Physico-Chemical Investigations of the Deeper Osteoid Tissue

Bone tissue is responsible for skeletal structure and function. The tissue undergoes dynamic remodeling characterized by a complex matrix of mineralized collagen fibrils. Recent studies on sheep bone biopsies have investigated the deeper osteoid (1) at the interface between osteoid and mature bone. It was identified as a distinct zone exhibiting an acidic pH (~ 5-6). This deeper domain was named acidic osteoid, and was shown to be highly structured by collagen molecules rather than fibrils. Further, it was characterized as a mesophase (liquid crystal analogues) with a continuous birefringence texture similar to that of mature bone (2). This observation strongly suggests that this domain is responsible for the formation of the twisted plywood structure in bone (3). This work aims to characterize this specific domain in different bone pathologies.<br/><br/>To this end, we use sections from physiological and pathological human biopsies, as well as collagen matrices as a biomimetic model of this acidic osteoid. The synthetic acidic osteoid is synthesized using an injection/dialysis process (4) to understand the mechanisms involved in the formation of this zone. The collagen solution is implemented with organic additives identified in bone (i.e., glycosaminoglycans (5) and citrate (6)) and inorganic ions to precipitate carbonated apatite. In parallel, an in-depth investigation of this domain in a series of histological sections from different pathologies that are extracted from human iliac crests was carried out. Characterizations were conducted focusing on the acid osteoid density, composition, spreading, pH, and organization. Experimental characterizations imply an interdisciplinary approach, combining analytical techniques from the fields of biology and materials science including multi-scale microscopic observations (MOLP, SEM, and TEM) with different pathologist-related staining, microstructure characterization (SAXS/WAXS), and 1H and 31P environment analysis (solid-state NMR). Collaboration with medical experts and physicists was crucial for this original approach.<br/><br/>We show that models of the acidic osteoid domain can be synthesized in vitro in the presence of citrate and glycosaminoglycans. The additives tend to stabilize a lower pH and lead to differences in macroscopic, microscopic, and mineralization aspects. Moreover, analyses of the pathological histological sections revealed both qualitative and quantitative destabilization of the acidic non-fibrillar domain in some of the selected bone pathologies. Strikingly, this is also observed in the adjacent mature bone. The results strengthen the idea that the organization of mature bone directly depends on that of the deeper osteoid, which might be involved in the development of bone pathologies.<br/><br/>This interdisciplinary work contributes to a better understanding of the physico-chemical processes underlying bone formation. The aim is to validate the feasibility of a physico-chemical diagnosis that would complement the anatomo-pathological and genetic analyses that focus on bone quantity and quality. These advances could open up new prospects for the development of therapeutic targets (e.g., organ-on-chip).<br/><br/>(1) Raina V., J. Clin. Pathol. 1972, 25, 229.<br/>(2) Giraud-Guille M.M. et al. J Biomech. 2003, 36, 1571.<br/>(3) Robin M. et al. Adv. Sci. 2024, 11, 2304454.<br/>(4) Wang et al. Soft Matter. 2011, 7, 9659.<br/>(5) Fornasier V. L., Hum. Pathol. 1977, 8, 243.<br/>(6) Costello L. C. et al. Open Bone J. 2012, 4, 27.