192. Dynamic Changes In Temporospatial Phosphate Equilibrium By Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials Induces Osteogenesis Via Pit-1 And Pit-2

PURPOSE: Fabrication of biomaterials that emulate properties of tissue-specific extracellular matrix (ECM) and consequently regulate progenitor cell fate are of particular interest in the development of a “materials-only” regenerative application for osseous defects. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is a synthetic material that promotes in vitro osteogenic differentiation and in vivo skull regeneration without exogenous growth factors and ex-vivo progenitor cell seeding. The mineral content of MC-GAG is a critical distinguishing property that may be responsible for its capabilities, as non-mineralized collagen glycosaminoglycan (Col-GAG) materials are limited in promoting osteogenesis. Understanding the molecular mechanisms underlying the transmission of the biochemical properties of MC-GAG and subsequent intracellular signaling is particularly interesting as these are potentially tunable components. Particularly intriguing is the contribution of inorganic phosphate to MC-GAG activity, as phosphate has been described as essential for osteogenic differentiation. Sensing and transport of phosphate has been linked to the type III high affinity, sodium-dependent phosphate transporters, PiT-1 and PiT-2. In this work, we evaluated the contribution of phosphate signaling via PiT-1/2 on MC-GAG-induced osteogenesis. METHODS: Col-GAG and MC-GAG scaffolds were incubated cell-free or seeded with primary bone marrow-derived human mesenchymal stem cells (hMSC) in growth media. Phosphate concentrations in the growth media were measured at a 3-day interval for 21 days. hMSCs were cultured on Col-GAG and MC-GAG with growth media (baseline phosphate) or media supplemented with 10 mM β-glycerophosphate with or without phosphonoformic acid (PFA), an inhibitor of sodium phosphate cotransporters. hMSCs were transfected with small interfering RNAs (siRNAs) targeting PiT-1, PiT-2, and PiT-1/2, and scrambled control and were subsequently seeded onto Col-GAG and MC-GAG scaffolds. Osteogenic gene and intracellular mediator protein expression were measured using RT-PCR and western blot analyses, respectively. Mineralization was evaluated by Alizarin Red staining and micro-computed tomographic (CT) analyses. RESULTS: While Col-GAG maintained a soluble phosphate concentration identical to growth medium, MC-GAG exhibited a temporal relationship with soluble phosphate demonstrating elution early in culture shifting to absorption after 12 days with or without hMSCs. Unlike Col-GAG, MC-GAG induced mineralization of hMSCs in both growth medium and phosphate-supplemented medium, whereas Col-GAG required culture in phosphate-supplemented medium in order to display any mineralization. In the presence of PFA, mineralization was reduced across all scaffolds. hMSCs cultured on both scaffolds demonstrated an increased fold change in gene expression of PiT-1 and PiT-2 with no differences between the materials. For Col-GAG materials, siPiT-1 transfection demonstrated no effect on any of the osteogenic markers tested whereas both early (ALP and RUNX2) and late (BSP2 and OCN) osteogenic markers were reduced in hMSCs on MC-GAG. No additive effects were noted with siPiT-1/2 double knockdown. Mineralization on MC-GAG scaffolds was significantly reduced with knockdown of PiT-1, PiT-2, or both transporters, whereas no effects were evident on Col-GAG. CONCLUSIONS: The mineral content of MC-GAG alters phosphate concentrations within a local microenvironment resulting in osteogenic differentiation of progenitor cells via both PiT-1 and PiT-2 in a non-redundant, yet non-additive manner. This study contributes to the optimization of MC-GAG properties and design of second-generation scaffolds.