Exosomes derived from melatonin-modified bone marrow mesenchymal stem cells promote osteogenesis of bone marrow mesenchymal stem cells

doi:10.12307/2022.759

Abstract

Abstract: BACKGROUND: Osteoporosis is the most prevailing bone disease, which is characterized by the decrease in bone mass and deterioration of bone microarchitecture. Current treatments, although being used clinically for a long time, still have many side effects. Bone marrow mesenchymal stem cell-derived exosomes are expected to become a new method for the treatment of osteoporosis due to their non-immunogenicity and other advantages.
OBJECTIVE: To explore the effects of exosomes derived from melatonin (MT)-modified bone marrow mesenchymal stem cells on the osteogenesis of bone marrow mesenchymal stem cells and to analyze its feasibility as a cell-free method to promote osteogenic treatment of osteoporosis.
METHODS: Primary bone marrow mesenchymal stem cells were isolated and characterized by whole bone marrow adherent method. Exosomes derived from bone marrow mesenchymal stem cells (NC-Exos) and MT pretreated bone marrow mesenchymal stem cells (MT-Exos) were extracted by ultracentrifuge and identified. The uptaking of exsomes into bone marrow mesenchymal stem cells was observed under a confocal microscope. Bone marrow mesenchymal stem cells were cultured in osteogenic induction medium. Bone marrow mesenchymal stem cells were treated with MT-Exos and NC-Exos, separately. Bone marrow mesenchymal stem cells were treated with PBS as a blank control group. Alkaline phosphatase activity and alizarin red S staining were employed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells in each group. The mRNA and protein expression levels of alkaline phosphatase and Runt related transcription factor 2 were measured through RT-PCR and western blot assay, respectively. Western blot assay was utilized to detect the protein expression of Wnt1 and β-catenin in bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION: (1) Bone marrow mesenchymal stem cells had a typical spindle-like morphology and had the ability to differentiate into three lines. The majority of exosomes was smaller than 150 nm in diameter and expressed CD9, CD63, and CD81. Exosomes were taken up by bone marrow mesenchymal stem cells. (2) At 7 days after osteoinduction, compared with the NC-Exos group, MT-Exos group presented higher alkaline phosphatase activity (P < 0.01), mRNA and protein expression of alkaline phosphatase and Runt related transcription factor 2 (P < 0.05), and Wnt1 and β-catenin protein expression (P < 0.001). (3) At 14 days after osteoinduction, compared with the NC-Exos group, MT-Exos group showed more red calcium nodules. (4) The present study has demonstrated that MT-Exos have the ability to promote osteogenic differentiation of bone marrow mesenchymal stem cells by activating Wnt1/β-catenin signaling pathway.

Key words: exosome, melatonin, bone marrow mesenchymal stem cells, osteogenesis, signaling pathway

CLC Number:

Shen Enpu, Huang Ba, Liu Danping, Qi Hui, Wu Zhiwen, Li Beibei. Exosomes derived from melatonin-modified bone marrow mesenchymal stem cells promote osteogenesis of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(30): 4800-4805.

