Extracellular calcium regulates the reciprocal osteogenic and adipogenic commitment of Stem Cells from Human...

This study investigated the in vitro effects of varying extracellular calcium concentrations on the proliferation and multilineage differentiation of Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs), aiming to elucidate the underlying molecular mechanisms.

SHEDs were cultured in induction media supplemented with extracellular calcium concentrations ranging from 1.8 mM to 16.2 mM. Proliferation was evaluated using cell viability assays. Osteogenic differentiation was quantified via Alizarin Red S assays, while adipogenic differentiation was assessed by Oil Red O staining. Quantitative real-time PCR (q-PCR) determined the transcript expression of osteogenic marker genes (RUNX2, COL1A1, ALP, OPN, OSX, OCN) and adipogenic marker genes (LPL and PPARγ). To identify the specific pathways involved, pharmacological inhibitors was executed using nifedipine (an L-type voltage-gated calcium channel [L-VGCC] blocker), NPS2143 (a calcium-sensing receptor [CaSR] antagonist), and U0126 (a MEK/ERK signaling inhibitor).

Extracellular calcium concentration did not significantly affect SHEDs proliferation. Regarding osteogenesis, elevated calcium concentrations significantly enhanced physical calcium deposition and matrix mineralization, whereas concurrently downregulated the expressions of RUNX2 and COL1A1. Pharmacological assays showed that L-VGCC blockade significantly abolished calcium induced matrix mineralization. Regarding adipogenesis, elevated calcium consistently downregulated both LPL and PPARγ expressions. Application of the CaSR antagonist completely reversed this suppression. Conversely, L-VGCC inhibition partially restored LPL expression, while calcium supplementation successfully rescued LPL expression under conditions of MEK/ERK pathway blockade.

Extracellular calcium exerts a dual, pathway specific regulatory control on SHEDs differentiation. It drives osteogenic mineralization predominantly through L-VGCC-mediated calcium influx while suppressing adipogenic lineage commitment via CaSR activation.