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.

Micro- and nano-CT for the study of bone ultrastructure.

Micro-computed tomography (micro-CT)-a version of X-ray CT operating at high spatial resolution-has had a considerable success for the investigation of trabecular bone micro-architecture. Currently, there is a lot of interest in exploiting CT techniques at even higher spatial resolutions to assess bone tissue at the cellular scale. After recalling the basic principles of micro-CT, we review the different existing system, based on either standard X-ray tubes or synchrotron sources. Then, we present recent applications of micro- and nano-CT for the analysis of osteocyte lacunae and the lacunar-canalicular network. We also address the question of the quantification of bone ultrastructure to go beyond the sole visualization.

Alterations of mass density and 3D osteocyte lacunar properties in bisphosphonate-related osteonecrotic human jaw bone, a synchrotron µCT study.

Osteonecrosis of the jaw, in association with bisphosphonates (BRONJ) used for treating osteoporosis or cancer, is a severe and most often irreversible side effect whose underlying pathophysiological mechanisms remain largely unknown. Osteocytes are involved in bone remodeling and mineralization where they orchestrate the delicate equilibrium between osteoclast and osteoblast activity and through the active process called osteocytic osteolysis. Here, we hypothesized that (i) changes of the mineralized tissue matrix play a substantial role in the pathogenesis of BRONJ, and (ii) the osteocyte lacunar morphology is altered in BRONJ. Synchrotron µCT with phase contrast is an appropriate tool for assessing both the 3D morphology of the osteocyte lacunae and the bone matrix mass density. Here, we used this technique to investigate the mass density distribution and 3D osteocyte lacunar properties at the sub-micrometer scale in human bone samples from the jaw, femur and tibia. First, we compared healthy human jaw bone to human tibia and femur in order to assess the specific differences and address potential explanations of why the jaw bone is exclusively targeted by the necrosis as a side effect of BP treatment. Second, we investigated the differences between BRONJ and control jaw bone samples to detect potential differences which could aid an improved understanding of the course of BRONJ. We found that the apparent mass density of jaw bone was significantly smaller compared to that of tibia, consistent with a higher bone turnover in the jaw bone. The variance of the lacunar volume distribution was significantly different depending on the anatomical site. The comparison between BRONJ and control jaw specimens revealed no significant increase in mineralization after BP. We found a significant decrease in osteocyte-lacunar density in the BRONJ group compared to the control jaw. Interestingly, the osteocyte-lacunar volume distribution was not altered after BP treatment.

Osteocytes exposed to far field of therapeutic ultrasound promotes osteogenic cellular activities in pre-osteoblasts through soluble factors.

Low intensity pulsed ultrasound (LIPUS) was reported to accelerate the rate of fracture healing. When LIPUS is applied to fractures transcutaneously, bone tissues at different depths are exposed to different ultrasound fields. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). Moreover, we have reported that the therapeutic effect of LIPUS is dependent on the axial distance of ultrasound beam in rat fracture model. However, the mechanisms of how different axial distances of LIPUS influence the mechanotransduction of bone cells are not understood. To understand the cellular mechanisms underlying far field LIPUS on enhanced fracture healing in rat model, the present study investigated the effect of ultrasound axial distances on (1) osteocyte, the mechanosensor, and (2) mechanotransduction between osteocyte and pre-osteoblast (bone-forming cell) through paracrine signaling. We hypothesized that far field LIPUS could enhance the osteogenic activities of osteoblasts via paracrine factors secreted from osteocytes. The objective of this study was to investigate the effect of axial distances of LIPUS on osteocytes and osteocyte-osteoblast mechanotransduction. In this study, LIPUS (plane; 2.2 cm in diameter, 1.5MHz sine wave, ISATA=30 mW/cm(2)) was applied to osteocytes (mechanosensor) at three axial distances: 0mm (near field), 60mm (mid-near field) and 130 mm (far field). The conditioned medium of osteocytes (OCM) collected from these three groups were used to culture pre-osteoblasts (effector cell). In this study, (1) the direct effect of ultrasound fields on the mechanosensitivity of osteocytes; and (2) the osteogenic effect of different OCM treatments on pre-osteoblasts were assessed. The immunostaining results indicated the ultrasound beam at far field resulted in more ß-catenin nuclear translocation in osteocytes than all other groups. This indicated that osteocytes could detect the acoustic differences of LIPUS at various axial distances. Furthermore, we found that the soluble factors secreted by far field LIPUS exposed osteocytes could further promote pre-osteoblasts cell migration, maturation (transition of cell proliferation into osteogenic differentiation), and matrix calcification. In summary, our results of this present study indicated that axial distance beyond near field could transmit ultrasound energy to osteocyte more efficiently. The LIPUS exposed osteocytes conveyed mechanical signals to pre-osteoblasts and regulated their osteogenic cellular activities via paracrine factors secretion. The soluble factors secreted by far field exposed osteocytes led to promotion in migration and maturation in pre-osteoblasts. This finding demonstrated the positive effects of far field LIPUS on stimulating osteocytes and promoting mechanotransduction between osteocytes and osteoblasts.

