Collagen structures of demineralized bone paper direct mineral metabolism

Bone is a dynamic mineralized tissue that undergoes continuous turnover throughout life. While the general mechanism of bone mineral metabolism is documented, the role of underlying collagen structures in regulating osteoblastic mineral deposition and osteoclastic mineral resorption remains an active research area, partly due to the lack of biomaterial platforms supporting accurate and analytical investigation. The recently introduced osteoid-inspired demineralized bone paper (DBP), prepared by 20-mu m thin sectioning of demineralized bovine compact bone, holds promise in addressing this challenge as it preserves the intrinsic bony collagen structure and retains semi-transparency. Here, we report on the impact of collagen structures on modulating osteoblast and osteoclast-driven bone mineral metabolism using vertical and transversal DBPs that exhibit a uniaxially aligned and a concentric ring collagen structure, respectively. Translucent DBP reveals these collagen structures and facilitates longitudinal tracking of mineral deposition and resorption under brightfield microscopy for at least 3 wk. Genetically labeled primary osteogenic cells allow fluorescent monitoring of these cellular processes. Osteoblasts adhere and proliferate following the underlying collagen structures of DBPs. Osteoblastic mineral deposition is significantly higher in vertical DBP than in transversal DBP. Spatiotemporal analysis reveals notably more osteoblast adhesion and faster mineral deposition in vascular regions than in bone regions. Subsequent osteoclastic resorption follows these mineralized collagen structures, directing distinct trench and pit-type resorption patterns. In vertical DBP, trench-type resorption occurs at an 80% frequency, whereas transversal DBP shows 35% trench-type and 65% pit-type resorption. Our studies substantiate the importance of collagen structures in regulating mineral metabolism by osteogenic cells. DBP is expected to serve as an enabling biomaterial platform for studying various aspects of cellular and extracellular bone remodeling biology. While the mechanisms of bone mineral metabolism are known, the role of underlying collagen structure in directing osteogenic cellular processes remains an active research area due to the difficulty of monitoring these processes inside the bone cavity. In this study, we demonstrate the significance of collagen structure in modulating osteoblastic mineral deposition and osteoclastic mineral resorption using demineralized bone paper (DBP) created by thin slicing of demineralized bovine bone matrix. DBP preserves the natural collagen structure and retains semi-transparency for microscopic imaging, displaying distinct patterns based on vertical and transversal sectioning directions. Osteoblast adhesion and mineral deposition occur following the collagen structure, with vertical DBP supporting higher mineral deposition compared with transversal DBP. Osteoclast mineral resorption also follows these collagen patterns, with vertical DBP exhibiting higher trench-type resorption, while transversal DBP shows more pit-type resorption. This study underscores the importance of collagen structure in directing bone metabolism. We envision that DBP as a valuable tool for studying bone mineral metabolism and related processes. Graphical Abstract