University of California Hillsborough, CA, United States
Thomas Ambrosi1, Mathew Murphy1, Charles KF Chan1 and Nancy Lane2, 1Stanford University, Stanford, CA, 2University of California Davis, Hillsborough, CA
Background/Purpose: Glucocorticoid (GC) induced osteoporosis remains a significant health issue. While GCs are known to reduce bone formation and bone mass, patients treated with GCs often fracture at normal bone mineral densities. To increase our understanding of GCs affects on bone cells, we performed a detailed analysis of the bone cells numbers, maturation activity and single-cell gene expression in mice treated with GCs, after recovery from GCs with and without PTH, or GC with PTH.
Methods: Balb/cJ male mice, age 9 weeks were randomized to placebo (PLB) for 28 or 56 days, GC (MP pellets of 2.5mg) for 28d, GC for 28d then recovery (29-56d) or GC for 56d, GC for 56d+ hPTH (1-34) 29d-56d or GC for 28d followed by hPTH (29-56d ). Outcome variables included measures of distal femur bone mass (microCT), bone strength, flow cytometry for skeletal stem cells (SSCs) , Bone/Cartilage progenitor cells (BCSPs), and vascular progenitors (CD31, Tie2); in vitro bone marrow cultures for osteogenesis, osteoclastogenesis and chondrogenesis, and scRNA sequencing.
Results: GCs reduced femoral bone mass and bone strength compared to PLB at day 28 and 56 (p< 0.05). The number of SSCs, BCSPs and vascular progenitor cells ( CD31+ and Tie2+) decreased with GC Rx at day 28 vs. PLB (p< 0,05), however returned to baseline levels by day 56 despite continued GCs, and with GC+ PTH or PTH alone (day 29-56). In vitro measures of osteogenesis and chondrogenesis at day 56 increased with GC+PTH, and GC followed by PTH vs. PLB day 56, while osteoclastogenesis was increased with GC only and GC followed by PTH compared to PLB at day 56 (p< 0.01) Single Cell RNA sequencing at day 56 showed GCs upregulated both endothelial (Fabp4, Flt1, Ahnak, Clic1,) and promyeloid (Ptg2, Lsp1, Syne1, Mmp9, Ccl4) signaling genes, while osteogenic (Prg4,Pthr1, Fn1, Alpl)) and chondrogenic (Acan, Col2a1, Sox9, Col9a1) genes were downregulated with GCs, however were increased with GC+ PTH and PTH after GCs.
Conclusion: GCs initially reduced the number of SSCs, BCSPs and vascular progenitors and with continued GC exposure there was some recovery of these precursor cells, however osteogenesis and to a lesser extent chondrogenesis remained inhibited. While PTH treatment had no effect on the endothelial cells, it overcame the GC induced inhibition of osteogenesis and to some extent chondrogenesis.
Disclosures: T. Ambrosi, None; M. Murphy, None; C. Chan, None; N. Lane, Amgen, GlaxoSmithKlein(GSK).
