RESUMO
BACKGROUND: Despite recent advances in bone tissue engineering, efficient bone formation and vascularization remains a challenge for clinical applications. HYPOTHESIS: The aim of this study was to investigate if the osteoblastic differentiation of human mesenchymal stromal cells (MSCs) can be enhanced by co-culturing them with peripheral blood (PB) mononuclear cells (MNCs), with and without vascular endothelial growth factor (VEGF), a coupling factor of bone formation and angiogenesis. MATERIALS AND METHODS: Human bone marrow (BM) derived MSCs were co-cultured with PB-MNCs in osteogenic medium with or without VEGF. Osteoblastic differentiation and mineral deposition were studied by staining for alkaline phosphatase (ALP), and von Kossa, respectively, and measurements for ALP activity and calcium concentration (Ca). Cell proliferation was assayed with Alamar blue. The mechanism(s) were further studied by Transwell(®) cell culture experiments. RESULTS: Both ALP and mineralization (von Kossa and Ca) were significantly higher in the MSC-MNC co-cultures compared to plain MSC cultures. VEGF alone had no effect on osteoblastic differentiation of MSCs, but further enhanced differentiation in co-culture settings. The mechanism was shown to require cell-cell contact between MSCs and MNCs and the factors contributing to further differentiation appear to be soluble. No differences were observed in cell proliferation. CONCLUSION: Our study demonstrates that the in vitro ALP activity and mineralization of human BM-MSCs is more efficient in the presence of PB-MNCs, and exogenously added VEGF further enhances the stimulatory effect. This indicates that PB-MNCs could be a potential cell source in development of co-culture systems for novel tissue engineering applications for enhanced bone healing.
Assuntos
Diferenciação Celular , Leucócitos Mononucleares/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fosfatase Alcalina/metabolismo , Proliferação de Células , Técnicas de Cocultura , Humanos , Engenharia TecidualRESUMO
BACKGROUND AND AIMS: In adult connective tissues, mesenchymal stem cells (MSCs) play a key role in normal tissue turnover and repair. MSCs can participate in these processes not only through proliferation and differentiation but also through paracrine/autocrine functions. These characteristics make MSCs the optimal target in the development of cell-based therapies. This study describes a novel interaction between human MSC and blood mononuclear cells (MNCs), resulting in formation of blood vessel-like structures. MATERIALS AND METHODS: Human marrow-derived MSCs and peripheral blood MNCs were co-cultured in monolayer cultures as well as in bovine collagen sponge up to 20 days. No exogenously supplied growth factors were applied. Morphological changes and formations of three dimensional structures were detected by light microscopy. The process was further stu-died for the expression of different endothelial cell markers. The expression of PECAM-1 and endoglin was studied by immunohistochemistry and the expression of vascular endothelial growth factor receptors 1 and 2 using quantitative real time PCR. RESULTS: In co-cultures of human MSCs and MNCs, the previously nonadherent cells attached and started to elongate and formed tube-like structures within one week. At day 10, elongated PECAM-1 and endoglin expressing cells were detected in co-cultures. At day 20, PECAM-1 and endoglin-positive vessel-like structures were observed. VEGFR1 was up-regulated in co-cultures after 10 days, and expression levels increased with time. No PECAM-1, endoglin or VEGFR1 expressing cells were discovered in MSC-cultures without MNCs at any time point. CONCLUSIONS: This study demonstrates induction of endothelial differentiation in co-cultures of human MSCs and MNCs, indicating a mechanism by which local application of MSCs could induce angiogenesis in vivo.