Abstract
Background. Hyaluronic acid (HA) is the major component of the extracellular matrix of human tissue, where it regulates processes such as osmotic pressure, water retention, cell migration, and differentiation. For these reasons, hyaluronans are currently used in regenerative medicine in different areas. Nevertheless, hyaluronans exist in different forms accordingly with molecular weight and degree of crosslinking, which can have a different and context-depended effects on cellular processes. Thus, picking the most appropriate form of hyaluronan turn out to be fundamental as it can make a huge difference in tissue regeneration. MSCs have attracted attention in tissue regeneration for their proliferation potential and ability to differentiate in several cytotypes. Among MSCs, human Dental Pulp Stem Cells (hDPSCs) were shown to be remarkably suitable for bone differentiation.In this study, we tested the capability to induce osteogenic differentiation in hDPSCs of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high (HHA), low molecular weight (LHA), and the recently stabilized hybrid cooperative complexes (HCC), containing both sizes.Methods. hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. CD44, the main receptor of the HA on cell surface and an upstream regulator of YAP/TAZ signaling, was analyzed by immunofluorescence. YAP/TAZ expression was measured by qRT-PCR. To confirm the involvement of YAP/TAZ pathway, YAP/TAZ inhibitor-1 was used and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence.Results. HCC was found to be the most impacting in inducing osteogenesis, with significant effects already at 7-14 days of treatment. HCC induced strong overexpression of osteocalcin, osteopontin, and bone sialoprotein, calcification nodule formation, and CD44 up-regulation.In addition, we showed that this biological process is associated to the activation of YAP/TAZ pathway and its target genes CTGF, ANKDR-1, RUNX-1, and RUNX-2.Conclusions. In conclusion, in this study we show that HA’s molecular weight can have a tremendous impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells.
Co-culture of Human Dental Pulp Stem Cells and Endothelial Cells Using Porous Biopolymer Microcarriers: A Feasibility Study for Bone Tissue Engineering
