Type I and type II collagen Regulation of Chondrogenic Differentiation in Mesenchymal Progenitor Cells

Journal

of

Orthopaedic

Research

ELSEVIER

Journal of Orthopaedic Research 23 (2005) 446453

www.elsevier.com/locate/orthres

Type I and I1 collagen regulation

of chondrogenic differentiation

by mesenchymal progenitor cells

C.W. Chen

Y.H.

Tsai

b7c,1,

W.P. Deng

a,

S.N.

Shih

e,

C.L. Fang

d,

J.G. Burch

f,

W.H.

Chen

a,

W.F.

Lai

a Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street. Taipei 110, Taiwan Institute of Biomedical Materials, Taipei Medical University, 2.50 Wu-Hsing Street, Taipei 110, Taiwan Institute of Cellular and Molecular Biology, Taipei Medical University. 250 Wu-Hsing Street, Taipei 110, Taiwan

Department of Pathology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan Orthopedic Department, Chang-Gung Memorial Hospital, Taoyuan 333, Taiwan

'

Department of Orthodontics, School of Dental Medicine, Nova Southeastern University, Ft. Lauderdale, FL 33328, USA Received 19 December 2003; accepted 3 September 2004

Abstract

Chondrogenic differentiation by mesenchymal progenitor cells (MPCs) is associated with cytokines such as transforming growth

factor-beta

1 (TGF-PI) and dexamethasone. Extracellular matrix (ECM) also regulates the differentiation by MPCs. To define

whether ECM plays a functional role in regulation of the chondrogenic differentiation by MPCs, an in vitro model was used. That

model exposed to dexamethasone, recombinant human TGF-@l(rhTGF-@I)

and collagens. The results showed that MPCs incorpo-

rated with dexamethasone and rhTGF-01 increased proliferation and expression of glycosaminoglycan (GAG) after 14 days. Type

11

collagen enhanced the GAG synthesis, but did not increase alkaline phosphatase (ALP) activity. When adding dexamethasone

and rhTGF-PI MPCs increased mRNA expression

of

sox9. Incorporation with type

I1

collagen, dexamethasone and rhTGF-Pl,

MPCs induced mRNA expression of aggrecan and enhanced levels of type

I1

collagen, and sox9 mRNA. In contrast, incorporation

with type

I collagen, dexamethasone and rhTGF-PI MPCs reduced levels of aggrecan, and sox9 mRNA, and showed no type

I1

collagen mRNA. Altogether, these results indicate that type I and

I1

collagen, in addition to the cytokine effect, may play a func-

tional role in regulating of chondrogenic differentiation by MPCs.

0

2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: Chondrogenic differentiation; Collagen; Dexamethasone; Extracellular matrix; Mesenchymal progenitor cells, Sox9; TGF-Dl

Introduction

Cartilage has little capacity for self-repair [lo]. An in-

jured joint predisposed to continued arthritic degenera-

tion [8]. Current therapies for cartilage regeneration

* Corresponding author. Tel.: +886 2 87800225/27361661~5210; E-mail address: [email protected] (W.F. Lai).

'

C.W. Chen and Y.H. Tsai contributed equally to this paper. fax: +886 2 27357714.

include placement

of

carbon plugs [5], periosteum [ 141,

or periochondrium [

151.

Autologous chondrocyte trans-

plantation [6,3 13, and subchondral drilling [4,16,26].

Success rates vary. Most methods of therapy have seri-

ous clinical limitations.

Structural, chemical, and mechanical properties of

regenerated cartilage are definitely not normal [43].

The regenerated tissue does not bond to adjacent tissue

[38].

New cartilage undergoes degenerative changes.

After one year this new cartilage is similar to that in

untreated defects [25]. Thus, most repair methods fail

0736-0266/$

-

see front matter 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved doi: 10.101 6/j.orthres.2004.09.002

C. W. Chen et al. I Journal of Orthopaedic Research 23 (2005) 4 4 6 4 5 3 447

to improve cartilage beyond what is found in natural re-

pair of untreated osteochondral defects.

The clinical need for improved treatment options for

patients with cartilage injuries has encouraged scientists

to investigate in vivo implantation of isolated MPCs.

MPCs can undergo differentiation to regenerate either

cartilage [13] or bone [7,44] in defects. Implantation of

MPCs has been demonstrated to affect tissue regenera-

tion. Then different environmental factors can be intro-

duced to trigger differentiation into specific phenotypes.

