In Vitro Effects of BMP-2, BMP-7, AND BMP-13 on Differentiated and Undifferentiated Mouse Mesenchymal Stem Cells

The need for fracture healing alternatives is increasing. The understanding of how fractures heal at the cellular level of fracture repair is lacking. The relevance of this research was to further elucidate the function of different osteo-inductive proteins on mesenchymal stem cells (MSCs) by evaluating undifferentiated and osteoblastic differentiated mesenchymal stem cells treated with bone morphogenic protein-2 (BMP-2), bone morphogenic protein-7 (BMP-7), and bone morphogenic-13 (BMP-13) for time dependent changes in the MSC surface markers CD90 and CD146 and bone specific markers alkaline phosphatase, osteocalcin, opteopotin and RUNX2. In addition, MSCs were also evaluated for time dependent changes in cell number, growth, viability, and cytological changes after administration of proteins.;The measurement of MSC surface markers revealed that both undifferentiated and differentiated groups were positive for CD90 and/or CD146 at 24 and 168 hours of culture and all treated groups were forming osteoblasts. As culture time increased, MSC markers decreased. The measurement of bone specific markers revealed a significant increase in alkaline phosphastase (ALP) in undifferentiated cells treated with BMP-2 and BMP-7 at 24 and 72 hours without the progression of abnormal cells as confirmed by morphology. Osteopontin (OPN) was increased significantly in the undifferentiated groups at 168 hours which suggests that treating MSCs with BMPs before differential ion, produces more osteoblasts as measured by ALP and OPN than treating differentiated MSCs with BMPs. As culture time increased, BMP-7 groups measured a decrease in OCN which may lead to the overgrowth of bone. The measurement of Runt-related transcription factor (RUNX2) in differentiated cells revealed that the control and BMP-7 groups expressed RUNX2 in nodules and peripheral cells at 168 hours. This finding suggests the use of BMPs is not beneficial to MSCs to stimulate osteoblasts after differentiation. MSCs treated with BMP-2 after differentiation demonstrated a lack of osteoblast formation and the RUNX2 marker suggesting again that MSCs should be treated at the MSC stage and not the osteoblast differentiation stage. MSCs when treated with BMP-13 after differentiation expressed chondrocytic morphology and suggest that MSCs can develop into a chondrocyic-like phenotype.;The measurement of cellular growth revealed significant differences at 120 hours between control and BMP-7 and between control and BMP-13. At 120 hours of culture, the control group continued to divide and multiply. BMP groups began to differentiate and were confirmed by H&E morphology. Cellular growth of undifferentiated MSCs in the control group was consistent in that undifferentiated cells proliferate more rapidly than differentiated cells. Cellular growth of undifferentiated MSCs treated with a combination of OM and 0.2 ng of BMP2, BMP-7. and BMP-13 revealed a significant difference at 168 hours of cellular growth between BMP-7 and BMP-13. The BMP-13 group was no longer proliferating and had begun to differentiate into chondrocytic like cells. Some cells were multinucleated. Cellular damage measured by MDA revealed no significant differences at all time points.;The evaluation of cytopathological changes in undifferentiated MSCs after exposure to 0.2 ng BMP-2. BMP-7, and BMP-13 revealed that MSCs treated early with BMP-2 and BMP-7 will differentiate into the osteoblastic lineage. BMP-13 differentiated into the chrondrocytic lineage. MSCs treated with both OM and BMPs simultaneously had a higher rate of multinucleated cells under these conditions. Osteoblasts treated with BMP-7 formed nodules and isolated cells were differentiated. Osteoblasts treated with BMP-2 demonstrated some abnormal cells suggesting that MSCs should be treated at the undifferentiated MSC stage and not the osteoblast differentiation stage. Treating MSCs with BMPs before differentiation produces more osteoblasts without the progression of abnormal cells than treating cells with BMPs after differentiation. This new data is essential for clinical use of these growth factors. The timing and growth factors added may be essential in reducing healing time.