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Stem Cell Reviews and Reports

, Volume 9, Issue 2, pp 132–139 | Cite as

Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro

  • Helle LysdahlEmail author
  • Anette Baatrup
  • Anna Bay Nielsen
  • Casper Bindzus Foldager
  • Cody Bünger
Article

Abstract

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.

Keywords

Human mesenchymal stem cells Culture media Phenol red Chondrogenic differentiation Osteogenic differentiation 

Notes

Acknowledgements

This project was granted from the Velux Foundation and the A.P. Møller Foundation for the Advancement of Medical Science.

Disclosure of Potential Conflict of Interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2012_9417_Fig10_ESM.jpg (19 kb)
Supplementary Fig. 1

DNA content after 2, 7, 14, and 21 days of osteogenic differentiation of hMSCs. Vertical axis represents the DNA content expressed by 700 nm channel intensity. Horizontal axis represents the medium type and time points. Data are expressed as mean ± SD (n = 12). (JPEG 19 kb)

12015_2012_9417_MOESM1_ESM.tif (78 kb)
High resolution image (TIFF 78 kb)
12015_2012_9417_Fig11_ESM.jpg (41 kb)
Supplementary Fig. 2

DNA content and alkaline phosphatase activity after 2, 7, 14, and 21 days culture of hMSCs. Vertical axes represent DNA content expressed by 700 nm channel intensity and alkaline phosphatase activity expressed by nmol nitrophenol x ml−1 x min−1, respectively. Horizontal axes represent medium type and time points. Data are expressed as mean ± SD (n = 12). * Significant difference between DMEM wPR and DMEM woPR within the given time point, P < 0.05. (JPEG 40 kb)

12015_2012_9417_MOESM2_ESM.tif (149 kb)
High resolution image (TIFF 148 kb)
12015_2012_9417_Fig12_ESM.jpg (82 kb)
Supplementary Fig. 3

Relative gene expression levels of runx2, alkaline phosphatase, collagen type 1, and osteocalcin after 2, 7, 14, and 21 days of osteogenic differentiation of hMSCs. Vertical axes represent the relative gene expression level and horizontal axes represent the medium type and time points. Data are expressed as mean ± SD (n = 6). * Significant difference between DMEM woPR + and DMEM woPR + D within the given time point, P < 0.05. Vitamin D significantly increased the gene expression of runx2 at all time points (P = 0.0000), alkaline phosphatase on day 2 and 7 (P = 0.0000), collagen type 1 at all time points (P = 0.000), and osteocalcin at all time points (P = 0.000) (Supplementary figure 3). On day 14, vitamin D significantly decreased the gene expression of alkaline phosphatase (P = 0.0019). (JPEG 82 kb)

12015_2012_9417_MOESM3_ESM.tif (278 kb)
High resolution image (TIFF 278 kb)

