Co-cultures and cell sheet engineering as relevant tools to improve the outcome of bone tissue engineering strategies

摘要

Taking into consideration the complex biology of bone tissue it is quite clear that theunderstanding of the cellular interactions that regulate the homeostasis andregeneration of this remarkable tissue is essential for a successful Tissue Engineeringstrategy. The in vitro study of these cellular interactions relies on co-culture systems, atremendously useful methodology where two or more cell types are cultured at thesame time. Such strategy increases the complexity of typical monoculture systems,allowing the in vitro settings to closely mimic the in vivo environment. Moreover, 2D coculturesystems have been extensively used by cell biologists to study cell interactionsas an attempt to understand specific cellular mechanisms and signalling pathways. Theinteraction between osteoblasts/ osteoprogenitor cells and different cell types relevantwithin the bone Tissue Engineering context, namely mononuclear cells from peripheralblood and umbilical cord blood and fibroblasts, has been addressed in the first part ofthis thesis. The different co-cultures showed that mononuclear cells from peripheralblood were capable of accelerating the osteogenic differentiation of human bonemarrow stromal cells by producing BMP-2. On the other hand, osteoblasts cultured oncarrageenan membranes were capable of supporting the culture of endothelialprogenitors cells present in the mononuclear fraction of umbilical cord blood thatcontributed to the in vivo angiogenesis after implantation in an inflammatory setting.Furthermore, fibroblasts, which are key players in the formation of fibrotic tissue after abiomaterial implantation, were shown to decrease the osteogenic activity of osteoblaststhrough gap junctional communication.A serious limitation of the paradigmatic use of scaffolds for bone Tissue Engineering isthe lack of oxygen and nutrient supply to the cells in the core of the engineeredconstruct leading to cell necrosis at the bulk of the constructs. Furthermore, foreignbody response to the implanted biomaterial is a frequent reaction of the host and has as a consequence the formation of fibrotic tissue surrounding the implant. The use ofcell sheet engineering for bone Tissue Engineering can potentially avoid thoseshortcomings. One of the explored strategies comprised the production of osteogeniccell sheets using this technology. Its potential for in vivo bone formation was analyzedand the formation of vascularized bone tissue with a marrow was originallydemonstrated by implanting a single osteogenic cell sheet in a nude mice model.Furthermore, in order to promote vascularization, co-cultured osteogenic cell sheetswith endothelial cells were also created. Endothelial cells, stacked in betweenosteogenic cell sheets, were proven to contribute to new vessel formation andincreased bone formation in vivoThis thesis demonstrates that monocytes/macrophages from peripheral blood canaccelerate the osteogenic differentiation of osteoprogenitor cells while fibroblasts, havea deleterious effect on the osteogenic phenotype of osteoblasts. In addition, within aninflammatory host reaction, cells from the mononuclear fraction of umbilical cord bloodwere capable of contributing to new blood vessel formation after co-culture withosteoblasts. Moreover, when using the cell sheet technology to fabricate a bone tissueengineering construct, endothelial cells were also shown to improve in vivo boneformation.