Collagen structures of demineralized bone paper direct mineral metabolism

Introduction: Bone is a dynamic mineralized tissue that undergoes continuous remodeling throughout life. While the general mechanism of bone mineral metabolism is documented, the role of collagen structures in regulating osteoblastic mineral deposition and osteoclastic mineral resorption remains an active research area due to the lack of biomaterial platforms to support the complex structure of the bone matrix. To address this, we introduced demineralized bone paper (DBP), a thin section of demineralized bovine bone that preserves intrinsic collagen structure. DBP features distinct collagen structures depending on the sectioning direction; uniaxially aligned collagen structure in vertically sectioned DBP (vDBP) and concentric ring structure in transversely sectioned DBP (tDBP). DBP is semi-transparent, which allows direct visualization of collagen organization and its role in regulating cellular processes during mineral deposition and resorption. Leveraging these unique features of DBP, we report how collagen structures modulate osteoblast and osteoclast-driven bone mineral metabolism. First, we studied osteoblast adhesion, proliferation, and mineralization by seeding primary mouse osteoblasts on DBPs. The deposited mineral structure and composition were analyzed using scanning electron microscopy with energy-dispersive X-ray spectroscopy. Then, we examined the impact of mineralized collagen structures on osteoclast resorption. Osteoblasts on DBP stimulated with vitamin D3 and prostaglandin E2, induced bone marrow mononuclear cells to differentiate into multinucleated osteoclasts. Comparative spatiotemporal analysis of mineral deposition and resorption between vDBP and tDBP highlights the importance of collagen structures in regulating osteoblast and osteoclast-mediated mineral metabolism. We envision DBP as a valuable platform for in-depth investigation of bone mineral metabolism.

Materials and

Methods: DBP was generated by sectioning demineralized bovine compact bone into 20 µm thin sheets using cryostats. Two sectioning directions were employed to generate distinct collagen alignments, vertically sectioned DBP (vDBP) and transversely sectioned DBP (tDBP). Prior to cell culture, DBPs were biopsy-punched into 6 mm diameter circular shapes and attached to a 96-well plate.
Primary osteoblasts were isolated from the femoral and tibial bone chips of DsRed reporter mice, enabling fluorescent tracking of cellular processes. Osteoblasts adhesion, proliferation, and mineralization were assessed using time-lapse imaging, phalloidin actin staining, Alizarin Red S staining, and calcein mineral staining. Mineral structure and composition were analyzed using scanning electron microscopy (SEM) with energy dispense X-ray spectroscopy (EDS).
Bone marrow mononuclear cells (BM-MNCs) were differentiated into osteoclasts in coculture with osteoblasts. Osteoblasts were cultured on DBPs to re-mineralize the surface. Subsequently, BM-MNCs were introduced and stimulated with vitamin D3 (VD3) and prostaglandin E2 (PGE2) to induce osteoclastogenesis in vitro. Osteoclast differentiation was monitored through time-course fluorescent imaging of osteoclast activity and further characterized by examining multinucleation and tartrate-resistant acid phosphatase (TRAP) activity using nucleus DAPI staining and TRAP staining. Time-course imaging facilitated the tracking and quantification of osteoclastic mineral resorption areas and patterns.

Results, Conclusions, and Discussions: Results and

Discussion: Osteoblasts exhibited a preference for adhering and proliferating along the underlying collagen structures of the DBP. Notably, osteoblastic mineral deposition was significantly higher on vDBP compared to tDBP. The spatiotemporal monitoring of the mineralization process demonstrated increased osteoblast adhesion and faster mineral deposition in vascular regions rich in type IV collagen and laminin, relative to regions predominantly consisting of type I collagen regardless of DBP structure. This observation was supported by SEM-EDX analysis, which showed higher calcium and phosphate ratios in vDBP than in tDBP. SEM-EDX analysis further confirmed that vascular regions had higher mineral content compared to bone regions in both vDBP and tDBP. Time-course fluorescent imaging of osteoclast activity revealed that osteoclasts are also responsive to collagen alignment cues, influencing their resorptive behavior. Osteoclasts appeared elongated on vDBP and more rounded on tDBP, aligning with the underlying collagen structure. Furthermore, more osteoclasts differentiated and were larger on vDBP than on tDBP. Time-course analysis of osteoclastic mineral resorption areas further indicated that vDBP consistently promoted larger resorption areas compared to tDBP, suggesting that osteoclasts on vDBP were more actively resorbing the mineralized matrix. Time-lapse imaging showed distinct patterns of osteoclastic resorption that aligned with the underlying collagen structure. Trenches, characterized by extended and linear mineral resorption along the collagen fibers, were primarily observed on vDBP, whereas pits, exhibiting rounded and localized resorptive behavior, were more commonly identified on tDBP. These results underscore the role of collagen structures in directing the functional activities of osteoblasts and osteoclasts, suggesting that collagen orientation significantly influences bone remodeling processes.

Conclusions: DBP provides a novel and effective platform for the detailed study of bone ECM's role in osteoblastic and osteoclastic activities. Our findings confirm that collagen structure profoundly affects both mineral deposition by osteoblasts and mineral resorption by osteoclasts. The ability of DBP to mimic natural bone conditions and reveal the dynamic interactions within bone ECM makes it an invaluable tool for advancing our understanding of bone biology. Future studies using DBP could further illuminate the mechanistic pathways of bone remodeling and potentially lead to improved therapeutic strategies for bone-related diseases.

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