Chondrogenic differentiation has been induced in

vitro by cytokines TGF-PI and dexamethasone in

MPCs derived from bone marrow of human [46] or ani-

mal models [19,27,45]. ECM has been demonstrated to

regulate the chondrogenic or osteogenic differentiation

induced by MPCs [18,29]. How the mechanisms of

ECM affect MPC differentiation are still not well under-

stood. This study was undertaken to further identify

how collagen regulated rhTGF-b 1’s and dexametha-

sone’s effects on chondrogenic differentiation. Differen-

tial expression of MPC-induced glycosaminoglycan

(GAG)

and alkaline phosphatase (ALP) exposed to dif-

ferent concentrations of dexamethasone, rhTGF-Pl, and

collagens was measured by spectrophotometry. The

levels of mRNA expression of Sox9, aggrecan, and type

I1 collagen were found to be identifiers

of

certain mech-

anisms of chondrogenic differentiation by MPCs.

Materials and methods

Subjects

Consenting bone marrow donors were selected from patients admitted to the Orthopedic Section of Taipei Municipal Chung-Hsin Hospital, Taipei, Taiwan. None had endocrine disease or was receiving hormone replacement therapy. Bone marrow was obtained from femur fracture site by proximal femur aspiration during surgical treatment procedures.

Isolation and cultivation of MPCs

MPCs were isolated from rabbit and human bone marrow. They were mixed with sodium-heparin, and diluted with equal five volumes of PBS. The cell suspension was fractionated on a Percoll gradient (40% initial density, Phamacia). The MPC-enriched interface fraction was collected and cultured in Dulbecco’s modified Eagle medium with 1 g/ml glucose (DMEM/LG, Sigma D5523), 10% fetal bovine serum, l00UIml penicillin, 100 pglml streptomycin, and 0.25 pglrnl fungizone under normal conditions. The medium was changed every four days. During the primary culture, adherent cells formed colonies that were passaged when cells proliferated subconfluently. Second- and third- passage cells were selected to identify the mechanisms of differentiation. mRNA extraction and reverse transcriptase polymerase chain reaelion ( R T - P C R )

Total RNA harvested from subconfluent monolayer cultures (approximately1 O6 MPCs) was extracted using T R I Z O I ~ Reagent (Invitrogen life technologies, Carlsbad, CA). Extracted RNA was dis- solved in sterilized ddHlO and stored at -80°C. Reverse transcription was performed with SuperScript” 111 (Invitrogen life technologies) and

Oligo d(T)12-18 primer. Four micrograms of RNA was added into a final solution of 21pl l0mM dNTP mix, 10 X RT buffer, 25mM MgCI2, 0.1 M DTT, RNase Inhibitor and RNase H. Six pg of RT products were used in PCR amplification in a final 50pl solution con- taining 2.5 mM dNTP, 25 mM MgC12, upstreamldownstream primers and Taq DNA polymerase (Invitrogen life technologies). Following an initial denaturation at 95°C for 5min, the DNA was amplified in the Touchgene Gradient (TECHINE, UK). Thirty-five cycles of 1 min at 94°C were applied for denaturation and 1 min at 72°C for extension. A final extension was applied at 72°C for 5min. PCR prod- ucts were visualized on 1% agarose gel (Agarose I, AMRESCO, Ohio) and stained with EtBr. PCR products were confirmed by size verifica- tion and analyzed using FloGel-I (Fluorescent Gel Image System, TOPBIO). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer sets were following:

( I )

Sox9-5’-GGCAGCTGTGAACTGGCCA-3’

(sense primer) and 5’- GCACACGGGGAACTTGTCC-3’ (antisense primer) which gives a 408 bp product;

(2) COL2A 1 -5’-CACGCAGAAGTTCACCAAGAA-3‘ (sense primer) and 5’-CTGCAGGATCAGCCATGGTAGA-3’ (antisense primer) which gives a 501 bp products;

(3)Aggracan-5‘-TGAGGAGTGGAACAAGTACC-3’(sense

pri-

mer) and S’-GGAGGTGGTAAlTGCAGGGAACA-3’ (antisense

primer) which gives 466 bp product;

(4) GADPH-5’-GCTCTCCAGAACATCATCCCTGCC-3‘ (sense pri-

mer) and 5’-CGTTGTCATACCAGGAAATGAGCTT-3‘ (anti-

sense primer) which gives 346 bp product. Preparation of type I and II collagens

Type I and I1 collagens were prepared as previously described in this laboratory [20,21]. Collagen was heat-denatured at 95°C for 30 min, and analyzed using SDS-PAGE. After breaking the peptide chain of a-subunits, the triple-helical structure of collagen was denatured.

