Osteogenic differentiation of human mesenchymal stromal cells is promoted by a leukocytes containing fibrin matrix

Abstract

Mesenchymal stem cells (multipotent human mesenchymal stromal cells, MSC) are currently the most promising cell type for regenerative medicine. For a clinical approach, it is necessary to develop and establish methods for expansion, differentiation, and delivery. A completely autologous plasma clot containing peripheral blood mononuclear cells (PBMC) was tested for the osteopromotive activity towards expanded human mesenchymal stem cells in vitro. The plasma clot was prepared from anticoagulated blood plasma after addition of isolated leukocytes and calcium chloride. Plasma clots after the gelation were added to subconfluently growing MSC or used in a transwell system. Cell proliferation, the activity of alkaline phosphatase, the release of osteoprotegerin, C-terminal procollagen peptide, as well as osteocalcin, the analysis of matrix mineralization as well as bone nodule formation were analyzed up to 3 weeks. In contrast to plasma clots with no exogenously added leukocytes, the presence of PBMC within the plasma clot significantly promoted osteogenic differentiation of MSC correlated to the time period of incubation. Proliferation of MSC was decreased at maximal mineralization time points. In addition, the osteopromotive activity was identified as soluble factor/factors by transwell assay system. There was a decrease in osteoprotegerin when the cells were cultured in the presence of plasma clots compared to control cell cultures without plasma clots. The osteocalcin expression was continuously higher after culture in the presence of plasma clots and significantly higher after 2- and 3-week after culture in the presence of leukocyte-containing plasma clots compared to 1-week cell culture. Differences in the concentration of the C-terminal procollagen peptide were not measured. The direct inoculation of an autologous mononuclear cell fraction (which contains leukocytes and MSC), e.g., isolated from a bone marrow aspirate or a different source into an autologous plasma gel, may be a further new strategy for bone fracture therapy.