Kasper C., van Griensven M., P?rtner R. (Eds.) Bioreactor Systems for Tissue Engineering II: Strategies for the Expansion and Directed Differentiation of Stem Cells

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Table 2 Occurrence and actions of growth factors in developing and mature cartilage, diseased cartilage tissue, and applications in cartilage cultivation

in vitro

Growth factor/

receptor

Role in the development of

cartilage tissue in vivo

Role in adult cartilage tissue Role in cartilage lesions or in

articular cartilage disease

In vitro applications and functions

FGF family

FGF-2 Present in the AER Bound to heparan sulfate in

the ECM, possible role in

mechanotransduction

[97–99]

Recruitment of MSCs to

cartilage defects

Stimulation of proliferation of

MSC in vitro [146]

Stimulation of Tenascin synthesis

in mesenchymal

condensations [147]

– Induction of MMP-13 [101] and

noggin [104]

Maintenance of differentiation

potential of MSCs [126, 127]

Stimulation of tenascin and

syndecan synthesis in

condensations limiting size of

condensations [64]

– Increased production in RA

[148]

Increased integrin alpha10-

expression on cultured MSCs

in vitro [128]

Activation by endothelial

proteases in endochondral

ossiﬁcation [149]

– – Maintenance of differentiation

potential of mammalian

chondrocytes in vitro [123]

– – – “Dedifferentiation” of

chondrocytes [33]

FGF-4 Occurrence in the AER,

chemoattractant for limb bud

mesenchymal cells [46]

–– –

Occurrence in the ZPA, role in

AP axis formation, controls

the formation of gap junctions

[150, 151]

–– –

FGF-8 Expression in the AER,

maintenance of fgf-10

expression in the underlying

mesenchyme

[47, 75, 77]

–– –

(continued)

Cartilage Engineering from Mesenchymal Stem Cells 169

Table 2 (continued)

Growth factor/

receptor

Role in the development of

cartilage tissue in vivo

Role in adult cartilage tissue Role in cartilage lesions or in

articular cartilage disease

In vitro applications and functions

FGF-10 Lateral plate mesenchyme: – – –

Proliferation, migration [73, 77]– – –

Expression in blastemas of

regenerating limbs of

amphibia [78]

–– –

FGF-18 Role in endochondral ossiﬁcation,

coordination of

chondrogenesis and

osteogenesis [152, 153]

–– –

Signaling via FGFR3, promotion

of chondrogenesis [154]

–– –

FGF receptors

FGFRI Mesenchymal cells of the limb

bud prior to condensation

[155, 156]

– – Expression in proliferating cells

of mesenchymal lineage

in vitro [157]

Absence of FGFR1 causes

apoptosis in limb buds [158]

– – Expression prior to differentiation

[130, 131]

– – – Expression in hypertrophic

cultures of MSCs [130, 131]

FGFRII Mesenchymal condensations

[156], essential for limb

outgrowth [74]

Adult articular cartilage

[130]

– Expression in condensations of

mesenchymal micromass

cultures [157]

Expression in limb bud

ectodermal epithelium [155]

– – Expression during condensation

in vitro [131]

Ectodermal FGF-2 and

FGF-8 inhibit chondrogenesis

through FGFRII signaling

[159]

– – Down regulation during

differentiation in vitro [130]

In prechondrocytic cells prior to

condensation [155]

–– –

170 C. Goepfert et al.

FGFRIII Growth plate chondrocytes [155,

160]

– – Prior to calciﬁcation of

mesenchymal micromass

cultures [157]

TGF-b superfamily

TGF-b1 Expression in condensations,

stimulates synthesis of

ﬁbronectin Tenascin, N-

CAM, N-cadherin [50, 64, 65]

Present in adult cartilage,

bound to cartilage matrix

[107]

Induction of OA-like changes

upon overexpression [111]

Induction of chondrogenesis of

bone marrow MSC [133] and

synovium derived MSC [137]

Fn positive areas precede

chondrogenic differentiation

[86]

Stimulation of ﬁbronectin

synthesis in cartilage

explants [161]

Stimulation of proteoglycan

synthesis by OA

chondrocytes [ 162, 163]

Inhibitory effect on primary

chondrocytes [164, 165]

Inhibition of terminal

differentiation of epiphyseal

chondrocytes [166]

TGF binding domain in

Procollagen type IIA [167]

– Upregulation of TGF-b

expression in OA [168]

–

Storage in growth plate matrix

bound to LTBP, activation by

matrix vesicles [107, 108]

–– –

Stimulation of PTHrP synthesis in

epiphyseal chondrocytes

[169]

–– –

Proliferation of undifferentiated

mesenchymal cells [79]

–– –

TGF-b2 Condensations Present in adult cartilage Proliferation Induction of matrix synthesis,

Endogenous synthesis by

ATDC-5 aggregate cultures

[170]

– – – Inhibitory effect on proliferation

of MSCs [

146]

(continued)

Cartilage Engineering from Mesenchymal Stem Cells 171

Table 2 (continued)

Growth factor/

receptor

Role in the development of

cartilage tissue in vivo

Role in adult cartilage tissue Role in cartilage lesions or in

articular cartilage disease

In vitro applications and functions

TGF-b3 – – Proliferation, stimulation of

matrix synthesis

Chondrogenic differentiation of

MSCs [ 10, 36]

– – Induction by IL-1 [112]–

BMP (general) Negative regulators of the AER

[81]

– – ATDC5 cell line: stage speciﬁc

expression of BMP-2, -4, -6,

-7 [171]

Feedback inhibition by Noggin

[172]

–– –

BMP signaling required for

maintenance of differentiated

phenotype, cell proliferation

and hypertrophy [173]

–– –

Inhibition of BMP signaling by

noggin inhibits condensation

and differentiation [91]

–– –

BMPs act as heterodimers [82]– – –

Co-localization of BMPs [174]– – –

Feedback inhibition of BMP

signaling by noggin [175]

–– –

BMP-2 Expression in the AER and ZPA – – Stimulation of N-cadherin

synthesis and chondrogenesis

in C3H10T1/2 cells [176]

Induction of collagen type X

synthesis and alkaline

phosphatase expression in

growth plate chondrocytes

[177]

– – Induction of chondrogenesis of

bone marrow MSC [178] and

synovium derived MSC [15]

and synovial explants [137]

172 C. Goepfert et al.

BMP-4 Induction of collagen type X

synthesis and alkaline

phosphatase expression in

growth plate chondrocytes

[177]

–– –

BMP-6 Transient expression in

prehypertrophic chondrocytes

[179, 180]

– – Chondrogenic differentiation of

MSCs, synthesis by

prehypertrophic cells in vitro

[171]

BMP-7

(OP-1)

Expression during

chondrogenesis

Synovial ﬂuid, cartilage

tissue

Reduced expression in aged

cartilage tissue [113]

Synthesis of collagen type II,

aggrecan, GAG

Induction of collagen type X

synthesis and alkaline

phosphatase expression in

growth plate chondrocytes

[177]

– – Induction of chondrogenesis in

ATDC5 cell line [181]

– – – Induction of chondrogenesis of

synovial explants [137]

– – – Redifferentiation of articular

chondrocytes [182, 183]

BMP receptors BMPRIB expression preﬁgures

the cartilage primordia [184]

–– –

BMPRIB present on

chondrogenic cells,

stimulation of BMPRIB

expression by TGF-b [65,

185]

–– –

Inhibition of BMPRIB expression

by FGF causes apoptosis [80]

–– –

GDFs Formation of joints and joint

spaces [186, 187]

–– –

(continued)

Cartilage Engineering from Mesenchymal Stem Cells 173

Table 2 (continued)

Growth factor/

receptor

Role in the development of

cartilage tissue in vivo

Role in adult cartilage tissue Role in cartilage lesions or in

articular cartilage disease

In vitro applications and functions

IGF family

IGF-I IGF promotes limb bud

outgrowth [188–190]

IGF-I is detected in articular

cartilage [115] and in

synovial ﬂuid [191]

