## 4 Discussion  
In this study, we explored the osteoinductive potential of WSM from nacre, particularly focusing on its ability to enhance the differentiation of pre-osteoblasts into mature osteoblasts. Previous studies have demonstrated that nacre and its extracts stimulate bone formation and differentiation of human bone marrow stem cells (hBMSC) without affecting the physical size of the nacre [8; 9; 10]. The osteoinductive properties of nacre have been attributed to an organic WSM consisting of polysaccharides, water-soluble protein, and glycosaccharides, which can be slowly released from the organic matrix of nacre and subsequently mediate osteoblastic differentiation [13; 14]. However, the challenge lies in the limited release of osteoinductive factors from natural nacre due to its dense and compact surface [25]. To address this issue, we synthesized nano-nacre particles with a size of approximately 600 nm, smaller than the intact aragonite crystals of nacre (greater than 50 um). This reduction in particle size facilitates the enhancement of the release of WSM, resulting in faster pre-osteoblast differentiation. Notably, while osteoblasts typically require 21 days to differentiate and form mineralized nodules in standard mineralizing media [32], our results showed that 0.1% \(w/v\) WSM significantly increased RUNX2, ALP mRNA, and OCN mRNA expression by 1.5- and 1.8-fold, respectively, compared to the control within 7 days, while 0.05% \(w/v\) WSM achieved the same within 14 days (Figure 4). Additionally, ARS also confirmed significant calcium deposits in MC3T3-E1 treated with 0.1% \(w/v\) WSM within 7 days, indicating hydroxyapatite mineralization (Figure 5). Meanwhile, the MC3T3-E1 pre-osteoblasts also entered the mature stage of differentiation earlier. Therefore, WSM may accelerate bone formation, completing the process in just 7 days in a dose-dependent manner. In our study, WSM showed a prominent protein band at approximately 75 and 25 kDa (as shown in Figure 2). This finding is consistent with the results reported by [PERSON] et al., who indicated the presence of a protein band at 25, 52, 60, and 72 kDa in acid-extracted nacreous protein (AEP) from _H. diversicolor_ shell [33]. The protein band at 25 kDa was described as sometsuke, an abalone mantle protein, which has biomineralization and calcium-binding properties [34; 35]. According to the composition of nacre, which has similarities to bones in both its organic and inorganic components, it plays a crucial role in mineralization, which is regulated by the mantle tissue in molluss [16; 26; 36]. Mineral deposition occurs in specific regions of the mantle, where epithelial cells influence the formation of the nacreous layer [37; 38]. For nacreous proteins to be deposited into the nacreous layer, they must exhibit characteristics typical of secretory proteins, such as a signal peptide, glycosylation sites, and cysteine residues forming disulfide bridges for proper conformation. The presence of such features was confirmed in molecular analyses of the _Hd_BMP2/4 transcript from the univalve _H. diversicolor_ and the _P_/BMP2 transcript from the bivalve _P. ficata_[18; 24]. As shown in Figure 2, we performed WB by using a polyclonal antibody against BMP-2/4, but there was no detection of the pre-protein (-47 kDa) or the secreted (mature) protein (-11 kDa) forms of BMP-2/4 in the WSM. However, we analyzed its competition with the HdBMP-2/4 peptide and the result showed that the protein band at 25 kDa was absent. One explanation for the lack of pre-BMP-2/4 (-47 kDa) protein may be that it is in a dimeric or multimeric form, bound together by disulfide bridges or by electrostatic interaction, as also reported for other nacreous proteins [39; 40], which are subsequently disrupted by reducing or high salt conditions. Therefore, our competition assay further supported that HdBMP2/4 peptides are some of the integral components of WSM. As shown in Figure 3, the osteoinductive mechanism of WSM appears to involve the canonical BMP signaling pathway.