Covid-19 Research

Research Article

HIF-1α Promotes Osteogenic-Angiogenic Coupling Response of BMSCs Cell Sheets

Journal of Biomedical Research & Environmental Sciences article abstract with citation details, DOI, publication dates, subject areas, full text links, and references.

View DOI Submit Manuscript

Article Details

Publication record, authors, dates, abstract, and full text access.

Open Access
Article Type Research Article
Subject Biology Group
OCLC JBRES Record
Dan Zhang*, Yonghui Teng, Wei Liu, Chang Han and Hanwen Zhang
Issue: Volume6-Issue10
Pages: 1444-1456
Received: 2025-10-14
Accepted: 2025-10-17
Published: 2025-10-18

Abstract

Background: There is a critical need for management of vascularization in bone tissue engineering. The purpose of this study was to use Hypoxia-Inducible Factor-1α (HIF-1α) transduced Bone Marrow Mesenchymal Stem Cells(BMSCs) to fabricate prevascularized osteogenic cell sheets In vitro and explore HIF-1α promoted osteogenic-angiogenic coupling response of BMSCs cell sheets.

Methods: HIF-1α was over-expressed by using a lentiviral vector which encoded Green Fluorescent Protein(GFP) and transduced steadily in Wistar rats BMSCs to form HIF-1α/BMSCs. While empty lentiviral transduced BMSCs(Empty/BMSCs) were as a negative control group and BMSCs without transduced were as a blank control group. Fluorescence microscope was used to detect GFP expressions, meanwhile Real-time quantitative and western blot were performed to assess the expression level of HIF-1α. Next, HIF-1α/BMSCs were cultured to form Osteogenic Cell Sheets (OCTs) with a density of 1 × 105/cm2 under osteogenesis medium. Then, Alkaline Phosphatase staining (ALP) at day 14 and Alizarin-red staining at day 21 were performed to check the characteristic of osteogenesis. Empty/BMSCs formed OCTs served as control. Simultaneously, HIF-1α/BMSCs were induced to differentiate into endothelial-like cells(i-ECs) for 14 days, and the conversion rates were performed by Flow Cytometry assay. At the same time, we used Transwell assay to detect whether HIF-1α could migrate i-ECs In vitro. Finally, i-ECs were seeded onto OCTs with a density of 5 × 104/cm2 to fabricate Prevascularized- Osteogenic Cell Sheets (P-OCTs). In order to detect the role of HIF-1α involved in osteogenic-angiogenic coupling response of P-OCTs, Immunofluorescent staining for CD31 was performed at 1,3,7,14 days to check i-ECs migration and networks formation, and western blot of Osteopontin (OPN) and Ssteocalcin (OCN) at 1,7,14 days to check bone formation. Meanwhile, Empty/BMSCs formed P-OCTs were as control.

Conclusion: BMSCs were transduced by Lenti-HIF-1α with optimal multiplicity of infection was 30(MOI = 30) and the GFP expression over 90%. At the same time, qPCR and western blot confirmed a high HIF-1α expression in HIF-1α/BMSCs group, which had a statistic significance among BMSCs group and Empty/BMSCs group (p < 0.05). Next, Flow Cytometric analysis results showed the conversion rate of HIF-1α/BMSCs to i-ECs was 92.43% in experimental group (p < 0.05), which indicated BMSCs transduced by HIF-1α had a great superiority to differentiate into endothelial cells under experimental conditions. Transwell assay showed that HIF-1α could recruit i-ECs in vitro, with an average of over 400 i-ECs migrating per field of view in HIF-1α/BMSCs group. Meanwhile no migrating i-ECs in Empty/BMSCs group (p < 0.05). On the other hand, ALP at day 14 and Alizarin-red staining at day 21 of OCTs in HIF-1α/BMSCs group showed an obviously osteogenic differentiation characteristic with more deep stained calcium nodules deposits than Empty/BMSCs group (p < 0.05). Finally, we fabricated P-OCTs and detected angiogenesis by Immunofluorescent staining for CD31 at day 1,3,7,14, which showed i-ECs migrated reticulated fast and formed a large number of lumens and networks in HIF-1α/BMSCs group. While i-ECs migrated slowly and the lumens and networks formation was limited in Empty/BMSCs group (p < 0.05). At the same time, the expressions of OCN, OPN at day 1,7,14 showed that HIF-1α could promote osteogenic response in P-OCTs significantly in vitro (p < 0.05). All in all, the over-expressed HIF-1α of BMSCs cell sheets strategy can provide a new promising method for bone engineering, which could promote osteogenic-angiogenic coupling response In vitro.

