Mechanistic Insights into Milk Minerals Driving Bone Development and Mineralization in Growing Rats

Objective: To investigate the effect of milk minerals on bone mineral density (BMD) and bone quality in growing rats and explore the underlying mechanisms related to calcium absorption, bone metabolism, and the gut–bone axis. Methods: Sixty healthy 4-week-old male Sprague-Dawley (SD) rats were randomly divided to five groups based on their body weight: Low-Calcium Control Group (Control), Low-Dose milk mineral Group (Low), Medium-Dose milk mineral Group (Medium), High-Dose milk mineral Group (High) and Calcium Carbonate Control Group (CaCO3), which received the same dose level (elemental calcium) as the High group. The milk mineral dosage was set at 5, 10, and 15 times the human recommended intake of elemental calcium. After 12 weeks of intervention, femurs were collected for analysis of BMD, bone microstructure, and bone mechanical strength. Additionally, analyses included calcium levels in the femur, feces, and diet; serum bone metabolism biomarkers; tissue protein expression; as well as gut microbiota composition and short-chain fatty acid content. Result: Milk mineral exhibited non-inferior efficacy to CaCO3 in increasing femoral calcium content, enhancing BMD, and improving bone microarchitecture. Notably, the Medium group achieved comparable bone-protective effects to the CaCO3 group despite a 20.8% lower calcium content, which was accompanied by a relatively high calcium absorption rate (90.9% vs. 86.5%). With respect to serum markers, milk mineral maintained bone formation while suppressing bone resorption, resulting in a net anabolic state comparable to that of CaCO3. Milk mineral significantly upregulated the protein expression of renal CYP27B1 and intestinal calcium ion transporters, and increased serum IGF-I levels. Furthermore, milk mineral promoted the enrichment of certain specific gut microbial genera, which showed a significant positive correlation with IGF-I, bone calcium content and BMD. Conclusions: Milk mineral supplementation appears to promote bone formation and mineralization in growing rats, accompanied by enhanced intestinal calcium absorption, enrichment of characteristic gut microbes and elevated microbial metabolite concentrations.