Osteoarthritis (OA) is a common disabling disease for which no effective pharmacological therapy exists. The progression of osteoarthritis is characterized by the loss of homeostasis in the cartilage. Since in the early stages of the disease, a phenotypic switch is seen in which articular chondrocytes become hypertrophic and promote degradation of the cartilage extracellular matrix, targeting this phenomenon might be the key to developing an effective therapy. To accelerate the identification of potential therapy, drug repurposing strategies are used. In this study we have used a novel approach by combining this with the signature reversing principle on single cell transcriptomics data aimed to reverse the hypertrophic phenotype of chondrocytes in osteoarthritic cartilage of patients. We identified 6 drugs predicted to reverse the hypertrophic phenotype of chondrocytes. Subsequent in vitro evaluation in human chondrocytes and cartilage explants demonstrated that Cobimetinib, a MEK1/2 inhibitor, indeed reduced chondrocyte hypertrophy-related and catabolic gene expression, such as SPP1 , COL10A1 , MMP13 and ADAMTS5 , while promoting collagen type 2 and aggrecan gene expression. Finally, single-cell RNA sequencing performed on osteoarthritic cartilage explants exposed to Cobimetinib ex vivo confirmed the anti-hypertrophic effect of the identified drug on hypertrophy-related gene expression and velocity analysis shows that cells are diverting toward a homeostatic cartilage cluster. This study is a proof of concept that open-access single cell omics data together with a drug repurposing strategy can identify drugs that target a specific cellular phenotype in diseases like osteoarthritis and could accelerate the drug discovery process.

A functional vascular system is a prerequisite for bone repair as disturbed angiogenesis often causes non-union. Paracrine factors released from human bone marrow derived mesenchymal stromal cells (BMSCs) have angiogenic effects on endothelial cells. However, whether these paracrine factors participate in blood flow dynamics within bone capillaries remains poorly understood. Here, we used two different microfluidic designs to investigate critical steps during angiogenesis and found pronounced effects of endothelial cell proliferation as well as chemotactic and mechanotactic migration induced by BMSC conditioned medium (CM). The application of BMSC-CM in dynamic cultures demonstrates that bioactive factors in combination with fluidic flow-induced biomechanical signals significantly enhanced endothelial cell migration. Transcriptional analyses of endothelial cells demonstrate the induction of a unique gene expression profile related to tricarboxylic acid cycle and energy metabolism by the combination of BMSC-CM factors and shear stress, which opens an interesting avenue to explore during fracture healing. Our results stress the importance of in vivo - like microenvironments simultaneously including biochemical, biomechanical and oxygen levels when investigating key events during vessel repair. Statement of significance: Our results demonstrate the importance of recapitulating in vivo - like microenvironments when investigating key events during vessel repair. Endothelial cells exhibit enhanced angiogenesis characteristics when simultaneous exposing them to hMSC-CM, mechanical forces and biochemical signals simultaneously. The improved angiogenesis may not only result from the direct effect of growth factors, but also by reprogramming of endothelial cell metabolism. Moreover, with this model we demonstrated a synergistic impact of mechanical forces and biochemical factors on endothelial cell behavior and the expression of genes involved in the TCA cycle and energy metabolism, which opens an interesting new avenue to stimulate angiogenesis during fracture healing.

Enterococciwere detected occasionally in 100 L samples ofwater abstracted froma shallowaquifer in a natural dune infiltration area for drinking water production. Enterococcus moraviensis was the species most frequently identified in these samples. Because there are no existing reports of faecal sources of E. moraviensis and the closely related E. hemoperoxidus, this study aimed to find such sources of these two species in the dunes. Faecal samples from various animal species living in the vicinity of abstraction wells, were analysed for enterococci on Slanetz and Bartley Agar. From these samples, enterococci isolates (1,386 in total) were subsequently identified using matrix assisted laser desorption ionizationtimeof flight (MALDI-TOF)mass spectrometry. E.moraviensiswas found in the faeces of geese, foxes and rabbits. Also, E. haemoperoxidus was isolated from goose faeces. Using hierarchical clustering, the species composition of Enterococcus spp. isolated from abstracted water formed one cluster with the species composition found in geese droppings. A sanitary survey supported the indication that feral geese may provide a substantial faecal load in particular parts of this dune infiltration area, close to the water abstraction system. This study confirms the faecal origin of E.moraviensis and E. haemoperoxidus from specific animals, which strengthens their significance as faecal indicators.