The authors regret that the original data availability statement of our manuscript was not included in the final submission and this oversight was not identified during proofreading. In the context of open-access publication, it is essential that the correct data availability information is provided. The correct statement is as follows: The data generated and analysed during the current study can be found in the Supplementary Data file with the exception of the single cell RNA sequencing data. The count matrices derived from the raw single cell RNA sequencing data have been uploaded to Gene Expression Omnibus under accession number GSE290973. Due to privacy considerations, the raw sequencing files have not been deposited in the Gene Expression Omnibus but can be obtained from the corresponding author upon reasonable request and completion of a Data Use Agreement. The authors would like to apologise for any inconvenience caused.

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.

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with β-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-β for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

Antisense oligonucleotides (ASOs) carry an enormous therapeutic potential in different research areas, however, the lack of appropriate carriers for their delivery to the target tissues is hampering their clinical translation. The present study investigates the application of novel biomimetic nano-vesicles, Nano-Ghosts (NGs), for the delivery of ASOs to human mesenchymal stem cells (MSCs), using a microRNA inhibitor (antimiR) against miR-221 as proof-of-concept. The integration of this approach with a hyaluronic acid-fibrin (HA-FB) hydrogel scaffold is also studied, thus expanding the potential of NGs applications in regenerative medicine. The study shows robust antimiR encapsulation in the NGs using electroporation and the NGs ability to be internalized in MSCs and to deliver their cargo while avoiding endo-lysosomal degradation. This leads to rapid and strong knock-down of miR-221 in hMSCs in vitro, both in 2D and 3D hydrogel culture conditions (>90% and > 80% silencing efficiency, respectively). Finally, in vivo studies performed with an osteochondral defect model demonstrate the NGs ability to effectively deliver antimiR to endogenous cells. Altogether, these results prove that the NGs can operate as stand-alone system or as integrated platform in combination with scaffolds for the delivery of ASOs for a wide range of applications in drug delivery and regenerative medicine.