immunoglobulins

5.3 Cell Adhesion to Biomaterials .......................... 5-5

The Role of Interfacial Chemistry in Cell Adhesion •

The Role of Interfacial Biochemistry in Cell Adhesion •

Biomimetic Approaches to Regulate Cell Adhesion •

The Role of Interfacial Topography in Cell Adhesion

5.4 Measurement of Cell Adhesion to Biomaterials ....... 5-7

Micropipette Aspiration • Centrifugation • Laminar Flow

Chambers • Rotating Disc • Interpretation of

Adhesion Data

5.5 Effect of Biomaterial on Physiological Behavior ....... 5-11

5.6 Summary ................................................. 5-11

References ....................................................... 5-12

5.1 Introduction

Adhesion plays a critical role in the normal function of mammalian cells by regulating proliferation,

differentiation, and phenotypic behavior. Although the molecular mechanism of adhesion involves several

families of transmembrane receptors as well as numerous structural and regulatory proteins, they can

be divided phenomenologically into two groups: those that mediate adhesive contacts to neighboring

cells, and those that mediate adhesive contacts with structural extracellular matrix (ECM) proteins.

In development, speciﬁc adhesive interactions between adjacent cells lead to the assembly of three-

dimensional tissue structures, while cell adhesion to ECM proteins (e.g., collagens, elastin, and laminin)

establishes cellular orientation and spatial organization of cells into tissues and organs. Consequently,

both types are necessary for proper function of mammalian cells and tissues.

Initial efforts to design biomaterials — either to replace damaged tissues, or as a temporary scaffolds

for the manufacture of engineered tissues and organs — have focused on achieving robust mechanical

interactions between the biomaterial and adjacent cells (i.e., integration). However, evidence is emer-

ging that cell/biomaterial interactions can affect cell/cell interactions, and consequently impact tissue

development and function. Therefore, successful outcomes of tissue engineering efforts may require the

development of advanced materials that can facilitate both cell/cell and cell/biomaterial adhesion.

This chapter discusses the phenomenon of cell adhesion both from a tissue perspective and from

a biomaterial perspective. Section 5.2 describes the cell biology of adhesion receptors and their rel-

evance to particular tissue functions. Section 5.3 addresses cell adhesion to biomaterials with speciﬁc

5-1

mikos: “9026_c005” — 2007/4/16 — 21:47 — page2—#2

5-2 Tissue Engineering

focus on how the chemistry, biochemistry, and topography of biomaterial interfaces affect cell adhesion.

Section 5.4 describes quantitative techniques to measure cell adhesion to biomaterials. Finally, Section 5.5

describes how cell spreading, proliferation, and migration depend on cell adhesion to biomaterials.

5.2 Adhesion Receptors in Tissue Structures

Mammalian cells establish and maintain adhesive contacts with extracellular structures and adjacent cells

through several types of adhesion receptors that are displayed on the cell surface. The three main families

of these receptors are integrins, cadherins, and members of the immunoglobulin superfamily [1–3].

Although they are involved in different adhesion-related tasks, they share several common features. First,

when associated with extracellular ligands (e.g., ECM proteins, receptors on adjacent cells) they frequently

assemble into clusters. Second, these transmembrane receptors are often linked to cytoskeletal structures

(e.g., actin ﬁlaments, microtubules), and thus both anchor the cellular skeleton to extracellular structures

and mediate the transmission of mechanical forces. Third, regulatory proteins (e.g., protein kinases)

are associated with receptor clusters, and are responsible for controlling cluster stability and initiating

signaling events that regulate cell proliferation and function.

5.2.1 Integrins

Integrins are a family of receptors that mediate cell adhesion to ECM proteins. They are found as het-

erodimers, consisting of α and β subunits, and to date at least 8 β and 18 α subunits and at least 24

different dimer combinations have been reported [4]. A short list of known integrin heterodimers and

their respective ligands (Table 5.1) reveals that individual integrins can have multiple ligands (e.g., α

binds collagen, ﬁbronectin, and laminin) and multiple integrins may bind the same ligand (e.g., laminin

binds integrins α

, α

) [1,5]. In addition, most cell types express multiple integrin

TABLE 5.1 Integrin Dimers and Their Known Ligands

β subunit α subunit Ligand/counterreceptor Binding site

LM (COL)

COL (LM) DGEA (in COL I)

FN, LM, COL RGD?

