Osteogenic Differentiation Assays of Human Adipose-derived Stem Cells on Nanofibrous Biomimetic Scaffolds. Analysis of 14-3-3 proteins specific variations.

LIVERANI, LILIANA; UHART, MARINA; FRONTINI LÓPEZ, YESICA ROMINA; ABRAHAM, GUSTAVO; BUSTOS, DIEGO MARTÍN; ALDANA, ANA AGUSTINA; BOCCACCINI, ALDO

CABA

Simposio; Frontiers in Bioscience 3; 2018

IBioBA (CONICET-Partnet Institute of the Max Planck Society)

Adipose-derived mesenchymal stem cells (ASCs) are particularly useful in the field of regenerative medicine because of their many advantages compared to other mesenchymal stem cell types. They appear to accelerate osteointegration of bone grafts and improve efficiency and uniformity in the formation of bone tissue, providing a practical and clinically attractive approach in tissue regeneration. The aim of our work was to study the effect of nanofibrous biomimetic matrices composed of poly-ε-caprolactone (PCL) and nanohidroxyapatite (nHA) on human ASCs (hASCs) osteogenic differentiation. hASCs were isolated from the stromal vascular fractions (SVF) of freshly harvested subcutaneous adipose tissue obtained from abdomen dermolipectomy surgery explants. As characterized by flow citometry, our populations are positive for CD105, CD90 and CD73 and negative for CD45, CD34, CD11b, CD19, and HLA-DR. Isolated cells were induced to osteogenic differentiation with a drug cocktail both in plate and within the biomimetic scaffolds. We first stained untreated and treated samples with Alizarin red to detect extracellular matrix calcification progression at different days. Then, we evaluated by Western blot the levels of crytical osteogenic differentiation markers (RUNX2, Osteocalcin, Osteopontin, CD9), and also of six 14-3-3 paralogs (postulated as new key regulators of the adipose/osteogenic differentiation process) of the hASCs grown in culture plates. We found changes on specific 14-3-3 isoforms, which seem to be intriguingly opposite to that of adipogenic differentiationcita. After that, we challenged the hASCs to differentiate over the PCL/PCL-nHA scaffolds and determine their adhesion, penetration and differentiation by alkaline phosphatase (ALP) activity. hASCs were capable to differentiate over the scaffolds, as depicted by enzymatic kinetics and the ALP staining. We also observed by transmission electron microscopy that cell grows They also penetrated the PCL-nHA scaffolds, as observed in cross sections analyzed by transmission electron microscopy.