Combining Collagenase Injections with Exercise-Induced Mechanical Overload: A Mouse Model Simulating Overload Stress Injury Knee Osteoarthritis.

Knee osteoarthritis (KOA) is a multifactorial degenerative joint disease driven by the complex interplay of biological tissue degradation and sustained mechanical overload. conventional surgical animal models often induce trigger post-traumatic joint destruction, which fails to accurately replicate the gradual and cumulative pathogenesis of human degenerative KOA. To bridge this translational gap, the present study establishes a novel and reproducible mouse model simulating overload stress-induced KOA by synergizing mild enzymatic cartilage degradation with sustained mechanical loading. Specifically, standardized intra-articular injections of type II collagenase were paired with daily mechanical overexertion via a strictly calibrated rotator-type fatigue apparatus over a four-week period. The efficacy of this dual-factor intervention was validated using comprehensive behavioral, histological, and molecular analyses. Automated gait analysis revealed severe locomotor deficits in the combined group. These were characterized by significant reductions in stride length, stride width, and peak stance paw area. Histomorphological evaluations using safranin O-fast green staining confirmed these functional deficits, a showing progressive cartilage structural damage, surface defects, and significantly elevated Osteoarthritis Research Society International (OARSI) scores that faithfully mirror the pathological hallmarks of early-to-mid stage human KOA. Furthermore, immunohistochemical evaluations demonstrated marked upregulation of the mechanosensitive channel Transient Receptor Potential Vanilloid 4 (TRPV4) and concurrent reduction in the crucial matrix protein Collagen Type II Alpha 1 (COL2A1) in articular chondrocytes. By integrating localized joint instability with controlled mechanical stress, this minimally invasive strategy avoids the acute trauma associated with surgical models. This protocol establishes a clinically relevant experimental platform for tracking dynamic molecular mechanisms, evaluating chronic pain phenotypes, and testing long-term therapeutic interventions for early-stage osteoarthritis.