Ⅱ-4 functionalized 2D and 3D structures based on supramolecular interactions for in-situ vascular regeneration

摘要

Objective: In-situ vascular regeneration is based on implantation of biodegradable synthetic grafts that act as a temporary substrate for blood cell infiltration and mature tissue formation. Upon implantation, biomaterials elicit a host immune response which might lead either to regeneration or scar tissue formation). Therefore, designing target-specific biomaterials triggering desired immunological outcomes and thus supporting the regeneration process is a critical need for vascular tissue engineering. Among all the immune cells involved in the immune response, macrophages play a predominant role, influencing the scaffold fate depending on their phenotype. Particularly, the pro-inflammatory phenotype M1 is responsible for matrix destruction and tissue re-organization, while the pro-healing phenotype M2 promotes tissue maturation. Therefore, we hypothesize that scaffolds activating M2 macrophage polarization enhance tissue regeneration. To this end, we designed immunomodulating 2D dropcast materials (as a proof of concept) and 3D electrospun scaffolds, both based on supramolecular immobilization of interieukin 4 (IL-4), an anti-inflammatory cytokine responsible for M2 activation and polarization. Material and Methods: Chain-extended ureido-pyrimidinone (UPy) modified polycaprolactone (ceUPyPCL) was used as a base material to design both 2D and 3D structures. The ceUPy-PCL (custom developed by SupraPolix, the Netherlands) was further modified with a heparin binding peptide sequence (GLRKKLGKA), which was coupled to the UPy-moiety (UPyHBP), purified and mixed with the UPy-PCL polymer (0.5% mol UPy-HBP). For dropcasting, ceUPyPCL and ceUPyPCLHBP were dissolved in hexafluoro-2-propanol (HFIP) at 50 mg/mL, by pipetting 300 μL of solution on chamberslides. The HFIP was evaporated overnight and residual solvent removed in vacuo at 40°C overnight. The IL-4 was diluted in heparin at 20 ng/mL and 0.2 ng/mL, The dropcast films were incubated with the IL-4-heparin complexes overnight. Monocytes isolated from human peripheral blood mononudear cells (hPBMCs) were seeded on the chambered cover glasses at a seeding density of 1.4×106 cells/cm~2. Monocytes were further stimulated towards non-polarized macrophages with 100ng/mL of macrophage colony stimulating factor (MCSF). At day 3 cell morphology was observed with light microscopy. For electrospinning, ceUPyPCL and ceUPyPCLHBP were dissolved in chloroform and HFIP (15% w/v polymer in CHCI3/ HFIP 80/20% v/v) and fibrous sheets with 10.5±0.7 μm fiber diameter were fabricated. The fibrous scaffolds were incubated with FITC-labelled heparin and heparin complexation investigated via fluorescence analysis. Subsequently, scaffolds were incubated with 0.2 ng/mL of IL-4 in heparin and the IL-4 release was measured via an IL-4 specific enzyme linked-immune-sorbent assay (ELISA) during 1 week. Results: In 2D cultures, differences in cell morphology were observed mainly elongated M2-type macrophages were observed on IL4 functionalized UPyPCLHBP films, while mainly rounded cells were observed in ceUPyPCL films. Successful adsorption of heparin was shown on the UPy-HBP activated scaffolds. Moreover, less than 15% of IL-4 was released after 1 week, proving successful and retained biofunctionalization of the scaffold. Discussion and Conclusions: We developed an IL4-complexed material that shows first indications of successful M2 macrophage polarization in 2D. Moreover, we have shown that the IL4 immobilization via heparin approach can be translated to 3D electrospun scaffolds. This study represents the first step in designing functionalized grafts that harness the regenerative behaviour of host macrophages towards tissue regeneration.