Molecular differences in collagen organization and in organic-inorganic interfacial structure of bones with and without osteocytes.

Bone is a fascinating biomaterial composed mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, and water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications for the mechanical properties of the particular bone. STATEMENT OF SIGNIFICANCE: Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites.

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.