Evaluation of Trabecular Microstructure of Cancellous Bone Using Quarter-Detector Computed Tomography.

Quarter-detector computed tomography (QDCT) is an ultra-high-spatial-resolution imaging technique. This study aimed to verify the validity of trabecular structure evaluation using a QDCT scanner in the diagnosis of osteoporosis. We used a cancellous bone specimen image of the second lumbar vertebrae of an adult male with moderate osteoporosis. To obtain QDCT images, we created a three-dimensional model from micro-CT images of the specimen. Statistical analysis was performed on the relationship between micro-CT and QDCT imaging modalities. The differences between micro-CT and QDCT were assessed based on their significance with respect to the calculated mean measurements using the Mann-Whitney test. Single regression analysis was performed using linear regression, with micro-CT and QDCT as the explanatory and objective variables, respectively, to determine the relationship of the measured values between the two modalities. By applying the necessary correction to the micro-CT measured values, it is possible to perform an analysis equivalent to micro-CT, which offers higher spatial resolution than QDCT. We found evidence that if QDCT can be used, trabecular structure evaluation may contribute to image diagnosis to evaluate practical bone fragility.

Influence of Teriparatide and Ibandronate on Cortical Bone in New Zealand White Rabbits: A HR-QCT Study.

Teriparatide (TPTD) is known to increase the cortical thickness and porosity. The purpose of the present study was to determine whether switching from TPTD to ibandronate (IBN) would be useful for improving cortical bone parameters as assessed using high-resolution quantitative computed tomography (HR-QCT) analyses in mature rabbits. Forty-two female New Zealand white rabbits (18-22 weeks old) were randomized into six groups of 7 animals each as follows: 4-week vehicle administration group, 4-week TPTD administration group (20 µg/kg, subcutaneously [s.c.], daily), 12-week vehicle administration group, 4-week TPTD administration + 8-week vehicle administration group, 4-week TPTD administration + 8-week lower-dose IBN administration group (20 µg/kg, s.c., every 4 weeks), and 4-week TPTD administration + 8-week higher-dose IBN administration group (100 µg/kg, s.c., every 4 weeks). After the 4- or 12-week experimental period, the cortical bone of the distal femoral diaphysis was processed for HR-QCT analysis. The 4-week TPTD administration increased the pore ratio, number, and density as well as the cortical area, thickness, and bone mineral content (BMC), without significant influencing the volumetric bone mineral density (BMD). The 4-week TPTD administration + 8-week vehicle administration decreased the pore ratio, number, and density as well as the cortical area and thickness, compared with the 4-week TPTD administration, but the pore ratio, cortical area, and thickness were still higher compared with the 12-week vehicle administration. The 4-week TPTD administration + 8-week higher-dose IBN administration, but not the 4-week TPTD administration + 8-week lower-dose IBN administration, increased the cortical area, thickness, BMC, and volumetric BMD and decreased the pore ratio, but not the pore number or density, compared with the 4-week TPTD administration + 8-week vehicle administration. These results suggest that higher-dose IBN after TPTD therapy has a beneficial effect on the BMC, volumetric BMD, cortical area, thickness, and porosity in mature rabbits.

Osteocyte-directed bone demineralization along canaliculi.

The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno-canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality.

Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus.

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through 'osteogenic capillaries' in a Fosl1-regulated manner.

Trabecular microfractures in the femoral head with osteoporosis: analysis of microcallus formations by synchrotron radiation micro CT.

Trabecular bone microfracture pathogenesis and associated healing processes are not well understood. We analyzed the microcalluses that form subsequent to microfractures in patients with osteoporosis (OP) using synchrotron radiation micro CT (SRCT). Subchondral bone columns were extracted from the femoral heads of 11 female patients with a femoral neck fracture. SRCT scanning was performed with 5.9×5.9×5.9 µm3 voxel size and the microcallus number was measured in a 5-mm cubic subchondral bone region. The trabecular bone microstructure was measured and its relationship to the microcallus number was analyzed. In addition, the degree of mineralization of the microcallus region and that of the rest of the trabecular bone were measured and compared. Microcallus formations were detected in all cases, with a mean microcallus number of 4.9 (range, 2-11). The microcallus number had a significantly negative correlation with bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and degree of mineralization, and had a positive correlation with specific bone surface (BS/BV). The degree of mineralization of the microcallus region was lower than that of the rest of the trabecular bone and had a wider range of values. Microcallus formations were frequently detected in patients with OP, and more prevalent in the bone with thinner trabeculae, suggesting microfractures might occur due to activities of daily living as the OP progresses. The degree of mineralization of microcallus might represent the process of bone healing from immature woven bone to mature trabecular bone.