V-CAM (FN-alt) REDV(in FN-alt)

FN RGD +PHSRN

LM RGD

FN RGD

ICAM-1,2

FB, C3bi

FB, C3bi GPRP

IIb

FB, FN, VN, VWF RGD (+PHSRN in FN)

VN, FB, VWF, TSP RGD

VN, FN RGD

FN RGD

Source: Adapted from Albelda, S.M. and Buck, C.A., FASEB J. 4, 2868, 1990. With

permission and Hynes, R.O. and Lander, A.D., Cell 68, 303, 1992. With permission.

Notes: LM laminin, COL collagens, FN ﬁbronectin, VN vitronectin, FB ﬁbrinogen,

VWF von Willebrand Factor, TSP thrombospondin, C3bi breakdown product of

third component of complement, FN-alt alternatively spliced ﬁbronectin. DGEA,

RGD, REDV, PHSRN, and GPRP are amino acid sequences.

mikos: “9026_c005” — 2007/4/16 — 21:47 — page3—#3

Cell Adhesion 5-3

tensin

paxillin

talin

FAK

Extracellular

matrix proteins

Actin

cytoskeleton

a-actinin

Cell membrane

vinculin

FIGURE 5.1 Illustration of a focal adhesion complex. A cluster of transmembrane integrin receptor heterodimers

is shown interacting with extracellular matrix proteins and cytoplasmic proteins vinculin, α-actinin, talin, and focal

adhesion kinase (FAK). This complex interacts with the actin cytoskeleton. (Adapted from Petit, V. and Thiery, J.-P.,

Biol. Cell. 92, 477, 2000. With permission; Yamada, K.M. and Miyamoto, S., Curr. Opin. Cell Biol. 7, 681, 1995. With

permission.)

subunits. For example, the pluripotent mesenchymal progenitor bone marrow stromal cells have been

shown to express integrin subunits α

, α

, and β

[6], while aortic smooth muscle cells express α

, α

, β

, and β

[7]. It should be noted that integrins do not exclusively mediate cell/ECM adhesion:

and α

, presented on activated leukocytes and neutrophils mediate cell/cell adhesion by binding

adhesion receptors of the immunoglobulin superfamily (e.g., vascular cell adhesion molecules [VCAM],

intracellular adhesion molecules [ICAMs]) on activated endothelial cells [1,8].

Binding of integrin receptors to their extracellular ligands initiates a process of receptor clustering and

the assembly of focal adhesion complexes [4,9–11] (Figure 5.1). Conserved sequences in the cytoplasmic

tail of β subunit are responsible for recruiting integrins into clusters (except with β

and β

which do not

aggregate), while the cytoplasmic tail of the α subunit plays a regulatory role to ensure aggregation of only

ligand-bound receptors. The cytoplasmic proteins that associate with integrins in focal adhesion complexes

can be divided into the categories of structural and regulatory proteins. The structural proteins — which

include talin, α-actinin, ﬁlamin, and vinculin — stabilize the receptor aggregates and mechanically

link the integrins to actin ﬁlaments, thus anchoring the cell cytoskeleton to extracellular structures.

In particular, talin and α-actinin possess binding domains for β integrins, while vinculin has bonding

domains for talin and actin. Regulatory proteins include paxillin, focal adhesion kinase (FAK), Src, and

members of the Rho and Ras families [4]. FAK and Src are kinases that regulate focal adhesion complex

assembly by phosphorylating structural proteins. In contrast, Rho and Ras are GTPases involved in

initiating intracellular signaling. Rho is thought to act through a number of secondary messengers to

stimulate focal adhesion assembly, actin stress ﬁber formation, and contraction, while Ras is thought to

regulate integrin activation [12]. In addition, there is evidence that Ras activates mitogen-activated protein

(MAP) kinase signaling cascades that are involved in regulating cell proliferation, apoptosis, migration,

and development [10].

