Microenvironmental influence on cartilage regenerative tissue

Title:

Cartilage- and subchondral bone derived factors regulate collagen production in MSC

Background

Functional repair of critical-size articular cartilage defects is still a considerable challenge in the field of tissue regeneration. To address the problems of in vivo regeneration of chondral and osteochondral defects a better understanding of microenvironmental cues and the modulating influence of neighbouring cells and tissue interfaces on chondrogenic differentiation of chondroprogenitor cells are essential. We intend to compare impact of cartilage / subchondral bone surfaces on chondrogenic differentiation of mesenchymal stem cells (MSC) and properties of regenerated tissue. This study is designed to identify microenvironmental conditions which promote, prevent or interfere with chondrogenic differentiation of MSC and to analyse their impact on hyaline cartilage like quality and biomechanical properties of regenerated tissue.

State of study

We established a reproducible coculture model which demonstrated that chondrogenic differentiation of MSC is affected by the microenvironmental conditions. We detected that coculture with cartilage and subchondral bone explants from OA-patients suppress collagen I, II and III gene expression and production in MSC. Additional, we found an inhibitory effect of subchondral bone explant coculture on glycosaminoglycan content of the MSC. Future studies with ovine tissue will delineate if healthy cartilage/subchondral bone provokes different responses in MSC compared to OA derived tissues.

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Figure 1: Culture regimen:

MSC, chondrocytes and mixed cultures (1:1) of MSC+chondrocytes embedded in fibringel were kept in three different culture regimens: 1. in coculture with cartilage explants, 2. in coculture with  subchondral bone explants and 3. in monoculture.

Fig. 2: ELISA for collagen I and II

(A) collagen I: MSC and mixed cultures MSC+chondrocytes (1:1) kept in coculture with cartilage (FC) or subchondral bone explants (FB) have a significant reduced collagen I content compared to monocultures (F).

(B) collagen II: MSC and chondrocytes kept in coculture with cartilage (FC) or subchondral bone explants (FB) have a significant reduced collagen II content compared to monocultures (F).

Fig. 3: Immunofluorescence staining for (A) collagen I and (B) collagen II on day 28 of culture time line. MSC cocultured with OA-cartilage or subchondral bone show reduced collagen I and II staining compared to MSC in monoculture.

Aims

·         Identification of responsible signalling factors derived from cartilage and subchondral bone explants which modulate matrix production of cocultured MSC/chondrocytes

• Comparison of molecular properties of newly generated tissues by cells cultured on the surface of cartilage versus subchondral bone explants

·         Determination of biomechanical properties of regenerated tissues under the different coculture regimen

• Analyse weather cartilage/chondrocytes of healthy (non-OA) joints have a beneficial impact on implanted MSC/chondrocytes and support formation of high quality and functional repair tissue with a stabilized chondrogenic phenotype.

Investigators (Exp. Orthopedics):

Prof. Dr. Susanne Grässel, principal investigator
Michaela Leyh, Dipl. Biologist
Lilly Weger, MTA

Drs. Johannes Beckmann and Robert Springorum (Orthopedic Surgery)

Collaborators :

Prof. Dr. Rainer Deutzmann, Dept. of Biochemistry 1, University of Regensburg

Prof. Dr. Wiltrud Richter, Exp. Orthopedics, Orthopedic Surgery, University of Heidelberg

Drs. Uta Delling and Henriette Jülke, Large Animal Clinic for Surgery, Faculty of Veterinary Medicine, University of Leipzig

Grants:

This work is supported by the Deutsche Forschungsgemeinschaft (grant GR 1301/8-1and GR 1301/11-1) and the DGOOC.