[Comparative study on the main biological characteristics of marrow-derived stromal cells and chondrocytes in vitro culture in rabbits]

Dong Zhang, Jing-xiang Huang, Xiao-qin Ha
Chinese Journal of Reparative and Reconstructive Surgery 2004, 18 (1): 53-7

OBJECTIVE: To observe the main biological characteristics and chondrogenesis potency of bone marrow-derived stromal cells (MSCs) after cytokines induction or gene modification in vitro.

METHODS: MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group (Group A, 15% FBS in DMEM), the induced group by cytokines (Group B), the transfected group (Group C) with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-beta 1 (TGF-beta 1, 10 ng/ml), basic fibroblast growth factor (bFGF, 25 ng/ml) and dexamethasone (DEX, 10(-7) mol/L) with 15%FBS in DMEM. Cartilage slices were obtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in II collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans (GAG), immunohistochemical staining for type I, II collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type I and II collagen was detected by RT-PCR.

RESULTS: Primary MSCs proliferated as short-spindle shape, while the 5th MSCs showed long-spindle shape. Positive stain of type I collagen could be found in groups A, B and C, while positive stain of type II collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them (P > 0.05), and there was significant difference between groups A and D(P < 0.05). No significant difference was noted in groups A, B and C on proliferation by MTT(P > 0.05), except that of at the fourth day after transfection between groups A and C(P < 0.05). RT-PCR demonstrated that MSCs always had higher levels of mRNA type I collagen in groups A, B and C. The expression of mRNA type II collagen was identified in groups B and D, and only low levels of mRNA type II collagen in group C.

CONCLUSION: The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.

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