Load regulates bone formation and Sclerostin expression through a TGFß-dependent mechanism.

Bone continually adapts to meet changing physical and biological demands. Osteoblasts, osteoclasts, and osteocytes cooperate to integrate these physical and biochemical cues to maintain bone homeostasis. Although TGFß acts on all three of these cell types to maintain bone homeostasis, the extent to which it participates in the adaptation of bone to mechanical load is unknown. Here, we investigated the role of the TGFß pathway in load-induced bone formation and the regulation of Sclerostin, a mechanosensitive antagonist of bone anabolism. We found that mechanical load rapidly represses the net activity of the TGFß pathway in osteocytes, resulting in reduced phosphorylation and activity of key downstream effectors, Smad2 and Smad3. Loss of TGFß sensitivity compromises the anabolic response of bone to mechanical load, demonstrating that the mechanosensitive regulation of TGFß signaling is essential for load-induced bone formation. Furthermore, sensitivity to TGFß is required for the mechanosensitive regulation of Sclerostin, which is induced by TGFß in a Smad3-dependent manner. Together, our results show that physical cues maintain bone homeostasis through the TGFß pathway to regulate Sclerostin expression and the deposition of new bone.

Prolonged unloading in growing rats reduces cortical osteocyte lacunar density and volume in the distal tibia.

Bone dynamically adapts its structure to the environmental demands placed upon it. Load-related stimuli play an important role in this adaptation. It has been postulated that osteocytes sense changes in these stimuli and initiate adaptive responses, across a number of scales, through a process known as mechanotransduction. While much research has focused on gross and tissue-level adaptation, relatively little is known regarding the relation between cellular-level features (e.g. osteocyte lacunar density, volume and shape) and loading. The increasing availability of high resolution 3D imaging modalities, including synchrotron-based techniques, has made studying 3D cellular-level features feasible on a scale not previously possible. The primary objective of this study was to test the hypothesis that unloading (sciatic neurectomy) during growth results in altered osteocyte lacunar density in the tibial diaphysis of the rat. Secondarily, we explored a potential effect of unloading on mean lacunar volume. Lacunar density was significantly (p<0.05) lower in immobilized bones (49,642 ± 11,955 lacunae per mm(3); n=6) than in control bones (63,138 ± 1956 lacunae per mm(3); n=6). Mean lacunar volume for immobilized bones (209 ± 72 µm(3); n=6) was significantly smaller (p<0.05) than that for the control bones (284 ± 28 µm(3); n=6). Our results demonstrate that extreme differences in loading conditions, such as those created by paralysis, do indeed result in changes in osteocyte lacunar density and volume. Further investigation is warranted to examine relations between these measures and more subtle variation in loading as well as pathological states, which have been linked to alterations in mechanotransduction.

Nanoscale imaging of the bone cell network with synchrotron X-ray tomography: optimization of acquisition setup.

