ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 1

Dedifferentiation of Aged Human Articular Chondrocytes in Monolayer Culture

Michaela Truppe, Stefan Marlovits, Brigitte Tichy, and Vilmos Vécsei

Ludwig Boltzmann Institute for Biomechanics and Cell Biology, Trauma Research Laboratories, Clinic for Traumasurgery, University of A-Vienna

INTRODUCTION

Articular chondrocytes cultured in monolayer are used for the treatment of cartilage defects in human joints. In monolayer culture the cells dedifferenciate and the synthesis of cartilage specific matrix proteins gets lost.

METHODS

Chondrocyte monolayer cultures were established from small cartilage samples from the macroscopic unchanged areas of femoral heads of ten patients with an average age of 87 years (range 73 to 99) after femoral neck fracture and implantation of hip hemiprosthesis. The phenotype of the cultivated chondrocytes was determined with immune histochemical stainings using monoclonal antibodies against human collagen type I and type II, protein S-100, keratansulfate and vimentin. Collagen I and Collagen II synthesis was proved by Western Blot analysis.

Equal amounts of pepsin digested protein extracts of chondrocytes cultured for different time periods were separated on 6% PAA gels. One gel was stained with Coomassie Brilliant Blue the other was blotted on nitrocellulose. Purified Collagen I and Collagen extract of cartilage were used as controls. Collagen specific bands detected with Collagen I and Collagen II specific antibodies in Western Blot were vizualised by a horseradish peroxidase chemiluminiscence system on X-ray film.

RESULTS

Western blot analysis with Collagen type II specific antibody shows a synthesis of Collagen type II until day 27 in monolayer culture. Detection with Collagen type I specific antibody indicates the beginning of Collagen I synthesis around day 21 in monolayer culture. At day 7 and day 14 only Collagen II specific bands appear, chondrocytes at day 37 and 44 produce only Collagen I. These data are in accordance with the data shown by immunocytochemical staining.

CONCLUSION

Freshly isolated human articular chondrocytes express cartilage-specific Collagen type II and continue to do so for several days in primary monolayer culture. The "switch" from Collagen type II to Collagen type I synthesis during monolayer culture of these aged cells was found around day 25 (range 21 to 30).

This loss of phenotype in monolayer culture is reversible if the cells are placed in suspension cultures such as agarose or alginate.