{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T02:31:55Z","timestamp":1789612315041,"version":"build-2803163510"},"reference-count":28,"publisher":"IMR Press","issue":"6","license":[{"start":{"date-parts":[[2025,6,23]],"date-time":"2025-06-23T00:00:00Z","timestamp":1750636800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["81972090"],"award-info":[{"award-number":["81972090"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Front. Biosci. (Landmark Ed)"],"abstract":"<jats:p>Background: The ossification of the posterior longitudinal ligament (OPLL) is a progressive spinal disorder predominantly observed in Asian populations. Unfortunately, there is a limited availability of conservative therapies to impede the progression of OPLL. This study investigates the effects of cobalt chloride (CoCl2), which simulates an in vitro hypoxic microenvironment, on OPLL and explores its potential mechanisms, aiming to enhance our understanding of the pathogenesis of OPLL. Methods: Ligament cells were extracted from patients with OPLL and normal posterior longitudinal ligament (PLL) tissues, confirming their mesenchymal stem cell (MSC)-like properties. To simulate hypoxia, cells were treated with varying concentrations of CoCl2, and the effects on hypoxia-inducible factor 1-alpha (HIF1A) expression and osteogenic differentiation were assessed. Real-time quantitative reverse transcription\u2013polymerase chain reaction (qRT-PCR) and Western blotting were used to quantify HIF1A and bone morphogenetic protein 4 (BMP4) expression. Immunohistochemistry was performed to visualize HIF1A in tissue samples. Osteogenic differentiation was evaluated through Alizarin Red S staining and alkaline phosphatase (ALP) assays, with optical density (OD) values measured using a microplate reader. Transcriptome sequencing was conducted to identify differentially expressed genes following CoCl2 treatment. Results: We found that HIF1A was differentially expressed in the OPLL. Both PLL cells and OPLL cells exhibited mesenchymal stem cell properties; however, OPLL cells demonstrated a greater capacity for osteogenic differentiation (p &lt; 0.05). Following stimulation with cobalt chloride, the expression of HIF1A increased, which correlated with an enhanced osteogenic differentiation ability in PLL cells. Biosignature analysis revealed that HIF1A plays a regulatory role in BMP4 expression. Notably, BMP4 was downregulated, and the degree of osteogenic differentiation decreased upon inhibition of HIF1A with siRNA in OPLL cells. Conclusions: Primary cells derived from normal PLL and OPLL exhibited MSC-like properties and demonstrated the capacity for osteogenic, adipogenic, and chondrogenic differentiation, with OPLL cells showing a greater propensity for osteogenic differentiation. This study reports the potential involvement of HIF1A in the development of OPLL and investigates the regulatory role of the HIF1A-BMP4 axis in this process.<\/jats:p>","DOI":"10.31083\/fbl37494","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T05:28:52Z","timestamp":1751434132000},"source":"Crossref","is-referenced-by-count":3,"title":["Cobalt Chloride-simulated Hypoxia Promoted Ossification of the Posterior Longitudinal Ligament Through the HIF1A\u2013BMP4 Axis"],"prefix":"10.31083","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-1714-4559","authenticated-orcid":true,"given":"Shihao","family":"Lu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junbin","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junzhe","family":"Sheng","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aochen","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zichuan","family":"Wu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiabin","family":"Yuan","sequence":"first","affiliation":[{"name":"Department of Spine Surgery, Changhai Hospital, Naval Medical University, 200433 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Changzheng Hospital, Naval Medical University, 200003 Shanghai, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"16242","published-online":{"date-parts":[[2025,6,23]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","unstructured":"Wu JC, Chen YC, Huang WC. Ossification of the Posterior Longitudinal Ligament in Cervical Spine: Prevalence, Management, and Prognosis. Neurospine. 