Low-intensity pulsed ultrasound induces apoptosis in osteoclasts: Fish scales are a suitable model for the analysis of bone metabolism by ultrasound

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3 h of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18 h of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in osteoclasts using the TUNEL (TdT-mediated dUTP nick end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3 h of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure. © 2016 Elsevier Inc.Embargo Period 12 month

Repeated freeze–thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze–thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze–thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze–thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze–thaw cycles. In patella tendons, the ultimate stress, Young’s modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze–thaw cycles. In conclusion, we identified that cells surviving after freeze–thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze–thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity

A novel underuse model shows that inactivity but not ovariectomy determines the deteriorated material properties and geometry of cortical bone in the tibia of adult rats

Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-αB ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain. © 2013 Zoological Society of Japan