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Eggshell membrane (ESM) is a rich source of bioactive compounds, including proteins, peptides, and antioxidants, contributing to its potential therapeutic benefits. These natural antioxidants might help neutralize reactive oxygen species (ROS) and modulate inflammatory responses, which are often linked with chondrocyte damage in osteoarthritis. In this study, we investigated the functional effects of ESM proteins on H2O2-induced oxidative stress in a neonatal canine chondrocytes. The isolated neonatal chondrocytes demonstrated a high proliferation rate and increased glycosaminoglycan (GAG) production during cultivation. In addition, the expression of key cartilage markers, including collagen types II and IX, and aggrecan, confirmed the retention of the chondrocyte phenotype. Under in vitro conditions, post-treatment with ESM improved chondrocyte viability, indicating that ESM may have a reparative role in mitigating oxidative damage. This significant therapeutic potential was validated through XTT assays, which measured cell metabolic activity at 24 h, and Real-time Cell Analysis (RTCA), providing continuous monitoring over 98 h. In contrast, the preventive effects of ESM against stress were observed exclusively in the XTT analysis. By investigating these aspects, we provide insight into the potential of ESM proteins to protect chondrocytes from oxidative damage, particularly in cartilage repair and joint health. This study is one of the first to create a vital platform based on canine neonatal chondrocytes for monitoring dietary supplements designed to prevent or repair dog cartilage damage. Thus, the study offers a valuable contribution to understanding how ESM bioactive compounds can be used therapeutically, bridging the gap between in vitro findings and practical applications in veterinary medicine. [ABSTRACT FROM AUTHOR]

Simple Summary: Osteoarthritis is a degenerative disease of synovial joints characterized by joint inflammation, damage of articular cartilage, and degenerative changes in periarticular tissues. The onset of lameness, pain, and joint stiffness are usually the first clinical signs observed by the owner. In recent years, many conventional therapies (NSAIDs, opioids, paracetamol, and gabapentinoids) for osteoarthritis have been described. Due to the complex mechanism of pain, multimodal analgesia in OA treatment is necessary. Unfortunately, conventional therapy does not offer the regenerative abilities of damaged tissue. However, mesenchymal stem cells have a definite advantage in chondrogenic, immunomodulatory, anti-apoptotic, and anti-fibrotic effects in joint environments in contrast to conventional therapies. The aim of the study was to investigate the potential of cell-based regenerative therapy for elbow joints affected by osteoarthritis. Interest was focused on two intra-articular applications of amnion-derived mesenchymal stem cells (A-MSCs) to a group of different breeds of dogs with elbow osteoarthritis (13 joints). Two injections were performed 14 days apart. We evaluated synovial fluid biomarkers, such as IFN-γ, IL-6, IL-15, IL-10, MCP-1, TNF-α, and GM-CSF, by multiplex fluorescent micro-bead immunoassay in the treated group of elbows (n = 13) (day 0, day 14, and day 28) and in the control group of elbows (n = 9). Kinematic gait analysis determined the joint range of motion (ROM) before and after each A-MSCs application. Kinematic gait analysis was performed on day 0, day 14, and day 28. Kinematic gait analysis pointed out improvement in the average range of motion of elbow joints from day 0 (38.45 ± 5.74°), day 14 (41.7 ± 6.04°), and day 28 (44.78 ± 4.69°) with statistical significance (p < 0.05) in nine elbows. Correlation analyses proved statistical significance (p < 0.05) in associations between ROM (day 0, day 14, and day 28) and IFN-γ, IL-6, IL-15, MCP-1, TNF-α, and GM-CSF concentrations (day 0, day 14, and day 28). IFN-γ, IL-6, IL-15, MCP-1, GM-CSF, and TNF- α showed negative correlation with ROM at day 0, day 14, and day 28, while IL-10 demonstrated positive correlation with ROM. As a consequence of A-MSC application to the elbow joint, we detected a statistically significant (p < 0.05) decrease in concentration levels between day 0 and day 28 for IFN-γ, IL-6, and TNF-α and statistically significant increase for IL-10. Statistical significance (p < 0.05) was detected in TNF-α, IFN-γ, and GM-CSF concentrations between day 14 and the control group as well as at day 28 and the control group. IL-6 concentrations showed statistical significance (p < 0.05) between day 14 and the control group. [ABSTRACT FROM AUTHOR]