A thorough understanding of cell response to combined culture configuration and mechanical cues is of paramount importance in vascular tissue engineering applications. Herein, we investigated and compared the response of vascular smooth muscle cells (vSMCs) cultured in different culture environments (2D cell monolayers and 3D cellularized collagen-based gels) in combination with mechanical stimulation (7% uniaxial cyclic strain, 1 Hz) for 2 and 5 days. When cyclic strain was applied, two different responses, in terms of cell orientation and expression of contractile-phenotype proteins, were observed in 2D and 3D models. Specifically, in 2D configuration, cyclic strain caused ∼50% of cell population to align nearly perpendicular (80-90 degrees) to the strain direction, while not influencing the contractile-phenotype protein expression, as compared to the 2D static controls. Conversely, the application of uniaxial strain to 3D constructs induced a ∼60% cell alignment almost parallel (0-10 degrees) to the strain direction. Moreover, 3D mechanical stimulation applied for 5 days induced a twofold increase of SM α-actin level and a 14-fold increase of calponin expression as compared to 3D static controls. Altogether these findings provide a new insight into the potential to drive cell behavior by modulating the extracellular matrix and the biomechanical environment. Biotechnol. Bioeng. 2016;113: 2254-2263. © 2016 Wiley Periodicals, Inc.

The acquisition of new thorough knowledge on the interactions existing between vascular cells would represent a step forward in the engineering of vascular tissues. In this light, herein we designed a physiological-like tri-culture in vitro vascular wall model using a planar cellularized collagen gel as the scaffold. The model can be obtained in 24 h and features multi-layered hierarchical organization composed of a fibroblast-containing adventitia-like layer, a media-like layer populated by smooth muscle cells and an intima-like endothelial cell monolayer. After 7 days of static culture, the compaction of the collagen matrix by the vascular cells was achieved, and the deposition of the vascular extracellular matrix components fibronectin, fibrillin-1 and tropoelastin was observed. The blood-compatible functionality of the endothelial cell monolayer was demonstrated by a blood clotting assay: after 7 days of maturation, clotting was prevented on the endothelialized constructs (more than 80% free hemoglobin maintained after 60 min of blood contact) but not at all on non-endothelialized ones (less than 20% free hemoglobin). In addition, western blotting results suggested that in the tri-culture model the loss of smooth muscle cell phenotype was delayed compared to what was observed in the mono-culture model, finally resulting in a behaviour more similar to the in vivo conditions. Overall, our findings indicate that this in vitro model has the potential to be used as an advanced system to examine vascular cell behavioural interactions, as well as for drug testing and the investigation of physiological and pathological processes.

A fast re-endothelialization, along with the inhibition of neointima hyperplasia, are crucial to reduce the failure of vascular bypass grafts. Implants modifications with molecules capable of speeding up the re-endothelialization process have been proposed over the last years. However, clinical trials of angiogenic factor delivery have been mostly disappointing, underscoring the need to investigate a wider array of angiogenic factors. In this work, a drug release system based on a type I collagen hydrogel has been proposed for the controlled release of Pleiotrophin (PTN), a cytokine known for its pro-angiogenetic effects. Heparin, in virtue of its ability to sequester, protect and release growth factors, has been used to better control the release of PTN. Performances of the PTN drug delivery system on endothelial (ECs) and smooth muscle cells (SMCs) have been investigated. Structural characterization (mechanical tests and immunofluorescent analyses of the collagen fibers) was performed on the gels to assess if heparin caused changes in their mechanical behavior. The release of PTN from the different gel formulations has been analyzed using a PTN-specific ELISA assay. Cell viability was evaluated with the Alamar Blue Cell Viability Assay on cells directly seeded on the gels (direct test) and on cells incubated with supernatant, containing the released PTN, obtained from the gels (indirect test). The effects of the different gels on the migration of both ECs and SMCs have been evaluated using a Transwell migration assay. Hemocompatibility of the gel has been assessed with a clotting/hemolysis test. Structural analyses showed that heparin did not change the structural behavior of the collagen gels. ELISA quantification demonstrated that heparin induced a constant release of PTN over time compared to other conditions. Both direct and indirect viability assays showed an increase in ECs viability while no effects were noted on SMCs. Cell migration results evidenced that the heparin/PTN-modified gels significantly increased ECs migration and decreased the SMCs one. Finally, heparin significantly increased the hemocompatibility of the collagen gels. In conclusion, the PTN-heparin-modified collagen here proposed can represent an added value for vascular medicine, able to ameliorate the biological performance, and integration of vascular grafts.