In addition to initiating signaling cascades, integrins also can stimulate signaling events initiated by

cytokine receptors and growth factor receptors [12]. For example, interleukin (IL)-1 activation of MAP

kinases requires integrin-mediated cell adhesion [13]. In addition, insulin and platelet-derived growth

factor (PDGF) receptors — when bound to their respective ligands — physically interact with the α

integrin, and this association enhances MAP kinase signaling and cell proliferation [14]. Integrin binding

also can modulate effects of other adhesion receptors. For example, the association of α

integrins with

ﬁbronectin has been shown to block the upregulation of metalloproteinase by α

binding to ﬁbronectin

mikos: “9026_c005” — 2007/4/16 — 21:47 — page4—#4

5-4 Tissue Engineering

b-catenin

plakoglobin

a-catenin

p120

Actin

cytoskeleton

Adherens

junction

Cell membrane

EC1

EC2

EC3

EC4

EC5

FIGURE 5.2 Illustration of cadherin receptors associated with actin cytoskeleton. Cadherin dimers are shown in a

linear zipper structure with antiparallel binding of the EC1 domains. Cytoplasmic tails of cadherins interact with actin

ﬁlaments through structural proteins β-catenin, p120

cas

catenin, plakoglobin, and α-catenin.

[15]. Similarly, integrin-meditated adhesion to the ECM downregulates cadherin-mediated intercellular

adhesion and cell clustering [16]. This last example illustrates a competition between integrin-mediated

adhesion to the ECM and cadherin-mediated intercellular adhesion [17].

5.2.2 Cadherins

Cadherins are a family of calcium-dependent transmembrane glycoproteins that mediate cell/cell adhe-

sion. Classical cadherins (e.g., E-, N-, P-, and VE-cad) possess ﬁve extracellular (EC) cadherin repeats,

assemble into dimers, and mediate primarily homotypic binding between adjacent cells (Figure 5.2)

[18–20]. Although the outermost repeat (EC1) is thought to be responsible for intercellular binding,

it has been shown that cadherins can interact in at least three antiparallel alignments [21]. In addition,

crystallography has revealed that cadherins may assemble into a linear zipper structure [22]. The cytoplas-

mic tails of classical cadherins interact with structural proteins β-catenin, p120

cas

catenin, plakoglobin,

and through them α-catenin and the actin cytoskeleton. The cytoplasmic tail of endothelial cell-speciﬁc

receptor VE-cad also can interact vimentin intermediate ﬁlaments [18,20]. The protein p120

cas

— a target

of the Src kinase — is thought to be involved in regulating lateral clustering of cadherins, which has

been shown to increase adhesive strength [23]. Thus, Src may be involved in regulating robust cadherin

contacts.

Although cadherins do not exclusively cluster, the formation of adherens junctions is important for

several tissue functions [18]. In epithelial tissues cadherins mediate zonula adherens junctions, which

partition the cell membrane into apical and basolateral surfaces. In neuronal cells they mechanically

stabilize synaptic junctions by mediating adhesion between the presynaptic and postsynaptic cells. In car-

diac myocytes they establish intercalated discs, which both stabilize gap junctional coupling and transmit

contractile forces.

In addition to classical cadherins, several other cadherin types have been identiﬁed [18,20]. Among

these, desmocollins and desmogleins are expressed in cells that experience mechanical stress (e.g., epi-

dermis, myocardium) and are the transmembrane proteins present in desmosomes [18,20]. Like classical

cadherins, they have ﬁve extracellular cadherin repeats (except desmoglein 1, which has four repeats),

but they form desmoglein/desmocollin heterodimers and are thought to mediate intercellular adhesion

through heterotypic desmoglein/desmocollin interactions between adjacent cells. The cytoplasmic tails

mikos: “9026_c005” — 2007/4/16 — 21:47 — page5—#5

Cell Adhesion 5-5

of these receptors interact, through structural proteins desmoglobin and desmoplakin, with the keratin

intermediate ﬁlaments. To date three desmocollins and desmogleins have been identiﬁed. Desmoglein 2

and desmocollin 2 are expressed in all epithelial tissues that form desmosomes, while desmocollin 1 and

desmoglein 1 are expressed predominantly in epidermal tissue.

