Abstract
Leukemia inhibitory factor (LIF) is known to play a major role in bone physiology. In the present study, we examined the in vitro effects of LIF on osteoblast differentiation of bone marrow stem cells (BMSCs) and explored in vivo effects of LIF on the bone repair capacity of BMSCs-loaded biphasic calcium phosphate (BCP) scaffolds in mouse calvarial bone defect model. The mRNA and protein expression levels in the BMSCs were determined by quantitative real-time PCR and western blot, respectively; the in vitro osteoblast differentiation of the BMSCs was evaluated by using Alizarin Red S staining. The bone volume and bone density in the repaired calvarial bone defect were determined by Micro-CT. Bone regeneration was also histologically evaluated by hematoxylin and eosin staining and Masson’s trichrome staining. Hypoxia treatment induced the up-regulation of Lif mRNA and LIF protein in the BMSCs. Lif overexpression up-regulated the mRNA expression levels of osteopontin and Runt-related transcription factor 2, and increased intensity of Alizarin Red S staining in the BMSCs; while Lif silence exerted the opposite effects. The in vivo studies showed that implantation of Lif-overexpressing BMSCs-loaded BCP scaffolds significantly increased the bone volume and bone density at 4 and 8 weeks after transplantation, and promoted the regeneration of bone tissues in the mouse calvarial bone defect at 8 weeks after transplantation when compared to the BMSCs-loaded BCP scaffolds group; while Lif-silencing BMSCs-loaded BCP scaffolds had the opposite effects. The present study for the first time demonstrated that LIF promoted the in vitro osteoblast differentiation of hypoxia-treated BMSCs; and further studies revealed that LIF exerted enhanced effects on the bone repair capacity of BMSCs-load BCP scaffolds in mouse calvarial bone defect model. However, future studies are warranted to determine the detailed mechanisms of LIF in the large-scale bone defect repair.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All the data in the manuscript are available upon reasonable request from the corresponding author.
References
Berner A, Reichert JC, Müller MB, Zellner J, Pfeifer C, Dienstknecht T, Nerlich M, Sommerville S, Dickinson IC, Schütz MA, Füchtmeier B (2012) Treatment of long bone defects and non-unions: from research to clinical practice. Cell Tissue Res 347(3):501–519. https://doi.org/10.1007/s00441-011-1184-8
Berry MF, Pirolli TJ, Jayasankar V, Morine KJ, Moise MA, Fisher O, Gardner TJ, Patterson PH, Woo YJ (2004) Targeted overexpression of leukemia inhibitory factor to preserve myocardium in a rat model of postinfarction heart failure. J Thorac Cardiovasc Surg 128(6):866–875. https://doi.org/10.1016/j.jtcvs.2004.06.046
Bozec A, Bakiri L, Hoebertz A, Eferl R, Schilling AF, Komnenovic V, Scheuch H, Priemel M, Stewart CL, Amling M, Wagner EF (2008) Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia. Nature 454(7201):221–225. https://doi.org/10.1038/nature07019
Bueno EM, Glowacki J (2009) Cell-free and cell-based approaches for bone regeneration. Nat Rev Rheumatol 5(12):685–697. https://doi.org/10.1038/nrrheum.2009.228
Christiansen BA (2016) Effect of micro-computed tomography voxel size and segmentation method on trabecular bone microstructure measures in mice. Bone Rep 5:136–140. https://doi.org/10.1016/j.bonr.2016.05.006