FullText HTML FullText PDF DOI: 10.37871/jbres2201 Crossmark

Certificate of Publication

Certificate of Publication

Copyright

© 2025 Zhang D, et al. Distributed under Creative Commons CC-BY 4.0 Creative CommonsAttribution

How to cite this article

Zhang D, Teng Y, Liu W, Han C, Zhang H. HIF-1α Promotes Osteogenic-Angiogenic Coupling Response of BMSCs Cell Sheets. J Biomed Res Environ Sci. 2025 Oct 14; 6(10): 1444-1456. doi: 10.37871/jbres2201, Article ID: JBRES2201, Available at: https://www.jelsciences.com/articles/jbres2201.pdf

Subject area(s)

Stem Cells

References

  1. McGovern JA, Griffin M, Hutmacher DW. Animal models for bone tissue engineering and modelling disease. Dis Model Mech. 2018 Apr 23;11(4):dmm033084. doi: 10.1242/dmm.033084. PMID: 29685995; PMCID: PMC5963860.
  2. Simunovic F, Finkenzeller G. Vascularization Strategies in Bone Tissue Engineering. Cells. 2021 Jul 11;10(7):1749. doi: 10.3390/cells10071749. PMID: 34359919; PMCID: PMC8306064.
  3. Ren L, Ma D, Liu B, Li J, Chen J, Yang D, Gao P. Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration. Biomed Res Int. 2014;2014:301279. doi: 10.1155/2014/301279. Epub 2014 Jul 8. PMID: 25110670; PMCID: PMC4119697.
  4. Kang Y, Ren L, Yang Y. Engineering vascularized bone grafts by integrating a biomimetic periosteum and β-TCP scaffold. ACS Appl Mater Interfaces. 2014 Jun 25;6(12):9622-33. doi: 10.1021/am502056q. Epub 2014 Jun 6. PMID: 24858072; PMCID: PMC4075998.
  5. Mercado-Pagán ÁE, Stahl AM, Shanjani Y, Yang Y. Vascularization in bone tissue engineering constructs. Ann Biomed Eng. 2015 Mar;43(3):718-29. doi: 10.1007/s10439-015-1253-3. Epub 2015 Jan 24. PMID: 25616591; PMCID: PMC4979539.
  6. Ren L, Ma D, Liu B, Li J, Chen J, Yang D, Gao P. Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration. Biomed Res Int. 2014;2014:301279. doi: 10.1155/2014/301279. Epub 2014 Jul 8. PMID: 25110670; PMCID: PMC4119697.
  7. Chen J, Zhang D, Li Q, Yang D, Fan Z, Ma D, Ren L. Effect of different cell sheet ECM microenvironment on the formation of vascular network. Tissue Cell. 2016 Oct;48(5):442-51. doi: 10.1016/j.tice.2016.08.002. Epub 2016 Aug 9. PMID: 27561623.
  8. Xu M, Li J, Liu X, Long S, Shen Y, Li Q, Ren L, Ma D. Fabrication of vascularized and scaffold-free bone tissue using endothelial and osteogenic cells differentiated from bone marrow derived mesenchymal stem cells. Tissue Cell. 2019 Dec;61:21-29. doi: 10.1016/j.tice.2019.08.003. Epub 2019 Aug 7. PMID: 31759403.
  9. de Silva L, Bernal PN, Rosenberg A, Malda J, Levato R, Gawlitta D. Biofabricating the vascular tree in engineered bone tissue. Acta Biomater. 2023 Jan 15;156:250-268. doi: 10.1016/j.actbio.2022.08.051. Epub 2022 Aug 28. PMID: 36041651.
