WNT3A‐loaded exosomes enable cartilage repair

Abstract

Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L-cells overexpressing WNT3a or control un-transduced L-cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B-GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects. K E Y WO R D S cartilage, drug delivery, exosomes, joint repair, WNT3a  INTRODUCTIONCartilage defects are present in 61% of patients undergoing knee arthroscopy (Curl et al., 1997;Hjelle et al., 2002), can be disabling, and have the potential to progress into osteoarthritis (Dell'accio & Vincent, 2010). Cell based approaches to cartilage repair are effective, but, due to the largely autologous nature of the transplanted cells, such procedures are costly and laborious. A number of novel biological agents are being developed which harness and support the endogenous capacity of joint to repair (Sherwood, 2019). One major limitation in delivering such agents (often macromolecules) is the avascular nature of the cartilage tissue and the dense and strongly negatively charged extracellular matrix. Antibodies only penetrate the very superficial layersThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Wnt3a knockdown promotes collagen type II expression in rat chondrocytes

“…In in vivo experiments, Bertrand et al ( 35 ) came to the same conclusion that Wnt3a can modulate cartilage matrix maker genes (Sox9, Col2 and ACAN) through LRP6 phosphorylation and stabilization of β-catenin. In addition, Thomas et al ( 36 ) reported that Wnt3a-loaded exosomes can promote cartilage repair in the knees of mice. These exosomes may regulate circumarticular synovial tissues in addition to cartilage.…”

Exosome-based strategy for degenerative disease in orthopedics: Recent progress and perspectives

“…As WNT3a has a high affinity with exosomes, exosomes from L-cells (ATCC CRL2647/CRL2648; American Type Culture Collection, Manassas, VA, USA) were employed to deliver WNT3a to prolong the effect and were injected into mouse joints to repair large osteochondral defects. After 8 weeks, WNT3a-loaded MSC-Exos had successfully improved osteochondral defect repair via WNT signalling activation, while WNT3a without vehicle failed to activate WNT signalling to promote cartilage regeneration [ 56 ]. Overall, exosomes are potential vehicles with good cartilage penetration and cargo preservation, promising the delivery of proteins and small molecules, especially for hydrophobic substances.…”

Mesenchymal Stem Cell-Derived Exosomes and MicroRNAs in Cartilage Regeneration: Biogenesis, Efficacy, miRNA Enrichment and Delivery

“…Due to this, the exosomes were administered weekly. Another study conducted by Thomas and colleagues was in favor of the use of semi-solid formulation to deliver the exosomes [193]. To demonstrate cartilage repair in C57BL/6 mice, exosomes were dispersed in liquid rat type I collagen gel.…”