Effects of nutritionally relevant milk basic protein supplementation during zoledronate treatment in ovariectomized rats

Bisphosphonates such as zoledronate are widely used for osteoporosis treatment; however, their primary effects are limited to suppressing bone resorption. Milk basic protein (MBP), a bioactive whey-derived component, has been proposed to promote bone formation and modulate bone resorption. This study investigated the effects of nutritionally relevant MBP supplementation in combination with zoledronate in an ovariectomized (OVX) rat model of osteoporosis. Twenty female Sprague–Dawley rats underwent ovariectomy (OVX; n = 15) or sham surgery ( n = 5) at 24 weeks of age. Eight weeks after surgery, the OVX rats were randomly assigned to the Control, zoledronate (ZA), and zoledronate plus MBP (ZA + MBP) groups ( n = 5/group); sham-operated rats constituted the Sham group ( n = 5). Zoledronate (0.1 mg/kg) was administered as a single subcutaneous injection, and MBP was provided as a fourfold-diluted solution as the sole drinking fluid for ad libitum intake for 10 weeks. Bone microarchitecture and bone mineral density (BMD) were assessed by micro-CT, histological and histomorphometric analyses of the distal femur were performed using haematoxylin and eosin (H&E)-stained sections, biomechanical properties were measured by three-point bending, and serum bone turnover markers were analysed by ELISA. Compared with Controls, the ZA + MBP group showed significantly higher trabecular bone volume (BV), and both the ZA and ZA + MBP groups exhibited significantly higher BV/TV and Tb.N. No significant differences were observed between the ZA and ZA + MBP groups for these parameters. Cortical bone parameters and BMD did not differ significantly among groups. Histomorphometric analysis did not demonstrate significant differences in tissue area, bone area, or bone area fraction among the Control, ZA, and ZA + MBP groups. No significant differences in biomechanical properties were observed among the groups, except for Young’s modulus, which was significantly lower in the Control and ZA groups than in the Sham group; the difference between the ZA + MBP and Sham groups was not statistically significant. No significant differences were detected in serum osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX-1), or their ratio. Under the present experimental conditions, no consistent additional skeletal benefit of nutritionally relevant MBP supplementation beyond zoledronate treatment was demonstrated in ovariectomized rats. Given the small sample size, these findings should not be interpreted as evidence of equivalence or absence of an MBP effect.