Cornish J, Callon K, King A, Edgar S, Reid IR (1993) The effect of leukemia inhibitory factor on bone in vivo. Endocrinology 132(3):1359–1366. https://doi.org/10.1210/endo.132.3.8440191
Covey MV, Levison SW (2007) Leukemia inhibitory factor participates in the expansion of neural stem/progenitors after perinatal hypoxia/ischemia. Neuroscience 148(2):501–509. https://doi.org/10.1016/j.neuroscience.2007.06.015
Dazai S, Akita S, Hirano A, Rashid MA, Naito S, Akino K, Fujii T (2000) Leukemia inhibitory factor enhances bone formation in calvarial bone defect. J Craniofac Surg 11(6):513–520. https://doi.org/10.1097/00001665-200011060-00002
Du J, Yang J, He Z, Cui J, Yang Y, Xu M et al (2020) Osteoblast and osteoclast activity affect bone remodeling upon regulation by mechanical loading-induced leukemia inhibitory factor expression in osteocytes. Front Mol Biosci 7:585056. https://doi.org/10.3389/fmolb.2020.585056
Finkenzeller G, Graner S, Kirkpatrick CJ, Fuchs S, Stark GB (2009) Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model. Cell Prolif 42(4):498–505. https://doi.org/10.1111/j.1365-2184.2009.00610.x
Gharib SA, Dayyat EA, Khalyfa A, Kim J, Clair HB, Kucia M, Gozal D (2010) Intermittent hypoxia mobilizes bone marrow-derived very small embryonic-like stem cells and activates developmental transcriptional programs in mice. Sleep 33(11):1439–1446. https://doi.org/10.1093/sleep/33.11.1439
Hung SP, Ho JH, Shih YR, Lo T, Lee OK (2012) Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells. J Orthop Res 30(2):260–266. https://doi.org/10.1002/jor.21517
Jeong CH, Lee HJ, Cha JH, Kim JH, Kim KR, Kim JH, Yoon DK, Kim KW (2007) Hypoxia-inducible factor-1 alpha inhibits self-renewal of mouse embryonic stem cells in vitro via negative regulation of the leukemia inhibitory factor-STAT3 pathway. J Biol Chem 282(18):13672–13679. https://doi.org/10.1074/jbc.M700534200
Kanda M, Nagai T, Takahashi T, Liu ML, Kondou N, Naito AT, Akazawa H, Sashida G, Iwama A, Komuro I, Kobayashi Y (2016) Leukemia inhibitory factor enhances endogenous cardiomyocyte regeneration after myocardial infarction. PLoS One 11(5):e0156562. https://doi.org/10.1371/journal.pone.0156562
Kruyt MC, Dhert WJ, Oner FC, van Blitterswijk CA, Verbout AJ, de Bruijn JD (2007) Analysis of ectopic and orthotopic bone formation in cell-based tissue-engineered constructs in goats. Biomaterials 28(10):1798–1805. https://doi.org/10.1016/j.biomaterials.2006.11.038
Lee S, Choi E, Cha MJ, Hwang KC (2015) Cell adhesion and long-term survival of transplanted mesenchymal stem cells: a prerequisite for cell therapy. Oxidative Med Cell Longev 2015:632902–632909. https://doi.org/10.1155/2015/632902
Lee YC, Chan YH, Hsieh SC, Lew WZ, Feng SW (2019) 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 20(20):5015. https://doi.org/10.3390/ijms20205015
Li X, Yuan Z, Wei X, Li H, Zhao G, Miao J, Wu D, Liu B, Cao S, An D, Ma W, Zhang H, Wang W, Wang Q, Gu H (2016) Application potential of bone marrow mesenchymal stem cell (BMSCs) based tissue-engineering for spinal cord defect repair in rat fetuses with spina bifida aperta. J Mater Sci Mater Med 27(4):77. https://doi.org/10.1007/s10856-016-5684-7
Liang Y, Zhou Y, Jiang T, Zhang Z, Wang S, Wang Y (2011) Expression of LIF and LIFR in periodontal tissue during orthodontic tooth movement. Angle Orthod 81(4):600–608. https://doi.org/10.2319/102510-622.1
Moshaverinia A, Chen C, Xu X, Akiyama K, Ansari S, Zadeh HH, Shi S (2014) Bone regeneration potential of stem cells derived from periodontal ligament or gingival tissue sources encapsulated in RGD-modified alginate scaffold. Tissue Eng Part A 20(3–4):611–621. https://doi.org/10.1089/ten.TEA.2013.0229