Analysis of chondrogenic differentiation in monolayer culture

Rabbit and human MPCs were seeded into six-well plates with 10% FBS and 50 pg/ml ascorbic acid in DMEMILG. After four days of cul- tivation, the medium of rabbit MPCs was changed with dexametha- sone (10-7M, Sigma D2915, St. Louis), rhTGF-PI (0.1, I, and IOngl ml, R & D Systems 240-B, Minneapolis, MN), and collagen (10, and 100 pg/ml). Six wells were employed for each condition. Differentiation was analyzed by GAG synthesis and ALP activity after 14 days. GAG synthesis and ALP activity were measured using spectrophotometry of the absorption of O D m and OD405 respectively after alcian-blue stain- ing [l]. The medium of human MPCs, after four days of cultivation, was changed with dexamethasone (10-7M), rhTGF-PI (1 ng/ml), and collagen (100pglml). Three wells were employed for each condition. The mRNA for aggrecan, type I1 collagen, and sox9 was analyzed using RT-PCR as described above.

Structural effects of collagen on chondrogenic diflerentiation by MPCs

To further identify the regulatory effect of collagen’s structure on chondrogenic differentiation, native and denatured collagens were tested in cell cultivation. Each collagen solution (2mglml) was heated at 95°C for 30min to denature the peptide chains of the collagen subunits. Degradation of collagen and the triple-helical structure was analyzed using 5% SDS-PAGE. Native or denatured collagen was individually added to the medium at a concentration of IOOpglml each. The expres- sion of GAG by MPCs in each condition was measured by alcian-blue assay.

Statistical analysis

Expressions of each GAG and ALP from chondrocyte cultures ex- posed to different concentrations of dexamethasone, rhTGF-PI, and collagens were analyzed using Mann-Whitney test. Dexamethasone-,

448 C. W. Clien et al. I Journul of Orthopaedic Research 23 (2005) 446-453 rhTGF-P1-1, and collagen-treated and untreated control cultures at

different concentrations were also compared. Data are reported as the mean f. SD.

P

< 0.05 was considered statistically significant.

Results

Type I and II collagen regulated rhTGF-PI and

dexamethasone effects on GAG expression of rabbit

MPCs

Significantly increased GAG expression was found in

the culture supplemented with 10 ng/ml rhTGF-01.

GAG expression was 2.6 k

0.2

times greater than those

of

untreated cells. However, GAG synthesis did not in-

crease with 0.1 and 1 ng/ml concentrations of rhTGF-PI.

Treatment with lop7 M dexamethasone alone increased

GAG expression 2.25

f

0.18 times in rabbit MPCs com-

pared to the untreated group. The increased rate of

GAG expression remained the same as in the untreated

group after adding 0.1 and 1 ng/ml concentrations of

rhTGF-Pl.

A

synergistic effect was found when incor-

porated with both 10ng/ml rhTGF-01 and lop7 M dexa-

methasone. The maximal GAG expression was

3.75

f

0.32

times higher than that

of

control cells (Fig.

1 (A)).

Type

I1

collagen alone at the concentration of 10

pg/ml did not significantly change GAG expression,

whereas at the concentration

of

100

pg/ml, GAG expres-

sion increased 2.5 times. Synergistic increases in GAG

expression by MPCs to be up to 5.8 times when adding

lOOpg/ml type I1 collagen (Fig. l(B)). When adding

either 10pg/ml or lOOpg/ml concentration of type I1

collagen (10 or lOOpg/ml), the osteogenic marker ALP

activity of MPCs did not significantly increase. In-

creases in ALP activity were only 1.2 and 1.4 times,

respectively, higher than the control. With treatment

of

10ng/ml TGF-01 and 10p7M dexamethasone, ALP

expression increased 2.4 times. Further addition of type

TGF-beta 1 (nglml) Type I I Collagen (microgramlml)

(A) GAG, glycosaminoglycan; DEX, dexamethasone; TGF, hTGF-pl (B) GAG, glycosaminoglycan; DEX, dexamethasone; TGF, rhTGF-pl

(c)

ALP, alkaline phosphatase; DEX, dexamethasone; TGF, rhTGF-p1 (D) GAG, glycosaminoglycan

