Aicher WK, (aicher@uni-tuebingen.de), Steinbach K, Mollenhauer J, Fritz J, Ashammakhi N, Gaissmaier C.

University of Tuebingen Medical Centre, Hoppe-Seyler-Str. 3, 72076 Tuebingen (FRG)

Introduction:
For ACT chondrocytes are expanded in monolayer culture. During expansion the cells dedifferentiate characterized by changes in the expression of various genes encoding matrix proteins, growth factors, receptors and signal transduction molecules *1,2*. Cultivation of expanded chondrocytes in suitable scaffolds may partially restore the specific phenotype required for ACT; characterized by e.g. high expression of type II collagen *1,2*. Therefore we were interested in establishing molecular quality standards of expanded chondrocytes to ensure a high quality transplant and to be able to correlate the phenotype of these cells prior ACT to clinical outcome after ACT.

Methods:
Patients were selected for ACT according to the ICRS criteria. Chondrocytes were isolated from articular cartilage of patients undergoing ACT after informed consent (n=25). Cells were expanded for 10-12 days in primary culture in DMEM/F12 medium enriched with autologous serum. Cells were harvested for ACT by enzymatic detachment and an aliquot (105cells) was used for RNA extraction, cDNA synthesis and real time quantitative PCR (LightCyclerÒ). Clinical outcome of the ACT was evaluated after 6 weeks, 6 months and 12 months by medical check-up and/or MRT. In cases of doubt, second look arthroscopy was performed.

Results:
Ex vivo and in primary culture individual differences of gene expression patterns were found when the different patient samples were compared under identical conditions. In all cases analysed, high copy numbers encoding type II collagen correlated with high copy numbers encoding BMP-2 and IL-10. In 22/25 samples, low copy numbers encoding BMP-4, GDF-5, IL-1 and IL-18 were found. During subsequent cultivation, type II collagen, BMP-2 and IL-10 encoding mRNA decreased and a2 type I collagen, BMP-4, GDF-5 and IL-18 mRNA increased. However, recultivation of expanded chondrocytes in alginate recovered elevated IL-10 expression over IL-18 as well as type II collagen and BMP-2. Independent of the culture conditions, the variations of gene expression patterns were correlated with clinical outcome. We report that success of ACT correlated with chondrocytes expressing high levels of type II collagen, BMP-2 and IL-10. In all those samples where elevated IL-1 expression was detected in primary culture chondrocytes prior to ACT, the clinical outcome was not satisfying (n=3/25).

Conclusions:
Chondrocytes retain a phenotype suitable for ACT during primary culture in autologous serum for at least a 12-day expansion period. High expression of type II collagen, BMP-2 and IL-10 correlated with a high cell quality suitable for ACT. Samples expressing IL-1 may indicate a risk of ACT failure. As our cell culture conditions do not induce IL-1 expression in vitro, elevated IL-1 titers found in some samples may reflect an inflammatory and catabolic articular environment in situ prior to biopsy. Therefore, a prominent expression of type II collagen, BMP-2 and IL-10 in absence of IL-1 encoding mRNA may be useful molecular markers to predict chondrocyte qualities sufficient for ACT.

References:
1. Dell`Accio F, De Bari C, Luyten FP. Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo. Arthritis Rheum 2001, 44(7):1608-19. 2. Zaucke F, Dinser R, Maurer P, Paulsson M. Cartilage oligomeric protein (COMP) and collagen IX are sensitive markers for the differentiation state of articular primary chondrocytes. Biochem J 2001, 358: 17-24.