Differential Effects of Collagen Hydrolysate Digesta on Osteoclast and Osteoblast Responses In Vitro

Background/Objectives: Osteoarthritis (OA) is partly driven by an imbalance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs). Collagen hydrolysates (CHs) are widely used for OA symptom management, with potential benefits attributed to bioactive peptides (BAPs) released during digestion. However, their direct effects on bone cells remain unclear. Methods: Two bovine-sourced CHs, CH-GL and CH-OPT, were digested in vitro and tested on primary murine OC and OB cultures. Osteoclastogenesis was evaluated mainly under standard RANKL conditions (50 ng/mL), with exploratory analysis under high RANKL conditions (100 ng/mL). OC cultures were exposed to CH digesta at 0.01, 0.05, 0.1, or 0.5 mg/mL, while OB cultures were exposed to 0.01 or 0.1 mg/mL. Results: Under standard RANKL conditions, CH-GL significantly reduced OC area by over 50% across all tested concentrations, while OC number was not significantly altered. This coincided with decreased expression of selected osteoclastogenic markers, including Nfatc1, Ctsk, Dc-stamp, and Oscar. In OB cultures, CH-GL increased Runx2, Osterix, and Alp expression at 0.1 mg/mL and reduced Mmp9 expression at both tested doses. It also modestly increased mineralization-related signal intensity and collagen-associated matrix area. For CH-OPT, selected reductions in Dc-stamp and Oscar expression were observed, while OC number, OC area, and most OB-related outcomes were not significantly altered. Conclusions: CH digesta modulated selected OC- and OB-related outcomes in primary murine cells. These preliminary in vitro findings support further investigation of CH-derived bioactives in models that more closely reproduce OA-associated joint pathology.