Abstract
Osteoblastic induction is commonly studied using the colony-forming unit-fibroblastic (CFU-f) assay, in which bone marrow stromal cells (BMC) are grown in a tissue culture environment permissive for osteoblastic differentiation (DMEM containing dexamethasone, ascorbic acid and β-glycerophosphate). These cells form colonies, which express alkaline phosphatase, and form a collagenous matrix that becomes calcified. However, these same cells originate in the bone marrow where under normal circumstances they do not proliferate or differentiate despite being subjected to many of the same growth factors and hormones present within the tissue culture environment. We show here that phenol red, present within tissue culture medium as a pH indicator, may itself be a factor that permits osteoblastic recruitment. BMC cultured in the presence of the bone anabolic agents PGE2, PGA2, or bFGF, but in the absence of phenol red, failed to respond to these agents in terms of total or osteoblastic colony number. This effect was dose dependent, with low (2.5 mg/l) and high (15–20 mg/l) doses of phenol red being nonpermissive for the stimulatory effects of PGE2 whereas doses of 5–10 mg/l were permissive. Furthermore, the effects observed in the absence of phenol red could not be abrogated by the addition of 17β-estradiol indicating that these effects cannot be attributed to estrogenic impurities within the phenol red preparation. This indicates that phenol red itself can affect the differentiation of BMC by a mechanism not previously described.
Similar content being viewed by others
References
C Maniatopoulos J Sodek AH Melcher (1988) ArticleTitleBone formation in vitro by stromal cells obtained from bone marrow of young rats. Cell Tissue Res 254 317–330 Occurrence Handle1:STN:280:BiaD28zjtlM%3D Occurrence Handle3197089
PS Leboy JN Beresford C Devlin ME Owen (1991) ArticleTitleDexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures. J Cell Physiol 146 370–378 Occurrence Handle1:CAS:528:DyaK3MXit1WlsL4%3D Occurrence Handle2022691
JN Beresford JH Bennett C Devlin PS Leboy ME Owen (1992) ArticleTitleEvidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 102 341–351 Occurrence Handle1:CAS:528:DyaK38XlsVekt7w%3D Occurrence Handle1400636
K Satomura M Nagayama (1991) ArticleTitleUltrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro. Acta Anat 142 97–104 Occurrence Handle1:STN:280:By2C2c3mvFE%3D
SA Kuznetsov AJ Friedenstein PG Robey (1997) ArticleTitleFactors required for bone marrow stromal fibroblast colony formation in vitro. Br J Haematol 97 561–570 Occurrence Handle1:CAS:528:DyaK2sXktFartrk%3D Occurrence Handle9207401
AJ Friedenstein NV Latzink YuF Gorskaya EA Luria IL Moskvina (1992) ArticleTitleBone marrow stromal colony formation requires Stimulation by haemopoietic cells. Bone Miner 18 199–213 Occurrence Handle1:STN:280:ByyD3crpslA%3D Occurrence Handle1392694
S Gronthos PJ Simmons (1995) ArticleTitleThe growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 85 929–940 Occurrence Handle1:CAS:528:DyaK2MXjs1OjtL0%3D Occurrence Handle7849315
KR Dobson L Reading M Haberey X Marine A Scutt (1999) ArticleTitleCentrifugal Isolation of bone marrow from bone: an improved method for the recovery and quantitation of bone marrow osteoprogenitor cells from rat tibiae and femurae. Calcif Tissue Int 65 411–413 Occurrence Handle10.1007/s002239900723 Occurrence Handle1:CAS:528:DyaK1MXnsV2msrw%3D Occurrence Handle10541770
K Still A Scutt (2001) ArticleTitleStimulation of CFU-f formation by prostaglandin E(2) is mediated in part by its degradation product, prostaglandin A(2). Prostaglandins 65 21–31 Occurrence Handle10.1016/S0090-6980(01)00117-4 Occurrence Handle1:CAS:528:DC%2BD3MXltFKktLw%3D Occurrence Handle11334639
K Dobson L Reading A Scutt (1999) ArticleTitleA cost-effective method for the automatic quantitative analysis of fibroblastic colony-forming units. Calcif Tissue Int 65 166–172 Occurrence Handle10.1007/s002239900677 Occurrence Handle1:CAS:528:DyaK1MXksl2qu7c%3D Occurrence Handle10430652
A Scutt P Bertram (1995) ArticleTitleBone marrow cells are targets for the anabolic actions of PGE2: induction of a transition from non-adherent to adherent osteoblast precursors. J Bone Miner Res 10 474–487 Occurrence Handle1:CAS:528:DyaK2MXmvVWnsLc%3D Occurrence Handle7785470
