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

BackgroundBasic 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 (PPi), an inhibitor of mineralization. The progressive ankylosis protein (gene ANKH; protein ANKH) and ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1/ENPP1) increase PPi and inhibit mineralization, while tissue-nonspecific alkaline phosphatase (ALPL; TNAP) is a critical pro-mineralization enzyme that hydrolyzes PPi. We hypothesized that regulation by bFGF of mineralization in SHEDs occurs by modulation of P-i/PPi-associated genes.MethodsCells 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.ResultsCells 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 10ng/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/PPi ratio and mineral deposition. Effects of bFGF on ALPL and ANKH expression were detected within 24h. Addition of 20mM fibroblast growth factor receptor (FGFR) inhibitor SU5402 revealed the necessity of FGFR-mediated signaling, and inclusion of 1g/ml cyclohexamide (CHX) implicated the necessity of protein synthesis for effects on ALPL and ANKH. Addition of exogenous 10m PPi 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 PPi 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.ConclusionThis study reveals for the first time the effects of bFGF on P-i/PPi regulators in SHEDs and implicates these factors in how bFGF directs osteo/odontogenic differentiation and mineralization by these cells.