Influence of three-dimensional culture in a type II collagen sponge on primary cultured and dedifferentiated chondrocytes.
BACKGROUND
Once articular cartilage is destroyed, the intrinsic reparative ability is poor. Therefore, various techniques have been developed to repair articular defects. Many kinds of scaffolds have been used for cultured chondrocyte transplantation. In this study, we developed a sponge consisting of type II collagen. We investigated the influence of three-dimensional culture on the maintenance of the chondrocyte phenotype and on the redifferentiation of dedifferentiated chondrocytes.


METHODS
Chondrocytes were isolated from the rib cartilage of rats and were cultured in plastic dishes for a week (P0). The cells were then dissociated with trypsin and subcultured for another 2 weeks (P1). Primary isolated chondrocytes were cultured in the type II collagen sponges for 3 weeks (S1). We compared the gene expression of S1 for chondrogenic markers with the expression of P0 and P1 by reverse transcription-polymerase chain reaction (RT-PCR). The cells were then dissociated with trypsin and subcultured for another 2 weeks (P1) and then another 6 weeks (P3). Cells of P1 were subsequently cultured in type II collagen sponges for 4 weeks (P1r). At each time point, gene expression of chondrogenic markers was examined by RT-PCR.


RESULTS
Gene expression of COL2A1, COL10A1, and aggrecan in S1 was the same as in P0. Gene expression of COL10A1 and aggrecan in P1r was higher than in P1 and P3. Gene expression of COL1A1, COL2A1, and SOX9 in P1r was lower than in P1 and P3. Gene expression of ALP and osteocalcin in P1r was detected.


CONCLUSIONS
These results show that culture in type II collagen sponges could maintain the chondrocyte phenotype; however, dedifferentiated chondrocytes differentiated to hypertrophic chondrocytes. These finding suggest that the complex of cells and scaffolds with primary cells was more useful than that with dedifferentiated chondrocytes in laboratory and clinical application.
Mukaida
Tomoyuki
T
Department of Orthopaedic Surgery, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara 228-8555, Japan.
Urabe
Ken
K
Naruse
Kouji
K
Aikawa
Jun
J
Katano
Motoaki
M
Hyon
Suong-Hyu
SH
Itoman
Moritoshi
M
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000502:physiology*; D024043:Collagen Type II; D015870:Gene Expression; D051381:Rats
D024043:Collagen Type II