Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation

Hruschka, Veronika, Saeed, Aram, Slezak, Paul, Cheikh Al Ghanami, Racha, Feichtinger, Georg Alexander, Alexander, Cameron, Redl, Heinz, Shakesheff, Kevin and Wolbank, Susanne (2015) Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation. Tissue engineering. Part A, 21 (1-2). pp. 310-319. ISSN 1937-335X

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Abstract

Tissue engineering (TE) strategies aim to imitate the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation and differentiation. Based on the idea of combining a fully degradable polymer (Poly(ε-caprolactone) with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for the ability to support C2C12 cells and human adipose derived stem cells (ASCs) to generate an expandable 3D construct for soft or bone TE. As a first step, biomaterial seeding was optimised and cellular attachment, survival, distribution and persistence within the 3D material were characterised. C2C12 cells were differentiated towards the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic or osteogenic differentiation media. Differentiation was examined using qRT-PCR for the expression of osteogenic, myogenic and adipogenic markers and by enzymatic- or immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3 ± 0.2) and alkaline phosphatase (ALP) activity (p<0.001) were increased compared to the control C2C12 cells. 3D osteogenic differentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation towards the myogenic or adipogenic lineage showed expression of specific markers in 3D at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (p<0.001) as well as myoD (p<0.001) were found in C2C12 cells. The adipogenic differentiation of one donor showed greater expression of peroxisome proliferative activated receptor gamma (PPARg) (p=0.004), fatty acid binding protein 4 (FABP4) (p=0.008) and adiponectin (p=0.045) in 3D compared to 2D culture. Leptin levels in the supernatant of the ASC cultures were elevated in the 3D cultures in both donors at day 14 and day 21. In conclusion, the thermoresponsive scaffold was found suitable for 3D in vitro differentiation towards soft tissue.

Item Type: Article
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Pure Connector
Date Deposited: 08 Oct 2014 08:42
Last Modified: 17 Mar 2020 20:11
URI: https://ueaeprints.uea.ac.uk/id/eprint/50307
DOI: 10.1089/ten.TEA.2013.0710

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