PURPOSE: The fundamental role of the osteocyte cell network in regulating the bone remodeling has become evident in the last years. This has raised the necessity to explore this complex three-dimensional interconnected structure, but the existing investigation methods cannot provide an adequate assessment. The authors propose to use parallel beam synchrotron radiation computed tomography at the nanoscale to image in three dimensions the osteocyte lacunocanalicular network. To this aim, the authors study the feasibility of this technique and present an optimized imaging protocol suited for the bone cell network. Moreover, they demonstrate the multifaceted information provided by this method. METHODS: The high brilliance of synchrotron radiation combined with state of art detectors permits reaching nanoscale spatial resolution. With a nominal pixel size of 280 nm, the parallel beam computed tomography setup at the ID19 experimental station of the ESRF is capable of imaging the bone lacunocanalicular network, considering that the reported diameter of canaliculi is in the range 300-600 nm. However, the actual resolution is limited by the detector and by the radiation dose causing sample damage during the scan. The authors sought to overcome these limitations by optimizing the imaging setup and the acquisition parameters in order to minimize the necessary radiation dose to create the images and to improve the spatial resolution of the detector. RESULTS: The authors achieved imaging of the osteocyte cell network in human bone. Due to the optimization of the imaging setup and acquisition parameters, they obtained simultaneously a radiation dose reduction and an increase of the signal to noise ratio in the images. This permitted the authors to generate the first three-dimensional images of the lacunocanalicular network in an area covering several osteons, the fundamental functional units in the bone cortex. The method enables assessment of both architectural parameters of the microporosity and of mineralization degree in the bone matrix. The authors found that the cell network is dense and connected inside osteonal tissue. Conversely, the cell lacunae are sparse, unorganized, and disconnected in interstitial tissue. CONCLUSIONS: The authors show that synchrotron radiation computed tomography is a feasible technique to assess the lacunocanalicular network in three dimensions. This is possible due to an optimal imaging setup in which the detector plays an important role. The authors could establish two valid setups, based on two different insertion devices. These results give access to new information on the bone cell network architecture, covering a number of cells two orders of magnitude greater than existing techniques. This enables biomedical studies on series of samples, paving the way to better understanding of bone fragility and to new treatments for bone diseases.

Radiological and histologic studies of the mandibular cortex of ovariectomized monkeys.

OBJECTIVE: The objective was to study the radiological and histologic changes in the mandibular cortices of ovariectomized monkeys. STUDY DESIGN: Twelve female, adult, Cynomolgus monkeys (Macaca fascicularis) were used. Under anesthesia, 1 group was bilaterally ovariectomized (OVX), and the other (control group) underwent sham surgery. Seventy-six weeks after surgery, the monkeys were humanely killed, their mandibles were excised, and their mandibular inferior cortices (MIC) and adjacent cortices were examined histologically and with panoramic radiographs and micro computed tomography. RESULTS: Striped shadows were seen on the endosteal side of the OVX cortices on panoramic radiographs. Histologic observation revealed many enlarged pores with eroded surfaces and calcein labeling (indicating osteon remodeling) in the OVX cortices. CONCLUSIONS: In the MIC and adjacent cortices of OVX monkeys, enlarged Haversian canals were seen and there were indications of a high rate of bone turnover. The enlarged Haversian canals resulted in striped shadows and unclear endosteal margins on radiographic images.

Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone.

Aging decreases the human femur's fatigue resistance, impact energy absorption, and the ability to withstand load. Changes in the osteocyte distribution and in their elemental composition might be involved in age-related bone impairment. To address this question, we carried out a histomorphometric assessment of the osteocyte lacunar distribution in the periosteal and endosteal human femoral cortexes of 16 female and 16 male donors with regard to age- and sex-related bone remodeling. Measurements of the bone mineral density distribution by quantitative backscattered electron imaging and energy dispersive X-ray analysis were taken to evaluate the osteocyte lacunar mineral composition and characteristics. Age-dependent decreases in the total osteocyte lacunar number were measured in all of the cases. This change signifies a risk for the bone's safety. Cortical subdivision into periosteal and endosteal regions of interest emphasized that, in both sexes, primarily the endosteal cortex is affected by age-dependent reduction in number of osteocyte lacunae, whereas the periosteal compartment showed a less pronounced osteocyte lacunar deficiency. In aged bone, osteocyte lacunae showed an increased amount of hypermineralized calcium phosphate occlusions in comparison with younger cases. With respect to Frost's early delineation of micropetrosis, our microanalyses revealed that the osteocyte lacunae are subject to hypermineralization. Intralacunar hypermineralization accompanied by a decrease in total osteocyte lacunar density may contribute to failure or delayed bone repair in aging bone. A decreased osteocyte lacunar density may cause deteriorations in the canalicular fluid flow and reduce the detection of microdamage, which counteracts the bone's structural integrity, while hypermineralized osteocyte lacunae may increase bone brittleness and render the bone fragile.

Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT.