A Scutt P Bertram (1999) ArticleTitleThe effect of basic fibroblast growth factor on colony formation, expansion and osteoblastic differentiation by bone marrow stromal cells. Calcif Tissue Int 64 69–77 Occurrence Handle10.1007/s002239900581 Occurrence Handle1:CAS:528:DyaK1MXktVyruw%3D%3D Occurrence Handle9868287
KS Stewart S Walsh J Screen CM Jefferiss J Chainey GR Jordan JN Beresford (1999) ArticleTitleFurther characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells. J Bone Miner Res 14 1345–1356 Occurrence Handle1:CAS:528:DyaK1MXltlyltr8%3D Occurrence Handle10457267
BO Oyajobi A Lomri M Hott PJ Marie (1999) ArticleTitleIsolation and characterization of human clonogenic osteoblast progenitors immunoselected from fetal bone marrow stroma using STRO-1 monoclonal antibody. J Bone Miner Res 14 351–361 Occurrence Handle1:STN:280:DyaK1M7lt1egug%3D%3D Occurrence Handle10027900
JN Beresford CJ Joyner C Devlin JT Triffitt (1994) ArticleTitleThe effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro. Archs Oral Biol 39 941–947 Occurrence Handle1:CAS:528:DyaK2MXjs12ks70%3D
Y Berthois JA Katzenellenbogen BS Katzenellenbogen (1986) ArticleTitlePhenol red in tissue culture medium is a weak estrogen: implications concerning the study of estrogen- responsive cells in culture. Proc Natl Acad Sci USA 83 2496–2500 Occurrence Handle1:CAS:528:DyaL28XitF2nsrY%3D Occurrence Handle3458212
WV Welshons MF Wolf CS Murphy VC Jordan (1988) ArticleTitleEstrogenic activity of phenol red. Mol Cell Endocrinol 57 169–178 Occurrence Handle10.1016/0303-7207(88)90072-X Occurrence Handle1:CAS:528:DyaL1cXktVWjtrk%3D Occurrence Handle3402660
M Ernst C Schmid ER Frosch (1989) ArticleTitlePhenol 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. J Steroid Biochem 33 907–914 Occurrence Handle10.1016/0022-4731(89)90239-2 Occurrence Handle1:CAS:528:DyaK3cXntVagsQ%3D%3D Occurrence Handle2601335
RD Bindal KE Carlson BS Katzenellenbogen JA Katzenellenbogen (1988a) ArticleTitleLipophilic impurities, not phenolsulfonphthalein, account for the estrogenic activity in commercial preparations of phenol red. J. Steroid Biochem 31 287–293 Occurrence Handle1:CAS:528:DyaL1cXmtVGjurs%3D
RD Bindal JA Katzenellenbogen (1988b) ArticleTitleBis(4-hydroxyphenyl)-[2-(phenoxysulfonyl)-phenyl]methane: isolation and structure elucidation of a novel estrogen from commercial preparations of phenol red (phenolsulfonphthalein). J Med Chem 31 1978–1983 Occurrence Handle1:CAS:528:DyaL1cXlt1ynurY%3D
LH Grady DJ Nonneman GE Rottinghaus WV Welshons (1991) ArticleTitlepH-dependent cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells. Endocrinology 129 3321–3330 Occurrence Handle1:CAS:528:DyaK38XjsFymsw%3D%3D Occurrence Handle1954908
SS Greenberg A Johns J Kleha J Xie Y Wang J Bianchi K Conley (1994) ArticleTitlePhenol red is a thromboxane A2/prostaglandin H2 receptor antagonist in canine lingual arteries and human platelets. J Pharmacol Exp Ther 268 1352–1361 Occurrence Handle1:CAS:528:DyaK2cXis1Sqtr0%3D Occurrence Handle8138950
MM Walsh-Reitz FG Toback (1992) ArticleTitlePhenol red inhibits growth of renal epithelial cells. Am J Physiol 262 F687–F69L Occurrence Handle1:CAS:528:DyaK38XktVeksb0%3D Occurrence Handle1566882
PR Kym KL Hummert AG Nilsson M Lubin JA Katzenellenbogen (1996) ArticleTitleBisphenolic compounds that enhance cell cation transport are found in commercial phenol red. J Med Chem 39 4897–4904 Occurrence Handle10.1021/jm960300k Occurrence Handle1:CAS:528:DyaK28XmvVWnu7w%3D Occurrence Handle8960548
A Takeshita N Koibuchil N Oka M Taguchi N Shishiba Y Ozawa (2001) ArticleTitleBisphenol-A, an environmental estrogen, activates the human orphan nuclear receptor, steroid and xenobiotic receptor-mediated transcription. Eur J Endocrinol 145 513–517 Occurrence Handle1:CAS:528:DC%2BD3MXns1Gisr0%3D Occurrence Handle11581012
HM Wright CB Clish T Mikami S Hauser K Yanagi R Hiramatsu CN Serhan BM Spiegelman (2000) ArticleTitleA synthetic antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte differentiation. J Biol Chem 21 1873–1877 Occurrence Handle10.1074/jbc.275.3.1873
Acknowledgements
This work was funded by Strakan Pharmaceuticals Ltd, UK.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Still, K., Reading, L. & Scutt, A. Effects of Phenol Red on CFU-f Differentiation and Formation . Calcif Tissue Int 73, 173–179 (2003). https://doi.org/10.1007/s00223-002-2076-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00223-002-2076-4