| Designing 3D constructs with adequate properties to instruct and guide cells is one of the major focuses of tissue engineering. The features influencing cell viability, the mechanical load bearing capability and cell instruction are distributed along the nano and macro scale of the construct. This calls on new methodologies integrating bottom-up and top-down approaches, known as integrative, for the development of controlled multiscale 3D constructs. The main goal of this thesis is to develop new multiscale and cell instructive constructs by using an integrative approach and cost-effective natural resources. Using a Bioplotter, top-down polycaprolactone (PCL) constructs with controlled geometry were prepared. Layer-by-layer assembling (LbL) was chosen as the bottom-up methodology so that a tunable incorporation of instructive polymers/proteins could be achieved. First, this combination of techniques was explored as a way to create coatings and nano/micro fibrils on the prototyped scaffolds, adding new cell-anchorage points and a potential system to modulate cell behavior. The sequential use of rapid prototyping, LbL and freeze-drying allowed the creation of nanocoatings and/or fibrils inside the 3D scaffolds using alginate and chitosan polyelectrolytes (PEs). Those structures could be selectively added by controlling the LbL parameters. Once the concept was proven, the more adequate biochemistry that could be introduced with the LbL step was investigated. Inspired by the natural extracellular matrix (ECM), different marine-origin sulfated and aminated PEs, were selected: carrageenans (Car's) and chitosan (Chi), respectively. Thus, thin coatings with controlled sulfur and nitrogen content were prepared on polycaprolactone 2D surfaces, physicochemically characterized and their effect on osteoblast-like cells studied. Biomineralization increased with the presence of the coatings being significantly higher on iCar/Chi. This suggested the sulfate groups interact positively with molecules nvolved in the osteoblastic activity. Consequently, the potential of those PEs for the incorporation of human platelet lysate (PL -- source of multiple growth factors, GFs) was investigated: in 2D, on human adipose derived stem cells (hASCs) and human umbilical vein cord endothelial cells (HUVECs), in short-term cultures; and in 3D, for long-term cultures, to investigate the osteogenic differentiation of hASCs. These PEs allowed different degrees of growth factors (GFs) to be incorporated, where iota-Car showed the ability to highly incorporate all the GFs quantified. The sulfated PEs/PL coatings were shown to be efficient in promoting morphological changes, serum-free adhesion and proliferation of high passage hASCs (P>5). The more sulfated nanocoatings activated HUVECs, inducing the formation of tube-like structures and pro-angiogenic gene expressions. Moreover, the iCar/PL/iCar/Chi coatings and fibrillar structures, created into PCL constructs, induced the osteogenic differentiation of hASCs. In the absence of dexamethasone (osteogenic inducer), 10 tetralayers with PL induced hASCs into the osteoblastic lineage, while with 30 (or without PL) this did not occur, which emphasized the importance of a controlled PL incorporation. LbL is a suitable method to tune the incorporation of PL and tune its instruction on several cell types. Moreover, the proposed integrative and sequential approach promise new osteoinductive multiscale 3D construct, and nanocoatings for several tissue engineering applications (e.g., cell expansion and angiogenesis). |
