Underwater Fabrication of Carbon Nanotube/Coacervate Composites.

Soft conductive materials are of interest for a wide range of applications, but their syntheses have remained difficult. Herein, we present a convenient route for underwater fabrication of a composite made of carbon nanotubes (CNTs) and mussel-inspired complex coacervates. The key to success of this method is that CNTs were solubilized very effectively in protocoacervates, which are high-concentration solutions of polyelectrolytes at a pH where only one of them is charged, thereby impeding coacervate formation. Composite materials were formed by the simple injection of CNT-dispersed protocoacervate solutions into water under ambient conditions. The method is simple, fast, and ecofriendly, and composites of CNT-complex coacervate in the form of films or bulk materials were obtained. The composites demonstrated electrical conductivity and tunable mechanical properties, which depended on the concentration of polyelectrolytes and the CNT:protocoacervate ratio. Hence, the composites can be manipulated to attain diverse properties, for examples, tunable reduced modulus (15 to 32 GPa) and hardness (0.3 to 0.7 GPa) as well as an electrical conductivity of up to 4 × 103 S m-1.

No Signature of Osteocytic Osteolysis in Cortical Bone from Lactating NMRI Mice.

The roles of osteocytes in bone homeostasis have garnered increasing attention since it has been realized that osteocytes communicate with other organs. It has long been debated whether and/or to which degree osteocytes can break down the bone matrix surrounding them in a process called osteocytic osteolysis. Osteocytic osteolysis has been indicated to be induced by a number of skeletal challenges including lactation in CD1 and C57BL/6 mice, whereas immobilization-induced osteocytic osteolysis is still a matter of controversy. Motivated by the wish to understand this process better, we studied osteocyte lacunae in lactating NMRI mice, which is a widely used outbred mouse strain. Surprisingly, no trace of osteocytic osteolysis could be detected in tibial or femoral cortical bone either by 3D investigation by synchrotron nanotomography, by studies of lacunar cross-sectional areas using scanning electron microscopy, or by light microscopy. These results lead us to conclude that osteocytic osteolysis does not occur in NMRI mice as a response to lactation, in turn suggesting that osteocytic osteolysis may not play a generic role in mobilizing calcium during lactation.

Canalicular Junctions in the Osteocyte Lacuno-Canalicular Network of Cortical Bone.

The osteocyte lacuno-canalicular network (LCN) is essential for bone remodeling because osteocytes regulate cell recruitment. This has been proposed to occur through liquid-flow-induced shear forces in the canaliculi. Models of the LCN have thus far assumed that it contains canaliculi connecting the osteocyte lacunae. However, here, we reveal that enlarged spaces occur at places where several canaliculi cross; we name these spaces canalicular junctions. We characterize them in detail within mice cortical bone using synchrotron nanotomography at two length scales, with 50 and 130 nm voxel size, and show that canalicular junctions occur at a density similar to that of osteocyte lacunae and that canalicular junctions tend to cluster. Through confocal laser scanning microscopy, we show that canalicular junctions are widespread as we have observed them in cortical bone from several species, even though the number density of the canalicular junctions was not universal. Fluid flow simulations of a simple model system with and without a canalicular junction clearly show that liquid mass transport and flow velocities are altered by the presence of canalicular junctions. We suggest that these canalicular junctions may play an important role in osteocyte communication and possibly also in canalicular fluid flow. Therefore, we believe that they constitute an important component in the bone osteocyte network.

Immobilization and long-term recovery results in large changes in bone structure and strength but no corresponding alterations of osteocyte lacunar properties.

The ability of osteocytes to demineralize the perilacunar matrix, osteocytic osteolysis, and thereby participate directly in bone metabolism, is an aspect of osteocyte biology that has received increasing attention during the last couple of years. The aim of the present work was to investigate whether osteocyte lacunar properties change during immobilization and subsequent recovery. A rat cortical bone model with negligible Haversian remodeling effects was used, with temporary immobilization of one hindlimb induced by botulinum toxin. Several complementary techniques covering multiple length scales enabled correlation of osteocyte lacunar properties to changes observed on the organ and tissue level of femoral bone. Bone structural parameters measured by µCT and mechanical properties were compared to sub-micrometer resolution SR µCT data mapping an unprecedented number (1.85 million) of osteocyte lacunae. Immobilization induced a significant reduction in aBMD, bone volume, tissue volume, and load to fracture, as well as the muscle mass of rectus femoris. During the subsequent recovery period, the bone structural and mechanical properties were only partly regained in spite of a long-term (28weeks) study period. No significant changes in osteocyte lacunar volume, density, oblateness, stretch, or orientation were detected upon immobilization or subsequent recovery. In conclusion, the bone architecture and not osteocyte lacunar properties or bone material characteristics dominate the immobilization response as well as the subsequent recovery.

Osteocyte lacunar properties and cortical microstructure in human iliac crest as a function of age and sex.

