22. The Role of Orai1 in Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

• DOI: 10.1097/01.gox.0000937684.46113.c4
• Publication Date: 2023-05-18T20:02:17Z
• Authors: Meiwand Bedar; Xiaoyan Ren; Wei Chen; Kelly X. Huang; Shahrzad Moghadam; Jonnby LaGuardia; Justine C. Lee
• Keywords: Orai1

PURPOSE: Development of synthetic regenerative materials inspired by the extracellular matrix (ECM) has been a topic significant interest due to known properties ECM direct cell fate determination via tunable material composition and biomechanical properties. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is low density, open-cell foam that evaluated in our laboratory induce osteogenic differentiation primary osteoprogenitors vitro as well regeneration rabbit skull defects vivo without pre-seeding with progenitor cells or addition exogenous growth factors, which was not observed non-mineralized (Col-GAG) materials. Given critical difference between MC-GAG Col-GAG mineral content, spatial concentration inorganic ions on may be its This supported prior research demonstrating high calcium concentrations bone microenvironment stimulate osteoblastic differentiation. How these biochemical cues are transmitted activate downstream signaling pathways should further elucidated. Orai1 subunit store-operated release-activated channels (CRAC) implicated involved homeostasis. In this work, we contribution MC-GAG-mediated osteogenesis. METHODS: To examine role through channels, such Orai1, marrow-derived human mesenchymal stem (hMSCs), treated 2D cultures small molecule inhibitor entry (SOCE), MRS1845, for 14 days. hMSCs were subsequently seeded 7 days MRS1845. Baseline gene protein expression evaluated. Next, interfering RNAs (siRNAs) targeting ORAI1 scrambled control used specifically knockdown cultured MC-GAG. Osteogenic measured using quantitative RT-PCR western blot analyses, respectively. Mineralization Alizarin Red staining. RESULTS: understand whether essential differentiation, SOCE, found reduction depositions visualized staining when compared untreated hMSCs. 3D cultures, MRS1845 downregulated early marker ALP intracellular mediator Runx2 controls. As inhibitors lack channel specificity, knocked down siRNA transfection. When Col-GAG, baseline 1.8-fold higher (p=0.02) upregulated (p=0.003). reduced late BSP2 siRNA. significantly diminished knockdown, whereas no effects evident demonstrated CONCLUSIONS: ORAI1-mediated ion required MC-GAG-induced Further elucidation mechanisms refinement levels facilitate clinical application reconstruction osseous defects.