title: Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.
abstract: Tissue engineering (TE) strategies aim at imitating 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(ɛ-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 its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and 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 with the control C2C12 cells. Three-dimensional 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 toward 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) was found in C2C12 cells. The adipogenic differentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4 (FABP4) (p=0.008), and adiponectin (p=0.045) in 3D compared with 2D culture. Leptin levels in the supernatant of the ASC cultures were elevated in the 3D cultures in both donors at day 14 and 21. In conclusion, the thermoresponsive scaffold was found suitable for 3D in vitro differentiation toward soft tissue.
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , AUVA Research Centre, Austrian Cluster for Tissue Regeneration, Vienna, Austria .
authors.forename: Veronika
authors.initials: V
authors.lastname: Hruschka
authors.forename: Aram
authors.initials: A
authors.lastname: Saeed
authors.forename: Paul
authors.initials: P
authors.lastname: Slezak
authors.forename: Racha
authors.initials: R
authors.lastname: Cheikh Al Ghanami
authors.forename: Georg Alexander
authors.initials: GA
authors.lastname: Feichtinger
authors.forename: Cameron
authors.initials: C
authors.lastname: Alexander
authors.forename: Heinz
authors.initials: H
authors.lastname: Redl
authors.forename: Kevin
authors.initials: K
authors.lastname: Shakesheff
authors.forename: Susanne
authors.initials: S
authors.lastname: Wolbank
mesh_terms: D050156:Adipogenesis / Q000187:drug effects; D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018914:Cells, Immobilized / Q000166:cytology; D006801:Humans; D051379:Mice; D024510:Muscle Development / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects; D011091:Polyesters / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology*; D013696:Temperature; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D011091:Polyesters; C016240:polycaprolactone