Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells

Document Type : Original Article


1 aDepartment of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011,China

2 Department of Orthopaedics, Peking University Third Hospital, Beijing 100191,China

3 Departmentof Orthopedics, Clinical College of Yangzhou University, Yangzhou 225001, China


Objective(s): This research is to study the influences of different concentrations of simvastatin on the biological activities of nucleus pulposus-derived mesenchymal stem cells (NPMSC).
Materials and Methods: NPMSC were cultured with different concentrations of simvastatin (0, 0.01, 0.1, and 1 μM) and assessed to determine the possible effects of simvastatin. The cell proliferation was assessed with CCK-8 assay. The flowcytometry and multilineage differentiation were also performed to identify the stem characterization of the cells. The mRNA expressions of aggrecan, collagen type II, glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by qRT-PCR.
Results: The results demonstrated that the cells isolated from nucleus pulposus of healthy Sprague-Dawley (SD) rat met the criteria of MSC. NPMSC could form sunflower-like colonies and strongly expressed stem cell-related genes. In addition, NPMSC showed strong ability of chondrogenic, adipogenic and osteogenic differentiation. Simvastatin at certain range concentrations (0.01 μM-0.1 μM)) significantly promoted colony-forming rate and cell proliferation, and inhibited cell apoptosis. Simvastatin could promote expressions of aggrecan, collagen type II, HIF-1α, VEGF and GLUT-1, while 0.1 μmol/l concentration reached the maximum effect. Our study further demonstrated that HIF-1α-intermediated signaling pathway might participate in regulating the biological activities of NPMSC.
Conclusion: Proper concentration of simvastatin can promote the biological behavior of NPMSC, and HIF-1α-intermediated signaling pathway might participate in the mechanism.


