DNA cytofluorometric analysis of chondrocytes in human articular cartilages under normal aging or arthritic conditions.
OBJECTIVE
Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN).


METHODS
The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization.


RESULTS
The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S- G2 index, in diseased cartilages (OA, RA and AN) was significantly high (P< 0.0001). In OA cartilage, the S-G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate.


CONCLUSIONS
These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.
Kusuzaki
K
K
Department of Orthopedic Surgery, Kyoto Prefectural University of Medicine, Hirokoji, Kyoto, Japan. kusu@basic.kpu-m.ac.jp
Sugimoto
S
S
Takeshita
H
H
Murata
H
H
Hashiguchi
S
S
Nozaki
T
T
Emoto
K
K
Ashihara
T
T
Hirasawa
Y
Y
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology*; D001172:Arthritis, Rheumatoid / Q000473:pathology*; D002455:Cell Division / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002648:Child; D002675:Child, Preschool; D019902:Chondrocytes / Q000166:cytology*; D020732:Cytogenetic Analysis / Q000379:methods; D004171:Diploidy; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000473:pathology*; D010020:Osteonecrosis / Q000473:pathology*