Effect of different cell seeding concentrations on chondrogenic differentiation of adipose derived sromal cells in three-dimensional culture

Zhiyong YU; Qin FU; Tao ZHANG

ABSTRACT

BACKGROUND:

The implanted cartilage calls can synthesize cartilage matrix as cartilage in cartilage tissue enginsedng, and the density of implanted cells is the key point.

OBJECTIVE:

To evaluate the effect of cell seeding concentration on the chondrogenic differentiation of the adipose dadved sromal cells (ADSCs).DESIGN, TIME AND

SETTING:

The in vitro cellular-scaffold observation was performed at the cytobiological laboratory of China Medical University from November 2007 to July 2008.MATERIALS Six male SD rata with clean grade were supplied by the Experimental Animal Center of China Medical University.

METHODS:

Totally 5 g/L type ; collagen solution and 20 g/L chitosan was mixed in a mould with volume ratio of 73, after lyophillization, it was cut into pieces with 5 mm ～ 5 mm x 2 mm, followed by crosslinking with ethanol contained of 2% chondroitic acid at room temperature. After washing with double distilled water and freeze drying, the chitosan-collagen-chondroitin sulfate copolymar matrices scaffolds were harvested. ADSCs isolated from rat inguinal fat pads were digested with collagenase and trypsase. The prepared scaffolds were randomly divided into 3 groups, and the third passage cells with density of 2×10 9/L,2×10 109/L, and 2×10 11/L were seeded into chitosan-coflagen-chondroitin sulfate scaffolds, and cultured in chondrogenic medium for 3 weeks.MAIN OUTCOME

MEASURES:

The expression of cartilage specificity gene was detected by hematoxylin-eosin staining, type Ⅱ collagen immunohistochemical staining and RT-PCR.

RESULTS:

Hematoxylin-eosin staining showed that after 3 weeks of culture, the cell proliferated and differentiated well, especially in 2x101～/L group, more extrocelluer matrices were produced and cartilage lacuna-structure could be seen. The type Ⅱ collagen was positive expressed in each group, which showed a gradually increasing tendency with the cell seeding concentration increasing. RT-PCR showed that the expression of proteoglycen and type Ⅱ collagen mRNA were slowly increased. However the expression of Ⅹ collagen mRNA was decreased with increasing cell seeding concentration.

CONCLUSION:

The chitosan-collagen-chondroitin sulfate copolymer matrices can provide an appropdate environment for the generation of cartilage-like tissues and high call seeding concentration of 2×1010/L facilitate ADSCs to differentiate into cartilage.