Nguyen TB, Lee BT (2014) A combination of biphasic calcium phosphate scaffold with hyaluronic acid-gelatin hydrogel as a new tool for bone regeneration. Tissue Eng Part A 20(13–14):1993–2004. https://doi.org/10.1089/ten.TEA.2013.0352
Nyimi BF, Yifang Z, Liu B (2019) The changing landscape in treatment of cystic lesions of the jaws. J Int Soc Prev Community Dent 9(4):328–337. https://doi.org/10.4103/jispcd.JISPCD_180_19
Paul K, Linh NTB, Kim B-R, Sarkar SK, Choi H-J, Bae S-H et al (2015) Effect of rat bone marrow derived–stem cell delivery from serum-loaded oxidized alginate–gelatin–biphasic calcium phosphate hydrogel for bone tissue regeneration using a nude mouse critical-sized calvarial defect model. J Bioact Compat Polym 30(2):188–208. https://doi.org/10.1177/0883911515569008
Peng J, Wen C, Wang A, Wang Y, Xu W, Zhao B, Zhang L, Lu S, Qin L, Guo Q, Dong L, Tian J (2011) Micro-CT-based bone ceramic scaffolding and its performance after seeding with mesenchymal stem cells for repair of load-bearing bone defect in canine femoral head. J Biomed Mater Res B Appl Biomater 96(2):316–325. https://doi.org/10.1002/jbm.b.31770
Pereira H, Fatih Cengiz I, Gomes S, Espregueira-Mendes J, Ripoll PL, Monllau JC, Reis RL, Oliveira JM (2019) Meniscal allograft transplants and new scaffolding techniques. EFORT Open Rev 4(6):279–295. https://doi.org/10.1302/2058-5241.4.180103
Ruggeri A, Sun Y, Labopin M, Bacigalupo A, Lorentino F, Arcese W, Santarone S, Gülbas Z, Blaise D, Messina G, Ghavamzadeh A, Malard F, Bruno B, Diez-Martin JL, Koc Y, Ciceri F, Mohty M, Nagler A (2017) Post-transplant cyclophosphamide versus anti-thymocyte globulin as graft- versus-host disease prophylaxis in haploidentical transplant. Haematologica 102(2):401–410. https://doi.org/10.3324/haematol.2016.151779
Sallum EA, Ribeiro FV, Ruiz KS, Sallum AW (2019) Experimental and clinical studies on regenerative periodontal therapy. Periodontol 79(1):22–55. https://doi.org/10.1111/prd.12246
Santos GC, Silva DN, Fortuna V, Silveira BM, Orge ID, de Santana TA, Sampaio GL, Paredes BD, Ribeiro-dos-Santos R, Soares MBP (2020) Leukemia inhibitory factor (LIF) overexpression increases the Angiogenic potential of bone marrow Mesenchymal stem/stromal cells. Front Cell Dev Biol 8:778. https://doi.org/10.3389/fcell.2020.00778
Tao H, Han Z, Han ZC, Li Z (2016) Proangiogenic features of mesenchymal stem cells and their therapeutic applications. Stem Cells Int 2016:1314709–1314711. https://doi.org/10.1155/2016/1314709
Titsinides, S., Karatzas, T., Perrea, D., Eleftheriadis, E., Podaropoulos, L., Kalyvas, D., Katopodis C., Agrogiannis G. (2020). Osseous healing in surgically prepared bone defects using different grafting materials: An experimental study in pigs. Dent J (Basel) 8(1), doi:https://doi.org/10.3390/dj8010007
Wagegg M, Gaber T, Lohanatha FL, Hahne M, Strehl C, Fangradt M, Tran CL, Schönbeck K, Hoff P, Ode A, Perka C, Duda GN, Buttgereit F (2012) Hypoxia promotes osteogenesis but suppresses adipogenesis of human mesenchymal stromal cells in a hypoxia-inducible factor-1 dependent manner. PLoS One 7(9):e46483. https://doi.org/10.1371/journal.pone.0046483
Wang MT, Fer N, Galeas J, Collisson EA, Kim SE, Sharib J, McCormick F (2019) Blockade of leukemia inhibitory factor as a therapeutic approach to KRAS driven pancreatic cancer. Nat Commun 10(1):3055. https://doi.org/10.1038/s41467-019-11044-9