Multidetector-row computed tomography is useful to evaluate the therapeutic effects of bisphosphonates in glucocorticoid-induced osteoporosis.

Osteoporosis is one of the major complications of glucocorticoid therapy. Osteoporosis is usually defined by the levels of bone mineral density (BMD) assessed by dual energy X-ray absorptiometry (DEXA); however, glucocorticoids often induce fractures in patients with normal BMD. Thus, novel diagnostic approaches are required. In this study, we examined whether multidetector-row computed tomography (MDCT) is useful to assess the bone status in glucocorticoid-induced osteoporosis (GIO). Because bisphosphonates have been proven to prevent bone fracture in GIO, we tried to detect the therapeutic effects of bisphosphonates in GIO by MDCT. Fifteen Japanese patients with immunoglobulin A nephropathy who had normal renal function were enrolled in this open-label randomized trial. Patients were randomly divided into three groups-calcitriol (VD), menatetrenone (VK), or bisphosphonate (Bis). Bone conditions were analyzed twice by three different methods-bone turnover markers, DEXA, and MDCT-at the start and 6 months after the start of therapy. Both bone markers and DEXA could not detect significant differences among the therapeutic groups; however, MDCT-based analyses detected the preventive effects of bisphosphonates in GIO. Compared to VD, Bis improved structural indices, such as bone volume fraction, trabecular separation, marrow star volume, and structure model index whereas the difference between VD and VK was not significant. Finite element analysis revealed that simulated fracture load in the Bis group was significantly improved. These findings suggested that MDCT-based assessment is superior to bone markers and/or DEXA in assessing the therapeutic effect of bisphosphonates on GIO.

Talbot-defocus multiscan tomography using the synchrotron X-ray microscope to study the lacuno-canalicular network in mouse bone.

The three-dimensional network of lacunae and canaliculi that regulates metabolism in bone contains osteocytes and their dendritic processes. We constructed a synchrotron radiation X-ray microscope for sequential tomography of mouse tibia first by using a Talbot interferometer to detect the degree of bone mineralization and then by using absorption contrast under a slightly defocused setting to enhance outline contrast thereby visualizing structures of the osteocyte lacuno-canalicular network. The resultant pair of tomograms was precisely aligned with each other, allowing evaluation of mineral density in the vicinity of each osteocyte lacuna and canaliculus over the entire thickness of the cortical bone. Thus, multiscan microscopic X-ray tomography is a powerful tool for analyzing bone mineralization in relation to the lacuno-canalicular network at the submicron resolution level.

Three-dimensional ultrastructural study of oil and astaxanthin accumulation during encystment in the green alga Haematococcus pluvialis.

Haematococcus pluvialis is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of H. pluvialis. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%). In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%), in which the total chloroplast volume was markedly decreased (9.7%). Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage.

An approach to normalizing micro-CT depth profiles of mineral density for monitoring enamel remineralization progress.

To indicate the possibility of a new approach to creating mineral density profiles, and to examine longitudinal changes in 'the rate of remineralization (RA)' and 'the mineral density (DAs) at 4 different depths' (surface zone: SZ, lesion body: LB, middle zone: MZ, deep zone near to sound area: DZ) in enamel subsurface lesions, eight demineralized bovine enamel-dentin blocks were remineralized for 1 to 4 week and investigated using Micro-focus X-ray CT (micro-CT). After CT scanning, mineral density profiles were created.Mineral densities at each depth after demineralization were SZ is approximately equal to LBMZ>DZ. This study indicated a new approach to create a mineral density profile and suggested the greater the value of the mineral density before the remineralization, the smaller the mineral density increments.

[Osteocytic osteolysis : measurements of the volume of osteocytic lacunae].

