Experimental study on improvement of osteonecrosis of femoral head with exosomes derived from miR-27a-overexpressing vascular endothelial cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Gensheng ¹ , LIU Ruiyu ² , DANG Xiaoqian ² , LIU Jichao ¹ ,  JIAO Haibin ¹

1. Department of Orthopaedics, 3201 Hospital of Xi’an Jiaotong University Health Science Center, Hanzhong Shaanxi, 723000, P.R.China;
2. Department of Orthopaedics, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Shaanxi, 710004, P.R.China;

Corresponding?author：

JIAO Haibin, Email: jiaohaibin2001@163.com

Keywords：

MiR-27a; exosomes; osteogenesis; osteonecrosis of femoral head; human umbilical vein endothelial cells; gene targeted therapy

DOI：

10.7507/1002-1892.202011026

Video：

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Objective To investigate whether exosomes derived from miR-27a-overexpressing human umbilical vein endothelial cells (HUVECs)—exo (miR-27a) can promote bone regeneration and improve glucocorticoids (GC) induced osteonecrosis of femoral head (ONFH) (GC-ONFH).Methods The exo (miR-27a) were intended to be constructed and identified by transmission electron microscopy, nanoparticle tracking analysis, Western blot, and real-time fluorescent quantitative PCR (qRT-PCR). qRT-PCR was used to evaluate the effect of exo (miR-27a) in delivering miR-27a to osteoblasts (MC3T3-E1 cells). Alkaline phosphatase staining, alizarin red staining, and qRT-PCR were used to evaluate its effect on MC3T3-E1 cells osteogenesis. Dual-luciferase reporter (DLRTM) assay was used to verify whether miR-27a targeting Dickkopf WNT signaling pathway inhibitor 2 (DKK2) was a potential mechanism, and the mechanism was further verified by qRT-PCR, Western blot, and alizarin red staining in MC3T3-E1 cells. Finally, the protective effect of exo (miR-27a) on ONFH was verified by the GC-ONFH model in Sprague Dawley (SD) rats.Results Transmission electron microscopy, nanoparticle tracking analysis, Western blot, and qRT-PCR detection showed that exo (miR-27a) was successfully constructed. exo (miR-27a) could effectively deliver miR-27a to MC3T3-E1 cells and enhance their osteogenic capacity. The detection of DLRTM showed that miR-27a promoted bone formation by directly targeting DDK2. Micro-CT and HE staining results of animal experiments showed that tail vein injection of exo (miR-27a) improved the osteonecrosis of SD rat GC-ONFH model.Conclusion exo (miR-27a) can promote bone regeneration and protect against GC-ONFH to some extent.

Citation： ZHANG Gensheng, LIU Ruiyu, DANG Xiaoqian, LIU Jichao, JIAO Haibin. Experimental study on improvement of osteonecrosis of femoral head with exosomes derived from miR-27a-overexpressing vascular endothelial cells. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(3): 356-365. doi: 10.7507/1002-1892.202011026 Copy