  10. Rücker C, Kirch H, Pullig O, Walles H. Strategies and First Advances in the Development of Prevascularized Bone Implants. Curr Mol Biol Rep. 2016;2(3):149-157. doi: 10.1007/s40610-016-0046-2. Epub 2016 Aug 15. PMID: 27617188; PMCID: PMC4996880.
  11. Tsiklin IL, Shabunin AV, Kolsanov AV, Volova LT. In Vivo Bone Tissue Engineering Strategies: Advances and Prospects. Polymers (Basel). 2022 Aug 8;14(15):3222. doi: 10.3390/polym14153222. PMID: 35956735; PMCID: PMC9370883.
  12. Zou D, Han W, You S, Ye D, Wang L, Wang S, Zhao J, Zhang W, Jiang X, Zhang X, Huang Y. In vitro study of enhanced osteogenesis induced by HIF-1α-transduced bone marrow stem cells. Cell Prolif. 2011 Jun;44(3):234-43. doi: 10.1111/j.1365-2184.2011.00747.x. Erratum in: Cell Prolif. 2021 Jan;54(1):e12952. doi: 10.1111/cpr.12952. PMID: 21535264; PMCID: PMC6496451.
  13. Sun J, Shen H, Shao L, Teng X, Chen Y, Liu X, Yang Z, Shen Z. HIF-1α overexpression in mesenchymal stem cell-derived exosomes mediates cardioprotection in myocardial infarction by enhanced angiogenesis. Stem Cell Res Ther. 2020 Aug 28;11(1):373. doi: 10.1186/s13287-020-01881-7. PMID: 32859268; PMCID: PMC7455909.
  14. Forster R, Liew A, Bhattacharya V, Shaw J, Stansby G. Gene therapy for peripheral arterial disease. Cochrane Database Syst Rev. 2018 Oct 31;10(10):CD012058. doi: 10.1002/14651858.CD012058.pub2. PMID: 30380135; PMCID: PMC6517203.
  15. Chung SH, Sin TN, Ngo T, Yiu G. CRISPR Technology for Ocular Angiogenesis. Front Genome Ed. 2020 Dec 22;2:594984. doi: 10.3389/fgeed.2020.594984. PMID: 34713223; PMCID: PMC8525361.
  16. de Silva L, Bernal PN, Rosenberg A, Malda J, Levato R, Gawlitta D. Biofabricating the vascular tree in engineered bone tissue. Acta Biomater. 2023 Jan 15;156:250-268. doi: 10.1016/j.actbio.2022.08.051. Epub 2022 Aug 28. PMID: 36041651.
  17. You J, Liu M, Li M, Zhai S, Quni S, Zhang L, Liu X, Jia K, Zhang Y, Zhou Y. The Role of HIF-1α in Bone Regeneration: A New Direction and Challenge in Bone Tissue Engineering. Int J Mol Sci. 2023 Apr 28;24(9):8029. doi: 10.3390/ijms24098029. PMID: 37175732; PMCID: PMC10179302.
  18. Zhang D, Gao P, Li Q, Li J, Li X, Liu X, Kang Y, Ren L. Engineering biomimetic periosteum with β-TCP scaffolds to promote bone formation in calvarial defects of rats. Stem Cell Res Ther. 2017 Jun 5;8(1):134. doi: 10.1186/s13287-017-0592-4. PMID: 28583167; PMCID: PMC5460346.
  19. Bai H, Wang Y, Zhao Y, Chen X, Xiao Y, Bao C. HIF signaling: A new propellant in bone regeneration. Biomater Adv. 2022 Jul;138:212874. doi: 10.1016/j.bioadv.2022.212874. Epub 2022 May 18. PMID: 35913258.
  20. Tao J, Miao R, Liu G, Qiu X, Yang B, Tan X, Liu L, Long J, Tang W, Jing W. Spatiotemporal correlation between HIF-1α and bone regeneration. FASEB J. 2022 Oct;36(10):e22520. doi: 10.1096/fj.202200329RR. PMID: 36065633.
