Investigating the Osteoregenerative Properties of Juglans regia L. Extract on Mesenchymal Stem Cells and Osteoblasts Through Evaluation of Bone Markers: A Pilot Study.

Bone tissue regeneration is a complex process that takes place at the level of osteoblasts derived from mesenchymal cells and occurs under the action of multiple signaling pathways and through the expression of osteoregenerative markers. The leaf extract of Juglans regia L. (JR) is rich in polyphenols with demonstrated osteoregeneration effects. In the present study, we investigated the extract's effects on three types of cells with various stages of differentiation: adult mesenchymal stem cells (MSCs), osteoblasts at low passage (O6) and osteoblasts at advanced passage (O10). To assess the efficacy of the walnut leaf extract, in vitro treatments were performed in comparison with ellagic acid (EA) and catechin (CAT). The osteoregenerative properties of the leaf extract were evaluated in terms of cell viability, bone mineralization (by staining with alizarin red) and the expression of osteogenesis markers such as osteocalcin (OC), osteopontin (OPN), dentin matrix acidic phosphoprotein 1 (DMP1) and collagen type 1A. Another compound implicated in oxidative stress response, but also a bone homeostasis regulator, nuclear factor erythroid 2-related factor 2 (NRF2), was studied by immunocytochemistry. Together with collagen amount, alkaline phosphatase (ALP) activity and NF-kB levels were measured in cell lysates and supernatants. The obtained results demonstrate that JR treatment induced osteogenic differentiation and bone mineralization, and it showed protective effects against oxidative stress.