锶离子外泌体促间充质干细胞成骨分化和内皮细胞血管生成的实验研究

口腔生物医学 ›› 2024, Vol. 15 ›› Issue (2): 66-74.

锶离子外泌体促间充质干细胞成骨分化和内皮细胞血管生成的实验研究

杨思敏¹,殷实²,周名亮¹,江飞³,蒋欣泉⁴

1. 上海交通大学医学院附属第九人民医院
2. 上海交通大学医学院附属第九人民医院口腔修复科
3. 南京医科大学附属口腔医院
4. 上海交通大学医学院附属第九人民医院口腔修复科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室

收稿日期:2024-01-05 修回日期:2024-01-24 出版日期:2024-04-25 发布日期:2024-04-29
通讯作者: 蒋欣泉 E-mail:xinquanjiang@aliyun.com
基金资助:
国家自然科学基金创新研究群体;国家自然科学基金重点项目;国家自然科学基金青年项目

The study of strontium-induced MSC-derived exosomes in promoting osteogenic differentiation of mesenchymal stem cells and angiogenesis of endothelial cells

Received:2024-01-05 Revised:2024-01-24 Online:2024-04-25 Published:2024-04-29
Contact: Xin-Quan JIANG E-mail:xinquanjiang@aliyun.com

摘要/Abstract

摘要： 目的：探究锶离子诱导间充质干细胞（MSC）分泌的外泌体（以下简称为锶离子外泌体）对成骨分化和血管生成功能的影响。方法：向骨髓间充质干细胞（BMSC）中加入锶离子并通过超速离心法提取锶离子外泌体，以未经处理的BMSC外泌体作为对照。通过外泌体荧光标记观察其细胞内化作用；采用碱性磷酸酶（ALP）染色、ALP活性测定、实时荧光定量PCR等实验探究分离的外泌体对BMSC成骨分化的影响；采用细胞划痕实验、迁移实验、内皮细胞成管实验等方法评价外泌体对人脐静脉内皮细胞（HUVEC）体外血管形成能力的调控作用；利用miRNA测序探讨锶离子外泌体促进成骨和成血管作用的潜在分子机制。结果：锶离子刺激不会影响MSC分泌外泌体，且分泌的锶离子外泌体可以被BMSC摄取内化，与对照组外泌体相比，锶离子外泌体可以增强BMSC的ALP活性（P＜0.05），上调其ALP和Runt相关转录因子2（Runx2）的转录（P＜0.05）；同时锶离子外泌体可以促进HUVEC细胞迁移和小管形成（P＜0.05），并提高血管内皮生长因子（VEGF）、促血管生成素1（ANG1）和成纤维细胞生长因子（FGF）的转录（P＜0.05）；外泌体miRNA测序发现锶离子刺激会引起BMSC外泌体内miRNA的差异性表达，差异表达的miR-125b-5p、miR-132-3p、miR-31a-5p以及miR-450b可能参与锶离子外泌体促成骨和成血管的功能发挥。结论：锶离子诱导MSC分泌的外泌体具有促进成骨分化和血管生成的双重功能。

关键词: 骨组织工程, 锶离子, 外泌体, 成骨分化, 血管生成

Abstract: Objective:To explore the effect of mesenchymal stem cell (MSC) derived exosomes induced by strontium on osteogenic differentiation and angiogenesis, providing a new idea for the repair of vascularized bone defects. Methods:Strontium was added to bone marrow mesenchymal stem cell (BMSC) and strontium-exosomes were then extracted by ultracentrifugation, using untreated BMSC exosomes as control. The cell internalization was observed by fluorescent labeling of exosomes. Alkaline phosphatase (ALP) staining, ALP activity determination and qRT-PCR analysis were used to study the effects of exosomes on the osteogenic differentiation of BMSC. The effects of exosomes on vascular formation of endothelial cells in vitro were evaluated by wound healing assay, transwell assay, tube formation assay and other methods. Finally, the potential mechanism of strontium-exosomes in promoting osteogenesis and angiogenesis was discussed by the miRNA sequencing. Results:The stimulation of strontium ion could not affect the secretion of exosomes by BMSC, and the strontium-exosomes could be internalized by BMSC. Strontium-exosomes could enhance the ALP activity of BMSC and up-regulate the transcription of osteogenesis-related genes ALP and runt-related transcription factor 2 (Runx2). Strontium-exosomes could also promote the migration and the tubule formation of endothelial cells, and significantly increase the transcription of angiogenic-related genes of VEGF, ANG1 and FGF. The miRNA sequencing analysis revealed that the stimulation of strontium ions induced the differential expression of miRNA in exosomes derived from BMSC. The up-regulated expression of miR-125b-5p, miR-132-3p, miR-31a-5p and miR-450b may be then involved in promoting osteogenic differentiation and angiogenesis of cells. Conclusions:The strontium-exosomes have dual functions of promoting angiogenesis and osteogenic differentiation.

Key words: bone tissue engineering, strontium ion, exosome, osteogenic differentiation, angiogenesis

杨思敏殷实周名亮江飞蒋欣泉. 锶离子外泌体促间充质干细胞成骨分化和内皮细胞血管生成的实验研究[J]. 口腔生物医学, 2024, 15(2): 66-74.

