Department of Orthopedics, School of Pharmaceutical Science, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, People's Republic of China.
Department of Basic Science, School of Pharmaceutical Science, Jinzhou Medical University, Jinzhou, People's Republic of China.
Int J Nanomedicine. 2020 Jun 30;15:4705-4716. doi: 10.2147/IJN.S241163. eCollection 2020.
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.
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.
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.
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.
超小的金纳米簇（AuNCs）作为具有优异生物相容性的新兴荧光纳米材料，已被广泛研究用于体内生物医学应用。然而，它们在指导成骨分化方面的作用尚未得到研究，而这对于骨质疏松症的治疗和骨再生非常重要。在此，首次使用溶菌酶保护的 AuNCs（Lys-AuNCs）来刺激成骨分化，这为治疗骨疾病提供了新的可能。
MC3T3E-1 的增殖对于成骨分化很重要。首先，通过 Cell Counting Kit-8（CCK8）测定法研究 MC3T3E-1 的增殖率。PI3K/Akt 信号通路在控制全身增殖中起着核心作用。通过 Western blot（WB）和细胞内成像研究溶菌酶和溶菌酶保护的 AuNCs（Lys-AuNCs）中 PI3K/Akt 的表达，以探讨成骨分化的机制。此外，破骨细胞（OC）的形成对成骨细胞的分化起着负作用。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影响。用 tyloxapol 处理 Raw 264.7（鼠巨噬样）细胞和 NIH/3T3（鼠成纤维细胞）细胞，并评估细胞活力。还将 Raw 264.7 细胞用于体外研究，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 Western blot 研究破骨细胞形成的关键因素，如（NFATc-1、c-Fos、V-ATPase-2 和 CTSK）。
基于上述研究，发现 Lys-AuNCs 可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（保护模板）、AuNPs 或溶菌酶和 AuNPs 的混合物对成骨细胞分化几乎没有影响。
这项研究为开发用于促进成骨分化的新型金纳米材料开辟了新途径。可以预期，AuNCs 作为治疗骨质疏松症的纳米药物具有一定的应用前景。
