Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable human cardiac cells to be studied in vitro, although they use glucose as their primary metabolic substrate and do not recapitulate the properties of adult cardiomyocytes. Here, we have explored the interplay between maturation by stimulation of fatty acid oxidation and by culture in 3D. We have investigated substrate metabolism in hiPSC-CMs grown as a monolayer and in 3D, in porous collagen-derived scaffolds and in engineered heart tissue (EHT), by measuring rates of glycolysis and glucose and fatty acid oxidation (FAO), and changes in gene expression and mitochondrial oxygen consumption. FAO was stimulated by activation of peroxisome proliferator-activated receptor alpha (PPARα), using oleate and the agonist WY-14643, which induced an increase in FAO in monolayer hiPSC-CMs. hiPSC-CMs grown in 3D on collagen-derived scaffolds showed reduced glycolysis and increased FAO compared with monolayer cells. Activation of PPARα further increased FAO in cells on collagen/elastin scaffolds but not collagen or collagen/chondroitin-4-sulphate scaffolds. In EHT, FAO was significantly higher than in monolayer cells or those on static scaffolds and could be further increased by culture with oleate and WY-14643. In conclusion, a more mature metabolic phenotype can be induced by culture in 3D and FAO can be incremented by pharmacological stimulation.
Pore geometry (pore size and pore interconnectivity) and stiffness are important design requirements for 3D-scaffold fabrication. The required pore geometry allows the passage of growth factors for cell proliferation and removal of waste products, whereas the stiffness influences attachment of osteogenic cells. This work fabricates a 3D scaffold from collagen (Col) and snail shell hydroxyapatite (HApS) and examines the influence of the HApS on the scaffold pore geometry and stiffness. The scaffolds were fabricated using freeze-drying method. Col alone and Col-commercial hydroxyapatite (Col-HApC) scaffolds were used as controls. Scanning electron microscope (SEM) reveals well-interconnected pores for Col-HApS with a mean pore size of 246.9 ± 68.7 μm, which was statistically (p < 0.05) same as that of Col scaffolds 224.4 ± 85.7 μm and different (p < 0.05) from Col-HApC scaffolds 125.5 ± 26.7 μm. Mechanical testing showed a stiffness of 20.8 ± 0.4 kPa, 181.2 ± 11.8 kPa, and 206.9 ± 14.1 kPa for Col, Col-HApC, and Col-HApS, respectively. Uniquely, X-ray diffractometry (XRD) and Infrared (IR) spectroscopy of Col-HApS revealed phases and functional groups that were comparable to graphitic-like carbon nitride (g-C 3 N 4) polymeric structure. It was found that the structural change was responsible for the well-interconnected large pores and high stiffness of the scaffold. It is expected that the effect brings a wide range of functions (such as better cell attachment and nutrient transport) in the scaffold for osteogenesis. The findings indicate that Col-HApS scaffolds would promote osteogenic cell response more usefully than Col-HApC or Col scaffolds.
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