Promotion of extracellular matrix deposition by human vascular smooth muscle cells and monocytes within a biodegradable polyurethane scaffold

Abstract

Event Abstract Back to Event Promotion of extracellular matrix deposition by human vascular smooth muscle cells and monocytes within a biodegradable polyurethane scaffold Xiaoqing Zhang1, Kyle Battiston1, Rosalind Labow2, Craig Simmons1, 3, 4 and Paul Santerre1, 4 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Ottawa, Department of Biochemistry, Microbiology and Immunology, Canada 3 University of Toronto, Department of Mechanical and Industrial Engineering, Canada 4 University of Toronto, Faculty of Dentistry, Canada Introduction: Tissue engineered vascular grafts (TEVGs) have shown the potential to replace diseased arteries[1]. In developing TEVGs, it is desirable to incorporate contractile vascular smooth muscle cells (VSMCs) into biomaterial scaffolds and stimulate extracellular matrix (ECM) production allowing for fast maturation of tissue. It is also desired to have a polymeric scaffold which provides immunomodulatory capability early after implantation[2]. Monocytes are a predominant cell type at an implant site and play an important role in wound healing[3]. VSMC-monocyte co-culture on an immunomodulatory polyurethane (D-PHI) scaffold has previously demonstrated enhanced migration and proliferation of VSMCs, as well as a reduced pro-inflammatory monocytic state[4],[5]. The objective of this study was to evaluate the effect of varying VSMC:monocytes ratios on regulating ECM production in co-culture with D-PHI. Materials and Methods: D-PHI scaffolds were prepared as previously described[6]. Monocytes isolated from human peripheral blood (University of Toronto ethics approval #22203)[5] and human coronary artery VSMCs (Lonza CC-2583) (300k) were co-seeded in 50:50 RPMI:DMEM medium at VSMC:monocyte ratios of 1:2 (600k monocytes), 1:1 (300k monocytes), 2:1 (150k monocytes), 4:1 (75k monocytes), 8:1 (37.5k monocytes) and 1:0 (0 monocytes) on each D-PHI scaffold (6 mm diameter, 1 mm thickness) and maintained in culture for 4 weeks. Collagen production was assessed by hydroxyproline (OH-Pro) (accounts for 10% of total collagen[5]) and elastin expression was assessed by immunostaining with elastin-positive areas analyzed by ImageJ. Enzyme-linked immunosorbent assay (ELISA) was used to quantify insulin growth factor 1 (IGF-1, which promotes elastin production) and matrix metallo-proteinase 2 (MMP2, which degrades ECM proteins). Data were analyzed by analysis of variance (SPSS 17.0) using Tukey test for pair-wise comparisons. Statistical significance was reported for p < 0.05. Results and Discussion: Total OH-Pro quantification data (Fig. 1A) showed that collagen content was highest with 2:1, 4:1 and 8:1 VSMC:monocytes ratios, when compared to the previously reported co-culture ratio of 1:2 [5]. 2:1 and 8:1 ratios promoted more collagen deposition when compared to the 1:0 ratio. Immunostaining for elastin (Fig. 1B) demonstrated significantly more elastin on the scaffold for 2:1 and 4:1 vs. 1:2 and 1:0 VSMC:monocyte ratios. IGF-1 release was significantly higher in co-culture (1:2, 2:1 and 4:1 ratios) (Fig. 2A), however, a 1:2 co-culture ratio produced more MMP2 than other conditions (Fig. 2B) . Conclusion: This study demonstrates that the co-culture of VSMCs with a modest number of monocytes promotes ECM accumulation. The reduced ECM synthesis observed at the 1:0 and 1:2 co-culture ratios may be caused by lower matrix producing cytokine (IGF-1) (1:0 condition) and higher matrix degrading protein (MMP2) (1:2 condition) released respectively, in those conditions. Canadian Institutes of Health Research (CIHR) operating grant #230762.