Characterisation of human knee meniscus cell phenotype.
OBJECTIVE
Studies on the biology of the human meniscus cell are scarce. The objective of our studies was to assess survival/proliferation of human meniscus cells in different culture conditions and to characterize the extracellular matrix (ECM) produced by these cells in these artificial environments. The composition of this ECM offers a variable to define the distinct meniscus cell phenotype.


METHODS
Human meniscus cells were isolated enzymatically from visually intact lateral and medial knee menisci. Cells were cultured in monolayer conditions or in alginate gel. The composition of the cell-associated matrix (CAM) accumulated by the isolated cells during culture was investigated and compared to the CAM of articular chondrocytes cultured in alginate using flow cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies against type I collagen, type II collagen and aggrecan. Additional cell membrane markers analysis was performed to further identify the different meniscus cell populations in the alginate culture conditions and meniscus tissue sections. Proliferation was analyzed using the Hoechst 33258 dye method. In some experiments, the effect of TGFbeta1 on some of these variables was investigated.


RESULTS
The CAM of monolayer cultured meniscus cells is composed of high amounts of type I and II collagen and low amounts of aggrecan. A major population of alginate cultured meniscus cells on the other hand synthesized a CAM containing high amounts of type I collagen, low amounts of type II collagen and high amounts of aggrecan. This population is CD44+CD105+CD34-CD31-. In contrast, a minor cell population in the alginate culture did not accumulate ECM and was mainly CD34+. The CAM of alginate cultured articular chondrocytes is composed of low amounts of type I collagen, high amounts of type II collagen and aggrecan. The expression of aggrecan and of type II collagen was increased by the addition of TGFbeta1 to the culture medium. The proliferation of meniscus cells is increased in the monolayer culture conditions. Cell numbers decrease slightly in the alginate culture, but can be increased after the addition of TGFbeta1.


CONCLUSIONS
These results demonstrate that the human meniscus is populated by different cell types which can be identified by a distinct CAM composition and membrane marker expression. Unlike the monolayer culture conditions, the alginate culture conditions appear to favor a more fibrochondrocyte-like cell accumulating a CAM resembling the native tissue composition. This CAM composition is distinctly different from the CAM composition of phenotypically stable articular cartilage chondrocytes cultured in the same alginate matrix.
Department of Rheumatology, Ghent University, Belgium. gust.verbruggen@ugent.be
P C M
PC
Verdonk
R G
RG
Forsyth
J
J
Wang
K F
KF
Almqvist
R
R
Verdonk
E M
EM
Veys
G
G
Verbruggen
D000464:Alginates; D018929:Cell Culture Techniques; D005109:Extracellular Matrix / Q000378:metabolism; D005434:Flow Cytometry; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007719:Knee Joint / Q000166:cytology*; D008592:Menisci, Tibial / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000378:metabolism
D000464:Alginates; D006603:Hexuronic Acids; D016212:Transforming Growth Factor beta; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D020723:Glucuronic Acid