Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells
Abstract
:1. Introduction
2. Results
2.1. Three-Dimensional Fibrin Scaffold Characterization
2.2. The hTSPC Immunophenotyping and 3D Culture Establishment
2.3. Perfusion-Based Dynamic Culture Assures Long-Term Viability
2.4. Healthy and Pathological hTSPCs Display Different Collagen Gene Expression
2.5. Healthy and Pathological hTSPCs Show Differences in Tendon-Related Protein Expression
2.6. Pathological hTSPCs Show a Distinct Expression of Collagen Subtypes
2.7. Pathological hTSPCs Exhibit Reduced Overall Cytokine Expression
2.8. Healthy and Pathological hTSPCs Secrete Inflammatory Cytokines Differently
3. Discussion
4. Materials and Methods
4.1. Collection of Human Biopsies
4.2. The hTSPC Harvesting and Characterization
4.3. Flow Cytometry and Gating Strategy of hTSPCs
4.4. Fibrin Scaffold Drying, FE-SEM, and Pores Analysis
4.5. Assembly of 3D Bioengineered Scaffolds and Perfusion Culture System
4.6. Live and Dead Assays
4.7. RNA Isolation and Gene Expression Profiling
4.8. Immunohistochemical Assay
4.9. Hematoxylin and Eosin Staining
4.10. Sirius Red Staining
4.11. Detection of Cytokine Release
4.12. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target | Gene Bank Accession Number | Sequences | Product Size | Primer Efficiency |
---|---|---|---|---|
IL-6 | NM-000600.5 | Fwd: ACTTGCCTGGTGAAAATCAT Rev: CAGGAACTGGATCAGGACTT | 135 | 106 |
TNF | NM-000594.4 | Fwd: GCCCATGTTGTAGCAAACCC Rev: TATCTCTCAGCTCCACGCCA | 97 | 105 |
IL-12A | NM-000882.4 | Fwd: TCAGAATTCGGGCAGTGACT Rev: AGTCCCAQTCCTTCTTTCCCC | 163 | 110 |
IL-1β | NM-000576.3 | Fwd: GGAGAATGACCTGAGCACCT Rev: GGAGGTGGAGAGCTTTCAGT | 185 | 110 |
IL-10 | NM-000572.3 | Fwd: AAGACCCAGACATCAAGGCG Rev: AATCGATGACAGCGCCGTAG | 85 | 110 |
TGF-β1 | NM-000660.7 | Fwd: GCACTCGCCAGAGTGGTTAT Rev: AAGCCCTCAATTTCCCCTCC | 81 | 95 |
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Ciardulli, M.C.; Lovecchio, J.; Parolini, O.; Giordano, E.; Maffulli, N.; Della Porta, G. Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells. Int. J. Mol. Sci. 2024, 25, 9563. https://doi.org/10.3390/ijms25179563
Ciardulli MC, Lovecchio J, Parolini O, Giordano E, Maffulli N, Della Porta G. Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells. International Journal of Molecular Sciences. 2024; 25(17):9563. https://doi.org/10.3390/ijms25179563
Chicago/Turabian StyleCiardulli, Maria Camilla, Joseph Lovecchio, Ornella Parolini, Emanuele Giordano, Nicola Maffulli, and Giovanna Della Porta. 2024. "Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells" International Journal of Molecular Sciences 25, no. 17: 9563. https://doi.org/10.3390/ijms25179563