Whitney MJ, Lee A, Ylostalo J, Zeitouni S, Tucker A, Gregory CA (2009) Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells. Tissue Eng Part A 15(1):33–44. https://doi.org/10.1089/ten.tea.2007.0266
Yamachika E, Tsujigiwa H, Matsubara M, Hirata Y, Kita K, Takabatake K, Mizukawa N, Kaneda Y, Nagatsuka H, Iida S (2012) Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo. J Mol Histol 43(2):223–233. https://doi.org/10.1007/s10735-011-9385-8
Yang DC, Yang MH, Tsai CC, Huang TF, Chen YH, Hung SC (2011) Hypoxia inhibits osteogenesis in human mesenchymal stem cells through direct regulation of RUNX2 by TWIST. PLoS One 6(9):e23965. https://doi.org/10.1371/journal.pone.0023965
Yao B, He Y, Jie B, Wang J, An J, Guo C et al (2019) Reconstruction of bilateral post-traumatic midfacial defects assisted by three-dimensional craniomaxillofacial data in normal Chinese people-a preliminary study. J Oral Maxillofac Surg 77(11):2302.e2301–2302.e2313. https://doi.org/10.1016/j.joms.2019.04.030
Yu H, Yue X, Zhao Y, Li X, Wu L, Zhang C, Liu Z, Lin K, Xu-Monette ZY, Young KH, Liu J, Shen Z, Feng Z, Hu W (2014) LIF negatively regulates tumour-suppressor p53 through Stat3/ID1/MDM2 in colorectal cancers. Nat Commun 5:5218. https://doi.org/10.1038/ncomms6218
Yu X, Wan Q, Ye X, Cheng Y, Pathak JL, Li Z (2019) Cellular hypoxia promotes osteogenic differentiation of mesenchymal stem cells and bone defect healing via STAT3 signaling. Cell Mol Biol Lett 24:64. https://doi.org/10.1186/s11658-019-0191-8
Zhang Y, Lv J, Guo H, Wei X, Li W, Xu Z (2015) Hypoxia-induced proliferation in mesenchymal stem cells and angiotensin II-mediated PI3K/AKT pathway. Cell Biochem Funct 33(2):51–58. https://doi.org/10.1002/cbf.3080
Zhang C, Liu J, Wang J, Hu W, Feng Z (2020) The emerging role of leukemia inhibitory factor in cancer and therapy. Pharmacol Ther 107754:107754. https://doi.org/10.1016/j.pharmthera.2020.107754
Funding
This study was supported by the Shenzhen Science and Technology Innovative Project (No. JCYJ20180302144621755).
Author information
Authors and Affiliations
Contributions
RZ, XL and YL performed the study and analyzed the experimental data. ZL, LY, CC and XY performed the statistical analysis. RZ and YL designed the study and wrote the manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
All the in vivo experiments were performed in accordance with a protocol approved by the Research Ethic Committee of Sun Yat-sen University.
Consent for publication
Not applicable.
Conflict of interest
The authors have declared that no competing interest exists.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Supplemental Figure S1
Effects of hypoxia on the cell proliferation and OPN expression in BMSCs. (a) The cell proliferation of the control BMSCs and hypoxia-treated BMSCs was determined by CCK-8 assay. (b) The Opn mRNA expression levels inthe control BMSCs and hypoxia-treated BMSCs was determined by qRT-PCR. CON = control; N = 3, ***P < 0.05. (PNG 39 kb)
Rights and permissions
About this article
Cite this article
Liang, Y., Zhou, R., Liu, X. et al. Investigation into the effects of leukemia inhibitory factor on the bone repair capacity of BMSCs-loaded BCP scaffolds in the mouse calvarial bone defect model. J Bioenerg Biomembr 53, 381–391 (2021). https://doi.org/10.1007/s10863-021-09899-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10863-021-09899-z