Modulation of ossification and inflammatory pathways during dexamethasone-induced in vitro osteogenesis

Buetti-Dinh, A.; Siverino, C.; Ubeda Garrido, J.; Lanzillotti, C.; Pianta, E.; Grasso, G.; Haeckel, S.; Stoddart, M. J.; Della Bella, E.

doi:10.64898/2026.02.12.705508

bioRxiv · 10.64898/2026.02.12.705508

Modulation of ossification and inflammatory pathways during dexamethasone-induced in vitro osteogenesis

Buetti-Dinh, A.·Siverino, C.·Ubeda Garrido, J.·Lanzillotti, C.·Pianta, E.·Grasso, G.·Haeckel, S.·Stoddart, M. J.·Della Bella, E.

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Abstract

BackgroundDexamethasone (DEX) is used in vitro to promote osteogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). In clinical use, however, glucocorticoids induce osteoblast and osteocyte apoptosis while increasing osteoclast survival, leading overall to osteoporosis and high fracture risk. The overall impact of DEX on the differentiation of human progenitor cells remains contradictory and not fully understood, highlighting the need for further investigation using sequencing approaches as in vitro results will naturally influence further translational research. MethodshBMSCs were induced to osteogenic differentiation for 7 days using different concentrations of either DEX or the nonsteroidal glucocorticoid receptor agonist (+)-ZK216348. cDNA library preparation and RNA sequencing (RNAseq) were performed using Oxford Nanopore Technologies. Differentially expressed genes and pathways associated to the transactivation or transrepression activity of DEX were identified. Sequencing results were validated by qPCR, protein analysis, and with a functional assay on peripheral blood mononuclear cells to determine the overall effect of the BMSC supernatant. ResultsHierarchical clustering of RNAseq data identified eight subclusters with shared regulatory patterns. Enrichment analysis revealed that both upregulated and downregulated genes are involved in ossification and extracellular matrix organization pathways. Several pro- and anti-inflammatory genes were differentially regulated. qPCR analysis validated the upregulation of CXCL1, CXCL8, IL18, and COL8A1, while MMP1 and CXCL12 expression decreased in response to DEX. Comparing DEX results with those obtained using (+)-ZK216348 helped distinguish the potential mechanisms regulating the expression of specific genes. Notably, CXCL8 upregulation occurred through transactivation, whereas COL8A1 upregulation is downstream of a transrepressed gene. Further in vitro experiments confirmed that DEX significantly increased CXCL8 expression and IL-8 secretion. However, hPBMC responses indicated no significant pro- or anti-inflammatory effects from hBMSC conditioned medium. ConclusionsIn conclusion, the effects of DEX on the transcriptome of hBMSCs in a pro-osteogenic environment do not fully replicate the acquisition of an osteogenic phenotype. Several genes associated with ossification, extracellular matrix organization, and inflammation were dysregulated. The unique expression patterns of pro-inflammatory cytokines and collagen types warrant further investigation to elucidate their roles in osteogenic differentiation and bone homeostasis.

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Buetti-Dinh, A., Siverino, C., Ubeda Garrido, J., Lanzillotti, C., Pianta, E., Grasso, G., Haeckel, S., Stoddart, M. J., Della Bella, E.. 2026-02-13. Modulation of ossification and inflammatory pathways during dexamethasone-induced in vitro osteogenesis. https://doi.org/10.64898/2026.02.12.705508

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