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2.	Zhao D, Zhang F, Wang B, et al. Guidelines for clinical diagnosis and treatment of osteonecrosis of the femoral head in adults (2019 version). J Orthop Translat, 2020, 21: 100-110.
3.	Kubo T, Ueshima K, Saito M, et al. Clinical and basic research on steroid-induced osteonecrosis of the femoral head in Japan. J Orthop Sci, 2016, 21(4): 407-413.
4.	Zhang QY, Li ZR, Gao FQ, et al. Pericollapse stage of osteonecrosis of the femoral head: A Last chance for joint preservation. Chin Med J (Engl), 2018, 131(21): 2589-2598.
5.	Liu LH, Zhang QY, Sun W, et al. Corticosteroid-induced osteonecrosis of the femoral head: Detection, diagnosis, and treatment in earlier stages. Chin Med J (Engl), 2017, 130(21): 2601-2607.
6.	Hardy RS, Zhou H, Seibel MJ, et al. Glucocorticoids and bone: Consequences of endogenous and exogenous excess and replacement therapy. Endocr Rev, 2018, 39(5): 519-548.
7.	Weinstein RS, Hogan EA, Borrelli MJ, et al. The pathophysiological sequence of glucocorticoid-induced osteonecrosis of the femoral head in male mice. Endocrinology, 2017, 158(11): 3817-3831.
8.	Wang A, Ren M, Wang J. The pathogenesis of steroid-induced osteonecrosis of the femoral head: A systematic review of the literature. Gene, 2018, 671: 103-109.
9.	Fang SH, Chen L, Chen HH, et al. MiR-15b ameliorates SONFH by targeting Smad7 and inhibiting osteogenic differentiation of BMSCs. Eur Rev Med Pharmacol Sci, 2019, 23(22): 9761-9771.
10.	劉立華, 孫偉, 王云亭, 等. 頭頸部開窗減壓治療 L1 型激素性股骨頭壞死: 單中心前瞻性臨床研究. 中國組織工程研究, 2021, 25(6): 906-911.
11.	中國醫師協會骨科醫師分會骨循環與骨壞死專業委員會, 中華醫學會骨科分會骨顯微修復學組, 國際骨循環學會中國區. 中國成人股骨頭壞死臨床診療指南 (2020). 中華骨科雜志, 2020, 40(20): 1365-1376.
12.	Barile L, Vassalli G. Exosomes: Therapy delivery tools and biomarkers of diseases. Pharmacol Ther, 2017, 174: 63-78.
13.	Liao W, Du Y, Zhang C, et al. Exosomes: The next generation of endogenous nanomaterials for advanced drug delivery and therapy. Acta Biomater, 2019, 86: 1-14.
14.	Qin Y, Sun R, Wu C, et al. Exosome: A novel approach to stimulate bone regeneration through regulation of osteogenesis and angiogenesis. Int J Mol Sci, 2016, 17(5): 712.
15.	Masaoutis C, Theocharis S. The role of exosomes in bone remodeling: implications for bone physiology and disease. Dis Markers, 2019, 2019: 9417914.
16.	Behera J, Tyagi N. Exosomes: mediators of bone diseases, protection, and therapeutics potential. Oncoscience, 2018, 5(5-6): 181-195.
17.	Qi X, Zhang J, Yuan H, et al. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells repair critical-sized bone defects through enhanced angiogenesis and osteogenesis in osteoporotic rats. Int J Biol Sci, 2016, 12(7): 836-849.
18.	Zhao P, Xiao L, Peng J, et al. Exosomes derived from bone marrow mesenchymal stem cells improve osteoporosis through promoting osteoblast proliferation via MAPK pathway. Eur Rev Med Pharmacol Sci, 2018, 22(12): 3962-3970.
19.	Zhang J, Liu X, Li H, et al. Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway. Stem Cell Res Ther, 2016, 7(1): 136.
20.	Lu J, Wang QY, Sheng JG. Exosomes in the repair of bone defects: next-generation therapeutic tools for the treatment of nonunion. Biomed Res Int, 2019, 2019: 1983131.
21.	Song H, Li X, Zhao Z, et al. Reversal of osteoporotic activity by endothelial cell-secreted bone targeting and biocompatible exosomes. Nano Lett, 2019, 19(5): 3040-3048.
22.	Zheng H, Liu J, Tycksen E, et al. MicroRNA-181a/b-1 over-expression enhances osteogenesis by modulating PTEN/PI3K/AKT signaling and mitochondrial metabolism. Bone, 2019, 123: 92-102.
23.	Lin Z, He H, Wang M, et al. MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate. Cell Prolif, 2019, 52(6): e12688.
24.	Zhang L, Tang Y, Zhu X, et al. Overexpression of MiR-335-5p promotes bone formation and regeneration in mice. J Bone Miner Res, 2017, 32(12): 2466-2475.
