Osteoarthritis is a major cause of disability and has no cure. Loss of cartilage is a key driver of this disease, making cartilage regeneration an attractive therapeutic strategy. Now, two papers in Science Translational Medicine from the laboratory of Francesco Dell’Accio and collaborators highlight blockade of receptor tyrosine kinase-like orphan receptor 2 (ROR2) and administration of agrin as two potential approaches to promote cartilage formation.

Cartilage is made by cells called chondrocytes, which secrete and become embedded in a dense extracellular matrix. Unlike bone, cartilage has a low turnover and often fails to repair after injury.

In their recent study, Thorup et al. focused on ROR2 as this receptor is known to have a central developmental role in cartilage. In mice, osteoarthritis induced by joint destabilization led to ROR2 upregulation in cartilage 1 week later. Moreover, microarray analysis of patient samples revealed increased levels of ROR2 signalling mediators in osteoarthritis versus controls.

In a mesenchymal cell line, overexpression of ROR2 inhibited chondrocyte differentiation in response to bone morphogenetic protein 2 (BMP2), whereas RNA-mediated ROR2 silencing promoted chondrocyte differentiation, even in the absence of BMP2. Mechanistic studies showed that the chondrogenic effects of ROR2 inhibition were mediated by suppression of yes-associated protein (YAP) signalling.

To test the efficacy of ROR2 silencing in vivo, the researchers conjugated ROR2-targeted siRNA (ROR2i) to an atelocollagen gel to enable delivery to cartilage, which is avascular and highly negatively charged. They performed intra-articular injection of the ROR2i preparation every 5 days, 2 weeks after menisco-ligament injury in mice. After 5 weeks, unlike control mice, ROR2i-treated mice did not develop pain, as measured by weight loading across limbs, and had reduced structural damage to cartilage.

Next, Thorup et al. sought to investigate whether the findings in mice would translate to the human setting. They transduced human articular chondrocytes with lentivirus ROR2-shRNA and subcutaneously implanted these cells in nude mice. The resultant cartilage organoids were found to be more differentiated than those formed from chondrocytes transduced with control shRNA.

In the other study, Eldridge et al. found that the signalling proteoglycan agrin is upregulated after mechanical injury to human articular cartilage explants and after exposure of human chondrocytes to inflammatory cytokines associated with injury. Overexpression of agrin in mesenchymal stem cells that normally reside in the joint promoted differentiation into chondrocytes and increased cartilage production — but, interestingly, did not have this effect on bone marrow-derived stem cells.

Canonical WNT/β-catenin signalling is known to suppress chondrogenesis. In cell culture, Eldridge et al. showed that agrin activates chondrogenesis by inhibiting canonical WNT signalling, and this process involved CREB-dependent transcription.

To test the therapeutic potential of agrin in vivo, the researchers performed surgical injury to the knees of mice and sheep, followed by a single intra-articular injection of a collagen gel containing either human agrin or GFP. After 8 weeks, cartilage regeneration and the size of the residual bone defect were significantly better in agrin-treated mice than in control-treated mice. In sheep, after 6 months, the agrin group had improved joint repair and spent more time playing and less time resting than the control sheep.

The authors note that unlike previous attempts to target specific signalling pathways involved in joint patterning, which led to ectopic cartilage and bone formation, exogenous agrin reinitiates morphogenesis and preserves the architecture of the native tissue.

The researchers are working to optimize ROR2-targeted RNA and a smaller, soluble agrin mutant for therapeutic use and are seeking industry partners to advance these strategies into clinical testing.