The effect of incubation of mesenchymal stem cell derived osteoblants with spongious allografts on osteogenic activity

Purpose: Allografts are widely used in Orthopedics. The use of mesenchymal stem cells and combined grafting are increased with the increase use of mesenchymal stem cells in orthopedic surgery. But, there is limited knowledge about behavior of mesenchymal stem cells in combined grafting. Our object was to compare osteoblastic activities of bone marrow and fat derived stem cells and to find the differences of osteoblastic activities after incubation with fresh allografts.Material and Method: Bone marrow derived stem cells were isolated from a 23 years-old patients iliac crest aspiration materials who undergone pseudoarthrosis surgery of femur, and fat derived stem cells were isolated from a 32 years-old patients aspiration material who undergone abdominal liposuction surgery. After isolation, cells were multiplied in a growing medium for 10 days. After that, each group in respect to their origin were divided into two groups. One group was incubated in an osteogenic medium and the other in an osteogenic medium with bone graft for 21 days. These four culture groups? cell proliferations were evaluated with MTS at 1,7,14 and 21st days, osteogenic potentials were evaluated with the measurement of alkaline phosphatase levels at 7,14 and 21st days, and mineralization were evaluated with Von Kossa and Alizarin Red staining at 14 and 21st days.Results: There were no statistically significant difference in proliferation levels between days in each group without bone graft, but at 7th and 21st days, there were statistically significant difference in favour of bone marrow derived stem cells (p<0.05). This result preoccupy that, bone marrow derived stem cells have better osteoblastic proliferation due to their natural line. There were no statistically significant difference in alkaline phosphatase levels between each group at 7,14 and 21st days. There were no significant difference in Von Kossa and Alizarin Red stainings with microscopic evaluation even no quantitative measurement made. When groups with and without bone graft were compared, in the group without bone graft, an increase in cell count was observed except 14th day that specification occurs, but in the group with bone graft, cell count was decreased after 14th day at both two groups. At the 21st day the previously described decrease reached to a statistically significant level (p<0.05). There were no statistically significant difference in alkaline phosphatase levels between groups with and without bone graft at 7, 14 and 21st days. There were statistically significant decrease of mineralization with Von Kossa and Alizarin Red stainings at group with bone graft.Conclusions: Fresh spongios allografts inhibits the osteogenic activity of bone marrow and fat derived mesenchymal stem cellsFurther Perspectives: The decrease of cell count seen with MTS analysis at the late specification phase of mesenchymal stem cells could be evaluated with another study with DNA or protein analysis for more reliable results. The decrease of mineraliation at the groups with bone graft seen at Von Kossa and Alizarin Red stainigs could be also supported with calcium measurement for quantitative evaluation of mineralization. Furthermore, the inhibition at the late phases of specification could be supported with late phase markers of osteogenic activity like; Osteonectine, Osteopontine, Osteocalcine. Some molecules that possibly have role in inhibition like Sclerostin and DKK1 and pathways of these molecules could also been evaluated.