A comparative study on the effects of TGF-β1 and BMP-6 on the chondrogenic differentiating potential of canine adult mesenchymal stem cells derived from bone marrow and adipose tissue.

Abstract

Abstract Cartilage is a strong yet flexible connective tissue capable of withstanding large amounts of force and mechanical stress. These attributes are appropriate for this tissue that functions as a shock absorber that tolerates and transfers variable pressing loads between articulating bones in a joint. Unfortunately is has been well established that articular cartilage has an extremely limited healing capacity. Regenerative medicine aims to repair, replace and regenerate damaged tissues through the use of manipulated autologous mesenchymal stem cells (MSCs). Adult mesenchymal stem cells can be found throughout the body and have the ability to differentiate into multiple cell lineages. Unfortunately this multi-potent ability to differentiate into certain cell lineages differs from where the cells are derived (tissue of origin). In this study we looked into the chondrogenic differentiating abilities of canine mesenchymal stem cells from two sources namely, adipose tissue (cAT-MSC) and bone marrow derived (cBM-MSC). In this study we hypothesize BMP-6 has an additive effect on TGF-β1 regarding the chondrogenic differentiation potential of canine MSCs. The MSCs used in this study were harvested from 13 canine donor tissues. A three-dimensional pellet culture was chosen for the chondrogenic induction of 7 cBM-MSC donor tissues and 6 cAT-MSC donor tissues, the formed pellets were then cultured under 3 different conditions for 35 days. Namely a control condition with no added growth factors, a medium with 10 ng/ml TGF-β1 and finally a medium with TGF-β1 and BMP-6 both at 10 ng/ml. After 35 days in culture the pellets were collected and the level of chondrogenic differentiation was analyzed by means of quantitative PCR, histological staining with Safranin-O/ fast green + hematoxylin/ eosin and data analysis. Results of the histological staining revealed 4 cBM-MSC cultures that stained positively, these pellets showed cells with chondrocyte like morphology and a clear deposition of cartilage like extra cellular matrix in the center. Unfortunately none of the cAT-MSCs exhibited any positive staining. Statistical analysis demonstrated significant differences in gene expression of SOX-9, ACAN, Col2a1 between the two cell cultures (cAT-MSC and cBM-MSC) in all culture conditions. A basal difference in gene expression was established between the cAT-MSCs and the cBM-MSCs that may explain the different culture needs of these cell types. The results of this study lead us to conclude that BMP-6 at a concentration of 10 ng/ml has no additive effect on TGF-β1 regarding the chondrogenic differentiation potential of canine MSCs.