Autophagy Inhibition Impedes Stem Cell Mineralization on Fluorapatite (FA) Modified Scaffolds

Objectives: As a major intracellular degradation and recycling process, autophagy plays an important role for maintaining cellular homeostasis and remodeling during tissue development. The aim of this study is to discover whether the autophagy is involved in and coordinated with the osteogenic differentiation process of the stem cells grown on the FA modified electrospun polycaprolactone (PCL) scaffolds.
Methods: The STEMPRO^® Human Adipose-Derived Stem Cells (ASCs) were seeded on both PCL+FA and PCL scaffolds to measure the osteogenic inductive effects of FA crystals on the cells and the autophagic activities of ASCs cultured on these 3D models.
Results: The PCL+FA but not PCL only scaffold induced obvious mineral nodule formation at day 21, which was further confirmed by the enhanced ALP activity, stronger Alizarin Red and Von Kossa staining of the cells grown on the PCL+FA scaffold compared to PCL only scaffold. The autophagy marker, the microtubule-associated proteins 1 light chain 3A/B-II (LC3-II), was shown dynamic changes when the ASCs were grown under 2D and 3D environments at 6h, 12h, 1, 3, 7, 14, and 21 days. The LC3-II reached to its peak value at day 7 which was coordinated with the phenotypic changes of the ASC osteogenic differentiation indicated by the significantly elevated ALP activity and osteopontin (OPN) expression. Furthermore, when the autophagy inhibitors (3-MA, Balfimycin A1 and NH₄Cl) were applied after cell seeding for up to 3 days, all the osteogenic related markers (OPN, ALP and Alizarin Red staining and Von Kossa staining) were down regulated compared to the control group.
Conclusions: This study indicated that autophagy played a vital role in the ASC osteogenic differentiation process both on the 2D and the FA modified 3D models, which will enhance our knowledge in facilitating the application of these FA modified scaffolds in future dental and orthopedic regenerative applications.