25.	Gu C, Xu Y, Zhang S, et al. miR-27a attenuates adipogenesis and promotes osteogenesis in steroid-induced rat BMSCs by targeting PPARγ and GREM1. Sci Rep, 2016, 6: 38491.
26.	Wu X, Gu Q, Chen X, et al. MiR-27a targets DKK2 and SFRP1 to promote reosseointegration in the regenerative treatment of peri-implantitis. J Bone Miner Res, 2019, 34(1): 123-134.
27.	Sriram M, Sainitya R, Kalyanaraman V, et al. Biomaterials mediated microRNA delivery for bone tissue engineering. Int J Biol Macromol, 2015, 74: 404-412.
28.	Arriaga MA, Ding MH, Gutierrez AS, et al. The application of microRNAs in biomaterial scaffold-based therapies for bone tissue engineering. Biotechnology Journal, 2019, 14(10): e1900084.
29.	黎牧帆, 張二洋, 呂雷鋒, 等. 維生素 E 對大鼠早期激素性股骨頭缺血性壞死的作用及機制研究. 中國修復重建外科雜志, 2018, 32(11): 1421-1428.
30.	Velmurugan BK, Bharathi Priya L, Poornima P, et al. Biomaterial aided differentiation and maturation of induced pluripotent stem cells. J Cell Physiol, 2019, 234(6): 8443-8454.
31.	Bakhshandeh B, Zarrintaj P, Oftadeh MO, et al. Tissue engineering; strategies, tissues, and biomaterials. Biotechnol Genet Eng Rev, 2017, 33(2): 144-172.
32.	Jewell CM, Collier JH. Biomaterial interactions with the immune system. Biomater Sci, 2019, 7(3): 713-714.
33.	Sato Y, Bando H, Di Piazza M, et al. Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider. Cytotherapy, 2019, 21(11): 1095-1111.
34.	Colao IL, Corteling R, Bracewell D, et al. Manufacturing exosomes: A promising therapeutic platform. Trends Mol Med, 2018, 24(3): 242-256.
35.	Adamiak M, Cheng G, Bobis-Wozowicz S, et al. Induced pluripotent stem cell (iPSC)-derived extracellular vesicles are safer and more effective for cardiac repair than iPSCs. Circ Res, 2018, 122(2): 296-309.
36.	Wang B, Jia H, Zhang B, et al. Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy. Stem Cell Res Ther, 2017, 8(1): 75.
37.	Zhang S, Liu X, Ge L L, et al. Mesenchymal stromal cell-derived exosomes improve pulmonary hypertension through inhibition of pulmonary vascular remodeling. Respir Res, 2020, 21(1): 71.
38.	Qing L, Chen H, Tang J, et al. Exosomes and their microRNA cargo: New players in peripheral nerve regeneration. Neurorehabil Neural Repair, 2018, 32(9): 765-776.
39.	Zuo R, Kong L, Wang M, et al. Exosomes derived from human CD34 stem cells transfected with miR-26a prevent glucocorticoid-induced osteonecrosis of the femoral head by promoting angiogenesis and osteogenesis. Stem Cell Res Ther, 2019, 10(1): 321.
40.	Qin Y, Wang L, Gao Z, et al. Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo. Sci Rep, 2016, 6: 21961.
41.	Duan Y, Tan Z, Yang M, et al. PC-3-derived exosomes inhibit osteoclast differentiation by downregulating miR-214 and blocking NF-B signaling pathway. Biomed Res Int, 2019, 2019: 8650846.
42.	Gonzalez-King H, García NA, Ontoria-Oviedo I, et al. Hypoxia inducible factor-1α potentiates jagged 1-mediated angiogenesis by mesenchymal stem cell-derived exosomes. Stem Cells, 2017, 35(7): 1747-1759.
43.	Liao W, Ning Y, Xu HJ, et al. BMSC-derived exosomes carrying microRNA-122-5p promote proliferation of osteoblasts in osteonecrosis of the femoral head. Clin Sci (Lond), 2019, 133(18): 1955-1975.
44.	Familtseva A, Jeremic N, Tyagi SC. Exosomes: cell-created drug delivery systems. Mol Cell Biochem, 2019, 459(1-2): 1-6.
45.	Burnouf T, Agrahari V, Agrahari V. Extracellular vesicles as nanomedicine: Hopes and hurdles in clinical translation. Int J Nanomedicine, 2019, 14: 8847-8859.
46.	Zhou B, Peng K, Wang G, et al. miR-483-3p promotes the osteogenesis of human osteoblasts by targeting Dikkopf 2 (DKK2) and the Wnt signaling pathway. Int J Mol Med, 2020, 46(4): 1571-1581.
47.	Watson EC, Adams RH. Biology of bone: The vasculature of the skeletal system. Cold Spring Harb Perspect Med, 2018, 8(7): a031559.
48.	Sivaraj KK, Adams RH. Blood vessel formation and function in bone. Development, 2016, 143(15): 2706-2715.
49.	Pegtel DM, Gould SJ. Exosomes. Annu Rev Biochem, 2019, 88: 487-514.