In addition to regulating organized tissue functions, E-, N-, and VE-cadherin-mediated intercellular

contacts have been shown to suppress cell proliferation [24–26]. This phenomenon is thought to be a

consequence of cadherin-induced alterations in the cytoskeleton structure [27], and to contribute to the

abrogation of cell proliferation of conﬂuent tissue cultures.

5.2.3 Immunoglobulins

Members of the diverse immunoglobulin superfamily are characterized by the immunoglobulin (Ig) fold

motif, a 70–110 amino acid structure consisting of two sheets of antiparallel β-strands stabilized by a

disulﬁde bridge and numerous hydrophobic side chains [28,29]. Because the amine and carboxy termini

extend from opposite ends of this structure, members of the immunoglobulin family frequently possess

strings of Ig folds. Immunoglobulin proteins are most noted for their role in antigen presentation and

recognition functions of the immune system, but at least ten members of the superfamily are involved in

cell/cell adhesion. ICAM-1, ICAM-2, and VCAM, for example, are presented on the surface of activated

endothelial cells and mediate heterotypic binding to integrins on activated leukocytes and neutrophils

(Table 5.1) [8]. Neural cell adhesion molecules (NCAMs) and L1 — present in neurons and glia —

primarily mediated homotypic binding and are involved in axon guidance, neuronal migration, neurite

outgrowth, synapse formation, and maintenance of the integrity of myelinated ﬁbers [29,30]. Finally,

junctional adhesion molecules (JAMs) stabilize tight junctions, which regulate paracellular ion transport

across epithelial and endothelial layers [31].

5.3 Cell Adhesion to Biomaterials

The efﬁcacy of biomaterials — either as replacements for damaged tissues, or as a temporary scaffolds

for the manufacture of engineered tissues and organs — relies on the ability of the material surface to

regulate cell adhesion, and strategies to engineer this cell/biomaterial interaction have evolved over the

past few decades through three basic generations [32]. The ﬁrst generation is exempliﬁed by bulk materials

such as titanium, Dacron, and ultrahigh molecular weight polyethylene, which are durable and exhibit

good mechanical properties, but are nondegradable and essentially bioinert (i.e., they elicit minimal effects

in vivo). Second generation biomaterials are those that — when brought into contact with bodily ﬂuids and

cells — are modiﬁed chemically or biochemically to achieve more favorable biologic properties. Biochem-

ical modiﬁcations include the adsorption of ECM proteins to the biomaterial surface in conformations

that enhance cell and tissue behavior (e.g., adhesion [33], phenotype [34]), while examples of chemical

modiﬁcations include the ion-exchange reactions of bioactive glasses, dissolution and reorganization of

amorphous calcium phosphate, and hydrolytic degradation of polyurethanes and polyesters. Finally, third

generation biomaterials are those that incorporate biomimetic moieties (e.g., peptides sequences) that are

designed to interact with target proteins (e.g., adhesion receptors, growth factor receptors, matrix metal-

loproteinases) and elicit speciﬁc cell responses. Thus, third generation biomaterials are often referred to

as bioactive materials because they act on cells and tissues.

Cell adhesion to biomaterials may be characterized in terms of speciﬁc and nonspeciﬁc interactions.

Speciﬁc interactions entail cell receptor recognition and binding to proteins or biomimetic moieties

on the biomaterial surface, whereas nonspeciﬁc interactions encompass noncovalent attractive forces

(e.g., electrostatic interactions, hydrogen bonds, van der Waals forces) between the cell and biomaterial

that are not associated with a speciﬁc receptor. In general, both speciﬁc and nonspeciﬁc interactions

contribute to cell adhesion to biomaterials, and can be regulated through design of substratum chemistry

and biochemistry. The following subsections address (a) the role of substratum chemistry on nonspeciﬁc

mikos: “9026_c005” — 2007/4/16 — 21:47 — page6—#6

5-6 Tissue Engineering

adhesion, the roles of (b) proteins and (c) biomimetic moieties on speciﬁc adhesion, and (d) the effect of

substratum topography on cell adhesion.

