Novel bio-inspired synthetic composite materials as promising scaffolds for stem cell-mediated tissue regeneration supporting short term self-renewal of human pluripotent stem cells in feeder-free culture conditions

摘要

Human Pluripotent Stem Cells (hPSCs) hold great promise for cell therapy and tissue engineering, as well as drug screening . For a clinically valid development of stem cell-based therapies some biological and engineering challenges still need to be overcome, such as the design of engineered cell culture microenvironment that support hPSCs proliferation while maintaining their pluripotency. Recent studies on a number of different biological tissues demonstrated the great potential of stem cell-based tissue engineering strategies . In this study a novel composite synthetic scaffold was designed, inspired by the overall structure of tissue extracellular matrix (ECM), and the short-time expansion and self-renewal of human Embryonic Stem Cells (hESCs) and human Induced Pluripotent Stem Cells (hiPSCs) was investigated in view of potential application of the materials as scaffolds for stem cel-mediated tissue engineering applications. The scaffold was composed by a RGD-mimic polyamidoamine (PAA) AGMA1 hydrogel with embedded poly-L-lactic acid (PLLA) mat of continuous electrospun nanofibers with average diameter 570 ± 170 nm, mimicking the gel and fibrous components of ECM, respectively. The biomimetic properties and the softness of the hydrogel component were therefore combined with the strength of the nanofibrous PLLA mat. Strong matrix-fiber adhesion was achieved by N2 atmospheric pressure non-equilibrium plasma treatment of the PLLA mat. This treatment made the mat hydrophilic, therefore impregnable with aqueous solution, and introduced surface available amino groups on PLLA, able to covalently react with the acrylamide end-capped PAA chains via Michael addition. The scaffolds were characterized for their swelling and degradation behavior and their mechanical properties were investigated. Biological studies demonstrated that the scaffolds supported short term self-renewal of Human Pluripotent Stem cells in feeder-free conditions. Quantitative real-time polymerase chain reaction and immunofluorescence studies of undifferentiated markers demonstrated that the cells fully retained sternness for at least 7 days. In conclusion, novel composites were developed in this work, being endowed with a number of interesting properties that make them promising scaffolds for stem cell-mediated tissue regeneration. They are entirely synthetic, structurally defined, and they can be obtained by standardized procedures. Moreover, their mechanical properties and swelling and degradation behavior in aqueous media can be easily tuned by tailoring the crosslinking degree. Finally, their chemical structure is suitable for further modification and functionalization with peptides and bioactive molecules, that can be covalently incorporated in the PAA component in order to specifically modulate the signaling pathways of hiPSCs, demonstrating the high potential of the materials in the field of tissue engineering and regenerative medicine.