In vitro expression and in vivo osteogenic capability of pcDNA3-hBMP2-transfected marrow stromal cells in rabbits

Licheng WEI; Danping LIU; Qin PU

ABSTRACT

BACKGROUND:

Whether bone morphogenetic protein 2 (BMP-2) can be transduced into marrow stromal cells (MSCs) and produce osteogenic effects by viral or non-viral vector remains unclear?

OBJECTIVE:

To observe the expression of cultured rabbit MSCs transfected with pcDNA3-hBMP2 in vitro. Simultaneously, the MSCs were transfected but not screened and then transplanted into autologous muscle to investigate the osteogenic capability by X-ray. DESIGN, TIME AND

SETTING:

A controlled observation experiment was performed at the Department of Orthopedics, Liaoning Medical University between November 2004 and April 2005. MATERIALS Six adult New Zealand rabbits, of either gender, weighing 2.0-3.0 kg, were included for this study. BMP2 antibody was the product of Sanaka Company, USA. pcDNA3-hBMP2 was provided by Professor Pu Qin from the Department of Biochemistry, Fourth Military Medical University of Chinese PLA). Restriction enzyme was purchased from Takara biotechnology (Dalian) CO., LTD., China.

METHODS:

Super-purified plasmid pcDNA3-hBMP2 was extracted from E. coli. Bone marrow was taken from the adult rabbit femur for harvesting MSCs by density gradient separation. The MSCs were divided into the following 4 groups Group A, cells were transfected and screened by G418; Group B, cells were transfected by pcDNA3-hBMP2; Group C, cells were transfected by empty vector pcDNA3; Group D, only transfection reagent Fugene 6 was added.MAIN OUTCOME

MEASURES:

Transient BMP2 expression was analyzed by immunohistochemistry. Expression of osteocalcin and collagen I was examined by immunohistochemistry and in situ hybridization, respectively. Two weeks after transfection, MSCs from the group B were autologously transplanted into the muscle. Four weeks later, X-ray assay was used to observe bone formation.

RESULTS:

pcDNA3-hBMP2 was successfully transduced into MSCs and transiently expressed BMP2 100%. Four weeks after gene transfection, expression levels of osteocalcin and collagen I were significantly higher in the group A than in the groups C and D. X-ray results demonstrated new bone formation four weeks after MSCs transplanted into the muscle.

CONCLUSION:

pcDNA3-hBMP2 can safely and efficiently transfect MSCs and induce them to differentiate towards osteoblasts by secreting BMP2.