[Characteristics of osteoblastic differentiation in mesenchymal stem cells from porcine bone marrow in vitro].

OBJECTIVE

To observe the characteristics and related gene expression of osteoblastic differentiation in porcine bone marrow mesenchymal stem cells (MSCs) during.

METHODS

Bone marrow from 6 landrace pigs, 3-month-old about 50 kg, was aspirated from the medullary cavity of the proximal tibia. The MSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The MSCs from passage 1 were cultivated in DMEM with 1 x 10(-8) mmol/L dexamethasone (Dex), 10 mmol/L beta-glycerophosphate (beta-GP), 82 microg/ml ascorbic acid (Asc) and 10% inactivated fetal bovine serum (FBS) up to 21 days. The MSCs were cultivated in basic DMEM as a control. Cell morphology was observed by microscope. Cell proliferation was tested by using the fluorescent dye SYBR green I measurement. Osteoblastic differentiation was evaluated by alkaline phosphatase (ALP) histochemical staining, quantitative calcium deposit, and real-time PCR technology.

RESULTS

Characterization of primary MSCs: At day 1, most cells depicted round and floating hematopoietic cells. Colonies consisting of fibroblastlike cells were observed at day 3 after removal of non-adherent cells, colonies grew to various sizes at day 7. Thirteen population doublings took place in primary culture. Osteoblastic differentiation: During osteogenic stimulation, cellular morphology of MSCs changed from a fibroblastic shape to a cubical form. Cell proliferation had no impact in osteogenic medium compared to basic medium (P>0.05). At day 14, ALP staining presented strong positive. Calcium deposit pronouncedly increased at day 21 (P<0.01). Furthermore, the mRNA levels of core binding factor alpha1 (Cbfalpha1), osterix, ALP, collagen I (Col I ), osteonectin (ON) and osteocalcin (OC) increased gradually. Cbfalpha1, ON and ALP genes increased at early stage of osteoblastic differentiation. Osterix and OC at day 21 were significantly increased when compared with that at day 7 (P<0.05). Col I was increased at day 14 (P<0.05).

CONCLUSION

Porcine MSCs harvested from bone marrow by density gradient centrifugation are capable of osteoblastic differentiation in vitro. The potential of osteoblastic differentiation relies upon upregulation of genes specific to this lineage under the ostcogenic conditions.