Purpose: Ultra-small gold nanoclusters (AuNCs), as emerging fluorescent nanomaterials with excellent biocompatibility, have been widely investigated for in vivo biomedical applications. However, their effects in guiding osteogenic differentiation have not been investigated, which are important for osteoporosis therapy and bone regeneration. Herein, for the first time, lysozyme-protected AuNCs (Lys-AuNCs) are used to stimulate osteogenic differentiation, which have the potential for the treatment of bone disease.
Methods: Proliferation of MC3T3E-1 is important for osteogenic differentiation. First, the proliferation rate of MC3T3E-1 was studied by Cell Counting Kit-8 (CCK8) assays. Signaling pathways of PI3K/Akt play central roles in controlling proliferation throughout the body. The expression of PI3K/Akt was investigated in the presence of lysozyme, and lysozyme-protected AuNCs (Lys-AuNCs) by Western blot (WB) and intracellular cell imaging to evacuate the osteogenic differentiation mechanisms. Moreover, the formation of osteoclasts (OC) plays a negative role in the differentiation of osteoblasts. Nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling pathways are used to understand the negative influence of the osteogenic differentiation by the investigation of Raw 264.7 cell line. Raw 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the cell viability was assessed. Raw 264.7 cells have also been used for in vitro studies, on understanding the osteoclast formation and function. The induced osteoclasts were identified by TRAP confocal fluorescence imaging. These key factors in osteoclast formation, such as (NFATc-1, c-Fos, V-ATPase-2 and CTSK), were investigated by Western blot.
Results: Based on the above investigation, Lys-AuNCs were found to promote osteogenic differentiation and decrease osteoclast activity. It is noteworthy that the lysozyme (protected template), AuNPs, or the mixture of Lysozyme and AuNPs have negligible effects on osteoblastic differentiation compared to Lys-AuNCs.
Conclusion: This study opens up a novel avenue to develop a new gold nanomaterial for promoting osteogenic differentiation. The possibility of using AuNCs as nanomedicines for the treatment of osteoporosis can be expected.

Keywords: nanoclusters, bone, osteogenic differentiation, osteoporosis


中文翻译：

---

超小溶菌酶保护的金纳米簇作为纳米药物诱导成骨分化。

目的：超小型金纳米团簇（AuNCs）作为一种新兴的具有优异生物相容性的荧光纳米材料，已在体内生物医学应用中得到广泛研究。然而，尚未研究它们在指导成骨分化方面的作用，这对于骨质疏松症治疗和骨再生很重要。在此，溶菌酶保护的 AuNCs (Lys-AuNCs) 首次用于刺激成骨分化，具有治疗骨病的潜力。
方法：MC3T3E-1 的增殖对于成骨分化很重要。首先，通过 Cell Counting Kit-8 (CCK8) 测定研究 MC3T3E-1 的增殖率。PI3K/Akt 的信号通路在控制整个身体的增殖中起核心作用。在溶菌酶和溶菌酶保护的 AuNCs (Lys-AuNCs) 存在下通过蛋白质印迹 (WB) 和细胞内细胞成像研究 PI3K/Akt 的表达，以疏散成骨分化机制。此外，破骨细胞（OC）的形成在成骨细胞的分化中起负面作用。核因子 κB 配体 (RANKL) 和巨噬细胞集落刺激因子 (M-CSF) 信号通路用于通过研究 Raw 264.7 细胞系来了解成骨分化的负面影响。原始 264。7（鼠巨噬细胞样）细胞和NIH/3T3（小鼠成纤维细胞）细胞用泰洛沙泊处理，并评估细胞活力。原始 264.7 细胞也已用于体外研究，以了解破骨细胞的形成和功能。通过TRAP共聚焦荧光成像鉴定诱导的破骨细胞。通过蛋白质印迹研究了破骨细胞形成中的这些关键因素，例如（NFATc-1、c-Fos、V-ATPase-2 和 CTSK）。
结果：基于上述研究，发现Lys-AuNCs促进成骨分化并降低破骨细胞活性。值得注意的是，与 Lys-AuNCs 相比，溶菌酶（受保护的模板）、AuNPs 或溶菌酶和 AuNPs 的混合物对成骨细胞分化的影响可以忽略不计。
结论：本研究为开发促进成骨分化的新型金纳米材料开辟了一条新途径。可以预期使用 AuNCs 作为纳米药物治疗骨质疏松症的可能性。

关键词：纳米团簇，骨，成骨分化，骨质疏松