Rabbit nucleus pulposus cells induce the differentiation of mesenchymal stem cells after co-culture in a three-dimensional environment

ABSTRACT

BACKGROUND:

In the field of intervertebral disc tissue engineering, seed cells primarily consist of nucleus pulposus cells (NPCs) and mesenchymal stem cells (MSCs). NPCs have been known to have several shortcomings limited source, inconvenient collection, poor proliferative capacity, and difficult in vitro culture.

To investigate the proliferation of MSCs after co-culture with NPCs in alginate beads-simulated three-dimensional environment.DESIGN, TIME AND

A cell observation was performed at the Laboratory of Orthopedics, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology between December 2007 and October 2008.MATERIALS Six healthy New Zealand rabbits of 4 months old were provided by the Laboratory Animal Center, Tongji Medical College of Huazhong University of Science and Technology and were used for the present study.

Rabbit MSCs were isolated and purified using density gradient centrifugation and adherence methods. Rabbit NPCs were isolated and purified using collagenase Ⅱ digestion and adherence methods. Following liposome-mediated green fluorescent protein tranfection and G418 screening, MSCs of passage 3 were cultured either alone (control group) or with NPCs at a ratio of 11 (co-culture group) in alginate beads. After 14 days of culture, alginate beads were dissolved and MSCs were collected by flow cytometry sorting.MAIN OUTCOME

The expression of collagen Ⅱ and aggrecan in MSCs was detected by RT-PCR and Western blot techniques.

mRNA and protein expression of collagen Ⅱ and aggrecan was observed in the co-culture group, but not in the control group, after 14 days of culture.

MSC differentiation towards nucleus pulposus-like cells can be induced by co-culture with NPCs in a three-dimensional environment.