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Title: [Experimental study of resveratrol-solid lipid nanoparticles in promotion of osteogenic differentiation of bone marrow mesenchymal stem cells].
Author: Xiong F, Yao C, Zhou L, Li W, Wei B, Guan J, Mao Y.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2022 Sep 15; 36(9):1155-1165. PubMed ID: 36111480.
Abstract:
OBJECTIVE: To investigate the effect of solid lipid nanoparticles (SLNs) on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro by resveratrol (Res), and provide a method for the treatment of bone homeostasis disorders. METHODS: Res-SLNs were prepared by high-temperature emulsification and low-temperature solidification method, and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were co-cultured with different concentrations (0, 0.1, 1, 5, 10, 20 μmol/L) of Res and Res-SLNs. The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8 (CCK-8) and live/dead cell staining; the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining after osteogenic differentiation induction, and the optimal concentration of Res-SLNs for gene detection was determined. Anti-osteocalcin (OCN) immunofluorescence staining and real-time fluorescent quantitative PCR (RT-qPCR) were used to detect the effect of Res and Res-SLNs on osteoblast-related genes (ALP and OCN) of BMSCs. RESULTS: Live/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups; CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20 μmol/L (P<0.05), while Res-SLNs activity was not affected by Res concentration (P>0.05). After osteogenic differentiation, the staining intensity of ALP and ARS in both groups was dose-dependent. The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10 μmol/L and 1 μmol/L, respectively (P<0.05), and then decreased gradually; the most effective concentration of Res-SLNs was 1 μmol/L. The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group (P<0.05). CONCLUSION: Encapsulation of SLNs can improve the effect of Res on promoting osteogenesis, and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration, which is expected to be used in the treatment of bone homeostasis imbalance diseases. 目的: 探讨固体脂质纳米粒（solid lipid nanoparticles，SLNs）增强白藜芦醇（resveratrol，Res）促BMSCs体外成骨分化的效果，为骨稳态失衡疾病的治疗提供一种方法。. 方法: 通过高温乳化-低温固化法制备Res-SLNs，然后将分离培养的第2～3代SD大鼠BMSCs与不同浓度（0、0.1、1、5、10、20 μmol/L）Res和Res-SLNs共培养，通过细胞计数试剂盒 8（cell counting kit 8，CCK-8）和活/死细胞染色检测Res和Res-SLNs对BMSCs细胞活性的影响；通过成骨诱导分化后的ALP染色和茜素红S（alizarin red S，ARS）染色检测Res和Res-SLNs对BMSCs成骨分化的影响，并确定用于基因检测的Res-SLNs最适浓度；通过抗骨钙素（osteocalcin，OCN）免疫荧光染色和实时荧光定量PCR（real-time fluorescent quantitative PCR，RT-qPCR）检测Res和Res-SLNs对BMSCs成骨相关基因（ALP和OCN）的影响。. 结果: 活/死细胞染色示，Res和Res-SLNs各浓度组内的死细胞数量无明显差异；CCK-8检测示，浓度为20 μmol/L时，Res组BMSCs活性显著降低（P<0.05）；而浓度对Res-SLNs活性无影响（P>0.05）。成骨分化诱导后两组ALP和ARS染色强度均呈浓度依赖性，Res组和Res-SLNs组ALP阳性染色面积百分比和矿化结节面积百分比分别在10 μmol/L和1 μmol/L浓度时达最大值（P<0.05），然后逐渐减小；确定Res-SLNs最适浓度为1 μmol/L。成骨诱导分化培养14 d OCN免疫荧光染色和RT-qPCR检测示Res-SLNs组OCN表达量及ALP、OCN mRNA相对表达量均显著高于Res组（P<0.05）。. 结论: SLNs包封处理可以提高Res促进成骨的药物效果，在更低浓度达到BMSCs成骨分化的最佳效果，有望用于骨稳态失衡疾病的治疗。.

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