Basic fibroblast growth factor regulates phosphate/pyrophosphate regulatory genes in stem cells isolated from human exfoliated deciduous teeth

Abstract

Background: Basic fibroblast growth factor (bFGF) regulates maintenance of stemness and modulation of osteo/odontogenic differentiation and mineralization in stem cells from human exfoliated deciduous teeth (SHEDs). Mineralization in the bones and teeth is in part controlled by pericellular levels of inorganic phosphate (P_i), a component of hydroxyapatite, and inorganic pyrophosphate (PP_i), an inhibitor of mineralization. The progressive ankylosis protein (gene ANKH; protein ANKH) and ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1/ENPP1) increase PP_i and inhibit mineralization, while tissue-nonspecific alkaline phosphatase (ALPL; TNAP) is a critical pro-mineralization enzyme that hydrolyzes PP_i. We hypothesized that regulation by bFGF of mineralization in SHEDs occurs by modulation of P_i/PP_i-associated genes.

Methods: Cells were isolated from human exfoliated deciduous teeth and characterized for mesenchymal stem cell characteristics. Cells were treated with bFGF, and the osteogenic differentiation ability was determined. The mRNA expression was evaluated using real-time polymerase chain reaction. The mineralization was examined using alizarin red S staining.

Results: Cells isolated from primary teeth expressed mesenchymal stem cell markers, CD44, CD90, and CD105, and were able to differentiate into osteo/odontogenic and adipogenic lineages. Addition of 10 ng/ml bFGF to SHEDs during in vitro osteo/odontogenic differentiation decreased ALPL mRNA expression and ALP enzyme activity, increased ANKH mRNA, and decreased both P_i/PP_i ratio and mineral deposition. Effects of bFGF on ALPL and ANKH expression were detected within 24 h. Addition of 20 mM fibroblast growth factor receptor (FGFR) inhibitor SU5402 revealed the necessity of FGFR-mediated signaling, and inclusion of 1 μg/ml cyclohexamide (CHX) implicated the necessity of protein synthesis for effects on ALPL and ANKH. Addition of exogenous 10 μm PP_i inhibited mineralization and increased ANKH, collagen type 1a1 (COL1A1), and osteopontin (SPP1) mRNA, while addition of exogenous P_i increased mineralization and osterix (OSX), ANKH, SPP1, and dentin matrix protein 1 (DMP1) mRNA. The effects of PP_i and P_i on mineralization could be replicated by short-term 3- and 7-day treatments, suggesting signaling effects in addition to physicochemical regulation of mineral deposition.

Conclusion: This study reveals for the first time the effects of bFGF on P_i/PP_i regulators in SHEDs and implicates these factors in how bFGF directs osteo/odontogenic differentiation and mineralization by these cells.

Keywords: Basic fibroblast growth factor; Phosphate; Pyrophosphate; Stem cells isolated from human exfoliated deciduous teeth.

Fig. 1

SHED cell isolation and characterization. Cells harvested from primary teeth were analyzed by flow cytometry and found to express MSC markers, a CD44, b CD90, and c CD105, but lack expression of hematopoietic stem cell marker, d CD45. e MSC markers are expressed in more than 90% of cells, with CD45 expressed in only about 1% of cells. f Significant upregulation of ALPL and BGLAP mRNA expression is observed after osteogenic induction of SHED cells for 7 days. g Increased mineral deposition by SHED cells under osteogenic induction is evident after 14 days. h Upregulation of LPL and PPARγ is observed after adipogenic induction of SHED cells for 8 days. i Intracellular lipid accumulation is observed after adipogenic induction for 16 days. GM growth medium, OM osteogenic induction medium, AM adipogenic induction medium. Asterisks indicated the statistically significant difference compared to the control (p < 0.05)

Fig. 2

bFGF inhibits in vitro mineralization and alters osteogenic/odontogenic gene expression in SHEDs. a Expression of bFGF is reduced 50% (p < 0.05) in SHED cells during osteogenic induction over 14 days. Inclusion of 10 ng/mL bFGF in osteogenic induction medium significantly attenuates mineral deposition at 14 days (b, c) and reduces ALP enzymatic activity at 7 days (d). e–j Addition of bFGF reduces mRNA expression of RUNX2 (day 1), COL1A1 (days 1, 3, and 7), and BGLAP (day 3) (p < 0.05 for all), but does not significantly affect expression of OSX, SPP1, or DMP1. The Mann-Whitney U test was employed for comparisons of two groups. The Kruskal-Wallis test followed by pairwise comparison was used for comparison of three groups. Bars indicated the statistically significant difference (*p < 0.05, **p < 0.01). GM growth medium, OM osteogenic induction medium