Fig. I. GAG and ALP expression of MPCs exposed to varying concentrations of rhTGF-PI, DEX, and collagen, measured by spectrophotometry. Means f S.D; (n = 6). (A) TGF-PI and dexamethasone induced an increase in GAG by MPCs. An asterisk indicates a significant (p < 0.05) difference in DEX compared to the untreated control. A diamond indicates a significant (p < 0.05) difference among different TGF-PI concentrations. (B) Type I1 collagen up regulated GAG synthesis with and without dexamethasone. An asterisk indicates a significant (p < 0.05) difference in DEX compared to the Untreated control. A diamond indicates a significant (p < 0.05) difference among different type I1 collagen concentrations. (C) Type I1 collagen, in contrast to dexamethasone, did not significantly increase ALP activity by mesenchymdl progenitor cells. An asterisk indicates a significant

(p < 0.05) difference in TGF-PI and DEX compared to the untreated control. (D) Only type I1 collagen significantly increased GAG synthesis, but not cell proliferation, whereas type I collagen did not change GAG synthesis or cell proliferation. An asterisk indicates a significant (p < 0.05) difference in type I1 collagen compared to type I collagen and the untreated control.

C. W. Chen et al. I Journal of Orthopaedic Research 23 (2005) 446453 449

I1

collagen ALP caused the same increased expres-

sion of 2.4 times (at 10pg/ml concentration) and 2.7

times (at 100 pg/ml concentration). Thus, type I1 colla-

gen did not significantly regulate the ALP activity of

MPCs with

or

without TGF-P1 and dexamethasone

(Fig. l(C)).

Incorporated with lOOpg/ml

of

type I1 collagen, the

population of MPCs was reduced by 40%. However,

GAG expression increased 2.5 times. In contrast, with

the addition of 100 pg/ml of type I collagen, the popula-

tion and GAG expression of MPCs appeared to not sig-

nificantly change (Fig. l(D)).

Type

I

and II collagen regulated rhTGF-Bl und

dexamethasone effects on aggrecan,

Sox9

and collagen

mRNA expression

RT-PCR using RNA obtained from human MPCs

showed no signal for aggrecan, type

I1

collagen, and

Sox9. An mRNA expression

of

type

I1

collagen and

Sox9 was induced when treated with dexamethasone.

With dexamethasone and rhTGF-P 1 MPCs increased

mRNA expression of type

I1

collagen and Sox9. In addi-

tion to dexamethasone and rhTGF-Pl, type

I1

collagen

induced mRNA expression

of

aggrecan and enhanced

(A)

GAG, glycosaminoglycan;

DEX,

dexamethasone;

TGF, rhTGF-Pl

(6)

Fig. 2. Structural effects of collagen on chondrogenic differentiation by MPCs. (A) Native type I1 collagen remarkably increased 4.3 times GAG synthesis, the increase of GAG was reduced to 2.9 times with the denatured type I1 collagen. Noted no significant difference of GAG synthesis among groups of native type I collagen, denatured type I and I1 collagens, and dexamethasone and rhTGF-PI treated. (B) SDS-polyacrylamide gel electrophoresis of native and denatured collagens. Note the clearly identifiable bands of type I native collagen in lane 1 and of type I1 collagen in lane 3. The denatured type I collagen showed a smear phenomenon in lane 2, and small fragments of denatured type I1 collagen was showed in lane 4.

450 C. W. Chen et al. I Journal of Orihopaedic Research 23 (2005) 446453

SOX9- -408 bp

Type I1 collagen- -501 bp

Aggrecan- -350 bp

GAPDH- -346 bp

Marker MSC DEX DEX VEX DEX

+

+ +

+ +

TGF-PI TGF-PI TGF-PI

C O l 1 Call1

Fig. 3. Type I and I1 collagens regulated rhTGF-BI and dexameth- asone effects on aggrecan, sox9 and collagen mRNA expression. Total RNA was isolated from MPCs and used for PT-PCR to detect expression of type 11 collagen and aggregan. Expression of GADPH was used as control. Lane 1: DNA standards; lane 2: MPCs showed no signal for aggrecan, type I1 collagen, and sox9; lane 3: With dexamethasone MPCs induced mRNA expression of type I1 collagen and sox9; lane 4 With dexamethasone and rhTGF-01 MPCs increased mRNA expression of type I1 collagen and sox9; lane 5: Cultured with type I collagen, dexamethasone and rhTGF-PI MPCs showed no type I1 collagen mRNA; lane 6: Cultured with type I1 collagen. dexameth- asone and rhTGF-01 MPCs induced mRNA expression of aggrecan and enhanced levels of sox9 and type I1 mRNA.

levels of Sox9 mRNA. In contrast, incorporation with

type

I

collagen, dexamethasone and rhTGF-Pl MPCs

reduced levels of aggrecan, and Sox9 mRNA, showed

no type I1 collagen mRNA (Fig. 3).