5.3.1 The Role of Interfacial Chemistry in Cell Adhesion

Although cells express receptors for ECM proteins and adjacent cells, they have been shown to adhere

through nonspeciﬁc interactions to a variety of biomaterials. For example, a comprehensive study by

Schakenraad et al. [35] demonstrated cell adhesion and spreading on 13 commercially available polymers.

In particular, this study showed that cell spreading was markedly greater on materials with surface free

energies greater than 50 erg/sec (hydrophilic surfaces) relative to low energy (hydrophobic) surfaces

[35]. With the development of self-assembling methods (e.g., alkanethiolates on gold and alkylsilanes

on glass), chemically well-deﬁned model surfaces have been constructed and used to characterize cell

adhesion [36–41]. These model studies indicate that terminal amine (NH

) and carboxylic acid (COOH)

groups produce superior adhesion relative to hydrophobic methyl-(CH

)-terminated surfaces [36,38–40].

In contrast, surfaces presenting poly(ethylene glycol) subunits have been shown to inhibit cell adhesion

[42]. In addition to organic polymers, alloys [43], and ceramics [44] have also been studied for their

capacity to support cell adhesion. These materials have proven more difﬁcult to characterize as the

chemistries are complex, and the grain size appears to play an important role [44,45]. Nevertheless,

a consistent theme that emerges from these studies is that cell adhesion increases with the material’s

capacity to promote adsorption of ECM proteins [33,38,40,45].

5.3.2 The Role of Interfacial Biochemistry in Cell Adhesion

A simple method to enhance cell adhesion to ﬁrst and second generation biomaterials is to adsorb ECM

proteins to the biomaterial surface from protein solutions (e.g., collagens [46], ﬁbronectin [46–48]),

serum, or serum-containing culture medium. Although albumin is the predominant protein in serum,

vitronectin and ﬁbronectin have been shown to deposit readily from serum onto both organic [49] and

ceramic [50] biomaterial surfaces. The choice of ECM protein can be important to regulating cell behavior;

cell adhesion and function have been shown to vary with the adhesive protein [46,49]. In addition, the

chemistry of the biomaterial surface — to which the ECM proteins are adsorbed — can also affect cell

adhesion and function [33,34,51] by altering protein orientation and inducing conformational changes

(i.e., denaturation) [51–56] that undermine receptor binding. Conformational changes have been inferred

from an increase in the ratio of β-turn to β-sheet structures, and indicate a higher degree of protein

denaturation on hydrophobic methyl-terminated surfaces than on more hydrophilic carboxyl-terminated

surfaces [54]. In addition, protein denaturation is inversely related to the rate of protein adsorption [57],

and decreases as the protein concentration in solution is increased.

5.3.3 Biomimetic Approaches to Regulate Cell Adhesion

An attractive alternative to immobilization of entire ECM proteins at biomaterial interfaces is the use

of short peptide sequences that are speciﬁcally recognized by integrin adhesion receptors [58–60].

Usually these peptides are immobilized in a random, spatially uniform manner across the bioma-

terial surface using bioconjugate techniques [61]. However, enhanced cell adhesion and migration

has been reported when peptides are immobilized to surfaces in clusters that permit integrin cluster-

ing [62]. Particular peptide sequences that have been studied include argenine–glysine–aspartic acid

(RGD), tyrosine–isoleucine–glycine–serine–aspartic acid (YIGSR), isoleucine–lysine–valine–alanine–

valine (IKVAV), and arginine-glutamic acid–aspartic acid–valine (REDV). The RGD sequence — found

in collagens, ﬁbronectin, vitronectin, ﬁbrinogen, von Willebrand factor, osteopontin, and bone sialo-

protein [63–65] — is recognized by a large number of integrin receptors (Table 5.1), and consequently

has been exploited extensively to promote integrin-mediated adhesion to biomaterials. Interestingly, the

sequence proline–histidine–serine–arginine–asparagine (PHSRN) on ﬁbronectin has been shown to act

synergistically with RGD to enhance integrin-mediated adhesion and cell signaling via integrins α

and

mikos: “9026_c005” — 2007/4/16 — 21:47 — page7—#7

Cell Adhesion 5-7

11b

[66,67]. REDV is an integrin binding sequence found in the alternatively spliced type III connect-

ing segment region of ﬁbronectin and is recognized by α

integrin (Table 5.1) [68]. It has been studied

for its ability to support endothelial cell adhesion for development of engineered blood vessels [69–71].