Tomographic reconstructions of sections of human femoral bone were created from x-ray data sets taken using synchrotron radiation of 26.4 keV and with isotropic voxels 1.47 µm on a side. We demonstrate that it is possible to segment the data to isolate both the osteocyte lacunae and the Haversian canals in the bone as well as identifying osteon boundaries. From this information a wealth of data relating to bone structure becomes available. The data were used to map the spatial positions of the osteocyte lacunae, relative to the Haversian canals and of the osteon boundaries. The dimensions and volume of the imaged osteocyte lacunae were measured for close to 10,000 lacunae. When averaged over the 11 osteons measured, osteocyte densities varied from 4×10(4)per mm(3) close to the Haversian canals to about 9×10(4)per mm(3) at 80% of osteon radius. The nearest-neighbour distances varied from 10 µm to 40 µm with a peak at 23 µm and an approximately normal distribution. The distribution of lacunar long-axis length was also approximately normal with a small positive skew and the peak value was 8 µm with a range from 3 µm to 20 µm. The most significant finding from this study was that the distribution of the measured volumes of osteocyte lacunae had two distinct peaks, one at 200 µm(3) and a second at 330 µm(3).

Engineered osteochondral grafts using biphasic composite solid free-form fabricated scaffolds.

Tissue engineering has provided an alternative to traditional strategies to repair cartilage damaged by injury or degenerative disease. A successful strategy to engineer osteochondral tissue will mimic the natural contour of the articulating surface, achieve native mechanical properties and functional load-bearing ability, and lead to integration with host cartilage and underlying subchondral bone. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match articular geometry. The objective of this study was to utilize materials and biological factors in an integrated approach to regenerate a multitissue interface. Biphasic composite scaffolds manufactured by IBD and SFF fabrication were used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-L-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein 7 (BMP-7) in the ceramic phase and fully differentiated chondrocytes in the polymeric phase. After subcutaneous implantation into mice, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects.

Imaging of fine structure of bone sample with high coherent X-ray beam and high spatial resolution detector.

In this study, we observed bone specimens of the mouse using a very high coherence beam and high spatial resolution detector (zooming tube: approximately 0.7 micron resolution) and successfully obtained images of the Haversian canal, osteocytes, and osteoclasts.

Distinct anabolic response of osteoblast to low-intensity pulsed ultrasound.

Low-intensity pulsed ultrasound, a form of mechanical energy transmitted as high-frequency acoustical pressure waves, provides noninvasive therapeutic treatment for accelerating fracture repair and distraction osteogenesis. Relatively young osteoblasts respond to ultrasound by transiently upregulating message levels of immediate-early genes as well as that of osteocalcin and insulin-like growth factor I (IGF-I). Osteocytes derived from newborn rat tibia and calvaria responded to a lesser extent only in c-fos and cyclooxygenase-2 (COX-2) messages. Compared with the stretched osteocytes, which use stretch-activated and parathyroid hormone (PTH)-potentiated Ca2+ influx as an entry route to the protein kinase A (PKA) signal transduction pathways, there was no evidence of Ca2+ internalization by any of the cells tested on exposure to the ultrasound. On the other hand, inhibitors of p38 mitogen-activated protein kinase (MAPK) and upstream phosphoinositide 3-kinase (PI3K) blocked COX-2 and osteocalcin upregulation by the ultrasound-exposed ST2, murine bone marrow-derived cells. This is distinct from the aforementioned osteocytic response to low-frequency stretching and implies the involvement of integrins. Our findings suggested that accelerated fracture repair and distraction osteogenesis by the low-intensity pulsed ultrasound depend, at least in part, on the stimulation of osteoblastic cells at relatively early stages of osteogenic lineage. Bone is under control of multiple regulatory mechanisms so that diverse physical forces can be reflected to the microenvironment of each cell, in turn, to the entire bone.

[The pathogenesis of osteoradionecrosis]. / La pathogénie de l'ostéoradionécrose.

The microradiographic analysis of thick sections of fragments removed from irradiated patients suffering from osteoradionecrosis has made it possible to bring to the fore two types of bone resorption caused by cells: an osteoclastic one not followed by a relevant osteogenesis, and another, pathognomonic of postradic complications, linked with an altered activity of the osteocytes. Those cells, which have been affected, because of a progressive infection, are responsible for an irreversible widening of the osteoplasts, set in the properly vascularized bone regions, in particular in the wall of haversian canals. The coalescence of widened osteoplats causes polycylic cavities which is a typical feature of osteoradionecrosis. A third type of bone postradic damage consists in a massive demineralization, related to the presence of saliva or pus.