1.Hoy D, Bain C, Williams G, March L, Brooks P, Blyth F, et al. A systematic review of the global prevalence of low back pain. Arthritis Rheum 2012; 64:2028-2037.
2. Liu Y, Li Y, Huang ZN, Wang ZY, Nan LP, Wang F. The effect of intervertebral disc degenerative change on biological characteristics of nucleus pulposus mesenchymal stem cell: a study in rats. Connect Tissue Res 2019; 60:376-388.
3. Moradi-Lakeh M, Forouzanfar MH, Vollset SE, Bcheraoui CE, Daoud F, Afshin A, et al. Burden of musculoskeletal disorders in the Eastern Mediterranean Region. Ann Rheum Dis 2017; 76:1365-1373.
4. Zheng CJ, Chen J. Disc degeneration implies low back pain. Theor Biol Med Model 2015; 12:23-24.
5. Brinjikji W, Diehn F, Jarvik J, Carr C, Kallmes DF, Murad MH, et al. MRI findings of disc degeneration are more prevalent in adults with low back pain than in asymptomatic controls: a systematic review and meta-analysis. AJNR Am J Neuroradiol 2015; 36:2394-2399.
6. Noshchenko A, Hoffecker L, Lindley EM, Burger EL, Cain CM, Patel VV. Long-term treatment effects of lumbar arthrodeses in degenerative disk disease. J Spinal Disord Tech 2015; 28:493-521.
7. Tong W, Lu Z, Qin L, Mauck RJ, Smith HE, Smith LJ, et al. Cell therapy for the degenerating intervertebral disc. Transl Res 2017; 181:49-58.
8. Bydon M, Garza-Ramos RD, Macki M, Baker A, Gokaslan AK, Bydon A. Lumbar fusion versus nonoperative management for treatment of discogenic low back pain: a systematic review and meta-analysis of randomized controlled trials. J Spinal Disord Tech 2014; 27:297-304.
9. Sampara P, Banala RR, Vemuri SK, Subbaiah G. Understanding the molecular biology of intervertebral disc degeneration and potential gene therapy strategies for regeneration: a review. Gene Ther 2018; 1:1-4.
10.Zeng Y, Chen C, Liu W, Fu Q, Han Z, Li Y, et al. Injectable microcryogels reinforced alginate encapsulation of mesenchymal stromal cells for leak-proof delivery and alleviation of canine disc degeneration. Biomaterials 2015; 59:53-65.
11.Mochida J, Sakai D, Nakamura Y, Watanabe T, Yamamoto Y, Kato S. Intervertebral disc repair with activated nucleus pulposus cell transplantation: a three-year, prospective clinical study of its safety. Eur Cell Mater 2015; 29:202-212.
12.Gan Y, Li S, Li P, Xu Y, Wang L, Zhao C, et al. A controlled release codelivery system of MSCs encapsulated in dextran/gelatin hydrogel with TGF-β3-loaded nanoparticles for nucleus pulposus regeneration. Stem Cells Int 2016; 1:1-14.
13.Noriega DC, Ardura F, Hernández-Ramajo R, Martín-Ferrero MA, Sánchez-Lite L, Toribio B, et al. Intervertebral disc repair by allogeneic mesenchymal bone marrow cells: a randomized controlled trial. Transplantation 2017; 101:1945-1951.
14.Orozco L, Soler R, Morera C, Alberca M, Sánchez A, García-Sancho J. Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplantation 2011; 92:822-828.
15.Hang D, Li F, Che W, Wu X, Wan Y, Wang J, et al. One-Stage Positron Emission Tomography and Magnetic Resonance Imaging to Assess Mesenchymal Stem Cell Survival in a Canine Model of Intervertebral Disc Degeneration. Stem Cells Dev 2017; 26:1334-1343.
16.Huang YC, Leung VY, Lu WW, Luk KD, The effects of microenvironment in mesenchymal stem cell–based regeneration of intervertebral disc, Spine J  2013; 13:352-362.
17.Liang C, Li H, Tao Y, Zhou X, Li F, Chen G, et al. Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc, J Transl Med 2012; 10:48-49.
18.Wang F, Shi R, Cai F, Wang YT, Wu XT. Stem cell approaches to intervertebral disc regeneration: obstacles from the disc microenvironment. Stem Cells Dev 2015; 24:2479-2495.
19.Clouet J, Fusellier M, Camus A, Visage CJ, Guicheux J. Intervertebral disc regeneration: From cell therapy to the development of novel bioinspired endogenous repair strategies. Adv Drug Deliver R 2018; 1:1-12.
20.Huang Z, Zhang L, Feng X, Chen T, Bi S. A new in vivo method to retard progression of intervertebral disc degeneration through stimulation of endogenous stem cells with simvastatin. Med Hypotheses 2017; 101:65-66.
21.Kosaraju R, Rennert RC, Maan ZN, Duscher D, Barrera J, Whittam AJ, et al. Adipose-derived stem cell-seeded hydrogels increase endogenous progenitor cell recruitment and neovascularization in wounds. Tissue Eng Part A2016; 22:295-305.
22.González EC, Shih YR, Nakasaki M, Liu M, Varghese S. Mineralized biomaterials mediated repair of bone defects through endogenous cells. Tissue Eng Part A 2018; 1:1-9.
23.Su G, Liu L, Yang L, Mu Y, Guan L. Homing of endogenous bone marrow mesenchymal stem cells to rat infarcted myocardium via ultrasound-mediated recombinant SDF-1α adenovirus in microbubbles. Oncotarget 2018; 9:477.
24.Blanco JF, Graciani IF, Sanchez-Guijo FM, Muntion S, Hernandez-Campo P, Santamaria C, Carrancio S, et al. Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: comparison with bone marrow mesenchymal stromal cells from the same subjects. Spine 2010; 35:2259-2265.
25.Sang C, Cao X, Chen F, Yang X, Zhang Y. Differential Characterization of Two Kinds of Stem Cells Isolated from Rabbit Nucleus Pulposus and Annulus Fibrosus. Stem Cells Int 2016; 82:325-327.