Lastly, YIGSR [72] and IKVAV [73] are integrin binding sequences found in laminin. Because laminin is

found in peripheral nerve tissue and the basal lamina of endothelial tissues, these peptide sequences hold

promise for mediating adhesion and orientation of engineered epithelial [74] and neural tissues [75,76].

5.3.4 The Role of Interfacial Topography in Cell Adhesion

Cell adhesion to biomaterials is also inﬂuenced by topographical features, which restrict sites for cell

adhesion. Studies performed nearly a century ago using spiderwebs revealed that adherent cells would

spread with an orientation that could be dictated by the substratum topography [77]. This phenomenon,

referred to as contact guidance, holds promise as a means to enhance engineered tissue function by induc-

ing cell alignment [78,79]. Using microfabrication techniques (e.g., micromachining, photolithography)

model substrates have been constructed to characterize the effect of topographical surface features on cell

adhesion and function. For example, surfaces textured with parallel grooves can restrict focal adhesions

to the raised edges of the substrate [80], while cytoskeletal elements — including microtubules, vimentin

intermediate ﬁlaments, and actin ﬁlaments — are oriented parallel to the grooves [81]. Further, these

effects are limited to feature sizes of 0.3 [81] to 2 µm [80]. When spacing between parallel grooves

becomes too large, cells are able to attach to the depressed regions of the substratum and orientation is

lost. Substrates exhibiting random topographies (e.g., acid-etched, sand blasted) also affect the behavior of

adherent cells. Such substrates have been shown to enhance cell adhesion [82], synthesis of ECM proteins

[82,83], and phenotypic behavior [83]. However, the effect of roughness on cell proliferation remains

ambiguous: increasing roughness has been shown to decrease proliferation of osteoblasts [83], but may

increase proliferation of endothelial and smooth muscle cells [84,85].

5.4 Measurement of Cell Adhesion to Biomaterials

Over the past two decades several devices have been developed to measure and characterize the adhesive

interaction of cells with biomaterials, particles, and other cells. These devices share the common exper-

imental strategy that nonadherent spherical cells are allowed to establish adhesive contacts to the test

material under quiescent conditions and then are subjected to a well-deﬁned distractive force. From the

examination of large numbers of cells over a range of distractive forces, a probability distribution for cell

adhesion as a function of distractive force can be constructed (Figure 5.3). From this distribution, an

adhesion characteristic (e.g., τ

, the shear stress necessary to detach 50% of the cells [47,86–88] may be

determined. The primary differences between the various devices for measuring cell adhesion is the type

of distractive force that is applied (e.g., membrane tension, buoyancy, and hydrodynamic shear stress)

and the direction of the force relative to the plane of cell/surface contacts.

5.4.1 Micropipette Aspiration

Micropipette aspiration is a sensitive technique capable of measuring the strength of individual

receptor/ligand adhesion complexes [89,90]. The advantage of this technique is its ability to probe cell

contacts and adhesive structures with a range of forces (10

−3

to 10

pN) that are capable of deforming

the cell membrane (<0.1 pN) and extracting lipid-anchored receptors (∼20 pN), but not breaking actin

ﬁlaments (>100 pN) [90].

In this approach a cell is held at the end of micropipette by a small amount of suction. The cell is then

brought into contact with a test surface, adhesive contacts are allowed to form, and then suction is applied

to break this contact (Figure 5.4). The underlying principle is that suction, P, used to draw a spherical

cell of radius R

into the end of micropipette tip of radius R

creates a membrane tension, T

, at the