## 1 Introduction  
Osteoporosis, a common condition in aging societies, is particularly severe among postmenopausal women due to estrogen deficiency. This deficiency accelerates bone turnover, disrupting the balance between bone formation and resorption, leading to a decrease in bone density and an increased risk of fractures, especially in the spine and hips. These fractures are a leading cause of morbidity and mortality among the elderly population [1, 2]. Current pharmaceutical treatments, such as bisphosphonates, selective estrogen receptor modulators (SERMs), and newer agents like denosumab and romosozumab, are effective in reducing fracture risks but can have significant side effects, including infection risks, venous thromboembolism, and bone loss after discontinuation [3, 4]. Estrogen therapy,while beneficial in reducing bone loss and fracture risk, is also associated with cardiovascular events and certain types of cancer [5]. Consequently, there is a pressing need for new treatment options that can effectively stimulate bone density recovery and restore bone structure. Nacre, or mother of pearl, is a composite material primarily composed of calcium carbonate crystals in aragonite form, along with an organic WSM consisting of polysaccharides, proteins, and glycoproteins [6]. This composition is similar to bone, as both structures utilize organic matrices to guide controlled mineral deposition in biomineralization processes [7]. Moreover, studies suggest that nacre has strong osteoinductive properties. For example, implants of fragmented nacre from _Pinctada maxima_ (P. maxima) have been shown to stimulate bone formation in animal models, suggesting its potential for promoting bone healing [8; 9; 10; 11; 12]. In vitro studies also show that WSM extracted from nacre can induce human bone marrow stromal cells (hBMSCs) and pre-osteoblasts to differentiate into osteoblasts. These cells exhibit osteogenic markers, such as alkaline phosphatase and osteocalcin, indicative of active bone tissue formation [13; 14]. Nacre's organic matrix contains bioactive proteins like nacrein, MSI60, and perlucin, which are critical for its osteoinductive effects. These proteins are non-immunogenic, making nacre biocompatible and suitable for use in bone repair without causing adverse immune reactions [15; 16; 17; 18; 19; 20; 21; 22; 23; 24]. Studies have shown that fragmented nacre releases bioactive molecules into culture media, further promoting osteoblast differentiation and mineralization. However, achieving the release of WSM from natural nacre has been challenging due to its solid and compact surface [25]. Recent advances, such as the development of nano-nacre particles, have helped to enhance the release of WSM and calciumions by increasing the surface area [14]. However, it is well established that the osteoinductive effect of nacre is attributed to the organic components within the WSM. Therefore, the molecules responsible for promoting bone formation need to be identified and elucidated. In this study, nano-nacre particles were developed with a smaller diameter than the intact aragonite crystal of nacre to enhance the release of the organic matrix and identify potential osteogenic molecules within the WSM. This approach improves the bioavailability of nacre's active components, making it more effective in promoting osteogenesis. This innovation is particularly significant for the development of therapeutic strategies for osteoporosis and bone regeneration.