Osteocytes are suggested to play a central role in bone remodeling. Evaluation of iliac crest biopsies is a standard procedure for evaluating bone conditions in the clinical setting. Despite the widespread use of such biopsies, little is known about the population of osteocytes in the iliac crest from normal individuals. Contradicting results have been reported on osteocyte lacunar properties in human bone. Hence, a solid understanding of the osteocyte population in healthy bone and the effect of age and sex is needed as good reference data are lacking. Furthermore, the role of cortical bone in bone quality has recently been suggested to be more important than previously realized. Therefore, the present study assesses osteocyte lacunar properties and cortical microstructure of the iliac crest as a function of age and sex. A total of 88 iliac crest bone samples from healthy individuals (46 women, aged 18.5-96.4years and 42 men, aged 22.6-94.6years) with an even age-distribution were examined using synchrotron radiation µCT and in house µCT, with >5×10(6) osteocyte lacunae measured and analyzed. The study revealed that osteocyte lacunar volumes were unaffected by both age and sex. Osteocyte lacunar density did not differ between women and men, and only showed a significant decrease with age when pooling data from both sexes. Cortical porosity and Haversian canal density increased while cortical thickness decreased with age, with cortical thinning dominating the age-related cortical bone loss. None of the cortical microstructural parameters showed any sex dependency. Only weak links between osteocyte lacunar properties and cortical microstructural properties in iliac crest bone were found. Interestingly, the Haversian canal diameters were significantly but weakly negatively correlated with osteocyte lacunar volumes.

Organ and tissue level properties are more sensitive to age than osteocyte lacunar characteristics in rat cortical bone.

Modeling and remodeling induce significant changes of bone structure and mechanical properties with age. Therefore, it is important to gain knowledge of the processes taking place in bone over time. The rat is a widely used animal model, where much data has been accumulated on age-related changes of bone on the organ and tissue level, whereas features on the nano- and micrometer scale are much less explored. We investigated the age-related development of organ and tissue level bone properties such as bone volume, bone mineral density, and load to fracture and correlated these with osteocyte lacunar properties in rat cortical bone. Femora of 14 to 42-week-old female Wistar rats were investigated using multiple complementary techniques including X-ray micro-computed tomography and biomechanical testing. The body weight, femoral length, aBMD, load to fracture, tissue volume, bone volume, and tissue density were found to increase rapidly with age at 14-30 weeks. At the age of 30-42 weeks, the growth rate appeared to decrease. However, no accompanying changes were found in osteocyte lacunar properties such as lacunar volume, ellipsoidal radii, lacunar stretch, lacunar oblateness, or lacunar orientation with animal age. Hence, the evolution of organ and tissue level properties with age in rat cortical bone is not accompanied by related changes in osteocyte lacunar properties. This suggests that bone microstructure and bone matrix material properties and not the geometric properties of the osteocyte lacunar network are main determinants of the properties of the bone on larger length scales.

Osteocyte lacunar properties in rat cortical bone: Differences between lamellar and central bone.

Recently, the roles of osteocytes in bone maintenance have gained increasing attention. Osteocytes reside in lacunae that are interconnected by canaliculi resulting in a vast cellular network within the mineralized bone matrix. As the structure of the lacuno-canalicular network is highly connected to osteocyte function, osteocyte lacunar properties such as volume, shape, orientation, and density are now frequently reported in studies investigating osteocyte activity. Despite this increasing interest in lacunar morphometrics, many studies show a large spread in such values, suggesting a large inter-species but also inter-site variation in lacunar properties. Here, osteocyte lacunae in rat cortical bone have been studied using synchrotron radiation micro computed tomography (SR µCT) and backscattered electron (BE) microscopy. Quantitative lacunar geometric characteristics are reported based on the synchrotron radiation data, differentiating between circumferential lamellar bone and a central, more disordered bone type. From these studies, no significant differences were found in lacunar volumes between lamellar and central bone, whereas significant differences in lacunar orientation, shape and density values were observed. The 3D nature of the SR µCT data sets furthermore revealed that lacunae in central bone, which appear to be poorly aligned in transverse 2D cross sections, are in fact highly aligned along the bone long axis. These results demonstrate the importance of using 3D methods to investigate anisotropic biological materials such as bone and that the appropriate choice of subregions for high resolution imaging is not trivial.

Calcified cartilage islands in rat cortical bone.

Rats display little to no haversian remodeling of cortical bone. This fact, combined with the endochondral formation of cortical bone, means that rat femoral cortical bone contains highly mineralized cartilage islands in a central band of mid-femoral cross sections. We demonstrate that these islands have a significantly higher degree of mineralization than the surrounding bone, using quantitative backscattered electron imaging. The cartilaginous nature of the islands was verified by immunostaining for collagen type II. Toluidine blue staining of longitudinal sections and three-dimensional synchrotron radiation X-ray tomographic microscopy confirmed that the islands are elongated along the femoral long axis. Nanoindentation revealed significantly higher values of both reduced modulus and hardness in the islands compared to the surrounding bone, reflecting a higher degree of mineralization. The calcified cartilage islands were distributed in a central zone of the bone, from the growth plates through the mid-femoral bone. The presence of these cartilage islands and their possible effect on mechanical properties could be an additional reason why haversian remodeling is observed in higher-order species.