Vitamin K2 and Bone: Focus on Osteogenesis

Vitamin K2 (VitK2) is a lipid-soluble vitamin that regulates calcium homeostasis in “bone vascular cross talk” acting as cofactor for the enzyme γ-glutamyl carboxylase (GGCX). Recently, it has been shown that it plays a main role in this crosstalk by reducing the calcium deposit in the arteries and increasing it in the bone tissue (“calcium paradox”) through carboxylation and then activation of specific Gla proteins, Matrix Gla protein and osteocalcin (OC) respectively. This evidence made VitK2 a potential osteoinductive factor for bone tissue regeneration. Since little is known about the potential effect of VitK2 in the osteogenesis of mesenchymal stem cells, aim of our study was to investigate the possible role of VitK2 in the modulation of osteogenesis in a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) both in two-dimensional (2D) and three-dimensional (3D) in vitro culture systems. In 2D culture system, using Alizarin Red S staining, we observed that VitK2 significantly enhanced hAFMSCs osteogenic differentiation compared to cells treated with osteogenic medium alone. These data were confirmed by VitK2 increased expression of specific osteogenic markers such as alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), OC, alpha-1type I collagen (COL1A1) and osteopontin (OPN) (real time PCR and immunocytochemistry). Notably, the GGCX downregulation (by RNA interference) reverted the osteoinductive effects of VitK2, confirming the relevant role of this vitamin as cofactor for the enzymatic activity of GGCX. hAFMSCs were also cultured in 3D dynamic model represented by RCCS™ (Rotary Cell Culture System), generating sizeable aggregates that exhibited the same improvement in osteogenic markers following VitK2 treatment. Finally, to reproduce in vitro the “bone remodeling unit” we co-cultured hAFMSCs and human monocyte (hMCs) osteoclast precursors in RCCS™. Our preliminary data suggest that: 1. the cells are able to form viable aggregates with a well organized architecture characterized by the ability of hAFMSCs to sustain osteoclastogenic differentiation; 2. the presence of VitK2 significantly supports osteogenic process (monitored by immunohistochemical analysis of typical osteogenic markers and by Alizarin Red staining of mineralized matrix); 3. osteoclastogenic process, the number and the functionality of mature osteoclasts are negatively affected by Vitk2, as demonstrated by TRAP and Cathepsin K assays. The development of this 3D coculture system i. validates the positive role of Vitk2 in the anabolic process sustained by the osteoblastic cellular component, and ii. offers the possibility to improve the knowledge on the molecular mechanisms supporting the response of osteoblasts and osteoclasts to VitK2. These evidences suggest a possible employment of VitK2 in cell – based therapy for bone regenerative medicine. Moreover, our study support the use of VitK2 as food supplement in order to prevent bone related diseases.