Cross species applicable antler stem cell-secreted proteins repair canine knee cartilage

Canine articular cartilage injury acts as the initiating factor or critical link in their high-incidence joint diseases, severely impairing their quality of life. As the only mammalian bony organ capable of complete regeneration, deer antler achieves cartilage and bone tissue regeneration/repair relying on deer antler stem cells (ASCs). Given that most of the core repair-related secreted proteins in mammals exhibit high evolutionary conservation, we hypothesize that deer antler stem cell-secreted proteins (ASC-SPs) may possess strong cross-species cartilage repair potential. In vitro, ASC-SPs were added to culture medium to determine effects on proliferation, migration, apoptosis and cartilage markers of Canine articular chondrocytes (CACCs). In vivo, to reduce companion animal use, we first compared three ASC-SPs delivery methods (intra-articular injection, F127 hydrogel combined with ASC-SPs filling, intravenous injection) in rat cartilage injury model. Then, we selected the optimal method, and validated it in Beagles. Finally, ASC-SPs composition and mechanism were analyzed via proteomic sequencing. In vitro, ASC-SPs significantly enhanced proliferation and migration of CACCs, inhibited serum-free culture-induced CACCs apoptosis ( P < 0.001). It also upregulated mRNA and protein expressions of Collagen II, SOX9, and PCNA in CACCs ( P < 0.001). In the rat cartilage injury model, intra-articular injection showed the best therapeutic effect, with higher closure rate and better histological score compared with F127 hydrogel filling ( P < 0.05), while intravenous injection had no therapeutic effect ( P < 0.05). The intra-articular injection of ASC-SPs also significantly improved Beagle cartilage defect closure ( P < 0.001) and tissue repair ( P < 0.01). Proteomic sequencing indicated ITGB1 may interact with TGF-β1 and Collagen II via the TGF-β pathway to mediate repair. This study confirms that ASC-SPs can promote the proliferation, migration of CACCs and inhibit their apoptosis. Intra-articular injection of ASC-SPs exerts significant repair effects on canine articular cartilage defect. Protein sequencing reveals that the repair effect may be mediated through the ITGB1/TGF-β pathway. Notably, consistent efficacy in rat and canine models verifies the cross-species applicability of ASC-SPs.