References

  1. 1.
    Caplan, A. I. (2007). Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. Journal of Cellular Physiology, 213, 341–347.PubMedCrossRefGoogle Scholar
  2. 2.
    Caplan, A. I. (2005). Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Engineering, 11, 1198–1211.PubMedCrossRefGoogle Scholar
  3. 3.
    Jaiswal, N., Haynesworth, S. E., Caplan, A. I., & Bruder, S. P. (1997). Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. Journal of Cellular Biochemistry, 64, 295–312.PubMedCrossRefGoogle Scholar
  4. 4.
    Pytlik, R., Stehlik, D., Soukup, T., et al. (2009). The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering. Biomaterials, 30, 3415–3427.PubMedCrossRefGoogle Scholar
  5. 5.
    Coelho, M. J., & Fernandes, M. H. (2000). Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials, 21, 1095–1102.PubMedCrossRefGoogle Scholar
  6. 6.
    Coelho, M. J., Cabral, A. T., & Fernande, M. H. (2000). Human bone cell cultures in biocompatibility testing. Part I: osteoblastic differentiation of serially passaged human bone marrow cells cultured in alpha-MEM and in DMEM. Biomaterials, 21, 1087–1094.PubMedCrossRefGoogle Scholar
  7. 7.
    Vater, C., Kasten, P., & Stiehler, M. (2011). Culture media for the differentiation of mesenchymal stromal cells. Acta Biomaterialia, 7, 463–477.PubMedCrossRefGoogle Scholar
  8. 8.
    Nakamura, S., Yamada, Y., Baba, S., et al. (2008). Culture medium study of human mesenchymal stem cells for practical use of tissue engineering and regenerative medicine. Biomedical Materials and Engineering, 18, 129–136.PubMedGoogle Scholar
  9. 9.
    Berthois, Y., Katzenellenbogen, J. A., & Katzenellenbogen, B. S. (1986). Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proceedings of the National Academy of Sciences of the United States of America, 83, 2496–2500.PubMedCrossRefGoogle Scholar
  10. 10.
    Still, K., Reading, L., & Scutt, A. (2003). Effects of phenol red on CFU-f differentiation and formation. Calcified Tissue International, 73, 173–179.PubMedCrossRefGoogle Scholar
  11. 11.
    Myers, M. A. (1998). Direct measurement of cell numbers in microtitre plate cultures using the fluorescent dye SYBR green I. Journal of Immunological Methods, 212, 99–103.PubMedCrossRefGoogle Scholar
  12. 12.
    Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper–Excel-based tool using pair-wise correlations. Biotechnology Letters, 26, 509–515.PubMedCrossRefGoogle Scholar
  13. 13.
    Bruder, S. P., Jaiswal, N., Ricalton, N. S., Mosca, J. D., Kraus, K. H., Kadiyala, S. (1998). Mesenchymal stem cells in osteobiology and applied bone regeneration. Clinical Orthopaedics and Related Research S247–256.Google Scholar
  14. 14.
    Ernst, M., Schmid, C., & Froesch, E. R. (1989). Phenol red mimics biological actions of estradiol: enhancement of osteoblast proliferation in vitro and of type I collagen gene expression in bone and uterus of rats in vivo. Journal of Steroid Biochemistry, 33, 907–914.PubMedCrossRefGoogle Scholar
  15. 15.
    Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., & Karsenty, G. (1997). Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell, 89, 747–754.PubMedCrossRefGoogle Scholar
  16. 16.
    Beresford, J. N., Joyner, C. J., Devlin, C., & Triffitt, J. T. (1994). The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro. Archives of Oral Biology, 39, 941–947.PubMedCrossRefGoogle Scholar
  17. 17.
    van Driel, M., Koedam, M., Buurman, C. J., et al. (2006). Evidence that both 1alpha,25-dihydroxyvitamin D3 and 24-hydroxylated D3 enhance human osteoblast differentiation and mineralization. Journal of Cellular Biochemistry, 99, 922–935.PubMedCrossRefGoogle Scholar
  18. 18.
    Viereck, V., Siggelkow, H., Tauber, S., Raddatz, D., Schutze, N., & Hufner, M. (2002). Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts. Journal of Cellular Biochemistry, 86, 348–356.PubMedCrossRefGoogle Scholar
  19. 19.
    Maehata, Y., Takamizawa, S., Ozawa, S., et al. (2006). Both direct and collagen-mediated signals are required for active vitamin D3-elicited differentiation of human osteoblastic cells: roles of osterix, an osteoblast-related transcription factor. Matrix Biology, 25, 47–58.PubMedCrossRefGoogle Scholar
  20. 20.
    Nasatzky, E., Schwartz, Z., Boyan, B. D., Soskolne, W. A., & Ornoy, A. (1993). Sex-dependent effects of 17-beta-estradiol on chondrocyte differentiation in culture. Journal of Cellular Physiology, 154, 359–367.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Helle Lysdahl
    • 1
    Email author
  • Anette Baatrup
    • 1
  • Anna Bay Nielsen
    • 1
  • Casper Bindzus Foldager
    • 1
    • 2
  • Cody Bünger
    • 1
  1. 1.Orthopaedic Research LaboratoryAarhus University HospitalAarhus CDenmark
  2. 2.Sports Trauma ClinicAarhus University HospitalAarhusDenmark

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