The influence of proepicardial cells on the osteogenic potential of marrow stromal cells in a three-dimensional tubular scaffold

Abstract It is well established that the process of neovascularization or neoangiogenesis is coupled to the development and maturation of bone. Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) comprise a heterogeneous population of cells that can be differentiated in vitro into both mesenchymal and non-mesenchymal cell lineages. When both rat BMSCs and quail proepicardia (PEs) were seeded onto a three-dimensional (3-D) tubular scaffold engineered from aligned collagen type I strands and co-cultured in osteogenic media, the maturation and co-differentiation into osteoblastic and vascular cell lineages were observed. In addition, these cells produced abundant mineralized extracellular matrix materials and vessel-like structures. BMSCs were seeded at a density of 2 × 106 cells/15 mm tube and cultured in basal media for 3 days. Subsequently, on day 3, PEs were seeded onto the same tubes and the co-culture was continued for another 3, 6 or 9 days either in basal or in osteogenic media. Differentiated cells were subjected to immunohistochemical, cytochemical and biochemical analyses. Phenotypic induction was analyzed at mRNA level by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Immunolocalization of key osteogenic and vasculogenic lineage specific markers were examined using confocal scanning laser microscopy. In osteogenic tube cultures, both early and late osteogenic markers were observed and were reminiscent of in vivo expression pattern. Alkaline phosphatase activity and calcium content significantly increased over the observed period of time in osteogenic medium. Abundant interlacing fascicles of QCPN, QH1, isolectin and α-smooth muscle actin (α-SMA) positive cells were observed in these tube cultures. These cells formed extensive arborizations of nascent capillary-like structures and were seen amidst the developing osteoblasts in osteogenic cultures. The 3-D culture system not only generated de novo vessel-like structures but also augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, this novel co-culture system provides a useful in vitro model to investigate the functional role and effects of neovascularization in the proliferation, differentiation and maturation of BMSC derived osteoblasts.