In situ cardiovascular tissue engineering : study of MCP-1 delivery by mesoporous silica nanoparticles and in vitro evaluation of response to chemokine releasing scaffolds

In situ tissue engineering is an emerging and promising approach to create living substitutes directly implanted inside the human body. To reach this goal is important to produce bioactive scaffolds that are able to recruit cells from surrounding tissues. This work arises in the field of cardiovascular regeneration, in particular for small-diameter pulmonary arteries. The aim is the development of a drug delivery system using Mesoporous Silica Nanoparticles (MSNs) to obtain a controlled release of monocyte chemoattractant protein-1 (MCP-1) and to evaluate how the monocytes attraction is influenced by this release mechanism. This chemokine is shown to mediate monocytes recruitment and to play a central role in immune response. To create a cardiovascular substitute with these requirements, three different methods were investigated to bind MSNs to the polymeric scaffolds: 1) MSNs in the fibrin gel, 2) coating of MSNs on the surface of the scaffolds and 3) MSNs incorporated into UreidoPyrimidinone-Polyethylene Glycol (UPy-PEG) fibers via direct electrospinning. For the production of the electrospun scaffolds, two polymers were used with dual electrospinning technique: Polycaprolactone (PCL) Bisurea (TPE) and a blend of UPy-PEG 10k and UPy-PEG 20k (hydrogels) synthesized at the Department of Biomedical Engeneering of Eindhoven University of Technology. For the third method these two different hydrogels were mixed in a different ratio to obtain different degradation time to incorporate MSNs and to assess cell infiltration. A 15 wt% of PCL Bisurea was prepared dissolving it in a solvent mixture composed of 85% of chloroform and 15% HexaFluoroIsoPropanol (HFIP). Different solution of 10 wt% UPy-PEG were prepared with different ratio of UPy-PEG 10k and UPy-PEG 20k (50:50 w/w, 70:30 w/w, 90:10 w/w, 100%), dissolving the two polymers into a solvent mixture composed of 99% of chloroform and 1% of methanol. The morphological characterization was performed using the scanning electron microscope (SEM), then the mechanical characterization was performed through biaxial tensile tests. From SEM observations it was possible to note that the scaffolds obtained from different ratio of UPy-PEG 10k and UPy-PEG 20k have different time of degradation, but the results of the degradation study in static and dynamic conditions (pulsatile flow) showed that there was no difference between the different ratio of the two polymers used for the blends. The pulsatile flow conditions were obtained through the use of a fluidic system (Ibidi GmbH) modified to reproduce the pulsatile flow in pulmonary conditions, while for the degradation study in static conditions the samples were placed into 1 mL of medium (RPMI 1640). Another experiment performed in this work was the evaluation of in vitro cellular infiltration using human monocytic leukemia (THP-1) cells, seeded on each scaffold using PCL Bisurea as a control. The results of cellular infiltration didn’t show differences between scaffolds with UPy-PEG and without UPy-PEG, because after its selective removal the porosity of the scaffolds didn’t seem to increase. About the MSNs, different type of nanoparticles were synthesized at the Ulm University (Germany). The morphological characterization of the MSNs was performed with SEM. To assess the in vitro MCP-1 release from different type of MSNs, ELISA assay was performed and the type of MSN that showed the optimal release of MCP-1 was used for the evaluation of the three methods aforementioned. About the release in static and dynamic conditions of the three methods, the results showed a low amount of MCP-1 not only after 4 hours but also in the next time points and because of the higher content of the chemokine released over time, the second method showed the best results among the other methods considered and it was selected to evaluate the effect of MCP-1 delivery on the monocytes attraction. To evaluate the recruitment of monocytes, chemotaxis assay and experiment in pulsatile flow conditions, both followed by DNA assay, were performed. The chemotaxis assay performed with a PET porous membrane with pore size of 3 micron showed no difference between the groups considered ( 1) scaffold of PCL Bisurea, 2) MCP-1 adsorbed on the surface of the scaffold, 3) MSNs coating on scaffold, 4) coating of MCP-1-loaded MSNs on the scaffold, 5) free MCP-1 in fibrin gel), while with pore size of 8 micron there was a difference between free MCP-1 in fibrin gel (burst release mechanism) and the first three conditions. Regarding the experiment in flow conditions after 4 hours there was a statistical difference between MCP-1 adsorbed on PCL Bisurea and MCP-1 released using MSNs (controlled release mechanism) showing a not good efficacy for the last one. Infact the results showed that the efficacy of that controlled release system on the monocytes recruitment was not better if it’s compared to the burst release system : no differences were shown between the two different mechanism neither for chemotaxis assay nor for flow experiments. In light of this it is possible to conclude that the controlled release developed in this work doesn’t show efficacy in monocyte recruitment, that there is no statistically significant difference between the two methods for the THP-1 cells migration.