Osteocytes are present in osteocytic lacunae in both cortical and trabecular bone, where they are interconnected by numerous dendrites in osteocytic canaliculi. In mammals, osteocytes are the most abundant bone cells, outnumbering osteoclasts and osteoblasts. Osteoclasts are the primary bone-resorbing cells ; however, the concept that osteocytes resorb bone by a process called osteocytic osteolysis has been postulated to explain dynamic calcium release from bone in conditions as diverse as parathyroid hormone (PTH) stimulation, hibernation, glucocorticoid stimulation, and lactation. Osteocytic osteolysis remains a controversial concept, mainly because it is difficult to demonstrate experimentally. Recently, novel functions of osteocytes in mineral metabolism and bone remodeling have been reported, and osteocytic osteolysis is being examined more closely experimentally. This review discusses published literature relevant to osteocytic osteolysis and compares 2D and 3D measurements of the volume of osteocytic lacunae, which serve as anatomical evidence for "periosteocytic osteolysis" .

Relationship between microstructure and degree of mineralization in subchondral bone of osteoarthritis: a synchrotron radiation µCT study.

We analyzed the microstructure and degree of mineralization of the subchondral trabecular bone in hip osteoarthritis (OA) using synchrotron radiation computed tomography (SRCT) to identify the relationship between bone structure and bone turnover. Subchondral bone samples were extracted from femoral heads of 10 terminal-staged hip OA patients. The SRCT scan was performed at 30 keV energy and 5.9 µm voxel size. Trabecular bone structure, bone cyst volume, and the degree of trabecular bone mineralization were measured, and correlations between bone structure and the degree of mineralization were analyzed. In addition, the trabecular bone was divided into the area immediately surrounding the bone cyst and the remaining area, and they were compared. The average cyst volume fraction in the whole region was 31.8%, and the bone volume fraction in the bone region was 55.6%. Cyst volume was the only structural parameter that had a significant correlation with the degree of mineralization. The degree of mineralization was diminished when the bone cyst was larger (r = -0.81, p = 0.004). The trabecular bone immediately surrounding the bone cyst had a lower degree of mineralization when compared with the remaining trabecular bone (p = 0.008). In the bone sclerosis of OA subchondral bone, there are many large and small bone cysts, which are expected to play a significant part in the high bone turnover of OA.

Wear of resin composites and primary enamel and their applicability to full crown restoration of primary molars.

PURPOSE: To determine whether resin composites are appropriate for full crown restoration of primary molars by evaluating their wear characteristics. Specifically, the wear properties of resin composite specimens and the opposing enamel surfaces were characterized by means of impacting-sliding wear testing. METHODS: Three types of light-cured resin composites (Estelite Sigma quick, Litefill IIP, and Metafil C), one type of chemical-cured resin composite (Clearfil FII), and a hybrid composite (Estenia C&B) were tested in this study. The enamel sample was used as the control. The hemispherically prepared specimens were subjected to impacting-sliding wear testing against the flattened enamel of primary molars. The worn surfaces were examined by laser scanning microscopy and scanning electron microscopy. The volumetric loss was estimated by using micro-CT images. The areas of worn enamel surfaces were measured by 3D color laser microscopy. On most of the worn enamel surfaces, cracks appeared. Scatter plot analyses between their width and depth were carried out. Data for each specimen were statistically analyzed by multiple comparisons among the means of treatment by Bonferroni's method (P< 0.01). RESULTS: Clearfil showed significantly higher surface area wear, volumetric loss, and worn enamel surface area than did the other resin composites and the control enamel (P< 0.01). There was no significant difference among the worn surface areas ofEstelite, Litefill, Metafil, and Estenia (P< 0.01). The control enamel showed significantly lower worn surface area than did the resin composites (P< 0.01). There was no significant difference in volumetric loss and worn enamel surface areas among Estelite, Litefill, Metafil, Estenia, and the control enamel (P< 0.01). Cracks larger than that on the control enamel were seen on the worn enamel surface opposing Estenia.

Multiple myeloma: microstructural analysis of lumbar trabecular bones in patients without visible bone lesions--preliminary results.