  21. Zhuang Y, Zhao Z, Cheng M, Li M, Si J, Lin K, Yu H. HIF-1α Regulates Osteogenesis of Periosteum-Derived Stem Cells Under Hypoxia Conditions via Modulating POSTN Expression. Front Cell Dev Biol. 2022 Feb 17;10:836285. doi: 10.3389/fcell.2022.836285. PMID: 35252198; PMCID: PMC8891937.
  22. Song S, Zhang G, Chen X, Zheng J, Liu X, Wang Y, Chen Z, Wang Y, Song Y, Zhou Q. HIF-1α increases the osteogenic capacity of ADSCs by coupling angiogenesis and osteogenesis via the HIF-1α/VEGF/AKT/mTOR signaling pathway. J Nanobiotechnology. 2023 Aug 7;21(1):257. doi: 10.1186/s12951-023-02020-z. PMID: 37550736; PMCID: PMC10405507.
  23. Guo Q, Yang J, Chen Y, Jin X, Li Z, Wen X, Xia Q, Wang Y. Salidroside improves angiogenesis-osteogenesis coupling by regulating the HIF-1α/VEGF signalling pathway in the bone environment. Eur J Pharmacol. 2020 Oct 5;884:173394. doi: 10.1016/j.ejphar.2020.173394. Epub 2020 Jul 27. PMID: 32730833.
  24. Mao J, Liu J, Zhou M, Wang G, Xiong X, Deng Y. Hypoxia-induced interstitial transformation of microvascular endothelial cells by mediating HIF-1α/VEGF signaling in systemic sclerosis. PLoS One. 2022 Mar 1;17(3):e0263369. doi: 10.1371/journal.pone.0263369. PMID: 35231032; PMCID: PMC8887755.
  25. Maes C, Araldi E, Haigh K, Khatri R, Van Looveren R, Giaccia AJ, Haigh JJ, Carmeliet G, Schipani E. VEGF-independent cell-autonomous functions of HIF-1α regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival. J Bone Miner Res. 2012 Mar;27(3):596-609. doi: 10.1002/jbmr.1487. PMID: 22162090.
  26. Liu K, Shi L, Wang S, Yalikun A, Hamiti Y, Yusufu A. [Effect of accordion technique and deferoxamine on promoting bone regeneration in distraction osteogenesis]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2024 Aug 15;38(8):1001-1009. Chinese. doi: 10.7507/1002-1892.202404073. PMID: 39175324; PMCID: PMC11335587.
  27. Rathinasamy VS, Paneerselvan N, Jagadeeshan S, Erusan RR, Manjunathan R. Cobalt Chloride-Induced Tissue Regeneration and Wound Healing Depend on HIF-1α and VEGF-A-Mediated Neovascularization. Zebrafish. 2025 Oct 15. doi: 10.1177/15458547251383496. Epub ahead of print. PMID: 41091550.
  28. Farooq M, Khan AW, Kim MS, Choi S. The Role of Fibroblast Growth Factor (FGF) Signaling in Tissue Repair and Regeneration. Cells. 2021 Nov 19;10(11):3242. doi: 10.3390/cells10113242. PMID: 34831463; PMCID: PMC8622657.
  29. Chen K, Rao Z, Dong S, Chen Y, Wang X, Luo Y, Gong F, Li X. Roles of the fibroblast growth factor signal transduction system in tissue injury repair. Burns Trauma. 2022 Mar 23;10:tkac005. doi: 10.1093/burnst/tkac005. PMID: 35350443; PMCID: PMC8946634.
  30. De Pieri A, Rochev Y, Zeugolis DI. Scaffold-free cell-based tissue engineering therapies: advances, shortfalls and forecast. NPJ Regen Med. 2021 Mar 29;6(1):18. doi: 10.1038/s41536-021-00133-3. PMID: 33782415; PMCID: PMC8007731.
  31. Bou-Ghannam S, Kim K, Grainger DW, Okano T. 3D cell sheet structure augments mesenchymal stem cell cytokine production. Sci Rep. 2021 Apr 14;11(1):8170. doi: 10.1038/s41598-021-87571-7. PMID: 33854167; PMCID: PMC8046983.
  32. Thummarati P, Laiwattanapaisal W, Nitta R, Fukuda M, Hassametto A, Kino-Oka M. Recent Advances in Cell Sheet Engineering: From Fabrication to Clinical Translation. Bioengineering (Basel). 2023 Feb 6;10(2):211. doi: 10.3390/bioengineering10020211. PMID: 36829705; PMCID: PMC9952256.
  33. Miar S, Pearson J, Montelongo S, Zamilpa R, Betancourt AM, Ram B, Navara C, Appleford MR, Ong JL, Griffey S, Guda T. Regeneration enhanced in critical-sized bone defects using bone-specific extracellular matrix protein. J Biomed Mater Res B Appl Biomater. 2021 Apr;109(4):538-547. doi: 10.1002/jbm.b.34722. Epub 2020 Sep 11. PMID: 32915522; PMCID: PMC8740960.
  34. Onishi T, Shimizu T, Akahane M, Omokawa S, Okuda A, Kira T, Inagak Y, Tanaka Y. Osteogenic extracellular matrix sheet for bone tissue regeneration. Eur Cell Mater. 2018 Aug 2;36:68-80. doi: 10.22203/eCM.v036a06. PMID: 30069865.
  35. He Y, Wang W, Lin S, Yang Y, Song L, Jing Y, Chen L, He Z, Li W, Xiong A, Yeung KWK, Zhao Q, Jiang Y, Li Z, Pei G, Zhang ZY. Fabrication of a bio-instructive scaffold conferred with a favorable microenvironment allowing for superior implant osseointegration and accelerated in situ vascularized bone regeneration via type H vessel formation. Bioact Mater. 2021 Aug 12;9:491-507. doi: 10.1016/j.bioactmat.2021.07.030. Erratum in: Bioact Mater. 2022 May 27;20:164. doi: 10.1016/j.bioactmat.2022.04.010. PMID: 34820585; PMCID: PMC8586756.
  36. Rao RR, Peterson AW, Ceccarelli J, Putnam AJ, Stegemann JP. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis. 2012 Jun;15(2):253-64. doi: 10.1007/s10456-012-9257-1. Epub 2012 Mar 2. PMID: 22382584; PMCID: PMC3756314.
  37. Perepletchikova D, Kuchur P, Basovich L, Khvorova I, Lobov A, Azarkina K, Aksenov N, Bozhkova S, Karelkin V, Malashicheva A. Endothelial-mesenchymal crosstalk drives osteogenic differentiation of human osteoblasts through Notch signaling. Cell Commun Signal. 2025 Feb 19;23(1):100. doi: 10.1186/s12964-025-02096-0. Erratum in: Cell Commun Signal. 2025 Mar 12;23(1):133. doi: 10.1186/s12964-025-02118-x. PMID: 39972367; PMCID: PMC11841332.
Publish with JBRES — Peer-reviewed, multidisciplinary Open Access with rapid review, DOI, and global visibility.
Double-Blind CrossRef DOI Discoverable
Submit Manuscript Membership