Biology subjects

Haeckel, S.

Publications and source records attributed to Haeckel, S..

3 recordsLinked to original sources

bioRxiv2026

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

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.

cell biology↗

bioRxiv2026

Dexamethasone-induced PPARG expression in osteogenic differentiation in vitro: impact on SOX9 and RUNX2 levels.

BackgroundThe effects of dexamethasone during in vitro human osteogenesis present a complex picture. On one side, dexamethasone promotes the osteogenic differentiation of human bone marrow mesenchymal stromal cells (BMSCs) by downregulating SOX9. On the other side, it simultaneously promotes adipogenesis through the upregulation of PPARG. The regulation of SOX9 and PPARG levels appears to be mediated by the transactivation function of the glucocorticoid receptor (GR), suggesting an indirect effect of dexamethasone on SOX9 downregulation. This study aims to determine whether PPAR-{gamma} affects the expression levels of SOX9, as suggested by several studies. MethodsHuman BMSCs were isolated from bone marrow and cultured in different osteogenic induction media containing 10 or 100 nM dexamethasone. Undifferentiated cells were used as control. Cells were treated either with a pharmacological PPAR-{gamma} inhibitor (T0070907) or with a PPARG-targeting siRNA. Differentiation markers or PPAR-{gamma} target genes were analysed by RT-qPCR. Mineral deposition was assessed by Alizarin Red staining. Two-way ANOVA followed by a Sidak multiple comparison test was used to compare the effects of treatments. ResultsPharmacological inhibition of PPAR-{gamma} had a mild effect on the expression of PPAR-{gamma} target genes but hindered adipocyte formation. Neither RUNX2 nor SOX9 expression were affected by T0070907. siRNA treatment successfully downregulated PPARG expression, as well as that of PPAR-{gamma} target genes LPL, LPAR1, and ADIPOQ. Contrary to expectations, RUNX2 was significantly downregulated by the PPARG-siRNA treatment during osteogenic differentiation both in the absence and presence of dexamethasone, while SOX9 levels were downregulated in undifferentiated cells. Overall, Alizarin Red staining analysis showed no change in mineralization levels when PPARG expression or activity was inhibited. ConclusionsUnderstanding how dexamethasone regulates human BMSC differentiation is crucial to refine current in vitro models. These results suggest that PPAR-{gamma} is not involved in SOX9 or RUNX2 repression during in vitro osteogenic differentiation of human cells.

cell biology↗

bioRxiv2021

Biofabrication of development-inspired scaffolds for regeneration of the annulus fibrosus macro- and microarchitecture

Annulus fibrosus (AF) tissue engineering is a promising strategy for repairing the degenerated intervertebral disc (IVD) and a research area that could benefit from improved tissue models to drive translation. AF tissue is composed of concentric layers of aligned collagen bundles arranged in an angle-ply pattern, an architecture which is challenging to recapitulate with current scaffold design strategies. In response to this need, we developed a strategy to print 3D scaffolds that induce cell and tissue organization into oriented patterns mimicking the AF. Polycaprolactone (PCL) was printed in an angle-ply macroarchitecture possessing microscale aligned topographical cues. The topography was achieved by extrusion through custom-designed printer nozzles which were either round or possessing circumferential sinusoidal peaks. Whereas the round nozzle produced extruded filaments with a slight uniaxial texture, patterned nozzles with peak heights of 60 or 120 m produced grooves, 10.87 {+/-} 3.09 m or 17.77 {+/-} 4.91 m wide, respectively. Bone marrow derived mesenchymal stem cells (BM-MSCs) cultured on the scaffolds for four weeks exhibited similar degrees of alignment within {+/-} 10 {degrees} of the printing direction and upregulation of outer AF markers (COL1, COL12, SFRP, MKX, MCAM, SCX and TAGLN), with no statistically significant differences as a function of topography. Interestingly, the grooves generated by the patterned nozzles induced longitudinal end-to-end alignment of cells, capturing the arrangement of cells during fibrillogenesis. In contrast, topography produced from the round nozzle induced a continuous web of elongated cells without end-to-end alignment. Extracellular collagen I, decorin and fibromodulin were detected in patterns closely following cellular organization. Taken together, we present a single-step biofabrication strategy to induce anisotropic cellular alignments in x-, y-, and z-space, with potential application as an in vitro model for studying AF tissue morphogenesis and growth.

bioengineering↗