Bone homeostasis is regulated by the functions of osteoblasts, osteocytes, and osteoclasts in bone tissue, and it plays a physiological role in maintaining new bone mass [[1], [2], [3], [4], [5], [6]]. This process is complexly regulated by systemic influences, including hormones, cytokines, growth factors, and stress with physical activity and weight bearing [5,6]. In addition, immune cells, including lymphocytes and macrophages, present in the bone marrow microenvironment contribute to the regulation of bone homeostasis [7]. The pathophysiological osteoporosis leading to bone loss in women is initially caused by estrogen deficiency with aging [8]. Severe bone loss is induced by cancer cells metastasizing to the bone [9], leading to the death of patients with fractures. In addition, inflammatory macrophages may contribute to the expression of bone disease [10]. Inflammatory cytokines, mainly tumor necrosis factor (TNF)-α, lead to osteoarthritis, osteoporosis, and cancer bone metastasis [[11], [12], [13]]. TNF-α, which is produced by inflammatory macrophages [12], has been shown to suppress osteoblastic mineralization and stimulate osteoclastic bone resorption via activation of NF-κB signaling [14], leading to bone loss.

RAW264.7 cells are a monocyte/macrophage-like cell lineage that has been well characterized concerning macrophage-mediated immune, metabolic, and phagocytic functions [12,13]. RAW264.7 cells are progressively more used and recognized as a cellular model for osteoclastogenesis, in which osteoclasts are differentiated from the monocyte-macrophage lineage [12,13]. The host immune system is stimulated by lipopolysaccharide (LPS), the major antigen of gram-negative bacteria [15]. LPS-activated inflammatory macrophages that produce TNF-α in the bone marrow microenvironment may play a pathogenic role in increasing bone loss. Clinically, the therapeutic factors may be important to prevent bone loss and fracture in various diseases, including inflammation and cancer bone metastasis.

The marine phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), originally found in the Pacific oyster Crassostrea gigas [16,17], has been shown to prevent oxidative stress as a radical scavenger in various cells [[16], [17], [18], [19], [20], [21]]. Interestingly, DHMBA is an excellent peroxyl radical scavenger, being about 15 times and 4 orders of magnitude better than Trolox for this purpose in lipid and aqueous media, respectively [22]. It has been suggested that this compound reacts more rapidly with HOO(−) than other known antioxidants such as resveratrol and ascorbic acid [22]. DHMBA may play a critical role in regulating cell function and maintaining health [[16], [17], [18], [19], [20], [21], [22]]. Notably, we demonstrated that DHMBA exhibited suppressive effects on the growth and metastatic activity of bone metastatic prostate cancer cells [23], suggesting a pharmacological role in bone disorder.

Osteogenic factors may be clinically important in reversing bone loss and preventing osteoporosis associated with aging and various diseases. Such factors need to be developed. This study elucidates whether DHMBA stimulates osteoblastic mineralization and prevents the impairment of osteoblastic mineralization associated with TNF-α produced by inflammatory macrophage RAW264.7 cells with LPS stimulation. Here, we find that culturing with DHMBA stimulated mineralization in mouse osteoblastic MC3T3-E1 cells in vitro and that DHMBA blocked the impairment of cell growth and mineralization in osteoblastic MC3T3-E1 cells via the signaling related to TNF-α produced in mouse inflammatory RAW264.7 cells activated by LPS stimulation in vitro. This study may provide a new strategy for the therapy of impaired osteoblastic bone formation and mineralization associated with inflammatory conditions in the bone marrow microenvironment.