Villars, F., B. Guillotin, T. Amé dé e, S. Dutoya, L. Bordenave, R. Bareille, and J. Amé dé e. Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication. Am J Physiol Cell Physiol 282: C775-C785, 2002; 10.1152/ajpcell.00310.2001.-Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This "juxtacrine signaling" could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18␣-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.coupling; intercellular messenger; connexin43; osteoblast; endothelial cells ANGIOGENESIS is a tightly regulated process involved in growth, repair, and bone remodeling (9, 11-13, 23, 28, 50, 54, 56). Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, in which systemic hormones and paracrine growth factors or cytokines play an active role (4,24,53,55). In a previous study (51), we showed that osteoblast progenitor cells [human bone marrow stromal cells (HBMSC)] behave differently in terms of proliferation and differentiation when cultured in association with endothelial cells [human umbilical vein endothelial cells (HUVEC)] in various coculture models (coculture with or without direct contact, conditioned medium) than when they are cultured alone. An increase in alkaline phosphatase activity (Al-P) was observed only when HBMSC were cocultured in direct contact with HUVEC. The enhancement of this early osteoblastic marker is not provided when HBMSC and HUVEC are cocultured separately with the use of a semipermeable membrane (51) or when HBMSC are seeded onto a matrix obtained from HUVEC cultures (51).Therefore, the intercommunication between endothelial cells and os...
Angiogenesis is a tightly regulated process involved in growth, repair, and bone remodeling. Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, where systemic hormones and paracrine growth factors play an active role. Angiogenesis is induced by a variety of growth factors; among them vascular endothelial growth factor (VEGF) may be an important mediator for the angiogenic process involved in bone physiology. We studied the VEGF effect on osteoblast progenitor cells (Human Bone Marrow Stromal Cells: HBMSE) cultured alone or associated with endothelial cells (Human Umbilical Vein Endothelial Cells: HUVEC) in different co-culture models (co-culture with or without direct contact, conditioned medium), to determine the influence of VEGF on these cells and on their relationship. In agreement with other studies, we show that HBMSC express and synthesize VEGF, HUVEC conditioned medium has a proliferative effect on them, and early osteoblastic marker (Alkaline phosphatase activity) levels increase when these cells are co-cultured with HUVEC only in direct contact. However, unlike previous studies, we did not find that VEGF increased these processes. These results suggest that the intercommunication between endothelial cells and osteoblastic-like cells requires not only diffusible factors, but also involving cell membrane proteins.
Research in biomaterials for bone reconstruction has led to elaborate osteogenic composites that combine porous ceramics with bone marrow stromal cells. The aim of this study was to evaluate the influence of direct vascularization of such composites on osteogenesis and the ability to produce a vascularized bone substitute transplant in an ectopic muscular site. Sixty-four coralline biomaterials were implanted in 32 Fisher rats under four conditions: (1) alone (reference group M, n = 16), (2) coated with bone marrow stromal cells (group MC, n = 16), (3) combined with a vascular pedicle (group MV, n = 16), or (4) coated with bone marrow stromal cells and combined with a vascular pedicle (MCV group, n = 16). The number of vessels in the pores (vessel-pore ratio) of the implants and the proportion of pores showing bone ingrowth (bone-pore ratio) were measured at 2, 4, 6, and 8 weeks on four implants of each group. Compared with the reference group, angiogenesis was higher when the biomaterial was combined with a vascular pedicle or was coated with osteoprogenitor cells. The association of both vascular pedicle and osteoprogenitor cells increased vascularization by 60 percent (p = 0.003) and osteogenesis by 62 percent (p < 0.001). A combination of both vascular pedicle and bone marrow osteoprogenitor cells in coralline implants enhances neovascularization and osteogenesis after implantation in ectopic intramuscular sites to a greater extent than either does alone.
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