Structural efsrcts of collagen on chondrogenic

differentiation induced by MPCs

To determine whether the structure of collagen affects

chondrogenic differentiation, native and denatured col-

lagens were introduced to MPCs. With pretreatment

using rhTGF-PI and dexamethasone, MPCs increased

GAG synthesis 4.3 times after adding native type

I1

col-

lagen. The enhancement of GAG synthesis was down-

graded to 2.9-fold increase, when denatured type

I1

collagen was added instead.

In

contrast, no significant

difference was noted between the increase in GAG syn-

thesis in the presence of native (2.5 times) or denatured

type I collagen (2.1 times) (Fig. 2(A)). Intact al(I), a2(I),

and al(I1) bands were clearly identified with native type

1 and I1 collagens. Contrarily, a smear phenomenon was

noted with denatured type I and I1 collagens (Fig. 2(B)).

These results indicate that type I1 collagen-triggered

chondrogenic differentiation is type-specific, and corre-

lates with collagen’s native triple-helical structure.

Type 11 and I collagen efects on morphological changes

in MPCs treated with dexamethasone and rh TGF-Pl

in rnonolayer cultures

MPC cultures of

all

six wells showed a phenomenon

of contact inhibition with fibroblast-like morphology

after 14 days (Fig. 4(A)). A mild pile-up was noted

in MPCs of all six wells when supplemented with

rhTGF-1 and dexamethasone (Fig. 4(D)). In all cultures

incorporating type I collagen, cells showed a shift to

elongated spindle-shaped fibroblastic appearance inde-

pendent of the absence or presence of

TGF-(31

and dexa-

methasone (Fig. 4(B) and (E)).

Incorporated with type I1 collagen, MPCs of all six

wells showed more cuboidal-, and less spindle-shaped

morphology (Fig. 4(C)). Semi-transparent fibrous like

substance was noted on cells. Cells predominantly ap-

peared rectangular after pretreatment with type

I1

colla-

gen, dexamethansone and rhTGF-(3 1. Cell-collagen

matrix aggregates were found in four of six wells (Fig.

4(F)). Abundant GAG, which found in the cartilage-like

cell-collagen matrix aggregate was then identified by

alcian-blue staining (Fig. 4(G)).

Discussion

Bone marrow-derived MPCs are highly proliferative,

multipotential cells that have been considered ideal cells

for use in repair of injured cartilage and fractures of

bone. It is known that repair tissue arises from differen-

tiation of local MPCs. Both periosteum and bone mar-

row contain these cells, which preserve the ability to

differentiate into both chondrocytes and osteoblasts

[ 11,24,28]. Chondrogenic differentiation can be trig-

gered, if the environmental factors such as ECM and

local cytokines are facilitative. Local MPCs enable

accumulation, proliferation, and terminal differentiation

into hypertrophic chondrocytes. If environmental fac-

tors are not facilitative; however, local MPCs differenti-

ate into fibrochondrocytes and form fibrocartilage

[9,381.

In addition to the potential for multidifferentiation,

MPCs are relatively easy to extract from bone marrow

and expand in culture. Development of an in vitro chon-

drogenic differentiation model of marrow-derived MPCs

presents an opportunity to explore the extracellular ma-

trix’s guiding effects on chondrogenesis that is of poten-

tial therapeutic utility.

Previous study showed that TGF-(31 induces prolifer-

ation of osteoblasts, chondrocytes, and mesenchymal

stem cells [40]. The present study also showed an

increase in the proliferation of mesenchymal progeni-

tors. Additionally, other study have shown that syn-

thesis of ECM was found to be enhanced by TGF-PI

[39]. Similarily, this study also showed a consistency

in this regard, in that TGF-PI increased synthesis of

GAG in MPCs.

TGF-PI controls the morphology

and differentiation of epithelial cells [47, 3,301. The

morphological changes in MPCs induced by TGF-P1

in this research were elongation and extension of cell

shapes.

