[Chondrogenesis of passaged chondrocytes induced by different dynamic loads in bioreactor].
OBJECTIVE
To investigate the effect of dynamic compression and rotation motion on chondrogenesis of the 3rd passage cell-loaded three-dimensional scaffold in a joint-specific bioreactor in vitro so as to provide theoretical basis of the autologous chondrocyte transplantation in clinical practice.


METHODS
Primary chondrocytes were isolated and cultured from the knee cartilage of 3-4 months old calves. The 3rd passage cells were seeded onto fibrin-polyurethane scaffolds (8 mm x 4 mm). Experiment included 5 groups: unloaded culture for 2 weeks (group A), direct load for 2 weeks (group B), unloaded culture for 4 weeks (group C), direct load for 4 weeks (group D), and unload for 2 weeks followed by load for 2 weeks (group E). The cell-scaffold was incubated in incubator (unload) or in a joint-specific bioreactor (load culture). At different time points, the samples were collected for DNA and glycosaminoglycan (GAG) quantification detect; mRNA expressions of chondrogenic marker genes such as collagen type I, collagen type II, Aggrecan, cartilage oligomeric matrix protein (COMP), and superficial zone protein (SZP) were detected by real-time quantitative PCR; and histology observations were done by toluidine blue staining and immunohistochemistry staining.


RESULTS
No significant difference was found in DNA content, GAG content, and the ratio of GAG to DNA among 5 groups (P > 0.05). After load, there was a large number of GAG in the medium, and the GAG significantly increased with time (P < 0.05). The mRNA expression of collagen type I showed no significant difference among 5 groups (P > 0.05). The mRNA expression of collagen type II in group B was significantly increased when compared with group A (P < 0.01), and groups D and E were significantly higher than group C (P < 0.01); the mRNA expression of Aggrecan in groups D and E were significantly increased when compared with group C (P < 0.01), and group E was significantly higher than group D (P < 0.01); the mRNA expression of COMP in group B was significantly increased when compared with group A (P < 0.01), and group E was significantly higher than group C (P < 0.01); and the mRNA expression of SZP in group E was significantly increased when compared with groups C and D (P < 0.05). The toluidine blue staining and immunohistochemistry staining displayed that synthesis and secretion of GAG could be enhanced after load; no intensity changes of collagen type I and collagen type II were observed, but intensity enhancement of Agrrecan was seen in groups D and E.


CONCLUSIONS
Different dynamic loads can promote chondrogenesis of the 3rd passage chondrocytes. Culture by load after unload may be the best culture for chondrogenesis, while the 3rd passage chondrocytes induced by mechanical load hold less capacity of chondrogenesis.
Department of Orthopedics, General Hospital of Chinese PLA, Beijing, 100853, P.R. China.
Ning
N
Wang
Jiying
J
Chen
Guoqiang
G
Zhang
Wei
W
Chai
D000818:Animals; D019149:Bioreactors; D002358:Cartilage, Articular / Q000166:cytology*; D002417:Cattle; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D020219:Chondrogenesis; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D007719:Knee Joint; D011140:Polyurethanes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry
D024042:Collagen Type I; D024043:Collagen Type II; D006025:Glycosaminoglycans; D011140:Polyurethanes; D012333:RNA, Messenger