1. Microsurgery Department of the Orthopedics Branch of the Chinese Medical Doctor Association. Chinese guideline for the diagnosis and treatment of osteonecrosis of the femoral head in adults. Orthop Surg, 2017, 9(1): 3-12.
2. Zhao D, Zhang F, Wang B, et al. Guidelines for clinical diagnosis and treatment of osteonecrosis of the femoral head in adults (2019 version). J Orthop Translat, 2020, 21: 100-110.
3. Kubo T, Ueshima K, Saito M, et al. Clinical and basic research on steroid-induced osteonecrosis of the femoral head in Japan. J Orthop Sci, 2016, 21(4): 407-413.
4. Zhang QY, Li ZR, Gao FQ, et al. Pericollapse stage of osteonecrosis of the femoral head: A Last chance for joint preservation. Chin Med J (Engl), 2018, 131(21): 2589-2598.
5. Liu LH, Zhang QY, Sun W, et al. Corticosteroid-induced osteonecrosis of the femoral head: Detection, diagnosis, and treatment in earlier stages. Chin Med J (Engl), 2017, 130(21): 2601-2607.
6. Hardy RS, Zhou H, Seibel MJ, et al. Glucocorticoids and bone: Consequences of endogenous and exogenous excess and replacement therapy. Endocr Rev, 2018, 39(5): 519-548.
7. Weinstein RS, Hogan EA, Borrelli MJ, et al. The pathophysiological sequence of glucocorticoid-induced osteonecrosis of the femoral head in male mice. Endocrinology, 2017, 158(11): 3817-3831.
8. Wang A, Ren M, Wang J. The pathogenesis of steroid-induced osteonecrosis of the femoral head: A systematic review of the literature. Gene, 2018, 671: 103-109.
9. Fang SH, Chen L, Chen HH, et al. MiR-15b ameliorates SONFH by targeting Smad7 and inhibiting osteogenic differentiation of BMSCs. Eur Rev Med Pharmacol Sci, 2019, 23(22): 9761-9771.
10. 劉立華, 孫偉, 王云亭, 等. 頭頸部開窗減壓治療 L1 型激素性股骨頭壞死: 單中心前瞻性臨床研究. 中國組織工程研究, 2021, 25(6): 906-911.
11. 中國醫師協會骨科醫師分會骨循環與骨壞死專業委員會, 中華醫學會骨科分會骨顯微修復學組, 國際骨循環學會中國區. 中國成人股骨頭壞死臨床診療指南 (2020). 中華骨科雜志, 2020, 40(20): 1365-1376.
12. Barile L, Vassalli G. Exosomes: Therapy delivery tools and biomarkers of diseases. Pharmacol Ther, 2017, 174: 63-78.
13. Liao W, Du Y, Zhang C, et al. Exosomes: The next generation of endogenous nanomaterials for advanced drug delivery and therapy. Acta Biomater, 2019, 86: 1-14.
14. Qin Y, Sun R, Wu C, et al. Exosome: A novel approach to stimulate bone regeneration through regulation of osteogenesis and angiogenesis. Int J Mol Sci, 2016, 17(5): 712.
15. Masaoutis C, Theocharis S. The role of exosomes in bone remodeling: implications for bone physiology and disease. Dis Markers, 2019, 2019: 9417914.
16. Behera J, Tyagi N. Exosomes: mediators of bone diseases, protection, and therapeutics potential. Oncoscience, 2018, 5(5-6): 181-195.
17. Qi X, Zhang J, Yuan H, et al. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells repair critical-sized bone defects through enhanced angiogenesis and osteogenesis in osteoporotic rats. Int J Biol Sci, 2016, 12(7): 836-849.
18. Zhao P, Xiao L, Peng J, et al. Exosomes derived from bone marrow mesenchymal stem cells improve osteoporosis through promoting osteoblast proliferation via MAPK pathway. Eur Rev Med Pharmacol Sci, 2018, 22(12): 3962-3970.
19. Zhang J, Liu X, Li H, et al. Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway. Stem Cell Res Ther, 2016, 7(1): 136.
20. Lu J, Wang QY, Sheng JG. Exosomes in the repair of bone defects: next-generation therapeutic tools for the treatment of nonunion. Biomed Res Int, 2019, 2019: 1983131.
21. Song H, Li X, Zhao Z, et al. Reversal of osteoporotic activity by endothelial cell-secreted bone targeting and biocompatible exosomes. Nano Lett, 2019, 19(5): 3040-3048.
22. Zheng H, Liu J, Tycksen E, et al. MicroRNA-181a/b-1 over-expression enhances osteogenesis by modulating PTEN/PI3K/AKT signaling and mitochondrial metabolism. Bone, 2019, 123: 92-102.
23. Lin Z, He H, Wang M, et al. MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate. Cell Prolif, 2019, 52(6): e12688.
24. Zhang L, Tang Y, Zhu X, et al. Overexpression of MiR-335-5p promotes bone formation and regeneration in mice. J Bone Miner Res, 2017, 32(12): 2466-2475.
25. Gu C, Xu Y, Zhang S, et al. miR-27a attenuates adipogenesis and promotes osteogenesis in steroid-induced rat BMSCs by targeting PPARγ and GREM1. Sci Rep, 2016, 6: 38491.
