[Redifferentiation of the dedifferentiated human articular chondrocytes by the bioreactor culturing].
OBJECTIVE
To examine the biological characteristic changes in the dedifferentiated human articular chondrocytes by the bioreactor culturing in vitro.


METHODS
The cartilage tissue was obtained from the joints of the adult human. The chondrocytes were isolated from the cartilage tissue with the type II collagenase digestion (0.2%, 37 degrees C, 3 h)and were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS) with 1 ng/ml of TGF-beta1, and 5 ng/ml of FGF-2. After about 20 passages by the monolayer culture, the cells were then transferred to the bioreactor culturing of the rotational cell culture system (RCCS) for a 3-week sequence culture. The cell counting was performed with the platelet counter, and the doubling time for each passage of the cells was determined. The frozen section was stained with HE. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry.


RESULTS
When the monolayer culture was performed without any growth factors, the chondrocytes were rapidly proliferated within 3 passages (average doubling time, 59 h), but at the same time, differentiation was also progressing rapidly. After the 4th passage, most of the cells were differentiated and the proliferation was decreased. With the growth factors (TGF-beta1/FGF-2), the speed of the expansion was accelerated (average doubling time, 47 h), but the speed of the dedifferentiation was slowed down. After 20 passages were performed with the monolayer culture, the dedifferentiated chondrocytes could be redifferentiated when they were cultured for 3 weeks with RCCS. Then, the Safranine-O staining was strongly positive for the cells, positive for aggrecan and collagen II, but negative for collagen I, with a well-regained phenotype.


CONCLUSIONS
The bioreactor culturing of the dedifferentiated human articular chondrocytes can regain the differentiated phenotype and it is a useful method of obtaining the human articular chondrocytes in large amounts and in a differentiated phenotype in vitro.
Yu
Fangyuan
F
Orthopedic Institute, Hospital of PLA, Beijing, 100853, PR China.
Guo
Quanyi
Q
Huang
Lihong
L
D000293:Adolescent; D000328:Adult; D000369:Aged, 80 and over; D019149:Bioreactors; D002358:Cartilage, Articular / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged