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Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture

Irina Okkelman (UGent) , Winter Vandenberghe (UGent) and Ruslan Dmitriev (UGent)
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Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture Irina A. Okkelman, Winter Vandenberghe, Ruslan I. Dmitriev Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University, Ghent 9000, Belgium Dental pulp stem cells (DPSC) are promising for bone tissue engineering and repair. To support angiogenesis and long-term viability within microfabricated and 3D-printed tissue constructs, they are often grown and differentiated together with endothelial cells in multicellular spheroids. However, this is often associated with the decline of endothelial cells and slow rate of DPSC differentiation towards osteoblasts. Interestingly, hypoxia affects cell viability and at the same time promotes angiogenesis with osteoblast differentiation. Here, we hypothesized that oxygen and glucose deprivation pre-conditioning can improve the process of DPSC differentiation in 3D culture with endothelial HUVEC cells. To do this, we performed oxygen and glucose deprivation experiments with 2D cultures of DPSC and HUVEC and tested effects on cell viability, redox potential and morphology. Interestingly, glucose deprivation did not significantly affect redox state of both HUVEC and DPSC. We therefore preconditioned these cell types with 0% O2, followed by 24 h reperfusion, and produced heterocellular spheroids. To understand the effects of preconditioning in this 3D culture, we performed fluorescence microscopy analysis of cell death (Sytox Green) together with monitoring of spheroid oxygenation (ratiometric red/near-infrared O2-sensing nanoparticles) during 7 days of differentiation. We found that cell death was not significantly different in the spheroids from non-differentiated and differentiated groups of cells. However, the periphery-to-core O2 gradients were significantly lower (p=0.05) in non-differentiated hDPSC 21% / HUVEC 0% and hDPSC 0% / HUVEC 0% groups in comparison to hDPSC 21% / HUVEC 21% spheroids. During differentiation, this difference in spheroid oxygenation was slightly ameliorated, keeping the same trend as in non-differentiated spheroids. Thus under the deeper hypoxia, the heterocellular spheroids formed with 0% O2-preconditioned endothelial cells demonstrated cell viability comparable to spheroids formed from non-preconditioned cells. This data correlates with the results on the alteration of redox potential in adherent (2D) DPSC and HUVEC cultures, where hypoxia-conditioned HUVEC cells demonstrated more profound response. We also found that preconditioned cells displayed stronger accumulation of hydroxyapatite and osteogenic differentiation. Collectively, our preliminary data indicates that hypoxia preconditioning, combined with imaging of oxygenation and cell death, can improve the quality of the spheroid-based ‘tissue building blocks’ in biofabrication. Funding: supported by the Special Research Fund (BOF) grant of Ghent University (BOF/STA/202009/003).

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MLA
Okkelman, Irina, et al. “Role of Preconditioning with Oxygen and Glucose Deprivation in Promoting Differentiation of Dental Pulp Stem Cells in 3D Culture.” MOLECULAR BIOLOGY OF THE CELL, vol. 34, no. 2, 2023, pp. 1212–1212.
APA
Okkelman, I., Vandenberghe, W., & Dmitriev, R. (2023). Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture. MOLECULAR BIOLOGY OF THE CELL, 34(2), 1212–1212.
Chicago author-date
Okkelman, Irina, Winter Vandenberghe, and Ruslan Dmitriev. 2023. “Role of Preconditioning with Oxygen and Glucose Deprivation in Promoting Differentiation of Dental Pulp Stem Cells in 3D Culture.” In MOLECULAR BIOLOGY OF THE CELL, 34:1212–1212.
Chicago author-date (all authors)
Okkelman, Irina, Winter Vandenberghe, and Ruslan Dmitriev. 2023. “Role of Preconditioning with Oxygen and Glucose Deprivation in Promoting Differentiation of Dental Pulp Stem Cells in 3D Culture.” In MOLECULAR BIOLOGY OF THE CELL, 34:1212–1212.
Vancouver
1.
Okkelman I, Vandenberghe W, Dmitriev R. Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture. In: MOLECULAR BIOLOGY OF THE CELL. 2023. p. 1212–1212.
IEEE
[1]
I. Okkelman, W. Vandenberghe, and R. Dmitriev, “Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture,” in MOLECULAR BIOLOGY OF THE CELL, Washington, DC, USA, 2023, vol. 34, no. 2, pp. 1212–1212.
@inproceedings{01GSA5ZE6E1NHPXW0E6QZBKGA1,
  abstract     = {{Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture
Irina A. Okkelman, Winter Vandenberghe, Ruslan I. Dmitriev
Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University, Ghent 9000, Belgium

Dental pulp stem cells (DPSC) are promising for bone tissue engineering and repair. To support angiogenesis and long-term viability within microfabricated and 3D-printed tissue constructs, they are often grown and differentiated together with endothelial cells in multicellular spheroids. However, this is often associated with the decline of endothelial cells and slow rate of DPSC differentiation towards osteoblasts. Interestingly, hypoxia affects cell viability and at the same time promotes angiogenesis with osteoblast differentiation. 
Here, we hypothesized that oxygen and glucose deprivation pre-conditioning can improve the process of DPSC differentiation in 3D culture with endothelial HUVEC cells. To do this, we performed oxygen and glucose deprivation experiments with 2D cultures of DPSC and HUVEC and tested effects on cell viability, redox potential and morphology. Interestingly, glucose deprivation did not significantly affect redox state of both HUVEC and DPSC. We therefore preconditioned these cell types with 0% O2, followed by 24 h reperfusion, and produced heterocellular spheroids. To understand the effects of preconditioning in this 3D culture, we performed fluorescence microscopy analysis of cell death (Sytox Green) together with monitoring of spheroid oxygenation (ratiometric red/near-infrared O2-sensing nanoparticles) during 7 days of differentiation. We found that cell death was not significantly different in the spheroids from non-differentiated and differentiated groups of cells. However, the periphery-to-core O2 gradients were significantly lower (p=0.05) in non-differentiated hDPSC 21% / HUVEC 0% and hDPSC 0% / HUVEC 0% groups in comparison to hDPSC 21% / HUVEC 21% spheroids. During differentiation, this difference in spheroid oxygenation was slightly ameliorated, keeping the same trend as in non-differentiated spheroids. Thus under the deeper hypoxia, the heterocellular spheroids formed with 0% O2-preconditioned endothelial cells demonstrated cell viability comparable to spheroids formed from non-preconditioned cells. This data correlates with the results on the alteration of redox potential in adherent (2D) DPSC and HUVEC cultures, where hypoxia-conditioned HUVEC cells demonstrated more profound response. We also found that preconditioned cells displayed stronger accumulation of hydroxyapatite and osteogenic differentiation. Collectively, our preliminary data indicates that hypoxia preconditioning, combined with imaging of oxygenation and cell death, can improve the quality of the spheroid-based ‘tissue building blocks’ in biofabrication. 
Funding: supported by the Special Research Fund (BOF) grant of Ghent University (BOF/STA/202009/003).}},
  articleno    = {{B621/P2999}},
  author       = {{Okkelman, Irina and Vandenberghe, Winter and Dmitriev, Ruslan}},
  booktitle    = {{MOLECULAR BIOLOGY OF THE CELL}},
  issn         = {{1059-1524}},
  language     = {{eng}},
  location     = {{Washington, DC, USA}},
  number       = {{2}},
  pages        = {{B621/P2999:1212--B621/P2999:1212}},
  title        = {{Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture}},
  url          = {{https://www.molbiolcell.org/doi/10.1091/mbc.E22-12-0555}},
  volume       = {{34}},
  year         = {{2023}},
}