Influence of intermittent hydrostatic pressure and low oxygen partial-pressure on the redifferentiation of dedifferentiated articular chondrocytes inalginate culture

One of the goals in the field of tissue engineering is the development of a rtificial cartilage for the treatment of cartilage defects. Therefore autol ogous chondrocytes are seeded on different artificial matrices to test thei r possible use as implants (resorption, antigenicity, toxicity and their in tegration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating l arge-scale defects or for comparing different matrices. An in-vitro-multipl ication of the cells is needed which causes the chondrocytes to dedifferent iate and become fibroblast-like. Therefore parameters which induce a rediff erentiation are of great interest The objective of this study was to determ ine the influence of intermittent hydrostatic pressure and low oxygen parti al pressure on the redifferentiation of dedifferentiated bovine articular c hondrocytes in monolayer and three-dimensional alginate bead culture. The r edifferentiation process was monitored by immunocytochemical detection of n ewly synthesized collagen type II. The viability of the cells was determine d by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20 %. After this they were subcultured in monolayer or three-dimensional alginate cult ure and subjected to three different stimuli for three weeks in order to re differentiate: 1.) 20 % O-2 (= 20,26 kPa P-O2) + 5 % CO2 + 75% N-2; 2.)5% O -2 (= 5,07 kPa P-O2) + 5% CO2 + 90% N-2; 3.) 5% O-2 (= 5,07 kPa P-O2) + 5 % CO2 + 90 % N-2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer the re was no detectable collagen type II found by immunocytochemistry under ei ther of the three culture conditions. Therefore a redifferentiation of dedi fferentiated chondrocytes was not possible in monolayer cultures with the t ested parameters. In the three-dimensional alginate culture there was no im munocytochemical staining of collagen type ii found in the beads cultured w ith 20 % oxygen. With 5 % oxygen we found a strong collagen type II-product ion by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5 % oxygen lead to a decreased collagen type II-pro duction compared to cells subjected to 5 % oxygen only. Also chondrocytes c loser to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chond rocytes in the alginate culture was close to 90 % after three weeks. Our ex periments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.