Figures/Tables 6

References

[1] LI T, JIANG S, LU C, et al. Melatonin: Another avenue for treating osteoporosis? J Pineal Res. 2019;66(2):e12548.
[2] 张智海,张智若,刘忠厚,等.中国大陆地区以-2.0SD为诊断标准的骨质疏松症发病率回顾性研究[J].中国骨质疏松杂志,2016, 22(1):1-8.
[3] COSMAN F, DE BEUR SJ, LEBOFF MS, et al. Clinician’s Guide to Prevention and Treatment of Osteoporosis. Osteoporos Int. 2014; 25(10):2359-2381.
[4] DAS S, CROCKETT JC. Osteoporosis - a current view of pharmacological prevention and treatment. Drug Des Devel Ther. 2013;7:435-448.
[5] AGHEBATI-MALEKI L, DOLATI S, ZANDI R, et al. Prospect of mesenchymal stem cells in therapy of osteoporosis: A review. J Cell Physiol. 2019;234(6): 8570-8578.
[6] ZUO R, LIU M, WANG Y, et al. Correction to: BM-MSC-derived exosomes alleviate radiation-induced bone loss by restoring the function of recipient BM-MSCs and activating Wnt/beta-catenin signaling. Stem Cell Res Ther. 2020;11(1):33.
[7] LUO ZW, LI FX, LIU YW, et al. Aptamer-functionalized exosomes from bone marrow stromal cells target bone to promote bone regeneration. Nanoscale. 2019;11(43):20884-20892.
[8] ZHANG L, YU D. Exosomes in cancer development, metastasis, and immunity. Biochim Biophys Acta Rev Cancer. 2019;1871(2):455-468.
[9] MA ZJ, YANG JJ, LU YB, et al. Mesenchymal stem cell-derived exosomes: Toward cell-free therapeutic strategies in regenerative medicine. World J Stem Cells. 2020;12(8):814-840.
[10] INGATO D, LEE JU, SIM SJ, et al. Good things come in small packages: Overcoming challenges to harness extracellular vesicles for therapeutic delivery. J Control Release. 2016;241:174-185.
[11] 綦惠,黄霸,杰永生,等.干细胞外泌体对骨质疏松大鼠骨生成的影响[J].中国矫形外科杂志,2021,29(8):726-730.
[12] HARDELAND R. Aging, Melatonin, and the Pro- and Anti-Inflammatory Networks. Int J Mol Sci. 2019;20(5):1223.
[13] LIU W, YU M, XIE D, et al. Melatonin-stimulated MSC-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway. Stem Cell Res Ther. 2020;11(1):259.
[14] ZHANG L, SU P, XU C, et al. Melatonin inhibits adipogenesis and enhances osteogenesis of human mesenchymal stem cells by suppressing PPARγ expression and enhancing Runx2 expression. J Pineal Res. 2010;49(4):364-372.
[15] AMSTRUP AK, SIKJAER T, MOSEKILDE L, et al. Melatonin and the skeleton. Osteoporos Int. 2013;24(12):2919-2927.
[16] 肖大伟,刘丹平,田大川,等.突变型低氧诱导因子-1α修饰骨髓间充质干细胞来源的外泌体联合软骨再生支架治疗家兔早期软骨缺损[J].中华实验外科杂志,2018,35(5):888-891.
[17] HUANG B, SU Y, SHEN E, et al. Extracellular vesicles from GPNMB-modified bone marrow mesenchymal stem cells attenuate bone loss in an ovariectomized rat model. Life Sci. 2021;272:119208.
[18] MIANEHSAZ E, MIRZAEI HR, MAHJOUBIN-TEHRAN M, et al. Mesenchymal stem cell-derived exosomes: a new therapeutic approach to osteoarthritis? Stem Cell Res Ther. 2019;10(1):340.
[19] BHATTI FU, MEHMOOD A, LATIEF N, et al. Vitamin E protects rat mesenchymal stem cells against hydrogen peroxide-induced oxidative stress in vitro and improves their therapeutic potential in surgically-induced rat model of osteoarthritis. Osteoarthritis Cartilage. 2017; 25(2):321-331.
[20] QAZI TH, MOONEY DJ, DUDA GN, et al. Biomaterials that promote cell-cell interactions enhance the paracrine function of MSCs. Biomaterials. 2017;140:103-114.
[21] LIU Z, GAN L, ZHANG T, et al. Melatonin alleviates adipose inflammation through elevating α-ketoglutarate and diverting adipose-derived exosomes to macrophages in mice. J Pineal Res. 2018;64(1). doi: 10.1111/jpi.12455.
[22] HUANG P, WANG L, LI Q, et al. Atorvastatin enhances the therapeutic efficacy of mesenchymal stem cells-derived exosomes in acute myocardial infarction via up-regulating long non-coding RNA H19. Cardiovasc Res. 2020;116(2):353-367.
[23] SATOMURA K, TOBIUME S, TOKUYAMA R, et al. Melatonin at pharmacological doses enhances human osteoblastic differentiation in vitro and promotes mouse cortical bone formation in vivo. J Pineal Res. 2007;42(3):231-239.
[24] RADIO NM, DOCTOR JS, WITT-ENDERBY PA. Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade. J Pineal Res. 2006;40(4):332-342.
[25] 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;10(1):335.
[26] BARON R, KNEISSEL M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med. 2013;19(2):179-192.