C. W. Chen et al. I Journul yf Ortliopardic Research 23 (2005) 446453 45 1

Fig. 4. Type I and I1 collagen effects on morphological changes in MPCs treated with dexamethasone and rhTGF-PI in monolayer culture ( 1 0 0 ~ ) . (A and D) MPCs appeared fibroblast-like morphology and mild pile-up 14 days after cultured with dexamethasone and TGF-PI. Incorporation with extracellular type I collagen cells showed an elongated spindle-shaped fibroblastic appearance in the absence (B) or presence (E) of rhTGF-PI and dexamethasone. (C) Cells showed more cuboidal-, and less spindle-shaped morphology after pretreatment with type I1 collagen. (F) Cells appeared predominantly rectangular after pretreated with type I1 collagen in addition to dexamethansone, and rhTGF-PI. (G) Note abundant G A G in the cartilaginous-like cell-collagen matrix aggregate (alcian-blue staining).

Chondrogenesis of chondroprogenitors can be stim-

TGF-P1 further enhances the process

of

the synthesis

ulated in the presence of the dexamethasone [12].

of extracellular matrix. Levels of mRNA for type

I1

Chondrogenesis was induced by dexamethasone. That

collagen and Sox9 were also increased when treated

was revealed by increased GAG expression. There

with dexamethasone in this study. This was correlated

was a synergistic effect with TGF-P1.

This

study imply

with previous study that an enhancement of

Sox

that dexamethasone may be the fundamental factor

mRNA by dexamethasone was showed in chondrocytes

that triggers chondrogenic differentiation, whereas

[36].

452 C.

W.

Chen et al. I Journal of Orthopaedic Research 23 (2005) 446453

The regulatory effect of collagen seems to be corre-

lated with its triple-helical structure. Our data showed

that enhancement of

GAG

synthesis by native type

I1

collagen (4.3 times) was significantly downgraded when

using denatured type

I1

collagen (2.9 times). These find-

ings agree with previous studies that denatured type

I1

collagen dimilished native type

I1

collagen effect on

chondrocyte regulation [33,4 1,421.

Exogenous type I1 collagen could maintain the phe-

notype of chondrocytes. This increased the syntheses

of type I1 collagen and

GAG

[37,32,33]. Cell-matrix

interactions via cell surface receptors transduce extracel-

lular signals inwards to regulate the cell phenotype [34].

In this research by adding type

I1

collagen the semi-

transparent fibrous-like substance became noted on

MPCs. This indicated the type

I1

collagen receptor

might be induced on cell surfaces. Integrin a2p1, a major

receptor for type

I1

collagen, plays important roles

during chondrogenic differentiation by MPCs [ 17,231.

Binding affinity and signaling of type

I1

collagen are

mediated by integrin receptor [35]; however, the signal-

ing of specific domains of

a

or

p

integrins for type 11 col-

lagen is not well understood. Further study will identify

the specific receptor for type

I1

collagen, their down-

stream signals, and how it regulates chondrogenic differ-

entiation of MPCs.

Type I collagen, in contrast to cartilage-specific type

I1

collagen, exists ubiquitously in bone, tendon, cornea,

and skin and acts as a structural protein in mammals

[22]. During cartilage repair of large osteochondral de-

fects, bone marrow-derived MPCs are induced to differ-

entiate into fibroblasts or osteoblasts within fibrin clots

containing fibronectin and type

I

collagen [2,38]. Consis-

tent with previous research, type

I

collagen treated

MPCs did not increase GAG expression, but induced

an elongated spindle-shaped fibroblastic appearance.

Therefore, type

I

collagen matrix may provide the suit-

able microenvironment, which is better for fibrogenic

differentiation. This matrix-guided mesenchymal pro-

genitor cell differentiation

in

situ predominantly induces

regeneration towards fibro-cartilage formation.

The current study substantiates the concept that

chondrogenic differentiation by MPCs can be synergisti-

cally triggered by cytokines and further regulated by the

ECM.

A

comprehensive understanding of the regulation

of chondrogenic differentiation by MPCs may enable the

intentionally engineering of cartilage development

in vitro. Subsequent results may yield further informa-

tion for the purpose of a direct repair of large cartilage

defects.

Acknowledgments

This work was supported by the National Science

Council, Taiwan, R.O.C. under project no. NSC91-

2314-B-038-033. The authors thank Prof C.Y. Yeh for

statistical advice, and Ching-Jin Tu and Ching-Fu Liao

for their technical help with histological preparations.

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