Dynamic monitoring of bone homeostasis by dual fluorescent imaging of osteoblast and osteoclast activity

Bone homeostasis is maintained through a dynamic balance between osteoblasts (OBs)-mediated bone formation and osteoclasts (OCs)-driven bone resorption. While numerous probes have been developed to detect either alkaline phosphatase (ALP) or extracellular protons (H⁺), which serve as key biochemical markers for OBs and OCs activity, respectively, no existing single molecular imaging platform enables the simultaneous, real-time monitoring of both bone formation and resorption in situ. In this study, we developed a dual-responsive near-infrared (NIR) fluorescence (FL) imaging platform for real-time, non-invasive monitoring of bone remodeling. Two NIR fluorescent probes, NIR-OB and NIR-OC, were designed to respond selectively to ALP and H⁺ via distinct "OFF-ON" FL emissions at 710 nm and 820 nm. These probes were chemically conjugated to TCP scaffolds fabricated with α-tricalcium phosphate and calvarial discs from mice, enabling both synaptic function and chemical sensing. In vitro and ex vivo experiments confirmed the responsiveness of the probes to OBs and OCs activity. The ovariectomized mice in vivo exhibited progressive FL enhancement in the femur and lumbar regions, correlating with bone loss validated by micro-computed tomography. Dual-channel FL imaging clearly delineated regions of bone formation and resorption, enabling precise and localized monitoring of bone remodeling, with the NIR probes demonstrating high photostability and biocompatibility throughout 16 weeks. Thus, these findings establish NIR-OB and NIR-OC as robust tools for dual-modal tracking of bone metabolism, with strong translational potential for early diagnosis and long-term monitoring of osteoporosis. STATEMENT OF SIGNIFICANCE: Bone remodeling results from the metabolic activities of osteoblasts and osteoclasts, yet no molecular imaging tools have enabled their simultaneous, real-time monitoring in situ. Here, we introduce a dual-responsive near-infrared fluorescence platform employing two probes (NIR-OB and NIR-OC) that selectively visualize osteoblast- and osteoclast-mediated activities with distinct emission signals (710 nm and 820 nm). This system demonstrated high specificity, biocompatibility, and longitudinal stability in vitro, ex vivo, and in vivo, with fluorescence signals correlating strongly with bone loss progression in osteoporotic models. By enabling precise, localized, and non-invasive tracking of bone formation and resorption, this platform provides a powerful new tool with strong translational potential for the early diagnosis and long-term monitoring of osteoporosis and related metabolic bone diseases.