参考文献 20

[1]	Szostakowski B, DeMaio M. Ideal xenograft or a perfect bone substitute?-A retrospective review and analysis of the historical concept of ivory implants in orthopaedics. Int Orthop. 2020 May; 44(5):1003-1009.
[2]	Kashirina A, Yao Y, Liu Y, et al. Biopolymers as bone substitutes: a review[J]. Biomaterials Science, 2019, 7.
[3]	Amghar-Maach S, Gay-Escoda C, Sánchez-Garcés Má. Regeneration of periodontal bone defects with dental pulp stem cells grafting: Systematic Review. J Clin Exp Dent. 2019 Apr 1; 11(4): e373-e381.
[4]	Lee YC, Chan YH, Hsieh SC, et al. Comparing the Osteogenic Potentials and Bone Regeneration Capacities of Bone Marrow and Dental Pulp Mesenchymal Stem Cells in a Rabbit Calvarial Bone Defect Model. Int J Mol Sci. 2019 Oct 10; 20(20):5015.
[5]	Lai S, Deng L, Liu C,et al. Bone marrow mesenchymal stem cell-derived exosomes loaded with miR-26a through the novel immunomodulatory peptide DP7-C can promote osteogenesis. Biotechnol Lett. 2023 Jul;45(7):905-919.
[6]	Saidak Z, Marie PJ. Strontium signaling: molecular mechanisms and therapeutic implications in osteoporosis. Pharmacol Ther. 2012 Nov; 136(2):216-26.
[7]	Marie PJ. Strontium ranelate: a dual mode of action rebalancing bone turnover in favour of bone formation. Curr Opin Rheumatol. 2006 Jun; 18 Suppl 1: S11-5.
[8]	Guo S, Yu D, Xiao X, et al. A vessel subtype beneficial for osteogenesis enhanced by strontium-doped sodium titanate nanorods by modulating macrophage polarization. J Mater Chem B. 2020 Jul 28; 8(28):6048-6058.
[9]	Chen Y, Zheng Z, Zhou R, et al. Developing a Strontium-Releasing Graphene Oxide-/Collagen-Based Organic-Inorganic Nanobiocomposite for Large Bone Defect Regeneration via MAPK Signaling Pathway. ACS Appl Mater Interfaces. 2019 May 1;11(17):15986-15997.
[10]	Li JJ, Dunstan CR, Entezari A, et al. A Novel Bone Substitute with High Bioactivity, Strength, and Porosity for Repairing Large and Load-Bearing Bone Defects. Adv Healthc Mater. 2019 Jul; 8(13): e1900641.
[11]	Guo SC, Tao SC, Yin WJ, et al. Exosomes from Human Synovial-Derived Mesenchymal Stem Cells Prevent Glucocorticoid-Induced Osteonecrosis of the Femoral Head in the Rat. Int J Biol Sci. 2016 Oct 17; 12(10):1262-1272.
[12]	Liang B, Liang JM, Ding JN, et al. Dimethyloxaloylglycine-stimulated human bone marrow mesenchymal stem cell-derived exosomes enhance bone regeneration through angiogenesis by targeting the AKT/mTOR pathway. Stem Cell Res Ther. 2019 Nov 20; 10(1):335.
[13]	Kuang MJ, Huang Y, Zhao XG, et al. Exosomes derived from Wharton's jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway. Int J Biol Sci. 2019 Jul 20; 15(9):1861-1871. (2019) 1861–1871.
[14]	Narayanan R, Huang CC, Ravindran S. Hijacking the Cellular Mail: Exosome Mediated Differentiation of Mesenchymal Stem Cells. Stem Cells Int. 2016; 2016:3808674.
[15]	Lai RC, Tan SS, Teh BJ, et al. Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome. Int J Proteomics. 2012; 2012:971907.
[16]	Kang Y, Xu C, Meng L, et al. Exosome-functionalized magnesium-organic framework-based scaffolds with osteogenic, angiogenic and anti-inflammatory properties for accelerated bone regeneration. Bioact Mater. 2022 Feb 18;18:26-41.
[17]	Qi L, Fang X, Yan J,et al. Magnesium-containing bioceramics stimulate exosomal miR-196a-5p secretion to promote senescent osteogenesis through targeting Hoxa7/MAPK signaling axis. Bioact Mater. 2023 Nov 4;33:14-29.
[18]	Wei P, Jing W, Yuan Z, et al. Vancomycin- and Strontium-Loaded Microspheres with Multifunctional Activities against Bacteria, in Angiogenesis, and in Osteogenesis for Enhancing Infected Bone Regeneration. ACS Appl Mater Interfaces. 2019 Aug 28;11(34):30596-30609.
[19]	Weng L, Boda SK, Teusink MJ, et al. Binary Doping of Strontium and Copper Enhancing Osteogenesis and Angiogenesis of Bioactive Glass Nanofibers while Suppressing Osteoclast Activity. ACS Appl Mater Interfaces. 2017 Jul 26;9(29):24484-24496.
[20]	Lin K, Xia L, Li H, et al. Enhanced osteoporotic bone regeneration by strontium-substituted calcium silicate bioactive ceramics. Biomaterials. 2013 Dec;34(38):10028-42.