Abstract
Nacre (mother of pearl) is a bioactive material capable of facilitating osteoblast proliferation and differentiation; however, further investigation into the mechanism underlying the effects of nacre on the stimulation of bone differentiation is required. The present study aimed to elucidate the effects of water‑soluble nano‑pearl powder (WSNNP) on osteoblast differentiation and to examine the underlying mechanisms. A MTT assay revealed that WSNNP (10, 25 and 50 µg/ml) may stimulate the viability of preosteoblastic MC3T3‑E1 cells and 50 µg/ml WSNNP exhibited the maximum stimulatory effect. Furthermore, WSNNP significantly enhanced the protein expression levels of differentiation markers, including collagen I, runt‑related transcription factor 2 (RUNX2), secreted phosphoprotein1 (SPP1) and alkaline phosphatase (ALP) in a dose‑dependent manner, which indicated that WSNNP may promote osteoblast differentiation. Subsequently, whether autophagy serves a role in WSNNP‑mediated differentiation of osteoblasts was investigated via western blotting and immunofluorescence. The results of the present study demonstrated that WSNNP treatment significantly evoked the expression of autophagy markers, including microtubule‑associated light chain 3 (LC3)II/I, Beclin1 and autophagy‑related 7 (ATG7), whereas the autophagy inhibitor 3‑methyladenine significantly inhibited WSNNP‑induced osteoblast differentiation. Furthermore, the role of WSNNP on the potential signaling pathways that activate autophagy was investigated. The present study reported that WSNNP may significantly upregulate the mitogen‑activated protein kinase kinase (MEK)/extracellular signal‑regulated kinase (ERK) signaling pathway. Treatment with the MEK inhibitor U0126 significantly inhibited the protein expression levels of WSNNP‑induced differentiation markers, including collagen I, RUNX2, SPP1 and ALP, and autophagy markers, including LC3II/I, Beclin1 and ATG7. Therefore, the findings of the present study suggested that WSNNP may contribute to osteoblast differentiation by enhancing autophagy via the MEK/ERK signaling pathway, thus suggesting a novel direction for optimizing the biological materials in bone implants.