Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response
Abstract
:1. Introduction
2. Results
2.1. Generation of Synthetic Modified Link N mRNA
2.2. Analysis of Transfectability of Human Primary Chondrocytes and SCP1 Cells with Synthetic mRNA
2.3. Exogenous Delivery of Link N mRNA Does Not Affect Cell Viability
2.4. Link N mRNA Transfection Augments Anabolic Effects in Human Primary Chondrocytes
2.5. Link N mRNA Transfection Upholds the Expression of ECM-Related Genes in SCP1 Cells
2.6. Transfectability of Cells Seeded on Knitted Titanium Scaffold with eGFP mRNA
2.7. Link N mRNA Transfection of Cells Seeded on Knitted Titanium Scaffold Triggers the Expression of Chondrocyte-Specific and ECM-Related Genes
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Generation of Modified Synthetic mRNA
4.3. Cy3 Labeling of Link N mRNA
4.4. Analysis of Transfection Efficiency Using Cy3 Labeled Link N mRNA
4.5. Analysis of Cell Viability
4.6. Cultivation of Cells
4.7. Transfection of Cells with Modified Synthetic Link N mRNA in 2D Cell Culture
4.8. Semi-Quantitative RT-PCR
4.9. Histochemical Analysis of ECM Production
4.10. Dot Blot Assay for Quantifying Link N Protein Post mRNA Transfection
4.11. Transient Cell Infections and Gene Reporter Assay
4.12. Preparation of Knitted Titanium Scaffolds and Cell Seeding
4.13. Transfection of Cells Seeded on Knitted Titanium Scaffold
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene Name | Accession Number | Forward Primer (5′-3′) | Reverse Primer (3′-5′) | Amplicon (bp) | Tm (°C) | Cycle No. |
---|---|---|---|---|---|---|
GAPDH | NM_002046 | GTCAGTGGTGGACCTGACCT | AGGGGTCTACATGGCAACTG | 420 | 56 | 30 |
SOX9 | NM_000346 | GAAGGACCACCCGGATTACA | GCCTTGAAGATGGCGTTGG | 120 | 60 | 35 |
ACAN | NM_001135 | CTTGGACTTGGGCAAACTGC | CACTAAAGTCAGGCAGGCCA | 143 | 60 | 35 |
COL X | NM_000493 | AAACCTGGACAACAGGGACC | CGACCAGGAGCACCATATCC | 125 | 60 | 35 |
COL II | NM_001844 | TGGATGCCACACTCAAGTCC | GCTGCTCCACCAGTTCTTCT | 254 | 60 | 35 |
BMP2 | NM_001200 | CCCCCTACATGCTAGACCTGT | CACTCGTTTCTGGTAGTTCTTCC | 150 | 60 | 35 |
BMP7 | NM_001719 | TAGCCATTTCCTCACCGACG | AGATCCGATTCCCTGCCCAA | 255 | 60 | 35 |
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Tendulkar, G.; Ehnert, S.; Sreekumar, V.; Chen, T.; Kaps, H.-P.; Golombek, S.; Wendel, H.-P.; Nüssler, A.K.; Avci-Adali, M. Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response. Int. J. Mol. Sci. 2019, 20, 1716. https://doi.org/10.3390/ijms20071716
Tendulkar G, Ehnert S, Sreekumar V, Chen T, Kaps H-P, Golombek S, Wendel H-P, Nüssler AK, Avci-Adali M. Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response. International Journal of Molecular Sciences. 2019; 20(7):1716. https://doi.org/10.3390/ijms20071716
Chicago/Turabian StyleTendulkar, Gauri, Sabrina Ehnert, Vrinda Sreekumar, Tao Chen, Hans-Peter Kaps, Sonia Golombek, Hans-Peter Wendel, Andreas K. Nüssler, and Meltem Avci-Adali. 2019. "Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response" International Journal of Molecular Sciences 20, no. 7: 1716. https://doi.org/10.3390/ijms20071716
APA StyleTendulkar, G., Ehnert, S., Sreekumar, V., Chen, T., Kaps, H.-P., Golombek, S., Wendel, H.-P., Nüssler, A. K., & Avci-Adali, M. (2019). Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response. International Journal of Molecular Sciences, 20(7), 1716. https://doi.org/10.3390/ijms20071716