In vitro chondrocyte differentiation of canine bone marrow stromal stem cells

ABSTRACT

BACKGROUND:

Bone marrow stromal stem cells(BMSCs)do not allow for single differentiation of chondrocytes due to their multi-directional differentiation,bone morphogenetic protein secreted from osteoblasts affect the non-differentiated precursor cells and promote their osteoblast differentiations,while those differentiated cells are bound to form tissues.

To in vitro induce canine BMSCs differentiate into chondrocytes,and to investigate the method and conditions of chondrocyte differentiation in vitro.DESIGN,TIME AND

Single sample observation was performed in the Laboratory of Tissue Engineering,Sun Yat-sen University between March 2005 and January 2006.MATERIALSOne male dog,aged 4 months,was involved to harvest BMSCs from the rib.

Rib BMSCs extracted from bone marrow of 2.0-3.0 mL were cultured in vitro. When cells reached a confluence at 8-11 days,trypsinization was conducted and then halted with L-DMEM synthesis culture solution containing 10% fetal bovine serum. Cellular suspension was collected and centrifuged,cells were rssuspended and incubated at a ratio of 13. The third generation of cells were cultured and amplified,10 μg/L basic fibroblast growth factor 2 mL was added to replenish culture medium twice,then 1 mg/L transforming growth factor β1 of 2 mL was applied to induce BMSCs differentiation into chondrocytes.MAIN OUTCOME

Toluidine blue and alcian blue stains were applied to determine cartilage matdx secretion,immunohistochemistTy was used for the detection of cartilage specific Ⅱ collagen expression.

After BMSCs were primarily cultured and subcultured in vitro,they were shown to grow well at the fourth generation,those induced by basic fibroblast growth factor and transforming growth factor β1 were positive for toluidine blue and aician blue staining;immunohistochemistry showed a positive outcome for type Ⅱ collagen,indicating the induced BMSCs exhibited chondrocyte's characteristics.

Utilizing basic fibroblast growth factor and transforming growth factor β1,the induced canine BMSCs could differentiate into chondrocytes,which is considered as an ideal seed cells for cartilage tissue engineering.