Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

J. Steinhagen; J. Bruns; O. Niggemeyer; M. Fuerst; W. Rüther; M. Schünke; B. Kurz

doi:10.1016/j.tice.2010.03.003

Tissue and Cell 42 (2010) 151–157

Contents lists available at ScienceDirect

Tissue and Cell

journal homepage: www.elsevier.com/locate/tice

Perfusion culture system: Synovial ﬁbroblasts modulate articular chondrocyte

matrix synthesis in vitro

J. Steinhagen

a,∗

, J. Bruns

a,1

, O. Niggemeyer

b,2

, M. Fuerst

b,2

, W. Rüther

a,1

,

M. Schünke

c,3

, B. Kurz

c,3

a

Department of Orthopaedics, University Medical Center Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany

b

Department of Orthopaedics, Klinikum Bad Bramstedt, Oskar-Alexander-Straße 26, D-24576 Bad Bramstedt, Germany

c

Department of Anatomy, University of Kiel, Otto-Hahn-Platz 8, D-24118 Kiel, Germany

article info

Article history:

Received 14 May 2009

Received in revised form 21 March 2010

Accepted 22 March 2010

Available online 28 April 2010

Keywords:

Co-culture

Perfusion chamber

Synovial ﬁbroblasts

Chondrocytes

abstract

Objective: To investigate the interactions of chondrocyte metabolism by synovial cells and synovial super-

natants in a new perfusion co-culture system.

Methods: Chondrocytes and synovial ﬁbroblasts were obtained from knee joints of slaughtered adult cat-

tle. For experimental studies chondrocytes and synovial ﬁbroblasts were placed together into a perfusion

chamber (co-culture) or were placed into two different perfusion culture containers, which were con-

nected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was

used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The

proteoglycan synthesis was quantiﬁed using

35

SO

4

2−

-labelling and the dimethylmethylene blue assay.

3

H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was

measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemi-

cally (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after

stimulation with hydrogen peroxide was measured.

Results: The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture

or in culture with synovial supernatants more than ﬁvefold within 12 days.

3

H-proline incorporation

as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with

synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the

35

SO

4

2−

incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a signiﬁcant

decrease of proteoglycan release and content. Quantiﬁcation of collagen type II by ELISA shows signiﬁcant

lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well

as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide

is reduced when chondrocytes are co-cultured with synovial ﬁbroblasts.

Conclusion: The co-culture perfusion system is a new tool to investigate interactions of different cell types

with less artiﬁcial interferences. Our results suggest that synovial supernatants and synovial ﬁbroblasts

modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility

to radical attack of reactive oxygen species.

1. Introduction

The physiological function of synovial joints is based on the

interactions of different connective tissues. Muscle tissue, adi-

pose tissue, ligaments, blood vessels and nerves are components

of synovial joints. However, articular cartilage and synovium are

supposed to be the most important tissues both in physiology and

∗

Corresponding author. Tel.: +49 40 7410 53670; fax: +49 40 7410 55018.

1

Tel.: +49 40 7410 53670; fax: +49 40 7410 55018.

2

Tel.: +49 4192 900; fax: +49 419290 2373.

3

Tel.: +49 431 880 2470; fax: +49 431 880 1557.

pathophysiology. Interactions of chondrocytes and synoviocytes

got into the focus of interest, since it has been shown that nutri-

tion of cartilage in particular is dependent on factors produced by

synoviocytes (Levick and McDonald, 1995). It is also known that

inﬂammatory processes of the synovium are a part of osteoarthri-

tis and rheumatoid arthritis (Katrib et al., 2002). However, little is

currently known about this cellular cross-talk between cartilage

and synovium.

In former investigations synovial supernatants were used to

study the modulation of chondrocyte metabolism in vitro (Lee et al.,

1997), but cellular interactions could not be detected by this experi-

mental setup. Further reports described several other techniques to

culture synoviocytes and chondrocytes together. Nevo et al. (1993)

doi:10.1016/j.tice.2010.03.003

Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Steinhagen, J.; Bruns, J.; Niggemeyer, O.; Fuerst, M.; Rüther, W.; Schünke, M.; Kurz, B.

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