2018; 15: 33\u201341.","DOI":"10.14245\/ns.1836084.042"},{"key":"ref2","doi-asserted-by":"publisher","unstructured":"Edwards DS, Clasper JC. Heterotopic ossification: a systematic review. Journal of the Royal Army Medical Corps. 2015; 161: 315\u2013321.","DOI":"10.1136\/jramc-2014-000277"},{"key":"ref3","doi-asserted-by":"publisher","unstructured":"Shore EM, Kaplan FS. Inherited human diseases of heterotopic bone formation. Nature Reviews. Rheumatology. 2010; 6: 518\u2013527.","DOI":"10.1038\/nrrheum.2010.122"},{"key":"ref4","doi-asserted-by":"publisher","unstructured":"Kim KS. Clinical Effectiveness of Posterior Cervical Decompression and Fusion in Terms of Reducing OPLL Growth Versus Cervical Motion Preservation. Neurospine. 2019; 16: 492\u2013493.","DOI":"10.14245\/ns.19edi.012"},{"key":"ref5","doi-asserted-by":"publisher","unstructured":"Chen LF, Tu TH, Chen YC, Wu JC, Chang PY, Liu L, et al. Risk of spinal cord injury in patients with cervical spondylotic myelopathy and ossification of posterior longitudinal ligament: a national cohort study. Neurosurgical Focus. 2016; 40: E4.","DOI":"10.3171\/2016.3.FOCUS1663"},{"key":"ref6","doi-asserted-by":"publisher","unstructured":"Bernstein DN, Prong M, Kurucan E, Jain A, Menga EN, Riew KD, et al. National Trends and Complications in the Surgical Management of Ossification of the Posterior Longitudinal Ligament (OPLL). Spine. 2019; 44: 1550\u20131557.","DOI":"10.1097\/BRS.0000000000003127"},{"key":"ref7","doi-asserted-by":"publisher","unstructured":"Xu R, Hu J, Zhou X, Yang Y. Heterotopic ossification: Mechanistic insights and clinical challenges. Bone. 2018; 109: 134\u2013142.","DOI":"10.1016\/j.bone.2017.08.025"},{"key":"ref8","doi-asserted-by":"publisher","unstructured":"Abiola R, Rubery P, Mesfin A. Ossification of the Posterior Longitudinal Ligament: Etiology, Diagnosis, and Outcomes of Nonoperative and Operative Management. Global Spine Journal. 2016; 6: 195\u2013204.","DOI":"10.1055\/s-0035-1556580"},{"key":"ref9","doi-asserted-by":"publisher","unstructured":"Huang Y, Wang X, Lin H. The hypoxic microenvironment: a driving force for heterotopic ossification progression. Cell Communication and Signaling: CCS. 2020; 18: 20.","DOI":"10.1186\/s12964-020-0509-1"},{"key":"ref10","doi-asserted-by":"publisher","unstructured":"Wang H, Lindborg C, Lounev V, Kim JH, McCarrick-Walmsley R, Xu M, et al. Cellular Hypoxia Promotes Heterotopic Ossification by Amplifying BMP Signaling. Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research. 2016; 31: 1652\u20131665.","DOI":"10.1002\/jbmr.2848"},{"key":"ref11","doi-asserted-by":"publisher","unstructured":"Lin L, Shen Q, Leng H, Duan X, Fu X, Yu C. Synergistic inhibition of endochondral bone formation by silencing Hif1\u03b1 and Runx2 in trauma-induced heterotopic ossification. Molecular Therapy: the Journal of the American Society of Gene Therapy. 2011; 19: 1426\u20131432.","DOI":"10.1038\/mt.2011.101"},{"key":"ref12","doi-asserted-by":"publisher","unstructured":"Kawao N, Yano M, Tamura Y, Okumoto K, Okada K, Kaji H. Role of osteoclasts in heterotopic ossification enhanced by fibrodysplasia ossificans progressiva-related activin-like kinase 2 mutation in mice. Journal of Bone and Mineral Metabolism. 2016; 34: 517\u2013525.","DOI":"10.1007\/s00774-015-0701-3"},{"key":"ref13","doi-asserted-by":"publisher","unstructured":"Mu\u00f1oz-S\u00e1nchez J, Ch\u00e1nez-C\u00e1rdenas ME. The use of cobalt chloride as a chemical hypoxia model. Journal of Applied Toxicology: JAT. 2019; 39: 556\u2013570.","DOI":"10.1002\/jat.3749"},{"key":"ref14","doi-asserted-by":"publisher","unstructured":"Rovetta F, Stacchiotti A, Faggi F, Catalani S, Apostoli P, Fanzani A, et al. Cobalt triggers necrotic cell death and atrophy in skeletal C2C12 myotubes. Toxicology and Applied Pharmacology. 2013; 271: 196\u2013205.","DOI":"10.1016\/j.taap.2013.05.005"},{"key":"ref15","unstructured":"Meng XL, Wang H, Yang H, Hai Y, Tian BP, Lin X. T allele at site 6007 of bone morphogenetic protein-4 gene increases genetic susceptibility to ossification of the posterior longitudinal ligament in male Chinese Han population. Chinese Medical Journal. 2010; 123: 2537\u20132542."},{"key":"ref16","doi-asserted-by":"publisher","unstructured":"Furushima K, Shimo-Onoda K, Maeda S, Nobukuni T, Ikari K, Koga H, et al. Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine. Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research. 2002; 17: 128\u2013137.","DOI":"10.1359\/jbmr.2002.17.1.128"},{"key":"ref17","doi-asserted-by":"publisher","unstructured":"Liu J, Chen Y, Shan X, Wang H. Investigation of the biomarkers involved in ectopic ossification: The shared mechanism in ossification of the spinal ligament. Frontiers in Genetics. 2022; 13: 991834.","DOI":"10.3389\/fgene.2022.991834"},{"key":"ref18","doi-asserted-by":"publisher","unstructured":"Ren Y, Feng J, Liu ZZ, Wan H, Li JH, Lin X. A new haplotype in BMP4 implicated in ossification of the posterior longitudinal ligament (OPLL) in a Chinese population. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 2012; 30: 748\u2013756.","DOI":"10.1002\/jor.21586"},{"key":"ref19","doi-asserted-by":"publisher","unstructured":"Chang J, Yang B, Zhou Y, Yin C, Liu T, Qian H, et al. Acute Methylmercury Exposure and the Hypoxia-Inducible Factor-1\u03b1 Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro. Environmental Health Perspectives. 2019; 127: 127006.","DOI":"10.1289\/EHP5139"},{"key":"ref20","doi-asserted-by":"publisher","unstructured":"Zheng H, Wang N, Li L, Ge L, Jia H, Fan Z. miR-140-3p enhanced the osteo\/odontogenic differentiation of DPSCs via inhibiting KMT5B under hypoxia condition. International Journal of Oral Science. 2021; 13: 41.","DOI":"10.1038\/s41368-021-00148-y"},{"key":"ref21","doi-asserted-by":"publisher","unstructured":"Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews. Genetics. 2009; 10: 57\u201363.","DOI":"10.1038\/nrg2484"},{"key":"ref22","doi-asserted-by":"publisher","unstructured":"Hrdlickova R, Toloue M, Tian B. RNA-Seq methods for transcriptome analysis. Wiley Interdisciplinary Reviews. RNA. 2017; 8:  https:\/\/doi.org\/","DOI":"10.1002\/wrna.1364"},{"key":"ref23","doi-asserted-by":"publisher","unstructured":"Zhang H, He L, Cai L. Transcriptome Sequencing: RNA-Seq. Methods in Molecular Biology (Clifton, N.J.). 2018; 1754: 15\u201327.","DOI":"10.1007\/978-1-4939-7717-8_2"},{"key":"ref24","doi-asserted-by":"publisher","unstructured":"Withanage MHH, Liang H, Zeng E. RNA-Seq Experiment and Data Analysis. Methods in Molecular Biology (Clifton, N.J.). 2022; 2418: 405\u2013424.","DOI":"10.1007\/978-1-0716-1920-9_22"},{"key":"ref25","doi-asserted-by":"publisher","unstructured":"Ayturk U. RNA-seq in Skeletal Biology. Current Osteoporosis Reports. 2019; 17: 178\u2013185.","DOI":"10.1007\/s11914-019-00517-x"},{"key":"ref26","doi-asserted-by":"publisher","unstructured":"Athanasios A, Charalampos V, Vasileios T, Ashraf GM. Protein-Protein Interaction (PPI) Network: Recent Advances in Drug Discovery. Current Drug Metabolism. 2017; 18: 5\u201310.","DOI":"10.2174\/138920021801170119204832"},{"key":"ref27","doi-asserted-by":"publisher","unstructured":"Li P, Zhang W, Zhang J, Liu J, Fu J, Wei Z, et al. Macrophage migration inhibitory factor promotes heterotopic ossification by mediating ROS\/HIF-1\u03b1 positive feedback loop and activating Wnt\/\u03b2-catenin signaling pathway. Bone. 2025; 190: 117331.","DOI":"10.1016\/j.bone.2024.117331"},{"key":"ref28","doi-asserted-by":"publisher","unstructured":"Chen C, Song C, Liu B, Wang Y, Jia J, Pang K, et al. Activation of BMP4\/SMAD pathway by HIF-1\u03b1 in hypoxic environment promotes osteogenic differentiation of BMSCs and leads to ectopic bone formation. Tissue & Cell. 2024; 88: 102376.","DOI":"10.1016\/j.tice.2024.102376"}],"container-title":["Frontiers in Bioscience-Landmark"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.imrpress.com\/journal\/FBL\/30\/6\/10.31083\/FBL37494","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T05:35:25Z","timestamp":1751434525000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.imrpress.com\/journal\/FBL\/30\/6\/10.31083\/FBL37494"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,23]]},"references-count":28,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2025]]}},"alternative-id":["S2768-6701(25)01736-8"],"URL":"https:\/\/doi.org\/10.31083\/fbl37494","relation":{},"ISSN":["2768-6701","2768-6698"],"issn-type":[{"value":"2768-6701","type":"print"},{"value":"2768-6698","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,23]]},"article-number":"37494"}}