Background: Equine osteoarthritis is a major cause of lameness in horses, directly reducing athletic performance and leading to significant economic losses in the equine industry. Studies have shown that platelet rich plasma (PRP) exhibited clinical benefits in equine patients with OA, but there is still lack of investigation on the effect of different types of PRP on OA. Objectives: This study aims to evaluate the effects of different concentrations and storage methods of PRP (fresh PRP and freeze-dried PRP) on alleviating inflammatory responses and regulating tissue metabolism in an equine osteoarthritis cartilage-synovial explant model. Study Design: An in vitro cartilage-synovial explant model was established to investigate osteoarthritis-related inflammatory responses and metabolic imbalances. Methods: Cartilage and synovium were co-cultured with 10 ng/mL interleukin-1β (IL-1β) for 48 hours to induce equine osteoarthritis. Fresh PRP and lyophilized PRP at concentrations of 25% and 50% were applied to the explants. Triamcinolone was applied as positive control. Inflammatory cytokines and metabolic pathways were analyzed, including nitric oxide levels and gene expressions of cyclooxygenase-2 (COX-2) and matrix metalloproteinase-13 (MMP-13). Results: The PRP treatment effectively suppressed inflammatory responses similar to triamcinolone, with fresh PRP demonstrating better inhibition of nitric oxide (NO) production. Metabolomic analysis revealed that both PRP storage forms could modulate glucose and purine metabolism, with fresh PRP exhibiting superior effects. The mean differences in inflammatory cytokines and metabolic pathways biomarkers were calculated with 95% confidence intervals. Main limitations: The study’s limitations include the model’s representativeness of natural complexity of osteoarthritis and the generalizability of findings to larger populations. Conclusions: The study demonstrated that both fresh and freeze-dried PRP possess significant anti-inflammatory effects and potential to regulate cartilage cell metabolism. Future research could further explore the mechanisms by which PRP influences metabolic pathways in equine osteoarthritis.