Highly Ordered Interstitial Water Observed in Bone by Nuclear Magnetic Resonance

Abstract

NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation. Introduction:The unique mechanical characteristics of bone tissue have not yet been satisfactorily connected to the exact molecular architecture of this complex composite material. Recently developed solid-state nuclear magnetic resonance (NMR) techniques are applied here to the mineral component to provide new structural distance constraints at the subnanometer scale. Materials and Methods: NMR dipolar couplings between structural protons (OH − and H 2 O) and phosphorus (PO 4 ) or carbon (CO 3 ) were measured using the 2D Lee-Goldburg Cross-Polarization under Magic-Angle Spinning (2D LG-CPMAS) pulse sequence, which simultaneously suppresses the much stronger protonproton dipolar interactions. The NMR dipolar couplings measured provide accurate distances between atoms, e.g., OH and PO 4 in apatites. Excised and powdered femoral cortical bone was used for these experiments. Synthetic carbonate (∼2-4 wt%)-substituted hydroxyapatite was also studied for structural comparison. Results: In synthetic apatite, the hydroxide ions are strongly hydrogen bonded to adjacent carbonate or phosphate ions, with hydrogen bond (O-H) distances of ∼1.96 Å observed. The bone tissue sample, in contrast, shows little evidence of ordered hydroxide. Instead, a very ordered (structural) layer of water molecules is identified, which hydrates the small bioapatite crystallites through very close arrangements. Water protons are ∼2.3-2.55 Å from surface phosphorus atoms. Conclusions: In synthetic carbonated apatite, strong hydrogen bonds were observed between the hydroxide ions and structural phosphate and carbonate units in the apatite crystal lattice. These hydrogen bonding interactions may contribute to the long-range stability of this mineral structure. The biological apatite in cortical bone tissue shows evidence of hydrogen bonding with an ordered surface water layer at the faces of the mineral particles. This structural water layer has been inferred, but direct spectroscopic evidence of this interstitial water is given here. An ordered structural water layer sandwiched between the mineral and the organic collagen fibers may affect the biomechanical properties of this complex composite material.

Measurements of mobile and bound water by nuclear magnetic resonance correlate with mechanical properties of bone

“…The socalled solid hydrogen band reflects protons of the collagen peptide and protons of the hydroxyapatite-related mineral, each with slightly different relaxation rates. Bound water band likely represents the so-called structural water layer bridging mineral and collagen that was reported to exist by Wilson et al [45] who applied a cross-polarization NMR technique to cortical bone and synthetic apatite. Still, it also likely represents the number of water bridges (via hydrogen bonding) that exist between and within triple helical peptides of collagen [20,46,47].…”

“…While both NMR and magnetic resonance imaging (MRI) have been used to characterize bone in a number of studies [40,45,[48][49][50][51][52][53][54][55][56], the present work is the first to quantify water distribution in bone for two age groups. A previous study had actually characterized water distribution in dentin samples from a 20 year, 35 year, and 50 year old donor [50].…”

Citrate bridges between mineral platelets in bone

“…In our model, water molecules in these interplatelet hydrated layers are within 2.3-2.55 Å of mineral phosphorus atoms, which model the presence of extra structured water in bone mineral compared with model carbonated apatites (10). In our model, these hydrated layers are lined with hydrogen phosphate groups (inherently disordered because of the disordered citrate anions among them), which would account for the high proportion of such species in bone mineral.…”

“…Second, up to 28% of the water associated with bone mineral is highly organized and very strongly bound to mineral, with water hydrogens of the order of ∼2.3-2.55 Å from mineral phosphorus atoms (10). This finding is in sharp contrast with <15% of such a water population for synthetic nanoscopic, carbonated apatite models of bone mineral (10), suggesting that bone mineral somehow has an extra capacity for strongly bound water compared with synthetic apatitic models. Moreover, there is significant NMR evidence for two distinct sites for structural water in bone mineral (11).…”

Bone Material Properties in Osteogenesis Imperfecta

“…Water may also play an important role in the interactions between matrix and mineral. (55) Layered water provides multiple hydrogen bonds at interfaces between mineral platelets and also allows interactions between platelets and fibrils. (56) Molecular dynamics simulations indicate that water can significantly alter the interactions between platelets and fibrils during loading.…”