PURPOSE: To evaluate the accuracy of trabecular analysis of the lumbar spine by using multidetector computed tomography (CT) in differentiating multiple myeloma patients without spinal fractures from control subjects. MATERIALS AND METHODS: This retrospective single-institution study was approved by the institutional review board, with a waiver of informed consent. Spinal microarchitecture was examined by using 64-detector row CT in 29 patients with multiple myeloma and 51 control subjects. The patients were 13 men and 16 women (mean ages, 71.1 and 69.2 years, respectively). By using multidetector CT data, eight parameters were calculated for the L3 vertebral trabeculae with a three-dimensional image analysis system. The χ(2) test was used to select a preliminary set of predictors for multiple myeloma. A multivariable generalized linear model was constructed to identify parameters that could be used to differentiate between patients and controls. Parameters with findings of P < .05 were included in the multivariable model. RESULTS: Generalized linear models showed that mean trabecular thickness (TbTh) (patients, 703.7 µm ± 46.7 [standard deviation]; controls, 661.1 µm ± 35.7) and fractal dimension (FD) (patients, 2.23 ± 0.24; controls, 2.41 ± 0.15) in men (P = .05 and .03, respectively) and degree of anisotropy (DA) (patients, 1.63 ± 0.23; controls, 1.38 ± 0.10) in women (P = .02) had significant effects in differentiation between patients and controls. CONCLUSION: Significant differences were observed in spinal microarchitecture between control subjects and multiple myeloma patients without pathologic fractures. Independent predictors of multiple myeloma included TbTh and FD in male patients and DA in female patients.

Impaired vibration of auditory ossicles in osteopetrotic mice.

In the middle ear, a chain of three tiny bones (ie, malleus, incus, and stapes) vibrates to transmit sound from the tympanic membrane to the inner ear. Little is known about whether and how bone-resorbing osteoclasts play a role in the vibration of auditory ossicles. We analyzed hearing function and morphological features of auditory ossicles in osteopetrotic mice, which lack osteoclasts because of the deficiency of either cytokine RANKL or transcription factor c-Fos. The auditory brainstem response showed that mice of both genotypes experienced hearing loss, and laser Doppler vibrometry revealed that the malleus behind the tympanic membrane failed to vibrate. Histological analysis and X-ray tomographic microscopy using synchrotron radiation showed that auditory ossicles in osteopetrotic mice were thicker and more cartilaginous than those in control mice. Most interestingly, the malleal processus brevis touched the medial wall of the tympanic cavity in osteopetrotic mice, which was also the case for c-Src kinase-deficient mice (with normal numbers of nonresorbing osteoclasts). Osteopetrotic mice showed a smaller volume of the tympanic cavity but had larger auditory ossicles compared with controls. These data suggest that osteoclastic bone resorption is required for thinning of auditory ossicles and enlargement of the tympanic cavity so that auditory ossicles vibrate freely.

Fra-1/AP-1 impairs inflammatory responses and chondrogenesis in fracture healing.

Inflammation inevitably follows injury of various tissues, including bone. Transgenic overexpression of Fra-1, a component of the transcription factor activator protein-1 (AP-1), in various tissues progressively and globally enhances bone formation, but little is known about the possible effects of Fra-1/AP-1 on fracture healing. We created a transverse fracture of the mouse tibial diaphysis and examined fracture healing radiologically, histologically, and immunologically. Strikingly, fracture union was delayed even though the bone formation rate in callus was higher in Fra-1 transgenic (Tg) mice. In these mice, chondrogenesis around the fracture site was impaired, resulting in accumulation of fibrous tissue, which interferes with the formation of a bony bridge across the callus. Curiously, immediately after fracture, induction of the inflammatory mediators TNF-alpha, interleukin (IL)-6, and Cox-2 was significantly suppressed in Fra-1 Tg mice followed, by the reduced expression of Sox-9 and BMP-2. Because serum prostaglandin E(2) (PGE(2)) levels were dramatically low in these mice, we administered PGE(2) to the fracture site using a slow-release carrier. The accumulation of fibrous tissue in Fra-1 Tg mice was significantly reduced by PGE(2) administration, and chondrogenesis near the fracture site was partially restored. These data suggest that the Fra-1-containing transcription factor AP-1 inhibits fracture-induced endochondral ossification and bony bridge formation presumably through suppression of inflammation-induced chondrogenesis.