Thaler, Roman

AimsTo characterize the effects of varying experimental parameters of transforming growth factor-beta 1 (TGF-β1) in primary knee fibroblasts as an in vitro model of arthrofibrosis.MethodsPrimary knee fibroblasts were isolated from one patient undergoing primary total knee arthroplasty (TKA) and one patient undergoing revision TKA for arthrofibrosis, respectively. Fibroblasts were stimulated with varying concentrations and durations of the pro-fibrotic cytokine TGF-β1. Picrosirius red staining (PSR) was conducted to assess collagen deposition, and real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine cellular gene expression levels. A collagen gel contraction assay was used to assess TGF-β1-induced fibroblast contraction.ResultsIn our experiments, TGF-β1-mediated collagen deposition was consistent across a vast concentration range (0.5 to 40 ng/ml). While α-smooth muscle actin (ACTA2) protein levels and SMAD2 phosphorylation were induced by low concentrations (1 ng/ml), robust ACTA2 transcription required higher concentrations (5 ng/ml) of TGF-β1. Evaluation of TGF-β1-mediated cell and cytoskeletal contractility shows that in the measured fibroblasts, this process occurs within three hours following TGF-β1 administration. Of note, TGF-β1-differentiated myofibroblasts exhibited greater contractile properties than naïve fibroblasts in the presence of TGF-β1.ConclusionTaken together, this study elucidates key TGF-β1 experimental parameters that will inform the development and interpretation of in vitro models of arthrofibrosis.Cite this article: Bone Joint Res 2025;14(6):527–538.

AimsTo evaluate the role of autophagy in primary knee fibroblasts undergoing myofibroblast differentiation as an in vitro model of arthrofibrosis, a complication after total knee arthroplasty characterized by aberrant intra-articular scar tissue formation and limited range of motion.MethodsWe conducted a therapeutic screen of autophagic-modulating therapies in primary human knee fibroblasts undergoing transforming growth factor-beta 1 (TGF-β1)-mediated myofibroblast differentiation. Autophagy was induced pharmacologically with rapamycin or by amino acid deprivation. Picrosirius red staining was performed to quantify collagen deposition. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were conducted to evaluate fibrotic gene expression levels.ResultsRapamycin, an mTOR complex 1 (mTORC1) inhibitor and autophagy inducer, reduced TGF-β1-mediated collagen deposition. Interestingly, we simultaneously report that myofibrogenic genes, including ACTA2, were highly upregulated following rapamycin-TGF-β1 treatment. When autophagy was induced through amino acid deprivation, we demonstrated suppressed extracellular matrix levels, fibrotic gene expression (e.g. ACTA2), and SMAD2 phosphorylation levels in TGF-β1-stimulated fibroblasts.ConclusionOur findings demonstrate that the induction of cellular autophagy suppresses TGF-β1-induced collagen deposition in primary human knee fibroblasts. Taken together, these data suggest that cellular autophagy may be prophylactic against the pathogenesis of arthrofibrosis.Cite this article: Bone Joint Res 2025;14(4):328–337.

AbstractOsteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone‐related genes, including those encoding extracellular matrix proteins or factors that control cell‐cell, and cell‐matrix interactions. Cell‐cell communication in the many skeletal pericellular micro‐niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by “A Disintegrin And Metalloproteinase” (ADAM) proteins. These cell membrane‐associated metalloproteinases support proteolytic release (“shedding”) of protein ectodomains residing at the cell surface. We analyzed microarray and RNA‐sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα‐signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (−0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic‐nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2‐Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Statins and bisphosphonates are increasingly recognized as anti-cancer drugs, especially because of their cholesterol-lowering properties. However, these drugs act differently on various types of cancers. Thus, the aim of this study was to compare the effects of statins and bisphosphonates on the metabolism (NADP+/NADPH-relation) of highly proliferative tumor cell lines from different origins (PC-3 prostate carcinoma, MDA-MB-231 breast cancer, U-2 OS osteosarcoma) versus cells with a slower proliferation rate like MG-63 osteosarcoma cells. Global gene expression analysis revealed that after 6 days of treatment with pharmacologic doses of the statin simvastatin and of the bisphosphonate ibandronate, simvastatin regulated more than twice as many genes as ibandronate, including many genes associated with cell cycle progression. Upregulation of starvation-markers and a reduction of metabolism and associated NADPH production, an increase in autophagy, and a concomitant downregulation of H3K27 methylation was most significant in the fast-growing cancer cell lines. This study provides possible explanations for clinical observations indicating a higher sensitivity of rapidly proliferating tumors to statins and bisphosphonates.