Increased synthesis of IGF-I and

IGFBP-3 in OA [192–194]

and RA [194]

Chondrogenic effect on MSCs in

the absence of insulin [195]

Co-expression of IGF-I and

IGFBP-2 in condensing

mesenchyme [196]

Autocrine effect [197]– –

Stimulation of IGF synthesis in

growth plate by growth

hormone [116]

–– –

Expression mainly in

proliferating chondrocytes of

the growth plate [121]

–– –

IGF-II Expression in chondrocyte

precursors, co-expression

with both types of IGF

receptors [ 95]

Autocrine effect [197]– –

IGF binding proteins

IGFBP-3 Perichondrium [94] Transport of IGF-I Inhibits of IGF-I induced matrix

synthesis

Antiproliferative effect of

IGFBP-3 on RCJ3.1C5.18 cell

line [198]

– – Increased in synovial ﬂuid of

OA and RA [194]

–

– – Increased synthesis in OA [192,

193]

–

IGFBP-4 Developing cartilage [95]– – –

IGFBP-5 Mesenchymal condensations [95]– – –

IGFBP-6 Mesenchymal condensations [95]– – –

IGF receptors – – Increased number of binding

sites in OA chondrocytes,

decreased responsiveness to

IGF [192]

–

174 C. Goepfert et al.

PTHrP

PTHrP Synthesis in epiphyseal

chondrocytes [ 67, 169]

– Prevention of collagen type X

synthesis by MSC of OA

donors [144]

Prevention of collagen type X

synthesis by bone marrow

MSC [143, 144] and by

adipose tissue derived MSC

[143]

Hedgehog proteins

Ihh Production by prehypertrophic

chondrocytes, induction of

PTHrP in epiphyseal cartilage

[68, 69]

–– –

Shh Expression in the ZPA, role in AP

axis formation [89, 199]

–– –

Growth factor interactions

FGF/TGF Stimulation of TGF synthesis by

FGF in progress zone =>

promotes transition of

precursor cells towards

differentiation [46]

– – FGF suppresses senescence

induced by TGF-b2[129]

– – – “Secondary progenitor cells”

from human articular

chondrocytes [35]

– – – Stimulation of chondrocyte

growth in vitro [203]

– – – Synergistic effect on chondrocyte

proliferation together with

PDGF-BB [124]

FGF/BMP FGF and BMP have opposite

effects on limb outgrowth

[208]

–– –

FGF and BMP interact to induce

apoptosis of the interdigital

mesenchyme [80]

–– –

IGF/TGF – – – Additive effect on chondrogenesis

of MSC [138]andon

synovium derived MSC [200]

(continued)

Cartilage Engineering from Mesenchymal Stem Cells 175

Table 2 (continued)

Growth factor/

receptor

Role in the development of

cartilage tissue in vivo

Role in adult cartilage tissue Role in cartilage lesions or in

articular cartilage disease

In vitro applications and functions

– – – Enhanced chondrogenesis of

periosteal explants [201]

– – – Synergistic effect of IGF-I and

TGF-b on redifferentiation of

human articular chondrocytes

[202]

IGF/BMP – – Synergistic effect on matrix

synthesis in OA

chondrocytes [ 204]

Synergistic effect on matrix

synthesis [204, 206]

– – Inhibitory effect on MMP-13

expression [205]

IGF/TGF/FGF – – – Enhance proliferation of

synovium derived stem cells

[200]

IGF/BMP/FGF – – – Inhibition of anabolic effects of

IGF-I and BMP-7 on

chondrocytes by FGF [105]

TGF/BMP – – – Induction of chondrogenesis of

adipose-derived stem cells

by TGF-b3 combined with

BMP-6 [135]

– – – Induction of chondrogenesis of

bone marrow and adipose

tissue derived MSC by

TGF-b2 in combination with

BMP-7 [136]

– – – Synergistic effect of TGF-b1 and

BMP-2 on the chondrogenesis

of bone marrow MSC [207]