26.Liu J, Tao H, Shen C, Liu X, Wang H, Dong F, et al. Biological behavior of human nucleus pulposus mesenchymal stem cells in response to changes in the acidic environment during intervertebral disc degeneration. Stem Cells Dev 2017; 12:901-911.
27.Tao YQ, Liang CJ, Li H, Zhang YJ, Li FC, Chen G, et al. Potential of co-culture of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in hyperosmotic microenvironment for intervertebral disc regeneration. Cell Biol Int 2013; 37:826-834.
28.Li H, Tao Y, Liang C, Han B, Li F, Chen G, et al. Influence of hypoxia in the intervertebral disc on the biological behaviors of rat adipose- and nucleus pulposus-derived mesenchymal stem cells. Cells, tissues, organs 2013; 98:266-277.
29.Chen X, Zhu L, Wu G, Liang Z, Yang L, Du Z. A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model. Int J Surg 2016; 28:77-82.
30.Sakai D, Nakamura Y, Nakai T, Mishima T, Kato S, Grad S, et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 2012; 3:1262-1264.
31.Jia Z, Yang P, Wu Y, Tang Y, Zhao Y, Wu J, et al. Comparison of biological characteristics of nucleus pulposus mesenchymal stem cells derived from non-degenerative and degenerative human nucleus pulposus. Exp Ther Med 2017; 13:3574-3580.
32.Zhao Y, Jia Z, Huang S, Wu Y, Liu L, Lin L, et al. Age-Related Changes in Nucleus Pulposus Mesenchymal Stem Cells: An In Vitro Study in Rats. Stem Cells Int 2017; 1:1-13.
33.Tu J, Li W, Zhang Y, Wu X, Song Y, Kang L, et al. Simvastatin Inhibits IL-1β-Induced Apoptosis and Extracellular Matrix Degradation by Suppressing the NF-kB and MAPK Pathways in Nucleus Pulposus Cells. Inflammation 2017; 40:725-734.
34.Zhang H, Lin CY. Simvastatin stimulates chondrogenic phenotype of intervertebral disc cells partially through BMP-2 pathway. Spine 2008; 33:E525-531.
35.Zhang C, Wu JM, Liao M, Wang J, Xu CJ. The ROCK/GGTase pathway are essential to the proliferation and differentiation of neural stem cells mediated by simvastatin. J Mol Neurosci 2016; 60:474-485.
36.Bing W, Pang X, Qu Q, Bai X, Yang W, Bi Y, et al. Simvastatin improves the homing of BMSCs via the PI3K/AKT/miR‐9 pathway. J Cell Mol Med 2016; 20:949-961.
37.Niu J, Ding G, Zhang L. Effects of simvastatin on the osteogenic differentiation and immunomodulation of bone marrow mesenchymal stem cells. Mol Med Rep 2015; 12:8237-8240.
38.Zhang H, Ma X, Zhang L, Guan X, Bai T, Xue C. The ability to form cartilage of NPMSC and BMSC in SD rats. Int J Clin Exp Med. 2015;8:4989-96.
39.Hatano H, Maruo A, Bolander ME, Sarkar G. Statin stimulates bone morphogenetic protein-2, aggrecan, and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes. J Orthop Sci 2003; 8:842-848.
40.Shen Q, Zhang L, Chai B, Ma X. Isolation and characterization of mesenchymal stem-like cells from human nucleus pulposus tissue. Sci China Life Sci 2015; 58:509-511.
41.Zanette DJ, Lorenzi JC, Panepucci RA, Palma PVB, Dos Santos DF, Prata KL, et al. Simvastatin modulates mesenchymal stromal cell proliferation and gene expression. PLoS One 2015; 10:120-137.
42.Lee OKS, Ko YC, Kuo TK, Chou SH, Li HJ, Chen WH, et al. Fluvastatin and lovastatin but not pravastatin induce neuroglial differentiation in human mesenchymal stem cells. J Cell Biochem 2004; 93:917-928.
43.Stoyanov JV, Gantenbein-Ritter B, Bertolo A, Aebli N, Baur M, Alini M, et al. Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells. Eur Cell Mater 2011; 21:533-547.
44.Ni L, Liu X, Sochacki KR, Ebraheim M, Fahrenkopf M, Shi Q, et al. Effects of hypoxia on differentiation from human placenta-derived mesenchymal stem cells to nucleus pulposus-like cells.  Spine J 2014; 14:2451-2458.
45.Zhang Z, Li F, Tian H, Guan K, Zhao G, Shan J, et al. Differentiation of adipose-derived stem cells toward nucleus pulposus-like cells induced by hypoxia and a three-dimensional chitosan-alginate gel scaffold in vitro. Chin Med J 2014; 127:314-321.
46.Han XB, Zhang YL, Li HC, Chen B, Chang B, Zhang W, et al. Differentiation of human ligamentum flavum stem cells toward nucleus pulposus-like cells induced by coculture system and hypoxia. Spine 2015; 40: E665.
47.Yueyi C, Xiaoguang H, Jingying W, Quansheng S, Jie T, Xin F, et al. Calvarial defect healing by recruitment of autogenous osteogenic stem cells using locally applied simvastatin. Biomaterials 2013; 34:9373-9380.
48.Park HS, Kim JH, Sun BK, Song SU, Suh W, Sung JH. Hypoxia induces glucose uptake and metabolism of adipose‑derived stem cells. Mol Med Rep 2016; 14:4706-4714.
49.Pons J, Huang Y, Arakawa-Hoyt J, Washko D, Takagawa J, Ye J, et al. VEGF improves survival of mesenchymal stem cells in infarcted hearts. Biochem Biophys Res Commun 2008; 376:419-422.
50.Liu X. Feng T, Cheng T, Bertolo A, Aebli N, Baur M. Effect of notch signaling pathway on VEGF promoting rat mesenchymal stem cell proliferation. Journal of Experimental Hematology 2014; 22:1068-1071.
51.Cheng J, Zhang F, Wang L, Yuan Y, Zhang Q, Ma j. Influence of simvastatin on the proliferation and paracrine functions of bone marrow-derived mesenchymal stem cells. Journal of Nanjing Medical University (Natural Sciences) 2010; 1:20-24.
52.Tamama K, Kawasaki H, Kerpedjieva SS, Guan J, Ganju RK, Sen CK. Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition. J Cell Biochem 2011; 112:804-817.