Exogenous Application of Proteoglycan to the Cell Surface Microenvironment Facilitates to Chondrogenic Differentiation and Maintenance
Abstract
:1. Introduction
2. Results
2.1. Effect on the Proliferation of ATDC5 Cells by Addition of ECM Constituting Factor
2.2. Effect of sPG on Cellular Proliferation-Related Receptor Activation
2.3. Effects of ECM Constituents on Chondrogenic Differentiation in ATDC5 Cells
2.4. Evaluation of Three-Dimensional Structure in Cartilage-Like Nodules Induced by sPG in ATDC5 Cells
2.5. Effect of sPG on Chondrogenic Differentiation-Related Factor in ATDC5 Cells
2.6. Influence of ECM Constituting Factors on Cartilage Calcification in ATDC5 Cells
2.7. Effect of sPG on Cartilage Calcification-Related Factor (mRNA)
2.8. Influence of sPG on Continued Calcification Conditions
2.9. Localization Analysis of sPG Applied to Living Cells
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cells and Cell Culture
4.3. Cell Proliferation Assay
4.4. Immunoblotting
4.5. Chondrogenic Differentiation
4.6. Cartilage Calcification
4.7. RNA Isolation and Reverse Transcription (RT)-PCR
4.8. Real-Time Quantitative PCR
4.9. Alcian Blue Staining
4.10. Alizarin Red Staining
4.11. Histology of Chondrogenic Differentiation
4.12. Fluorescent Labeling of sPG
4.13. Confocal Fluorescence Live Cell Imaging
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
BMP | Bone morphogenetic protein |
CLSM | Confocal laser scanning microscopy |
CS | Chondroitin sulfate |
ECM | Extracellular matrix |
EGFR | Epidermal growth factor receptor |
FGF | Fibroblast growth factor |
GAG | Glycosaminoglycan |
GDF5 | Growth differentiation factor 5 |
GlcN | Glucosamine hydrochloride |
HA | Hyaluronan |
IGF-1 | Insulin-like growth factor 1 |
IRS-1 | Insulin receptor substrate 1 |
MGP | Matrix gla protein |
OA | Osteoarthritis |
PCM | Pericellular matrix |
PG | Proteoglycan |
PTHrP | Parathyroid hormone-related protein |
Runx2 | Runt-related transcription factor 2 |
SOX9 | SRY-box transcription factor 9 |
TGF-β | Transforming growth factor beta |
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Masutani, T.; Yamada, S.; Hara, A.; Takahashi, T.; Green, P.G.; Niwa, M. Exogenous Application of Proteoglycan to the Cell Surface Microenvironment Facilitates to Chondrogenic Differentiation and Maintenance. Int. J. Mol. Sci. 2020, 21, 7744. https://doi.org/10.3390/ijms21207744
Masutani T, Yamada S, Hara A, Takahashi T, Green PG, Niwa M. Exogenous Application of Proteoglycan to the Cell Surface Microenvironment Facilitates to Chondrogenic Differentiation and Maintenance. International Journal of Molecular Sciences. 2020; 21(20):7744. https://doi.org/10.3390/ijms21207744
Chicago/Turabian StyleMasutani, Teruaki, Shuhei Yamada, Akira Hara, Tatsuji Takahashi, Paul G Green, and Masayuki Niwa. 2020. "Exogenous Application of Proteoglycan to the Cell Surface Microenvironment Facilitates to Chondrogenic Differentiation and Maintenance" International Journal of Molecular Sciences 21, no. 20: 7744. https://doi.org/10.3390/ijms21207744