The discontinuation of denosumab [antibody targeting receptor activator of nuclear factor kappa B ligand (RANKL)] therapy may increase the risk of multiple vertebral fractures; however, the underlying pathophysiology is largely unknown. In patients who underwent discontinuation after multiple injections of denosumab, the levels of tartrate-resistant acid phosphatase 5b increased compared to pretreatment levels, indicating a phenomenon known as “overshoot.” The rate of decrease in bone mineral density during the withdrawal period was higher than the rate of decrease associated with aging, suggesting that the physiological bone metabolism had broken down. Overshoot and significant bone loss were also observed in mice receiving continuous administration of anti-RANKL antibody after treatment was interrupted, resembling the original pathology. In mice long out of overshoot, bone resorption recovered, but osteoblast numbers and bone formation remained markedly reduced. The bone marrow exhibited a significant reduction in stem cell (SC) antigen 1- and platelet-derived growth factor receptor alpha-expressing osteoblast progenitors (PαS cells) and alkaline phosphatase-positive early osteoblasts. Just before the overshoot phase, the osteoclast precursor cell population expands and RANKL-bearing extracellular vesicles (EVs) became abundant in the serum, leading to robust osteoclastogenesis after cessation of anti-RANKL treatment. Thus, accelerated bone resorption due to the accumulation of RANKL-bearing EVs and long-term suppression of bone formation uncoupled from bone resorption leads to the severe bone loss characteristic of denosumab discontinuation.

Bone is mainly composed of type I collagen, hydroxyapatite, and water. Bone strength is increased in bone formation and reflects 70% bone mineral density (BMD) and 30% bone quality; however, BMD is clinically used to assess bone strength. In this work, we analyzed collagen fiber orientation in femur of different aged rats using infrared (IR) dichroism image due to assess consequences of collagen fiber orientation for bone strength. IR dichroism images indicated that the degree of collagen fiber orientation in rat femur was increased until 33 weeks old at least, and it can affect bone strength.

Fourier transform infrared (FTIR) imaging is a powerful tool for the assessment of bone quality; however, it requires the preparation of thin bone sections. Conventional poly(methyl methacrylate) (PMMA) embedding for the preparation of sections takes more than two weeks and causes denaturation of the bone. Development of a quick and easy sample preparation technique without denaturation is needed for accurate clinical evaluation of fresh calcified bone using FTIR imaging. Frozen sectioning allows the quick and easy preparation of thin sections without denaturation, but it requires a substrate with good chemical resistance and improved heat shock resistance. Polypropylene (PP) film afforded both good chemical resistance and greater heat shock resistance, and the 4-μm-thick PP film coated with glue was thin enough for the IR beam to pass through it, while the optical anisotropy of infrared bands overlapping with PO₄^3- band was negligible. The bone quality of femoral thin sections prepared by the conventional PMMA embedding and sectioning procedure (RESIN-S) or the newly developed frozen sectioning procedure (FROZEN-S) was evaluated by FTIR imaging. The mineral-to-matrix ratio and crystallinity in the RESIN-S sections were higher than those in the FROZEN-S sections, whereas the carbonate-to-phosphate ratio in the RESIN-S sections was lower than that in the FROZEN-S sections. In RESIN-S, the increased mineral-to-matrix ratio could be caused by dehydration, and the increased crystallinity and decreased carbonate-to-phosphate ratio might be consequence of dissolution of bone mineral during PMMA embedding. Therefore, the combined use of PP film coated with glue and the frozen sectioning procedure without denaturation appears well suited to the assessment of the bone quality of fresh calcified bone using FTIR imaging.

We successfully synthesised octacalcium phosphate with incorporated 3,3′-dithiodipropionate ions.