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Calcium And Phosphate Ion Signaling Triggers Osteogenic Differentiation On Nanoparticulate Mineralized Collagen Glycosaminoglycan Scaffolds
Allison C. Hu, BA1, Anthony B. Bertrand, MD, MBDA1, David Fouland, MD1, Xiaoyan Ren, MD, PhD1, Brian N. Dang, BS1, Candace H. Chan, BS1, Qi Zhou, PhD, DDS1, Brendan A.C. Harley, PhD2, Justine C. Lee, MD, PhD1.
1University of California, Los Angeles David Geffen School of Medicine, Los Angeles, CA, USA, 2Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

PURPOSE: The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, we demonstrated that a nanoparticulate mineralized collagen glycosaminoglycan material (MC-GAG) induced in vivo calvarial regeneration in the absence of exogenous growth factors or addition of exogenous progenitor cells via an autogenous activation of Smad1/5 in the canonical bone morphogenetic protein receptor (BMPR) signaling pathway in endogenous progenitor cells. We also demonstrated that this phenomenon was not found in non-mineralized collagen glycosaminoglycan (Col-GAG). In this work, we investigated the contribution of inorganic ion signaling as the trigger for osteogenic differentiation induced by MC-GAG.
METHODS: Primary bone marrow-derived human mesenchymal stem cells (hMSCs) were osteogenically differentiated on Col-GAG and MC-GAG materials for 1, 2, or 8 weeks in the absence and presence of 1 mM phosphonoformic acid (PFA), 10 ÁM NPS-2143 (NPS), or 10 ÁM nifedipine (Nif), inhibitors of the sodium phosphate symporters (PiT-1/2), calcium sensing receptor (CaSR), and L-type voltage gated calcium channels (L-VGCCs), respectively. Quantitative reverse transcriptase, western blot, micro-computed tomography (micro-CT), and histologic analyses were performed to assess gene expression, activation of intracellular signaling mediators, and mineralization.
RESULTS: PFA inhibited activation of Smad1/5 phosphorylation and mineralization of hMSCs on both Col-GAG and MC-GAG materials, suggesting that phosphate signaling is necessary for osteogenic differentiation regardless of material. In contrast, while calcium signaling was also necessary for osteogenic differentiation on either material, Col-GAG required CaSR while MC-GAG required L-VGCCs. In both circumstances, the activation of Smad1/5 was less pronounced than the activation of ERK1/2 phosphorylation. Finally, unlike Col-GAG which required addition of phosphate in growth media for hMSC differentiation, hMSCs cultured on MC-GAG underwent osteogenic differentiation in standard growth media.
CONCLUSION: Our current work suggests that the osteogenic activity of MC-GAG, an extracellular matrix-inspired regenerative material, is directly related to the nanoparticulate inorganic content via the activation of the sodium phosphate symporters and the L-type voltage gated calcium channels.


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