The influence of collagen-based scaffolds on cardiac differentiation and endothelial growth

Keywords: elastin
collagen
vascularisation
cardiac differentiation
hyaluronic acid
angiogenesis

Myocardial infarction is the leading cause of death worldwide due to insufficient long-term treatments or the unavailability of suitable donors. To make clinically appropriate therapy for cardiac patients, cardiac tissue engineering focuses on designing 3D constructs that compositionally and mechanically reflect native cardiac tissue by providing better cellular interactions. However, the complexity and the abundance of cell load in the cardiac tissue complicate the developing process.
This thesis reports novel composite scaffolds containing collagen type I, fibrillar elastin gel (FEG) and hyaluronic acid (HA) and their applications in vitro and in vivo. Highly porous (>97%) scaffolds with various ratios of collagen and FEG were fabricated by lyophilisation, and HA was infiltrated into these structures. The effect of chemically crosslinking and accompanying HA infiltration was also studied. Mechanical testing results showed a strong correlation between scaffold stiffness and the concentration of FEG and the improvement of mechanical properties by HA infiltration in the hydrated state. Despite crosslinking, comparable tensile properties were achieved between the collagen-FEG composites and native porcine heart tissue.
The HA-infiltrated scaffolds were tested in vitro using a novel approach enabling the culture and differentiation of human induced pluripotent stem cells (hiPSC) to cardiomyocytes (hiPS-CM) in 3D. The studies with the alamarBlue assay indicated that these scaffolds were favourable to culture hiPSCs and that there was no significant difference between scaffold types in terms of proliferation. The scaffolds were also studied to evaluate the endothelial growth potential of human umbilical vein endothelial cells (HUVEC) using alamarBlue and phalloidin-DAPI staining, showing that these cells proliferated better in collagen-based scaffolds (C100) compared to collagen-FEG composites, which suggests that collagen is a crucial regulator to support endothelialisation. The successful differentiation of hiPS-CMs in 3D was shown in gene expression level by cardiac-specific (MYL7, MYH6) markers, although the differentiated cells were found to be an immature form of cardiomyocytes by immunocytochemistry. Collagen-FEG composites significantly enhanced the gene expression of extracellular matrix (ECM)-specific (COL1A1, ELA) markers than that of C100 scaffolds. The increase in the concentration of FEG also increased the endothelial gene expression (CD31, CD105, VEGFR2 and ANG2) when compared to C100.
The last part of the thesis covers the in vivo biocompatibility of collagen and collagen-FEG composites at different pre-conditions using histological staining. Non-crosslinked scaffolds significantly had better ECM deposition and cell migration than crosslinked and HA-infiltrated groups. No type of scaffold was encapsulated within the host, and the presence of capillary blood vessels and glycosaminoglycans was also shown in each scaffold type, suggesting that design scaffolds were feasible in vivo.