Fig. 3

bFGF alters expression of phosphate/pyrophosphate regulatory genes in SHEDs. a SHED cells cultured in osteogenic medium over 14 days increase ALPL mRNA (day 7; p < 0.05), with no significant alterations in b ANKH, c ENPP1, or d SLC20A1 expression. Inclusion of 10 ng/mL bFGF in osteogenic induction medium e reduces ALPL mRNA (days 3 and 7), f increases ANKH mRNA (days 1, 3, and 7), g does not significantly affect ENPP1 mRNA, and h increases SLC20A1 mRNA (day 1). Addition of 20 mM FGFR inhibitor (SU5402) inhibits bFGF-induced changes in i ALPL and j ANKH on day 7. k Addition of 20 mM SU5402 in the absence of exogenous bFGF increases ALPL (p < 0.05) but does not affect ANKH expression. The Mann-Whitney U test was employed for comparison of two groups. The Kruskal-Wallis test followed by pairwise comparison was used for comparison of three or more groups. Bars indicated the statistically significant difference. OM osteogenic induction medium (*p < 0.05)

Fig. 4

Regulation of ALPL and ANKH expression by bFGF in SHEDs. Cells were maintained in normal growth medium in the presence or absence of 10 ng/mL bFGF for 6, 12, or 24 h. a Addition of bFGF significantly inhibits ALPL mRNA by 24 h. b The induction of ANKH mRNA by bFGF is observed at 6, 12, and 24 h. c, d For both ALPL and ANKH, inclusion of 20 mM FGFR inhibitor (SU5402) in growth medium abolishes effects of bFGF at 24 h. Addition of 1 μg/ml cyclohexamide (CHX) inhibits effects of bFGF on ALPL and ANKH expression, suggesting that protein translation is required. e In culture medium collected from SHEDs on days 1, 6, and 14 after osteogenic induction, bFGF significantly reduces P_i/PP_i ratio by day 14. f Addition of 0.05 U ALP rescues bFGF-attenuated ALP expression and mineral deposition in SHEDs. The Mann-Whitney U test was employed for comparisons of two groups. The Kruskal-Wallis test followed by pairwise comparison was used for comparisons of three or more groups. Bars indicate statistically significant differences (*p < 0.05, **p < 0.01). OM osteogenic induction medium

Fig. 5

PP_i regulates mineralization and gene expression in SHEDs. a, b Addition of 10 μM PP_i to osteogenic induction medium of SHED cells nearly completely inhibits mineral deposition by 14 days. c–l At 7 days, inclusion of PP_i increases COL1A1 (p < 0.05) and ANKH (p < 0.05), but does not significantly affect other genes associated with mineralization and P_i/PP_i regulation, compared to untreated controls. The Mann-Whitney U test was employed for comparisons of two groups, and bars indicated the statistically significant difference (*p < 0.05). OM osteogenic induction medium

Fig. 6

P_i regulates mineralization and gene expression in SHEDs. a, b Addition of 5 mM P_i to osteogenic induction medium of SHED cells increases mineral deposition at 7 and 14 days. c–l At 7 days, inclusion of P_i increases OSX, DMP1, SPP1, and ANKH (p < 0.05 for all), but does not significantly affect other genes associated with mineralization and P_i/PP_i regulation, compared to untreated controls. The Mann-Whitney U test was employed for comparisons of two groups, and bars indicated the statistically significant difference (*p < 0.05). OM osteogenic induction medium

Fig. 7

Regulation of mineralization in SHEDs by continuous and short-term P_i and PP_i treatment. a Cells were treated with 5 mM P_i or 10 μM PP_i in osteogenic medium, with no P_i or PP_i serving as negative control, and continuous administration of P_i or PP_i serving as the positive control, whereas conditions A, B, and C incorporated shorter-term P_i or PP_i treatments of 1, 3, or 7 days, respectively, followed by completion of the experiment in osteogenic medium. b Continuous treatment of SHEDs with P_i significantly increases (p < 0.05) mineral deposition while or continuous PP_i significantly decreases (p < 0.05) mineral deposition. c Cells under condition A show no significant differences in mineralization from negative controls. d Cells under condition B exhibit PP_i-mediated inhibition of mineralization by 3 days (p < 0.05). e Cells under condition C show increased mineralization from P_i treatment (p < 0.05) and decreased mineralization from PP_i treatment (p < 0.05). The Mann-Whitney U test was employed for comparisons of three or more groups, and bars indicated the statistically significant difference (*p < 0.05). OM osteogenic induction medium