176 C. Goepfert et al.

mesenchymal stem cells promotion of

lineage progression

differentiated cells

paracrine / autocrine

growth factors

extracellular

matrix

release of growth

factors from ECM

binding of growth

factors to ECM

proliferating /

differentiating cells

stage specific

synthesis of paracrine /

autocrine GF

GF promoting

lineage progression

stage specific

synthesis / remodelling

of ECM

feedback via

cell surface receptors

Fig. 1 Interaction of cellular differentiation, growth and differentiation factors, and speciﬁc

matrix molecules during histogenesis. Morphogenesis and histogenesis result from the interaction

of speciﬁc growth and differentiation factors acting in a stage dependent manner. Growth factor

actions are modulated by binding to ECM molecules and the expression of their receptors

depending on the developmental stage of the cells

proliferation and

differentiation

terminal

differentiation

calcification /

vascularization

ossificationproliferation

and migration

FGF-10

FGF-2,4,8

FGFR1

condensation

TGF-ß

FGF-2

shh

FGF-2/FGFR2

IGF-II

BMP-2,4,7

FGF-18/FGFR3

PTHrP

BMP-2,7

FGF-2/FGFR1

BMP-2,7

collagen I

collagen IIA collagen IIB

collagen X

collagen I

diaphysis

epiphysis

perichondral

bone collar

fibronectin

blood vessel

cartilaginous

model

N-

cadherin

aggrecan

tenascin

hyaluronan

growth

plate

Fig. 2 Development of the long bones under the control of growth factors. The different develop-

mental stages of the skeletal primordia are shown together with the speciﬁc growth and differenti-

ation factors (above the arrows) which promote the stage speciﬁc synthesis of matrix molecules

(below)[6, 144]

Cartilage Engineering from Mesenchymal Stem Cells 177

2.1 Migration and Proliferation

In the initial phase of limb development, mesenchymal cells of the lateral plate

mesenchyme and of the somitic mesenchyme migrate towar ds the limb ﬁeld [2].

The ectodermal cells are induced by the mesoderm to form a specialized epithelial

structure termed the apical ectodermal ridge (AER). The AER supports migration

and proliferation of the mesench ymal cells providing the cell mass for the forma-

tion of precartilage condensations [46, 47].

2.2 Cell Condensation

Cell condensation is characterized as a transient stage during the early morphogen-

esis which can be detected by PNA (peanut agglutinin) staining [48, 49]. During

cell condensation, cell density in the prospective limb regions is increased leading

to cell–cell contacts mediated by cell adhesion molecules such as N-cadherin and

N-CAM [50–52] and the formation of gap junctions [53, 54]. Cell adhesion

molecules are expressed speciﬁcally during the condensation phase and down

regulated subsequently upon chondrogenic differentiation of the prechondro genic

cells. Prior to cell condensation, the extracellular matrix in the prospective limb

regions contains high amounts of collagen type I and hyaluronan [55, 56]. Hyaluronan

is supposed to prevent cell–cell interactions prior to the condensation phase [56].

During the condensation phase, hyaluronidase activity is detected, suggesting that

matrix remodeling takes place allowing for cell–cell interaction. During the con-

densation phase, a speciﬁc splice variant of ﬁbronectin, FnEIIIA, is detected

throughout the condensations [57, 58]. Fibronectin was shown to be essential

for condensation and subsequent chondrogenesis. Furthermor e, ﬁbrone ctin distri-

bution during the condensation phase indicates the localization of skeletal elements

formed later on [55].

2.3 Chondrogenic Differentiation

Cellular differentiation is characterized by the increased synthesis of transcription

factors sox-5 and sox-6, and the appearance of the cartilage speciﬁc transcription

factor sox-9 [59–61]. Collagen type I and ﬁbronectin are synthesized in the ECM

prior to condensation and reach a maximum density at the time of cellular differen-

tiation [55]. Chondrogenic differentiation of the condensing cells is characterized

by the appearance of collagen types II, IX, and XI, the characteristic components of

collagenous network of cartilage tissue. As a result of chondrogenic differentiation,

178 C. Goepfert et al.