Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite
Abstract
1. Introduction
2. Results and Discussion
2.1. Structural Characterizations of 3D Scaffolds
2.1.1. X-ray Microtomography of Hydrogels in a Dry State
2.1.2. OCT of Hydrogels in a Hydrated State
2.2. Characterizations of MSC/HUVEC Spheroids Within Hydrogels
2.2.1. Spheroid Mapping
2.2.2. Spheroid Diameter
2.2.3. Spheroid Volume
2.3. Proliferation and Viability of the MSC/HUVEC Spheroids
2.3.1. Cell Proliferation Quantification
2.3.2. Viability Quantification and Mapping
2.4. 3D Spatial Reorganization of the MSCs and the HUVECs
2.4.1. Reorganization of Cells
2.4.2. The Detection of Clustering Patterns Across the Entire Diameter of the Spheroid
2.5. Computation of the WSS Within the Bioreactor and Oxygen Concentration Mapping in the Hydrogel
2.5.1. Hydrodynamics
2.5.2. Oxygen Diffusion
2.6. Osteogenic and Angiogenic Capacities of the MSC/the HUVEC Spheroids
2.6.1. Osteogenic Differentiation of Immortalized MSCs
2.6.2. Alkaline Phosphatase (ALP) Quantification
2.6.3. Mineralization of ECM Through Alizarin Red and Von Kossa Staining
2.6.4. Vasculogenic Capacity of the HUVECs in Co-Culture Spheroids
3. Conclusions
4. Materials and Methods
4.1. Preparation of Porous Polysaccharide-Based Hydrogel Scaffolds
4.2. X-ray Computed Microtomography Hydrogel Characterization
4.2.1. Image Acquisition
4.2.2. X-ray Computed Microtomography Image Reconstruction, Visualization, and Analysis
4.3. Cell Lines
SV40+hTERT-Immortalized Bone Marrow-Derived Mesenchymal Stem Cells Differentiation Capacity
4.4. Cell Seeding Within 3-D Macroporous Scaffold
4.5. Optical Coherence Tomography Hydrogel Characterization
4.5.1. Image Acquisition
4.5.2. Image Reconstruction, Visualization, and Analysis
4.6. Dynamic Culture in a Perfusion Bioreactor
4.7. The MSC and HUVEC Coculture
4.7.1. Cell Proliferation Assay
4.7.2. Cell Viability Assessment
4.7.3. Alkaline Phosphatase (ALP) Activity Assay
4.7.4. ALP Live Stain
4.7.5. Mineralization
- 1.
- Von Kossa staining
- 2.
- Alizarin red staining
- 3.
- Collagen type IV immunocytochemistry
4.8. Confocal Image Reconstruction, Visualization, and Spatial Analysis
4.8.1. 3D Plots
4.8.2. Ripley’s K Function
4.9. Computational Fluid Dynamics (CFD) Simulation of Porous Scaffold Perfusion
4.9.1. Modeling and Lattice Boltzmann Method (LBM) Implementation
4.9.2. Computational Configuration
4.10. Simulation of Oxygen Transport and Consumption Within a Porous Scaffold
4.10.1. Modeling and LBM Implementation
4.10.2. Computational Configuration
4.11. Data Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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CSD1 | CSD2 | CSD3 | CSD4 | |
---|---|---|---|---|
Cell Seeding Density (CSD)/scaffold | 400,000 | 600,000 | 800,000 | 1,000,000 |
Average spheroid volume (µm3) × 105 | 6.0 ± 0.8 | 7.6 ± 0.1 | 6.3 ± 0.4 | 10.9 ± 0.1 |
Spheroid number | 773 ± 252 | 805 ± 54 | 2384 ± 203 | 1368 ± 249 |
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El Hajj, S.; Ntaté, M.B.; Breton, C.; Siadous, R.; Aid, R.; Dupuy, M.; Letourneur, D.; Amédée, J.; Duval, H.; David, B. Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite. Gels 2024, 10, 666. https://doi.org/10.3390/gels10100666
El Hajj S, Ntaté MB, Breton C, Siadous R, Aid R, Dupuy M, Letourneur D, Amédée J, Duval H, David B. Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite. Gels. 2024; 10(10):666. https://doi.org/10.3390/gels10100666
Chicago/Turabian StyleEl Hajj, Soukaina, Martial Bankoué Ntaté, Cyril Breton, Robin Siadous, Rachida Aid, Magali Dupuy, Didier Letourneur, Joëlle Amédée, Hervé Duval, and Bertrand David. 2024. "Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite" Gels 10, no. 10: 666. https://doi.org/10.3390/gels10100666
APA StyleEl Hajj, S., Ntaté, M. B., Breton, C., Siadous, R., Aid, R., Dupuy, M., Letourneur, D., Amédée, J., Duval, H., & David, B. (2024). Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite. Gels, 10(10), 666. https://doi.org/10.3390/gels10100666