Influence of intermittent hydrostatic pressure and low oxygen partial-pressure on the redifferentiation of dedifferentiated articular chondrocytes inalginate culture
C. Domm et al., Influence of intermittent hydrostatic pressure and low oxygen partial-pressure on the redifferentiation of dedifferentiated articular chondrocytes inalginate culture, ORTHOPADE, 29(2), 2000, pp. 91-99
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.