L-Type Voltage Gated Calcium Channels and Sodium Phosphate Symporters are Required for Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Materials
David Foulad1, Xiaoyan Ren1, Qi Zhou1, Marley Dewey2, Brendan Harley2, Justine C. Lee1.
1UCLA Division of Plastic and Reconstructive Surgery, Los Angeles, CA, USA, 2University of Illinois Urbana Champaign, Urbana, IL, USA.
PURPOSE: The increasing understanding that modulation of extracellular matrix (ECM) components can instruct differential cell fate determination has resulted in significant interest in the synthesis of ECM-inspired materials for regeneration.Previously we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion via an autogenous activation of the bone morphogenetic protein receptor (BMPR) signaling pathway. Such activities were not induced in non-mineralized collagen glycosaminoglycan (Col-GAG) materials, suggesting that the upstream trigger is related to the inorganic content of the material. In this work, we evaluated the contributions of inorganic ion signaling to MC-GAG-induced osteogenic differentiation.
METHODS: Primary hMSCs were osteogenically differentiated on collagen glycosaminoglycan materials in the absence or presence of inhibitors to the L-type voltage gated calcium channels (L-VGCC), calcium sensing receptor (CaSR), and the sodium phosphate symporters (PiT-1/2) using nifedipine, NPS2143,and phosphonoformic acid (PFA), respectively. Western blots were performed for phosphorylated and total Smad1/5, ERK1/2, Akt, p38, JNK1/2, total Runx2, and actin. Expression of BMPs were assessed with immunofluorescent staining of scaffold sections. Mineralization was evaluated by Alizarin red staining and micro-computed tomographic analyses. Analyses of variance with posthoc comparisons were performed.
RESULTS: PFA diminished phosphorylated Smad1/5, BMP-2,and Runx2 expression as well as mineralization for both Col-GAG and MC-GAG scaffolds. While NPS2143 diminished Runx2 and mineralization on Col-GAG, no effects on osteogenic protein expression, BMP-2 expression, or mineralization were evident on MC-GAG. In contrast, no effects were mediated by nifedipine on Col-GAG,whereas nifedipine inhibited phosphorylated Smad1/5, phosphorylated ERK1/2, Runx2, BMP-2 staining, and mineralization on MC-GAG.
CONCLUSION: In combination, these data suggested that the autogenous activation of the BMPR signaling pathway induced by MC-GAGis firsttriggered by calcium and phosphate influxvia L-VGCCs and the sodium and phosphate symporters.
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