26. Wu X, Gu Q, Chen X, et al. MiR-27a targets DKK2 and SFRP1 to promote reosseointegration in the regenerative treatment of peri-implantitis. J Bone Miner Res, 2019, 34(1): 123-134.
27. Sriram M, Sainitya R, Kalyanaraman V, et al. Biomaterials mediated microRNA delivery for bone tissue engineering. Int J Biol Macromol, 2015, 74: 404-412.
28. Arriaga MA, Ding MH, Gutierrez AS, et al. The application of microRNAs in biomaterial scaffold-based therapies for bone tissue engineering. Biotechnology Journal, 2019, 14(10): e1900084.
29. 黎牧帆, 張二洋, 呂雷鋒, 等. 維生素 E 對大鼠早期激素性股骨頭缺血性壞死的作用及機制研究. 中國修復重建外科雜志, 2018, 32(11): 1421-1428.
30. Velmurugan BK, Bharathi Priya L, Poornima P, et al. Biomaterial aided differentiation and maturation of induced pluripotent stem cells. J Cell Physiol, 2019, 234(6): 8443-8454.
31. Bakhshandeh B, Zarrintaj P, Oftadeh MO, et al. Tissue engineering; strategies, tissues, and biomaterials. Biotechnol Genet Eng Rev, 2017, 33(2): 144-172.
32. Jewell CM, Collier JH. Biomaterial interactions with the immune system. Biomater Sci, 2019, 7(3): 713-714.
33. Sato Y, Bando H, Di Piazza M, et al. Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider. Cytotherapy, 2019, 21(11): 1095-1111.
34. Colao IL, Corteling R, Bracewell D, et al. Manufacturing exosomes: A promising therapeutic platform. Trends Mol Med, 2018, 24(3): 242-256.
35. Adamiak M, Cheng G, Bobis-Wozowicz S, et al. Induced pluripotent stem cell (iPSC)-derived extracellular vesicles are safer and more effective for cardiac repair than iPSCs. Circ Res, 2018, 122(2): 296-309.
36. Wang B, Jia H, Zhang B, et al. Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy. Stem Cell Res Ther, 2017, 8(1): 75.
37. Zhang S, Liu X, Ge L L, et al. Mesenchymal stromal cell-derived exosomes improve pulmonary hypertension through inhibition of pulmonary vascular remodeling. Respir Res, 2020, 21(1): 71.
38. Qing L, Chen H, Tang J, et al. Exosomes and their microRNA cargo: New players in peripheral nerve regeneration. Neurorehabil Neural Repair, 2018, 32(9): 765-776.
39. Zuo R, Kong L, Wang M, et al. Exosomes derived from human CD34 stem cells transfected with miR-26a prevent glucocorticoid-induced osteonecrosis of the femoral head by promoting angiogenesis and osteogenesis. Stem Cell Res Ther, 2019, 10(1): 321.
40. Qin Y, Wang L, Gao Z, et al. Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo. Sci Rep, 2016, 6: 21961.
41. Duan Y, Tan Z, Yang M, et al. PC-3-derived exosomes inhibit osteoclast differentiation by downregulating miR-214 and blocking NF-B signaling pathway. Biomed Res Int, 2019, 2019: 8650846.
42. Gonzalez-King H, García NA, Ontoria-Oviedo I, et al. Hypoxia inducible factor-1α potentiates jagged 1-mediated angiogenesis by mesenchymal stem cell-derived exosomes. Stem Cells, 2017, 35(7): 1747-1759.
43. Liao W, Ning Y, Xu HJ, et al. BMSC-derived exosomes carrying microRNA-122-5p promote proliferation of osteoblasts in osteonecrosis of the femoral head. Clin Sci (Lond), 2019, 133(18): 1955-1975.
44. Familtseva A, Jeremic N, Tyagi SC. Exosomes: cell-created drug delivery systems. Mol Cell Biochem, 2019, 459(1-2): 1-6.
45. Burnouf T, Agrahari V, Agrahari V. Extracellular vesicles as nanomedicine: Hopes and hurdles in clinical translation. Int J Nanomedicine, 2019, 14: 8847-8859.
46. Zhou B, Peng K, Wang G, et al. miR-483-3p promotes the osteogenesis of human osteoblasts by targeting Dikkopf 2 (DKK2) and the Wnt signaling pathway. Int J Mol Med, 2020, 46(4): 1571-1581.
47. Watson EC, Adams RH. Biology of bone: The vasculature of the skeletal system. Cold Spring Harb Perspect Med, 2018, 8(7): a031559.
48. Sivaraj KK, Adams RH. Blood vessel formation and function in bone. Development, 2016, 143(15): 2706-2715.
49. Pegtel DM, Gould SJ. Exosomes. Annu Rev Biochem, 2019, 88: 487-514.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study on improvement of osteonecrosis of femoral head with exosomes derived from miR-27a-overexpressing vascular endothelial cells

Abstract Full text Figures/Tables Video References Cited by

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